Polysaccharides of Microbial Origin: Biomedical Applications 3030422143, 9783030422141
This book provides a comprehensive analysis of microbial polysaccharides, their current uses, and highlights biomedical
127 72 30MB
English Pages 1320 [1289] Year 2022
Preface
Contents
About the Editors
Contributors
1 Introduction
1 Context
References
Part I: Bacterial Polysaccharides
2 Glucans
1 Introduction
2 Glucans and the Immune System: Firsts Experiments
3 Immune Responses
3.1 Basis of the Immune Response to Polysaccharides
3.2 Dectin-1 Receptor
3.2.1 Other Dectin-1 Triggers
3.3 Glucan as Immunoadjuvant
3.3.1 Oral Administration
4 Final Considerations
References
3 Levan
1 Introduction
2 Extraction, Isolation, Purification and Production Advanced Processes
2.1 Production by Fermentation
2.2 Enzymatic Production
3 Structural, Physicochemical, and Biological Properties
3.1 Structural Properties
3.2 Rheology
3.3 Degree of Branching
3.4 Molecular Weight
3.5 Adhesive
3.6 Anti-Inflammatory and Antioxidant Activity
3.7 Immunostimulant
3.8 Description of the Advanced Functionalization and Modification Methods
4 Applications and Uses in Medical and Pharmaceutical Fields
4.1 Healing Damaged Tissue
4.2 Cholesterol
4.3 Diabetes
4.4 Anticancer Activity
4.5 Calcium Absorption
4.6 Toxicity Decrease
4.7 Antimicrobial
4.8 Ulcer Treatment
4.9 Pharmaceuticals
5 Conclusions
References
4 Gums
1 Introduction
2 Production of Microorganism Gums
3 Antimicrobial Properties
4 Antioxidant Action
5 Anticancer Activity
6 Healing Capacity
7 Drug Delivery
8 Challenges in Microbial Gums
9 Conclusions
References
5 The Promise and Challenge of Microbial Alginate Production: A Product with Novel Applications
1 Introduction
2 Biosynthesis of Alginates and Their Biological Functions in Producing Bacteria
2.1 Postpolymerization Modification of Alginate
2.2 Biological Role of Alginate Production
3 Industrial Production of Microbial Alginate: Challenges and Opportunities
3.1 Technical Challenges in Alginate Production from A. vinelandii
3.2 Nonpathogenic Strains of Pseudomonas as Promising Alginate Producers
4 Applications of Alginate in Food, Pharmaceutical, and Biomedical Sectors
4.1 Alginate Applications in Food Sectors
4.2 Pharmaceutical and Biomedical Applications of Alginate
5 Conclusion
References
6 Kefiran
1 Introduction
2 Kefiran from Kefir Grains and Kefir
3 Kefiran Structure
4 Kefiran from Microorganisms Isolated from Kefir Grains
5 Kefiran Production for Industrial Application
6 Biological Activity of Kefiran
6.1 Antimicrobial Activity
6.2 Bifidogenic Activity
6.3 Modulation of Immune and Inflammatory Response
6.4 Antitumoral
6.5 Other Biological Activities
7 Physicochemical and Functional Properties of Kefiran
7.1 Kefiran as Thickener Agent
7.2 Kefiran Gels
7.3 Kefiran-Based Films
7.4 Kefiran in Emulsions
8 Biomedical Application of Kefiran
9 Conclusion
References
7 Microbial Glucuronans and Succinoglycans
1 Introduction
2 Glucuronans
2.1 Sources and Structures of Microbial Glucuronans (GP)
2.2 Enzymatically and Chemically Oxidized Glucans
2.3 Enzymes Acting on Glucuronans
3 Succinoglycans
3.1 Sources and Structures of Microbial Succinoglycans
3.2 Enzymes Acting on Succinoglycan
4 Rheology of Glucuronan and Succinoglycan
5 Conclusions
References
8 Cyanobacterial Extracellular Polymeric Substances (EPS)
1 Introduction
2 Cyanobacterial Extracellular Polymeric Substances (EPS) and Their Biological Roles
3 Biosynthesis
3.1 First Steps and Modification Enzymes
3.2 Assembly and Export
3.3 Regulatory Mechanisms
4 Optimization of Production and Isolation Procedures
5 Composition and Structure
5.1 Tailoring
6 Biotechnological Applications
6.1 Water Treatment and Soil Conditioning
6.2 Food, Personal Care, and Other Industries
6.3 Biomedical
7 Conclusions and Future Perspectives
References
9 Glycosaminoglycans
1 Introduction
2 Structural Features
2.1 Heparin (HEP) and Heparan Sulfate (HPS)
2.2 Chondroitin Sulfate (CS) and Dermatan Sulfate (DS)
2.3 Keratan Sulfate (KS)
2.4 Hyaluronan/Hyaluronic Acid (HA)
3 Biosynthesis
4 Industrial Production of GAGs
5 Bacterial Production of GAGs
5.1 Bacterial Hyaluronan
5.2 Bacterial Chondroitin
5.3 Bacterial Heparosan
6 Properties and Clinical Applications
6.1 Hyaluronan
6.2 Chondroitin Sulfate and Dermatan Sulfate
6.3 Heparin and Heparan Sulfate
6.4 Keratan Sulfate
7 Conclusions and Future Trends
References
Part II: Fungal and Microalgal Polysaccharides
10 Botryosphaeran
1 Introduction
2 Chemical Structure of Botryosphaeran
2.1 A Family of Botryosphaerans
2.2 Sulfonylation of Botryosphaeran: Anticoagulant and Antiviral Activities
3 Antimutagenicity and Chemopreventive Effect of Botryosphaeran
4 Anti-Obesogenic Activity of Botryosphaeran
5 Hypoglycemia and Hypocholesterolemia Exhibited by Botryosphaeran
5.1 Improving the Diabetic Condition
5.2 Improving the Dyslipidemic Condition
6 Anticancer Activity of Botryosphaeran
7 Conclusion and Future Perspectives
References
11 Chitin and Its Derivatives
1 Introduction
2 Chitin Extraction and Structure
3 Chitin and Chitosan Properties
4 Chitin and Chitosan-Based Matrices: Processability and Chemical Modification
5 Biomedical Applications of Chitin and its Derivatives
5.1 Skin Regeneration
5.2 Bone Repair
5.3 Cartilage Regeneration
5.4 Infectious Diseases
6 Conclusions
References
12 Chitosan
1 Fungi Chitosan
1.1 Production
1.2 Extraction and Isolation
1.3 Purification and Production Advanced Processes
2 Biochemical and Biological Characteristics
2.1 Physicochemical Properties
2.1.1 Degree of Deacetylation
2.2 Biological Properties
3 Biotechnology Applications
3.1 Applications and Uses in the Pharmaceutical Industry
3.2 Applications and Uses in Biomedical Fields
4 Conclusion
References
13 An Insight into Pullulan and Its Potential Applications
1 Introduction
2 Physicochemical Properties of Pullulan
3 Fermentative Production of Pullulan
3.1 Microbial Sources of Pullulan
3.2 Mechanism of Pullulan Synthesis
3.3 Upstream and Downstream Processing for Pullulan
3.4 Factors Influencing Pullulan Production
3.4.1 Fermentation Media Supplements
Carbon Source
Nitrogen Source
Supplements
3.4.2 Fermentation Type
3.4.3 Bioreactor Configuration and Operation
3.4.4 Fermentation Time
3.4.5 Microbial Culture
3.4.6 Oxygen Intensity
3.4.7 pH
3.4.8 Temperature
4 Characterization of Pullulan
4.1 Structural Characterization
4.2 Molecular Weight
4.3 Thermal and Mechanical Properties
5 Surface Derivatization Approaches for Pullulan
5.1 Polyionic Derivatives
5.2 Cross-Linking
5.3 Hydrophobic Modification
5.4 Grafting
6 Recent Applications and Uses in Biomedical and Pharmaceutical Fields
6.1 Pharmaceutical Formulations
6.2 Tissue Engineering
6.3 Targeted Drug Delivery
6.4 Gene Delivery
7 Future Perspectives
8 Conclusion
References
14 Scleroglucan and Schizophyllan
1 Introduction
2 Structural Description
3 Physicochemical Aspects
3.1 Properties of Scleroglucan
3.1.1 Properties of Scleroglucan Aqueous Solutions
3.1.2 Rheological Properties of Scleroglucan
3.1.3 Physiological Properties of Scleroglucan
3.2 Properties of Schizophyllan
3.2.1 Properties of Dilute Schizophyllan Solutions
3.2.2 Behavior of Concentrated Schizophyllan Solutions
3.2.3 Gelation Behavior of Schizophyllan
4 Production and Isolation of Scleroglucan and Schizophyllan
4.1 Production and Isolation of Scleroglucan
4.1.1 Biosynthesis of Scleroglucan
4.1.2 Fermentation Conditions for Scleroglucan Production
4.1.3 Downstream Processing for Recovery of Scleroglucan
4.2 Production and Isolation of Schizophyllan
5 Applications for Scleroglucan and Schizophyllan
5.1 Applications in the Food Industry
5.1.1 As Stabilizers, Viscosifiers/Thickeners, and Gelling Agents
5.1.2 Preparation of Edible Films
5.1.3 Production of Functional Foods
5.2 Biomedical/Pharmaceutical Applications
5.2.1 As Immunostimulants in Anticancer Activities
5.2.2 As Immunomodulators in Antitumor Activities
5.2.3 As Antiviral and Antimicrobial Agents
5.2.4 Hypocholesterolemic and Hypoglycemic Effects
5.2.5 Excipients
5.2.6 As Carriers for Targeted Delivery of Drugs and Bioactive Compounds
6 Conclusion/Perspectives
References
15 Sulfated Seaweed Polysaccharides
1 Introduction
2 Sulfated Polysaccharides: Their Properties
2.1 Fucoidan
2.2 Carrageenan
2.3 Agar
2.4 Ulvan
3 Bioactive/Biological Properties of Sulfated Polysaccharides
3.1 Fucoidan
3.2 Carrageenan
3.3 Agar
3.4 Ulvan
4 Biomedical Application
4.1 Fucoidan
4.2 Carrageenan
4.3 Agar
4.4 Ulvan
5 Conclusions and Final Remarks
References
16 Polysaccharides Produced by Microalgae
1 Introduction
2 General Structures of Polysaccharides from Commercial Microalgae
3 Commercial Microalgae
3.1 Species
3.2 Spirulina
3.3 Chlorella
3.4 Nannochloropsis
3.5 Tetraselmis
3.6 Isochrysis
3.7 Thalassiosira
3.8 Dunaliella
3.9 Phaeodactylum
3.10 Porphyridium
3.11 Botryococcus
3.12 Chaetoceros
3.13 Chlamydomonas
3.14 Pavlova
3.15 Scenedesmus
3.16 Synechococcus
3.17 Conclusion
4 Conclusions
References
17 Microalgae Polysaccharides with Potential Biomedical Application
1 Introduction
2 Polysaccharides and Extracellular Polymeric Substances Founded in Microalgae
3 The Cultivation Conditions that Increase Polysaccharides or Extracellular Polymeric Substances Production in Microalgae
4 Extraction and Purification of Microalgae Polysaccharides or Extracellular Polymeric Substances
5 Uses of Polysaccharides or Extracellular Polymeric Substances from Microalgae in Biomedical Applications
5.1 Antitumoral
5.2 Antioxidant
5.3 Antiviral
5.4 Anti-Inflammatory
5.5 Other Activities
6 Final Considerations
References
18 Fungal Polysaccharide Production for Dermatological Purposes
1 Introduction
1.1 Fungal Conservation to Protect Species of Biotechnological Interest
1.1.1 Ecological Role and Human Interaction
1.1.2 Agaricus subrufescens, and Important Biotechnological Species
2 Laboratory Fungal Cultivation and Scale-Up
2.1 Biomass Production
2.2 Culture Media and Inoculation
2.3 Bioreactors for Submerged Cultivation of Basidiomycetes
2.4 Operation of Bioreactors for the Cultivation of Basidiomycetes
2.5 Mechanical Dispersion of Pellets During Cultivation
3 Bioactive Polysaccharides
3.1 Dermatological Activities
3.2 Cosmeceuticals
3.3 Dermatological Use of Agaricus subrufescens Polysaccharides
4 Conclusion
References
Part III: Isolation, Extraction, Purification, and Production Processes
19 Exopolysaccharides from Lactic Acid Bacteria
1 Introduction
2 Physiological Roles of Exopolysaccharides
3 Chemical Classification of Exopolysaccharides
4 Biosynthesis of EPS
4.1 The Extracellular Synthesis Pathway
4.1.1 Levan Biosynthesis
4.1.2 Dextran Biosynthesis
4.2 The Wzx/Wzy-Dependent Pathway
5 Production and Isolation of Exopolysaccharides
6 Chemical Characterization of Exopolysaccharides
7 Techno-Functional Properties of Exopolysaccharides
8 Biological Properties of Exopolysaccharides
9 Industrial Applications and Commercialization of Exopolysaccharides
10 Interactions Between EPS and Proteins
11 Conclusion
References
20 Isolation of Microbial Polysaccharides
1 Introduction
2 Physical Properties and Chemical Structure
3 Polysaccharides Screening Approaches
3.1 Screening Approaches for Solid Media
3.1.1 Detection of EPS-Producing Phenotypes
3.1.2 Agar-Plates with Dyes
3.2 Screening Approaches for Liquid Media
3.2.1 Precipitation
3.2.2 Viscosity
3.2.3 Specific Carbohydrate Screening
4 Extraction of Polysaccharides from Microorganisms
4.1 Extraction of Exopolysaccharides Existing as Slime
4.2 Extraction of Exopolysaccharide Existing as a Capsule
5 Precipitation of EPS
5.1 Precipitation Methods
6 Determination of Precipitation Yield
7 Purification of EPS
8 Conclusions
References
21 Biosynthesis of Bacterial Polysaccharides
1 Introduction
2 Wzx/Wzy-Dependent Pathway
2.1 O-Specific Polysaccharides
2.2 Capsular Polysaccharides
3 ABC Transporter-Dependent Pathways
3.1 O-Specific Polysaccharides
3.2 Capsular Polysaccharides
3.3 Other Glycopolymers
4 Synthase-Dependent Pathway
4.1 Exopolysaccharides
4.2 Capsular Polysaccharides
4.3 O-Specific Polysaccharides
5 Single Sucrase Pathway
6 Conclusions
References
Part IV: Polysaccharide properties, Functionalizations, and Modifications
22 Polysaccharides of Fungal Origin
1 Introduction: A History of C. neoformans
2 Comparison of Bacterial and Fungal Polysaccharide Synthesis, Transport, and Attachment
3 Current Understanding of Cryptococcal Polysaccharides
3.1 Biophysical Characterization
3.2 Structural Characterization
3.3 Immunological Characterization
4 Future Application and Technologies for the Study of Cryptococcal Polysaccharides
5 Conclusion
References
23 Bioactivities of Bacterial Polysaccharides
1 Introduction
2 Polysaccharide Structure
2.1 Polysaccharide Synthesis
2.2 EPS Activities and Uses
3 Antibacterial Activity of Bacterial Polysaccharides
4 Antiviral Activity of Bacterial Polysaccharides
5 Anticancer activity of Bacterial Polysaccharides
6 Other Bioactivities
6.1 Cholesterol-Lowering Effect
6.2 Antioxidant Effect
6.3 Immunomodulator Effect
7 Production and Extraction of Polysaccharides of Microbial Origin
8 Conclusion
References
24 Surface Properties of Polysaccharides
1 Introduction
2 Production, Extraction, and Purification/Isolation of Exopolysaccharides
3 Physicochemical Properties of Microbial Polysaccharides
3.1 Dextran
3.2 Xanthan
3.3 Gellan
3.4 Cellulose
3.5 Chitin and Chitosan
3.6 Curdlan
3.7 Alginate
3.8 Other Examples
4 Surface Modification and Functionalization of Microbial Polysaccharides
4.1 Copolymerization
4.2 Cross-Linking
4.2.1 Physical Cross-Linking
4.2.2 Chemical Cross-Linking
4.3 Functionalization
5 Applications of Microbial Polysaccharides
5.1 Environmental Applications
5.2 Biological and Pharmaceutical Applications
5.3 Tissue Engineering Applications
5.4 Food Applications
6 Concluding Remarks and Future Perspectives
References
25 Antioxidant and Antibacterial Activities of Polysaccharides
1 Introduction
2 Types of In Vitro Antioxidant Activity
3 Antioxidant Activity of Polysaccharides
4 Mechanism and Factors Affecting the Antioxidant Activity of Polysaccharide
5 Antibacterial Activity of Polysaccharides
6 Mechanism and Factors Affecting the Antibacterial Activity of Polysaccharides
7 Conclusion
References
26 Gel Properties of Microbial Polysaccharides
1 Introduction
2 Microbial Polysaccharides
3 Gels
4 Hydrogels
4.1 Classification of Hydrogels
5 Gel Properties of Microbial Polysaccharides
5.1 Conditions of Gel Formation
5.2 Physical Properties
5.3 Stability of Polysaccharide Gels
5.4 Biological Properties
5.5 Swelling Properties
5.6 Rheological Behavior
5.7 Structure
5.8 Syneresis
5.9 Aging
5.10 Common Microbial Polysaccharides and Their Gels
5.10.1 Xanthan Gum
5.10.2 Gellan Gum
5.10.3 Dextran
5.10.4 Hyaluronic Acid
5.10.5 Levan
5.10.6 Alginate
5.10.7 Curdlan
5.10.8 Pullulan
6 Conclusions
References
27 Polysaccharides in Bacterial Biofilm
1 Introduction
2 Polysaccharides in Oral Biofilms
2.1 Intracellular and Extracellular Polysaccharides
2.2 Understanding the Role of Sucrose in Oral Biofilms
3 Extracellular Polysaccharides in Oral Biofilms
3.1 Glucosyltransferase (GTF) and Fructosyltransferase (FTF): Genes and Enzymes
3.2 Glucosyltransferase (GTF) and Fructosyltransferase (FTF): Mechanism of Action
3.3 Types of Extracellular Polysaccharides (EPS): Insoluble and Soluble
3.3.1 Insoluble EPS
α-(13) Glucan (Mutan)
3.3.2 Soluble EPS
α-(16) Glucan (Dextran)
β-(26) Fructan (Levan)
β-(21) Fructan (Inulin)
4 Intracellular Polysaccharides (IPS)
4.1 IPS Metabolism
4.2 The Role of IPS and Its Importance in Oral Biofilms
5 Conclusion
References
28 Bioactive Polysaccharides from Microalgae
1 Introduction
2 Polysaccharides from Microalgae
2.1 Cyanophyta
2.2 Chlorophyta
2.3 Rhodophyta
2.4 Select Chromistan Algae
3 Conclusions
References
29 Polysaccharides of Biomedical Importance from Genetically Modified Microorganisms
1 Introduction
2 Types of Polysaccharides
2.1 Food Storage Polysaccharides
2.2 Structural Polysaccharides
2.3 Mucoid Polysaccharides
3 Naturally Occurring Polysaccharides
4 Microbial Polysaccharides
4.1 Bacterial Exopolysaccharides
4.1.1 Bacterial Polysaccharides Used for Industrial Applications
Xanthan Gums
Gellan Gum
Dextran
Cellulose
5 Synthetic Polysaccharides
6 Biological Macromolecules
7 Biomedicine
7.1 Proteins
7.2 Carbohydrates
7.3 Lipids
7.4 Nucleic Acids
7.5 Synthetic Macromolecules in Biomedicine
7.6 Synthetic Plastics
7.7 Polyurethane
7.8 Silicon Gels
8 Fungi as a Source of Macromolecules
9 Genetically Modified Organisms
10 Polysaccharides Produced by Genetically Modified Microorganisms
11 Bacteria as the Most Preferred Models
12 Polymers Produced Through Genetic Engineering Technologies
13 Risks Associated with Genetic Engineered Polymers
14 Why Genetic Engineering?
15 The Downstreaming Process, Easy or Hard?
16 Conclusion
References
30 Sulfation of Microbial Polysaccharides
1 Introduction
2 Sulfation Reactions
2.1 Modification Reactions
2.2 Reaction of Sulfation
2.3 Tools to Support the Study of New and Modified Polysaccharides
3 Final Remarks
References
Part V: Pharmaceutical Applications
31 Dextran Pharmaceutical Applications
1 Introduction
2 Origin of Dextran
3 Structure and Physicochemical Properties
4 Reactivity
5 Pharmacokinetic Fate
6 Dextran Conjugates
6.1 Irreversible Dextran Conjugates
6.1.1 Dextran Enzyme Conjugates
6.1.2 Dextran Small-Molecule Complexes
6.1.3 Dextran Metal Complexes
6.2 Reversible Dextran Conjugates/Prodrugs
7 Modes of Covalent Drug Attachment to Dextran
7.1 Dextran Conjugates for Lowering Ulcerogenicity of Nonsteroidal Anti-Inflammatory Drugs
7.2 Dextran Prodrugs for Enhancement of Water Solubility
7.3 Dextran Prodrugs in Colon-Specific Delivery
7.4 Liver-Specific Drugs Using Dextran as a Carrier
7.5 Anticancer Dextran-Drug Conjugates
8 Drug Delivery Systems of Dextran Conjugates
8.1 Lymphocytokinin-Dextran Conjugate
8.2 Dextran-Magnetic Layered Composite Hydroxide Fluorouracil Targeting Lipid
8.3 Dextran Hydrogels
8.4 Bioadhesive Oral Delivery Systems of Dextran
8.5 Conjugates of Dextran in Production of Micelles
8.6 Functionalized Dextran Aldehyde-Drug Conjugate
9 Common Methods for Synthesis of Dextran Conjugates
9.1 Direct Esterification
9.2 Carbonyldiimidazole Activation Method
9.3 Carbonate or Carbamate Ester Method
9.4 Periodate Oxidation Method
9.5 Cyanogens Bromide Activation Method
9.6 Etherification of Dextran
10 Applications of Dextran and Its Derivatives
10.1 Medical Uses
10.1.1 Antithrombotic Effect
10.1.2 Usage in Intravenous Fluids
10.1.3 DNA Transfection
10.1.4 Iron Dextran
10.1.5 Hysteroscopy
10.1.6 Antimicrobial Activity
10.1.7 Anticoagulant Activity
10.1.8 Volume Expansion
10.1.9 Antioxidant Properties and Immunomodulatory Potential
10.1.10 Acute Dengue Infection
10.1.11 Dextran-Based Hydrogels as Drug Delivery Systems
10.1.12 Tissue Engineering
10.2 In Food Industry
10.2.1 Bakery Products
10.2.2 Confectionery
10.2.3 Ice Cream
10.2.4 Frozen and Dried Foods
10.2.5 Fermented Dairy Products
10.2.6 Reduced-Fat Cheese
10.2.7 Prebiotics
10.3 In Photographic Industry
10.4 In the Field of Cosmetics
10.5 Waste Water Management
10.6 Laboratory Uses
11 Toxic Effects of Dextran Sulphate
11.1 Alopecia
11.2 Changes in Nails
11.3 Diarrhoea
11.4 Reduction in Platelets
11.5 Hydrocephalus
12 Conclusion
References
32 Polysaccharides in Cancer Therapy
1 Introduction
2 Tiny Tale of Cancer Metastasis
3 Polysaccharides: Properties, Modifications, and Anticancerous Mechanisms
3.1 Relation Between Structure and Anticancer Potentiality of Polysaccharides
3.1.1 Molecular Weight and Solubility
3.1.2 Bands and Branching
3.1.3 3D Arrangement
3.1.4 Composition
3.2 Chemical Modifications
3.3 Anticancer Mechanisms of Polysaccharides
3.3.1 Polysaccharides Involve in Cell Cycle Arrest
3.3.2 Antioxidant Activity
3.3.3 Mitochondrial Dysfunction
3.3.4 Immunomodulation
3.3.5 Antiangiogenesis
4 Polysaccharides in Cancer Therapy
4.1 Anticancerous Polysaccharides in Clinical Trial
4.2 Polysaccharide-Based Prodrugs
4.3 Immune-Therapy with Polysaccharide-Based Vaccine
4.4 Iminosugars for Cancer Therapy
4.5 Polysaccharide-Based Cancer Diagnostics
5 Concluding Remarks and Future Perspective
References
33 Marine Polysaccharides in Pharmaceutical Uses
1 Introduction
2 Marine-Derived Assorted Polysaccharides
3 Marine Biomass: A Great Resource for Many Polysaccharides
4 Principals of Physicochemical Modification in Biopolymeric Milieu
5 Patents on Marine-Based Polysaccharides
6 Production, Applications, and Modification Approaches for Marine Polysaccharide
7 Biotechnology for Marine-Based Biomolecules
8 Compact Thrust into Marine Biotechnology Industry
9 Marine Polysaccharide-Based Hydrogels
10 Marine-Derived Polysaccharides for Bio-Adhesives and Muco-Adhesives
11 Tactical Modifications in Marine-Based Polysaccharides
11.1 Blending Actions
11.2 Physicochemical Alteration
11.3 Hydrophobic Modification
11.4 Depolymerization
11.5 Sulfation
12 Pharmaceutically Vital Polysaccharides
12.1 Alginate: (C6H9O7-)n
12.1.1 Structural Features
12.1.2 History and Chemistry
12.1.3 Property
12.1.4 Utility of Alginates
Drug Delivery by Alginates
Wound Healing with Alginates
Cell Immobilization Via Alginates
12.2 Chitin: (C8H13O5N)n
12.2.1 Structural Features
12.2.2 History and Chemistry
12.2.3 Occurrence and Properties
12.2.4 Chitin/Chitosan Synthesis
12.2.5 Solubility Pattern
12.2.6 Utility of Chitin/Chitosan
12.2.7 Chitosan in Biomedicals
12.3 Carrageenan: C42H74O46S6
12.3.1 Structural Features
12.3.2 History, Chemistry
12.3.3 Property
12.3.4 Utility of Carrageenan
13 Conclusion
References
34 Bacterial Polysaccharides: Cosmetic Applications
1 Introduction
2 Bacterial Polysaccharides
2.1 Main Properties
2.2 Biotechnological Importance
2.2.1 Applications of Bacterial Polysaccharides in the Food Sector
2.2.2 Applications of Bacterial Polysaccharides in the Health Sector
2.2.3 Applications of Bacterial Polysaccharides in the Agricultural Sector
2.2.4 Applications of Bacterial Polysaccharides in the Industrial Sector
2.3 Cosmetic Applications of Bacterial Polysaccharides
2.3.1 Functional Polysaccharides
Cationic Polysaccharides
Anionic Polysaccharides
Nonionic Polysaccharides
Amphoteric Polysaccharide
2.3.2 Bioactive Bacterial Polysaccharides
Bacterial Cellulose
Levan
Hyaluronic Acid
3 Skincare
3.1 Skin Structure
3.2 Skin Conditions
3.2.1 Psoriasis
3.2.2 Eczema
3.2.3 Acne
3.2.4 Rosacea
3.2.5 Hyperpigmentation and Skin Aging
4 Cosmetic Products
4.1 Definition and Categories of Cosmetics Products
4.2 Cosmetic Formulation
4.3 Main Cosmetic Vehicles
4.3.1 Emulsions
4.3.2 Suspensions
4.3.3 Hydrogels
4.3.4 Encapsulating Structures
4.4 Safety Requirements and Regulation
4.4.1 The Requirements of the EU Regulation
4.4.2 The Requirements of the United States
Cosmetic Ingredient Review (CIR) and Good Clinical Practices (GCPs)
5 Conclusions
References
35 Natural Polysaccharides for Skin Care
1 Introduction
2 The Specialty Bacterial Polysaccharides for Skin Care Cosmetics
2.1 Cellulose
2.1.1 Methylcellulose
2.1.2 Hydroxyethyl Cellulose
2.1.3 Hydroxypropyl Cellulose
2.2 Chitin and Chitosan
2.3 Hyaluronic Acid
2.4 Xanthan
2.5 Dextran
2.6 Curdlan
2.7 Gellan
2.8 Levan
2.9 Alginate
3 The Specialty Fungal Polysaccharides for Skin Care Cosmetics
3.1 Pullulan
3.2 Lentinus edodes
3.3 Ganoderma lucidum
3.4 Agaricus subrufescens
3.5 Grifola frondosa
3.6 Trametes versicolor
3.7 Pleurotus ostreatus
3.8 Poria cocos
4 Challenges and Future Prospects of Microbial Polysaccharides for Cosmetics
4.1 Biodegradable Material for Packaging
4.2 Delivery System
4.3 Source and Production
5 Conclusions
References
36 Immunogenicity and Vaccines of Polysaccharides
1 Gums/Polymers from Phytogenic Origin Used for Vaccine Delivery
1.1 Introduction
1.2 Plant Gums
2 Mucosal Adjuvants
2.1 Living Antigens
2.2 Adjuvants of Phytogenic Origin
2.3 Gums and Mucilages: Salient Features
2.4 Characterization of Gums and Mucilages from Excipient Analysis
2.4.1 Classification of Gums
2.5 Gums as Delayed Transit and Continuous Release Systems
2.6 Attempts at Use of Plant Polysaccharide for Vaccine Delivery
3 Conclusions
References
37 Bacterial Polysaccharides Versatile Medical Uses
1 New Developments of Well-Known, Commercial Bacterial Polysaccharides
1.1 Dextran
1.1.1 Dextran Derivatives as Drug Carriers
1.1.2 Dextran Derivatives in Systems for Topical Therapeutics
1.1.3 Dextran Derivatives in Gene and Cell Delivery
1.1.4 Dextran in Other Medical Applications
1.2 Hyaluronic Acid
1.2.1 Hyaluronic Acid in Wound Healing
1.2.2 Hyaluronic Acid in Drug and Gene Therapy
1.2.3 Hyaluronic Derivatives in Tissue Engineering
1.3 Xanthan
1.3.1 Xanthan as Drug Carrier
1.3.2 Xanthan in Tissue Engineering and Wound Healing
1.4 Gellan
1.4.1 Gellan Developments as Drug Carriers
1.4.2 Gellan in Wound Healing and Tissue Engineering
1.5 Bacterial Cellulose
1.5.1 Bacterial Cellulose Developments in Wound Healing with Antimicrobials Delivery and Tissue Engineering
1.5.2 Bacterial Cellulose in Drug Delivery
1.6 Levan
1.6.1 Biological Activities of Levan for Health
1.6.2 Levan in Tissue Engineering
1.6.3 Levan as Drug Carrier
2 New Discovered Bacterial Polysaccharides with Potential Medical Applications
3 Conclusions and Perspectives
References
38 Pharmaceutical and Biomedical Potential of Sulphated Polysaccharides from Algae
1 Introduction
2 Structural Characteristics of Sulphated Polysaccharides Produced by Algae
2.1 Macroalgae/Seaweed
2.1.1 Freshwater Algae (Macroalgae)
2.1.2 Marine Algae (Macroalgae)
2.2 Microalgae and Cyanobacteria
2.2.1 Freshwater Microalgae
2.2.2 Marine Microalgae
2.2.3 Cyanobacteria (Blue-Green Algae)
2.3 Other Sulphated Polysaccharides Produced by Animals, Plants, or Other Microorganisms
3 Bioactivity of Sulphated Polysaccharides from Algae. Relation with Chemical Features of Their Structures and Mechanisms of A...
3.1 Polysaccharides from Marine Algae
3.1.1 Green Algae Versus Red Algae
3.2 Bioactivities of Sulphated Polysaccharides from Algae
3.2.1 Antiviral, Antibacterial, and Antifungal Activities
3.2.2 Antiproliferative, Tumor Suppressor, Apoptotic, and Cytotoxicity Activities
3.2.3 Anticoagulant and Antithrombotic Activities
3.2.4 Antilipidemic Activities
3.2.5 Anti-Inflammatory and Immunomodulatory Activities
3.2.6 Antioxidant Activities and Sequestration of Free Radicals
Antiaging Activity
3.2.7 Other Biological Activities
3.3 Case Study: Atherosclerosis
4 Potential Medical/Biomedical Applications of Sulphated Polysaccharides from Marine Algae
4.1 Tissue Engineering
4.2 Drug Delivery
4.3 Anticancer Agents
4.4 Immune Function
4.5 Wound Healing
4.6 Antipathogenic and Anti-Inflammatory
5 Clinical Trials
6 Conclusions
References
39 Natural Polymers for Biophotonic Use
1 Introduction
2 Applications of Biophotonics
2.1 Photoacoustic Imaging
2.2 Surface-Enhanced Raman Spectroscopy
2.3 Optical Coherence Tomography
2.4 Photodynamic Therapy (PDT)
2.5 Photothermal Therapy
3 Natural Polymers in Biophotonics - Current Uses and Applications
3.1 Chitosan
3.2 Cellulose
3.3 Keratin
3.4 Silk
3.5 Agarose
4 Conclusion and Future Perspectives
References
Part VI: Tissue Engineering Applications
40 Polysaccharides-Based Biomaterials for Surgical Applications
1 Introduction
2 Polysaccharides
2.1 Plant-Origin Polysaccharides
2.2 Animal-Origin Polysaccharides
2.3 Microbe-Origin Polysaccharides
3 Processing and Modifications of Polysaccharides
3.1 Bacterial Cellulose
3.2 Dextran
3.3 Gellan Gum
3.4 Carrageenan
3.5 Pullulan
3.6 Xanthan Gum
3.7 Glucan
4 Surgical Applications of Polysaccharides
4.1 Orthopedic Surgery
4.2 Neural Surgery and Plastic Surgery
4.3 Wound Healing and Miscellaneous Applications
5 Conclusions
References
41 Kefiran in Tissue Engineering and Regenerative Medicine
1 Introduction
2 Kefiran Potential for TERM Applications
3 Kefiran Physicochemical and Biological Properties of Interest for TERM Applications
3.1 Kefiran Structural Properties
3.2 Kefiran Molecular Weight
3.3 Kefiran Thermal Properties
3.4 Kefiran Mechanical Properties
3.5 Kefiran Biological Characterization
4 Conclusions and Future Perspectives
References
42 Gums for Tissue Engineering Applications
1 Introduction
2 Natural Gums
2.1 Classification of Natural Gums
2.1.1 Gum Arabica
2.1.2 Gum Ghatti
2.1.3 Karaya Gum
2.1.4 Gum Tragacanth
2.1.5 Guar Gum
2.1.6 Pectin
2.1.7 Acemannan
2.1.8 Konjac Glucomannan
2.1.9 Xylan
2.1.10 Cellulose
2.1.11 Chitin
2.1.12 Chitosan
2.1.13 Chondroitin Sulfate
2.1.14 Hyaluronic Acid
2.1.15 Xanthan Gum
2.1.16 Gellan Gum
2.1.17 Dextran
2.1.18 Scleroglucan
2.1.19 Pullulan
2.1.20 Alginate
2.1.21 Agar
2.1.22 Agarose
2.1.23 Carrageenan
2.1.24 Ulvan
2.1.25 Fucoidan
2.1.26 Laminarin
2.1.27 Carboxymethyl Cellulose
3 Conclusion
References
43 Microbial Polysaccharides as Cell/Drug Delivery Systems
1 Introduction
2 Polysaccharides
2.1 Microbial Polysaccharides
2.2 Fungal Polysaccharide
2.3 Cellulose
2.4 Xanthan Gum
3 Cell Delivery
3.1 Dextran
3.2 Dextran-Based Cryogels
3.3 Dextran-Based Colon Cancer Therapy
3.4 Hydrogels
3.5 Gellan Gum
3.6 Salecan
3.7 Heparosan Polysaccharide
3.8 Pullulan
3.9 Schizophyllan
3.10 Curdlan
4 Conclusion
References
44 Injectable Polymeric System Based on Polysaccharides for Therapy
1 Introduction
2 Injectable Polymeric Systems
3 Applications of Injectable Materials Based on Microbial Polysaccharides
3.1 Drug and Cell Delivery
3.2 Tissue Engineering and Regenerative Medicine
4 Conclusion and Outlook
References
45 The Role of Hyaluronic Acid in Tissue Engineering
1 Introduction
1.1 Tissue Scaffolds
1.2 The Extracellular Matrix (ECM)
2 Degradation and Chemical Modifications of HA
2.1 Conjugation of HA: Chemical Modifications Through the Carboxylic Acid Group
2.1.1 Amidation
2.1.2 Ugi Condensation
2.1.3 Ester Formation
2.2 Chemical Modifications Through the Hydroxyl Group
2.2.1 Ether Formation
2.2.2 Ester Formation
2.3 Modification of the -NHCOCH3
2.4 Other HA-Crosslinking Strategies for Biomedical Applications
3 Potential HA-Based Scaffold-Processing Methods
3.1 Phase Separation
3.2 Supercritical Fluid Technology
3.3 Porogen Leaching
3.4 Electrospinning
3.5 Freeze Drying
3.6 Centrifugal Casting
3.7 Scaffold-Templating Techniques
3.8 Micropatterning Techniques
3.9 Rapid Prototyping: Solid Freeform Fabrication (SFF) and Bioprinting
4 HA in Scaffold Vascularization Strategies
5 Immunomodulatory Properties of HA-Based Scaffolds
6 HA in Tissue-Engineering Applications
6.1 Peripheral Nerve Regeneration (PNS)
6.2 Central Nervous System (CNS)
6.3 Skin
7 Antimicrobial Properties of HA for Tissue-Engineering Strategies
7.1 Antibacterial Activity of HA
7.2 Antifungal Activity of HA
7.3 Antiviral Activity of HA
7.3.1 Highlight on COVID-19 and Other SARS
8 Conclusions and Future Outlook
References
46 Natural Polysaccharides on Wound Healing
1 Introduction
2 Extracellular Matrix and Wound
2.1 Extracellular Matrix
2.2 Wound Formation: Acute and Chronic Wound
3 Wound Healing
3.1 Hemostasis
3.2 Inflammation
3.3 Proliferation
3.4 Maturation and Restructuring
4 The Role of Extracellular Matrix in Wound Healing
5 Uses of Natural Polymers in Wound Healing
5.1 Drawbacks
6 In Vivo and In Vitro Studies
7 Conclusion
References
47 Xanthan Gum for Regenerative Medicine
1 Introduction
2 Xanthan Gum Production
3 Properties of Xanthan Gum
4 Modification of Xanthan Gum
4.1 Physical Methods
4.2 Chemical Methods
4.3 Enzymatic Treatment and Plasma Irradiation
5 Application of Xanthan Gum in Regenerative Medicine
5.1 Skin
5.2 Articular Cartilage
5.3 Tendons
5.4 Neuronal Tissue
5.5 Bone and Periosteal Tissue
5.6 Periodontal Tissue
5.7 Delivery of Bioactive Agents
5.8 Other Applications
6 Conclusions
References
48 Cellulose and Tissue Engineering
1 Tissue Engineering: Definition and Requirements
2 Cellulose: Function, Structure, and Properties
3 Cellulose: Derivatives and Cellulose Nanostructures
4 Cellulose Applied to Bone Tissue Engineering
5 Cellulose Applied to Cartilage Tissue Engineering
6 Cellulose for Skin Tissue Engineering and Wound Healing
7 Conclusions, Future Directions, and Challenges
References
49 Glycoconjugate for Tissue Engineering
1 Introduction
2 Glycoconjugate: Definition, Structure, and Type
2.1 Glycoprotein
2.2 Glycolipids
3 Glycan-Based Polymer
3.1 Nature Glycopolymers
3.1.1 Chitin/Chitosan
3.1.2 Glycosaminoglycans
3.1.3 Agarosa
3.1.4 Gelatin
3.2 Synthetic Polymer
3.2.1 Polyethylene Glycol (PEG)
3.2.2 Polycaprolactone (PCL)
3.2.3 Polylactic Acid (PLA)
3.2.4 Poly Lactic-Co-Glycolic Acid (PLGA)
3.3 Natural, Synthetic, Natural-Synthetic Polymer Blends
4 Glycoconjugate and Scaffolding Material in Tissue Engineering
5 Scaffolds Based on Extracellular Matrix Promote Neural Tissue Regeneration
6 Glycopolimer on Cancer Immunotherapy
7 Conclusion and Outlook
References
50 3D Printing of Microbial Polysaccharides
1 Introduction
2 3D Printing Technologies
2.1 Concept and Definition
2.2 Main Applications in Food and Regenerative Medicine
3 Description of the Main Microbial Polysaccharides
3.1 Polysaccharides Definition
3.2 Bacterial Cellulose
3.3 Xanthan
3.4 Gellan
3.5 Alginate
3.6 Dextran
3.7 Others Bacterial Polysaccharides
4 Use of Polysaccharides As Bioink
4.1 Cellulose Applications
4.2 Alginate Applications
4.3 Other Examples
5 Conclusion
References
51 Chitosan-Based Gels for Regenerative Medicine Applications
1 Introduction
2 Mechanism of Chitosan Gelation
2.1 Physical Gelation
2.2 Self-Assembly
2.3 Radical Polymerization
2.4 Ionic Gelation
2.5 Coacervation
2.5.1 Simple Coacervation
2.5.2 Complex Coacervation
2.6 Cryogelation
3 Application of Chitosan Gel
3.1 Chitosan Nanogels
3.2 Chitosan Microgel
3.3 Chitosan Macrogel
4 Conclusion
References
Index
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Joaquim Miguel Oliveira Hajer Radhouani Rui L. Reis Editors
Polysaccharides of Microbial Origin Biomedical Applications
Polysaccharides of Microbial Origin
Joaquim Miguel Oliveira • Hajer Radhouani • Rui L. Reis Editors
Polysaccharides of Microbial Origin Biomedical Applications
With 257 Figures and 85 Tables
Editors Joaquim Miguel Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory Braga/Guimarães, Portugal
Hajer Radhouani 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory Braga/Guimarães, Portugal
Rui L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory Braga/Guimarães, Portugal
ISBN 978-3-030-42214-1 ISBN 978-3-030-42215-8 (eBook) ISBN 978-3-030-42216-5 (print and electronic bundle) https://doi.org/10.1007/978-3-030-42215-8 © Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface
This book overviews and discusses the polysaccharides produced and secreted by microorganisms, used in the most recent developments in biomedical applications. In fact, the authoritative reference work revises the sources, properties, characteristics, and biotechnology and biomedical applications of important microbial polysaccharides. The main goal of this book is to provide a comprehensive analysis of microbial polysaccharides and their current uses and highlight biomedical opportunities. Thus, the topics comprise the following: (a) the overview of the main bacterial, fungal, and microalgal polysaccharides; (b) their extraction, isolation, purification, and advanced production processes; (c) the characterization of their structural, physicochemical, and biological properties, among others, by several techniques; (d) the description of the advanced functionalization and modification methods for the polysaccharidebased material; and (e) their applications and uses in pharmaceutical and tissue engineering fields. Each chapter is written by world-renowned academics and practitioners on their field, being a reference in the area of biomedical and material engineering. Guimarães, Portugal March 2022
Joaquim Miguel Oliveira Hajer Radhouani Rui L. Reis
v
Contents
Volume 1 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joaquim Miguel Oliveira, Hajer Radhouani, and Rui L. Reis
Part 1
1
Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
2
Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cesar A. Tischer
9
3
Levan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Onur Kırtel and Joan Combie
23
4
Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Weslley Felix de Oliveira, Priscilla Barbosa Sales Albuquerque, Priscila Marcelino dos Santos Silva, Luana Cassandra Breitenbach Barroso Coelho, and Maria Tereza dos Santos Correia
43
5
The Promise and Challenge of Microbial Alginate Production: A Product with Novel Applications . . . . . . . . . . . . . . . . . . . . . . . . . Wael Sabra
79
6
Kefiran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Simonelli, N. Gagliarini, M. Medrano, J. A. Piermaria, and A. G. Abraham
99
7
Microbial Glucuronans and Succinoglycans . . . . . . . . . . . . . . . . . . P. Dubessay, P. Andhare, D. Kavitake, P. H. Shetty, A. V. Ursu, C. Delattre, G. Pierre, and P. Michaud
117
8
Cyanobacterial Extracellular Polymeric Substances (EPS) . . . . . . Rita Mota, Carlos Flores, and Paula Tamagnini
139
9
Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hajer Radhouani, Susana Correia, Cristiana Gonçalves, Rui L. Reis, and Joaquim Miguel Oliveira
167
vii
viii
Contents
Part II
Fungal and Microalgal Polysaccharides . . . . . . . . . . . . . . . .
185
10
Botryosphaeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert F. H. Dekker and Aneli M. Barbosa-Dekker
187
11
Chitin and Its Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simone S. Silva, J. M. Gomes, L. C. Rodrigues, and Rui L. Reis
205
12
Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anabelle Camarotti de Lima Batista, Weslley de Souza Paiva, and Francisco Ernesto de Souza Neto
229
13
An Insight into Pullulan and Its Potential Applications . . . . . . . . . C. Nagendranatha Reddy, Bishwambhar Mishra, Sanjeeb Kumar Mandal, Dinesh Chand Agrawal, and Chandana Kruthiventi
247
14
Scleroglucan and Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . Hafiz Ubaid ur Rahman, Waqas Asghar, and Nauman Khalid
279
15
Sulfated Seaweed Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . Ana Rita Inácio, Ana C. Carvalho, Catarina Oliveira, Lara Reys, Simone S. Silva, Nuno M. Neves, Albino Martins, Rui L. Reis, and Tiago H. Silva
307
16
Polysaccharides Produced by Microalgae . . . . . . . . . . . . . . . . . . . . Antonio Trincone
341
17
Microalgae Polysaccharides with Potential Biomedical Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michele Greque Morais, Gabriel Martins Rosa, Luiza Moraes, Ana Gabrielle Pires Alvarenga, Jacinta Lutécia Vitorino da Silva, and Jorge Alberto Vieira Costa
18
Fungal Polysaccharide Production for Dermatological Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carla Maísa Camelini, Márcio José Rossi, Francielle Tramontini Gomes de Sousa, and Admir Giachini
Part III Isolation, Extraction, Purification, and Production Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
363
381
413
19
Exopolysaccharides from Lactic Acid Bacteria Yousra Abid and Samia Azabou
...............
415
20
Isolation of Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . Namita Jindal, Jasvirinder Singh Khattar, and Davinder Pal Singh
439
21
Biosynthesis of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . Yuriy A. Knirel and Johanna J. Kenyon
453
Contents
ix
Part IV Polysaccharide properties, Functionalizations, and Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
481
22
Polysaccharides of Fungal Origin . . . . . . . . . . . . . . . . . . . . . . . . . . Maggie P. Wear and Arturo Casadevall
483
23
Bioactivities of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . Karina Cruz-Aldaco, Mayela Govea-Salas, Rafael Gomes-Araújo, Miriam Desiree Dávila-Medina, and Araceli Loredo-Treviño
505
24
Surface Properties of Polysaccharides Evrim Umut
......................
527
25
Antioxidant and Antibacterial Activities of Polysaccharides . . . . . S. Chandra Mohan and Anand Thirupathi
553
26
Gel Properties of Microbial Polysaccharides . . . . . . . . . . . . . . . . . Gizem Akan and Ebru Toksoy Oner
579
27
Polysaccharides in Bacterial Biofilm . . . . . . . . . . . . . . . . . . . . . . . . Bárbara Emanoele Costa Oliveira, Ana Carolina dos Santos Ré, Carolina Patricia Aires, and Antônio Pedro Ricomini Filho
599
28
Bioactive Polysaccharides from Microalgae . . . . . . . . . . . . . . . . . . Schonna R. Manning, Katherine A. Perri, and Karlin Blackwell
625
29
Polysaccharides of Biomedical Importance from Genetically Modified Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regina Sharmila Dass, K. R. Anand, Damini Saha, Joy Elvin Dhinakar, and Pooja Thorat
30
Sulfation of Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . Cristiana Gonçalves, Hajer Radhouani, Joaquim Miguel Oliveira, and Rui L. Reis
649
675
Volume 2 Part V
Pharmaceutical Applications
........................
693
31
Dextran Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . Suneela Dhaneshwar, Neha Bhilare, and Supriya Roy
695
32
Polysaccharides in Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . Banani Kundu, Rui L. Reis, and Subhas C. Kundu
723
33
Marine Polysaccharides in Pharmaceutical Uses . . . . . . . . . . . . . . Rajendra Sukhjadeorao Dongre
745
34
Bacterial Polysaccharides: Cosmetic Applications . . . . . . . . . . . . . Sílvia Baptista and Filomena Freitas
781
x
Contents
35
Natural Polysaccharides for Skin Care . . . . . . . . . . . . . . . . . . . . . Mayuree Kanlayavattanakul and Nattaya Lourith
823
36
Immunogenicity and Vaccines of Polysaccharides . . . . . . . . . . . . . Benjamin Obukowho Emikpe and Michael Ayodele Odeniyi
847
37
Bacterial Polysaccharides Versatile Medical Uses Misu Moscovici and Cristina Balas
.............
859
38
Pharmaceutical and Biomedical Potential of Sulphated Polysaccharides from Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcina M. M. B. Morais, Ana Alves, Decha Kumla, and Rui M. S. C. Morais
39
Natural Polymers for Biophotonic Use . . . . . . . . . . . . . . . . . . . . . . Rita Rebelo, Mariana Caldas, Miguel A. D. Neves, Subhas C. Kundu, Rui L. Reis, and Vitor Correlo
Part VI
Tissue Engineering Applications . . . . . . . . . . . . . . . . . . . . .
893
921
941
40
Polysaccharides-Based Biomaterials for Surgical Applications . . . Garima Agrawal and Anuj Kumar
943
41
Kefiran in Tissue Engineering and Regenerative Medicine . . . . . . Susana Correia, Joaquim Miguel Oliveira, Hajer Radhouani, and Rui L. Reis
975
42
Gums for Tissue Engineering Applications Pritisha S. Khillar and Amit Kumar Jaiswal
..................
997
43
Microbial Polysaccharides as Cell/Drug Delivery Systems . . . . . . . 1025 M. Ramesh, K. Sakthishobana, and S. B. Suriya
44
Injectable Polymeric System Based on Polysaccharides for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045 Guy Decante, Joaquim Miguel Oliveira, Rui L. Reis, and Joana Silva-Correia
45
The Role of Hyaluronic Acid in Tissue Engineering . . . . . . . . . . . 1063 Maurice N. Collins, Fernanda Zamboni, Aleksandra Serafin, Guang Ren, A. V. Thanusha, and Mario Culebras
46
Natural Polysaccharides on Wound Healing Salih Maçin
47
Xanthan Gum for Regenerative Medicine . . . . . . . . . . . . . . . . . . . 1133 Renata Francielle Bombaldi de Souza, Fernanda Carla Bombaldi de Souza, Cecília Buzatto Westin, Rafael Maza Barbosa, and Ângela Maria Moraes
. . . . . . . . . . . . . . . . . 1117
Contents
xi
48
Cellulose and Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . 1161 Paula Cristina de Sousa Faria-Tischer
49
Glycoconjugate for Tissue Engineering . . . . . . . . . . . . . . . . . . . . . 1187 Hevi Wihadmadyatami and Dwi Liliek Kusindarta
50
3D Printing of Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . 1213 V. Nalbantova, P. Lukova, G. Pierre, N. Benbasat, P. Katsarov, P. J. P. Espitia, C. A. Fuenmayor, A. Nesic, M. S. Carranza, P. Michaud, and C. Delattre
51
Chitosan-Based Gels for Regenerative Medicine Applications . . . . 1247 Deepti Bharti, Bikash Pradhan, Sarika Verma, Subhas C. Kundu, Joaquim Miguel Oliveira, Indranil Banerjee, and Kunal Pal
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1273
About the Editors
Dr. Joaquim Miguel Oliveira, BSc, PhD, is a Portuguese Principal Investigator with Habilitation who has focused his work on the field of biomaterials for tissue engineering, nanomedicine, stem cells, and cell/drug d e l i v e r y ( h t t p : / / w w w. 3 b s . u m i n h o . p t / u s e r s / migueloliveira). Since December 2018, he is VicePresident of I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics of the University of Minho. He is also Director of Pre-clinical Research at the FIFA Medical Center, Estádio do Dragão, Porto, PT, since February 2013. Currently, he is a lecturer in Doctoral Program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC) at UMinho, PT (since December 2013). He is also an invited lecturer at the Faculty of Medicine, U. Porto (since September 2013) and Department of Polymer Engineering, UM, PT (2009-present). Recently, he has been nominated for integrating the National Ethics Committee for Clinical Research (CEIC), Serviço Nacional de Saúde (SNS), Portugal, for the period of 2020–2023. He has been awarded several prizes including the prestigious Jean Leray Award 2015 from the European Society for Biomaterials for Young Scientists for Outstanding Contributions within the field of Biomaterials. In addition, he is member of the advisory board of the Journal of Materials Science: Materials in Medicine, International Journal of Tissue Engineering, Journal ISRN Biomaterials, the Journal of Experimental Orthopaedics, and one of the Editors-in-chief of In vitro Models (Springer), and is referee in more than 40 international journals. As result of his proficiency (as of November 2021), Dr. Oliveira has published more than 470 scientific contributions in scientific journals with referee (some in xiii
xiv
About the Editors
high impact factor journals), 16 of those papers produced under invitation. Dr. Miguel Oliveira is inventor of 22 patents and published 10 books (+3 in preparation), 9 special issues/topical collections in scientific journals, 124 book chapters in books with international circulation, in international encyclopaedias, and science dissemination. He has great experience in intellectual property rights and patent exploitation. He has participated in more than 630 communications in national/ international conferences. Due to his expertise, he participated as invited/keynote speaker in more than 77 plenary sessions. He made great contributions in the osteochondral field, namely by proposing bilayered scaffolds, work that has been highly cited by its peers. Dr. Miguel Oliveira (as of November 2021) has an h-index of 54, i10 of 183, and received more than 10,700 citations (Google Scholar), or an h-index of 45 and ~7710 total citations (Scopus). He has an RG46.09 (ResearchGate). Dr. Hajer Radhouani, Eng, MSc, PhD, is a postdoctoral researcher at the Portuguese Government Associate Laboratory ICVS/3B’s, University of Minho (https:// 3bs.uminho.pt/users/hradhouani). She graduated in Industrial Engineering in Agronomy (Agro-Industries and Biotechnologies) at Institut Supérieur Industriel Agronomique de Huy/Gembloux (Huy, Belgium) and obtained her European PhD in Technologic, Comparative and Molecular Genetics from the Universidade de Trás-os-Montes e Alto Douro (Vila Real, Portugal) in a collaboration with the Universidad de La Rioja (Logroño, Spain) and the Universidad de Vigo (Ourense, Spain). Since 2013, she has been working with biomaterials based on natural polysaccharides to treat different articular cartilage pathologies. Currently, she has been working on Kefiran polymer as a novel therapy for osteoarthritis treatment. She has published 54 publications listed in ISI Web of Knowledge, 3 international granted patents, and 8 book chapters, among others. She has a Scopus h-index of 22 and more than 1279 citations. She also presented more than 100 works (posters and oral presentations) in conferences and meetings. She has several scientific profiles such as E-1837-2011 (Researcher ID); 25724661600 (Scopus
About the Editors
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Author ID); orcid.org/0000-0003-1007-4073 (ORCID); FE1C-88E7-0A8A (Ciência ID). Prof. Rui L. Reis, PhD, DSc, Hon. Causa MD, Hon Causa PhD, FBSE, FTERM, member of NAE, FAIMBE, FEAMBES, was born in 1967 in Porto, Portugal. He is a Full Professor of Tissue Engineering, Regenerative Medicine, Biomaterials and Stem Cells at the University of Minho (UMinho), Director of the 3B’s Research Group – member of the I3Bs – Institute for Biomaterials, Biodegradables and Biomimetics, and Director of the ICVS/3B’s Associate Laboratory, both of UMinho. He is also the CEO of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, the Global Past-President of the Tissue Engineering and Regenerative Medicine International Society (TERMIS), and the founding Editor-in-Chief of the Journal of Tissue Engineering and Regenerative Medicine. He is a recognized World expert in the TERM and biomaterials fields who has edited several books and has more than 1375 published works listed on ISI Web of Knowledge with an h index of 95 (1260 works and h ¼ 100 in Scopus, and 2135 and h ¼ 119 in Google Scholar), being also an inventor of around 115 patents. Based on those, he co-founded several companies that raised important private investments. According to Google Scholar his work has been cited more than 63,000 times. He has been awarded many important international prizes, including among several others different innovation awards, the Jean Leray and George Winter Awards (ESB), the Clemson Award (SFB), the TERMIS-EU contributions to the literature Award, the TERMIS-EU Career Achievement Award, and in 2018 the UNESCO – International Life Sciences Award and the IET A. F. Harvey Engineering Research Prize. He is one of the most successful scientists working in Europe in the field of biomaterials, tissue engineering, and regenerative medicine and is the PI of projects with a budget totalizing more than 100 MEuros of which around 50 MEuros are UMinho funding. Researcher ID (A-8938-2008); Scopus Author ID 56861715700; ORCID 0000-0002-4295-6129.
Contributors
Yousra Abid ENIS, Laboratoire Analyse, Valorisation et Sécurité des Aliments, Université de Sfax, Sfax, Tunisia A. G. Abraham Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA); Facultad de Ciencias Exactas, Universidad Nacional de La Plata – CONICET CCT La Plata – CIC, La Plata, Argentina Área Bioquímica y Control de Alimentos – Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina Dinesh Chand Agrawal Department of Biotechnology, Faculty of Engineering and Technology, Rama University, Kanpur, Uttar Pradesh, India Garima Agrawal School of Basic Sciences, Indian Institute of Technology Mandi, Kamand, India Carolina Patricia Aires Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil Gizem Akan IBSB Industrial Biotechnology and Systems Biology Research Group, Department of Bioengineering, Marmara University, Istanbul, Turkey Priscilla Barbosa Sales Albuquerque Departamento de Medicina, Universidade de Pernambuco, Garanhuns, PE, Brazil Ana Gabrielle Pires Alvarenga Laboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Ana Alves CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal K. R. Anand Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry, India P. Andhare PDPIAS, CHARUSAT University, Anand, India xvii
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Contributors
Waqas Asghar School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan Samia Azabou ENIS, Laboratoire Analyse, Valorisation et Sécurité des Aliments, Université de Sfax, Sfax, Tunisia Cristina Balas National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania Indranil Banerjee Department of Bioscience and Bioengineering, IIT, Jodhpur, India Sílvia Baptista UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology, NOVA School of Science and Technology, Caparica, Portugal 73100 Lda., Lisboa, Portugal Rafael Maza Barbosa Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil R-Crio Cryogenics S.A., Campinas, Brazil Aneli M. Barbosa-Dekker Beta-Glucan Produtos Farmoquímicos - EIRELI, Lote 24A, Bloco Zircônia, Universidade Tecnológica Federal do Paraná, Avenida João Miguel Caram, Londrina, Paraná, Brazil N. Benbasat Department of Pharmacognosy and Pharmaceutical Chemistry, Medical University-Plovdiv, Plovdiv, Bulgaria Department of Pharmacognosy and Pharmaceutical Botany, Medical University Sofia, Sofia, Bulgaria Deepti Bharti Department of Biotechnology and Medical Engineering, NIT, Rourkela, India Neha Bhilare Department of Pharmaceutical Chemistry, Arvind Gavali College of Pharmacy, Satara, India Karlin Blackwell Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Fernanda Carla Bombaldi de Souza R-Crio Cryogenics S.A., Campinas, Brazil Renata Francielle Bombaldi de Souza Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil Mariana Caldas Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, I3B’s Research Institute on Biomaterials Biodegradables and Biomimetics, Universidade do Minho, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associated Laboratory, Braga/Guimarães, Portugal
Contributors
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Carla Maísa Camelini Sole Vitae ® Cosméticos Brazil and Chile, Palmital, SP, Brazil M. S. Carranza Graduate School of Engineering Science, Osaka University, Osaka, Japan College of Science, De La Salle University, Manila, Philippines Ana C. Carvalho 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Arturo Casadevall Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Luana Cassandra Breitenbach Barroso Coelho Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, Brazil Maurice N. Collins School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Health Research Institute, University of Limeric, Limerick, Ireland Joan Combie Montana Biopolymers Inc., Winnsboro, SC, USA Susana Correia 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Vitor Correlo Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, I3B’s Research Institute on Biomaterials Biodegradables and Biomimetics, Universidade do Minho, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associated Laboratory, Braga/Guimarães, Portugal Jorge Alberto Vieira Costa Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Bárbara Emanoele Costa Oliveira Graduate Program in Dentistry, University Ceuma, São Luís, Maranhão, Brazil Karina Cruz-Aldaco Department of Food Research (DIPA), School of Chemistry, Universidad Autónoma de Querétaro, Querétaro, Mexico Mario Culebras School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland
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Contributors
Jacinta Lutécia Vitorino da Silva Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Miriam Desiree Dávila-Medina School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico Regina Sharmila Dass Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry, India Anabelle Camarotti de Lima Batista Agriculture Department, CCHSA, Federal University of Paraiba, Bananeiras, PB, Brazil PROFBIO, CCEN, Federal University of Paraiba, João Pessoa, PB, Brazil Weslley Felix de Oliveira Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, Brazil Francielle Tramontini Gomes de Sousa Division of Infectious Diseases and Vaccinology, School of Public Health, University of California-Berkeley, Berkeley, CA, USA Paula Cristina de Sousa Faria-Tischer Glucom Biotech Ltda., Londrina, Brazil Francisco Ernesto de Souza Neto Nova Esperança, College of Mossoro, Mossoró, RN, Brazil Weslley de Souza Paiva RENORBIO, Federal University of Rio Grande do Norte, Natal, RN, Brazil Guy Decante 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, GMR, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Robert F. H. Dekker Universidade Tecnológica Federal do Paraná (UTFPR), Programa de Pós-Graduação em Engenharia Ambiental, Câmpus Londrina, Londrina, Paraná, Brazil C. Delattre Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, Clermont-Ferrand, France Institut Universitaire de France (IUF), Paris, France Suneela Dhaneshwar Department of Pharmaceutical Chemistry, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Noida, India
Contributors
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Joy Elvin Dhinakar Department of Studies in Biochemistry, University of Mysore, Mysore, India Rajendra Sukhjadeorao Dongre Department of Chemistry, RTM Nagpur University, Nagpur, India Maria Tereza dos Santos Correia Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, Brazil Ana Carolina dos Santos Ré Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo, Brazil Priscila Marcelino dos Santos Silva Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco, Recife, PE, Brazil P. Dubessay Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, Clermont-Ferrand, France Benjamin Obukowho Emikpe Departments of Veterinary Pathology, University of Ibadan, Ibadan, Nigeria P. J. P. Espitia Nutrition and Dietetics School, Universidad del Atlántico, Puerto Colombia, Atlántico, Colombia Carlos Flores i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Centre for Urological Biology, Department of Renal Medicine, University College London, London, UK Filomena Freitas UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology, NOVA School of Science and Technology, Caparica, Portugal 73100 Lda., Lisboa, Portugal C. A. Fuenmayor Institute of Food Science and Technology (ICTA), Universidad Nacional de Colombia, Bogotá, DC, Colombia N. Gagliarini Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA); Facultad de Ciencias Exactas, Universidad Nacional de La Plata – CONICET CCT La Plata – CIC, La Plata, Argentina Admir Giachini Ressacada de Pesquisas em Meio Ambiente, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
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Contributors
J. M. Gomes 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Rafael Gomes-Araújo School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico Cristiana Gonçalves 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Mayela Govea-Salas School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico Ana Rita Inácio 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Amit Kumar Jaiswal Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore Institute of Technology, Vellore, Tamil Nadu, India Namita Jindal Department of Botany, Punjabi University, Patiala, India Onur Kırtel Bioengineering Department, Industrial Biotechnology and Systems Biology Research Group – IBSB, Marmara University, İstanbul, Turkey Mayuree Kanlayavattanakul School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, Thailand Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University, Chiang Rai, Thailand P. Katsarov Department of Pharmaceutical Sciences, Medical University, Plovdiv, Bulgaria Research Institute at Medical University-Plovdiv, Medical University – Plovdiv, Plovdiv, Bulgaria D. Kavitake Department of Food Science and Technology, Pondicherry University, Pondicherry, India Johanna J. Kenyon Centre for Immunolgy and Infection Control University of Minho, (CIIC), School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia
Contributors
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Nauman Khalid School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan Jasvirinder Singh Khattar Department of Botany, Punjabi University, Patiala, India Pritisha S. Khillar Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore Institute of Technology, Vellore, Tamil Nadu, India Yuriy A. Knirel N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Chandana Kruthiventi Department of Biotechnology, Chaitanya Bharathi Institute of Technology, Hyderabad, Telangana, India Anuj Kumar School of Chemical Engineering, Yeungnam University, Gyeongsan, South Korea Decha Kumla CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Banani Kundu 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Subhas C. Kundu 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Dwi Liliek Kusindarta Department of Anatomy, Universitas Gadjah Mada, Yogyakarta, Indonesia Araceli Loredo-Treviño School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico Nattaya Lourith School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, Thailand Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University, Chiang Rai, Thailand P. Lukova Department of Pharmacognosy and Pharmaceutical Chemistry, Medical University-Plovdiv, Plovdiv, Bulgaria Salih Maçin Department of Medical Microbiology, Selçuk University, Konya, Turkey
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Contributors
Sanjeeb Kumar Mandal Department of Biotechnology, Chaitanya Bharathi Institute of Technology, Hyderabad, Telangana, India Schonna R. Manning Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Albino Martins 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal M. Medrano Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA); Facultad de Ciencias Exactas, Universidad Nacional de La Plata – CONICET CCT La Plata – CIC, La Plata, Argentina P. Michaud Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, Clermont-Ferrand, France Bishwambhar Mishra Department of Biotechnology, Chaitanya Bharathi Institute of Technology, Hyderabad, Telangana, India S. Chandra Mohan Division of Phytochemistry, Shanmuga Centre for Medicinal Plants Research, Thanjavur, Tamil Nadu, India Luiza Moraes Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Ângela Maria Moraes Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil Alcina M. M. B. Morais CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Michele Greque Morais Laboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Rui M. S. C. Morais CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Misu Moscovici National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania Rita Mota i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
Contributors
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V. Nalbantova Department of Pharmacognosy and Pharmaceutical Chemistry, Medical University-Plovdiv, Plovdiv, Bulgaria A. Nesic Department of Chemical Dynamics and Permanent Education, Vinca Institute for Nuclear Sciences, University of Belgrade, Belgrade, Serbia Miguel A. D. Neves Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, I3B’s Research Institute on Biomaterials Biodegradables and Biomimetics, Universidade do Minho, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associated Laboratory, Braga/Guimarães, Portugal Nuno M. Neves 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Michael Ayodele Odeniyi Department of Pharmaceutics and Industrial Pharmacy, University of Ibadan, Ibadan, Nigeria Catarina Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Joaquim Miguel Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Ebru Toksoy Oner IBSB Industrial Biotechnology and Systems Biology Research Group, Department of Bioengineering, Marmara University, Istanbul, Turkey Kunal Pal Department of Biotechnology and Medical Engineering, NIT, Rourkela, India Katherine A. Perri Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA J. A. Piermaria Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA); Facultad de Ciencias Exactas, Universidad Nacional de La Plata – CONICET CCT La Plata – CIC, La Plata, Argentina Área Bioquímica y Control de Alimentos – Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
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Contributors
G. Pierre Université Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, Clermont-Ferrand, France Bikash Pradhan Department of Biotechnology and Medical Engineering, NIT, Rourkela, India Hajer Radhouani 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Hafiz Ubaid ur Rahman School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan M. Ramesh Department of Mechanical Engineering, KIT-Kalaignarkarunanidhi Institute of Technology, Coimbatore, Tamil Nadu, India Rita Rebelo 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal C. Nagendranatha Reddy Department of Biotechnology, Chaitanya Bharathi Institute of Technology, Hyderabad, Telangana, India Rui L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Guang Ren School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Lara Reys 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Antônio Pedro Ricomini Filho Department of Biosciences, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba, São Paulo, Brazil L. C. Rodrigues 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal
Contributors
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ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Gabriel Martins Rosa Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil Márcio José Rossi Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil Supriya Roy Department of Pharmacology, Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Noida, India Wael Sabra Faculty of Life Sciences, Rhine-Waal University of Applied Sciences, Kleve, Germany Damini Saha Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry, India K. Sakthishobana Department of Biotechnology, KIT-Kalaignarkarunanidhi Institute of Technology, Coimbatore, Tamil Nadu, India Aleksandra Serafin School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland P. H. Shetty Department of Food Science and Technology, Pondicherry University, Pondicherry, India Simone S. Silva 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Tiago H. Silva 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal Joana Silva-Correia 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, GMR, Portugal N. Simonelli Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA); Facultad de Ciencias Exactas, Universidad Nacional de La Plata – CONICET CCT La Plata – CIC, La Plata, Argentina Davinder Pal Singh Department of Botany, Punjabi University, Patiala, India
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Contributors
S. B. Suriya Department of Biotechnology, KIT-Kalaignarkarunanidhi Institute of Technology, Coimbatore, Tamil Nadu, India Paula Tamagnini i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal A. V. Thanusha School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Anand Thirupathi Faculty of Sports Science, Ningbo University, Ningbo, China Pooja Thorat Fungal Genetics and Mycotoxicology Laboratory, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry, India Cesar A. Tischer Biotechnology and Glicoconjugate Laboratory, Department of Biochemistry and Biotechnology, State University of Londrina – UEL, Londrina, Brazil Antonio Trincone Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli (Naples), Italy Evrim Umut Department of Medical Imaging Techniques, Dokuz Eylul University, School of Healthcare, Izmir, Turkey A. V. Ursu Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, Clermont-Ferrand, France Sarika Verma Council of Scientific and Industrial Research Advanced Materials and Processes Research Institute (CSIR-AMPRI), Bhopal, India Maggie P. Wear Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Cecília Buzatto Westin Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil Hevi Wihadmadyatami Department of Anatomy, Universitas Gadjah Mada, Yogyakarta, Indonesia Fernanda Zamboni School of Engineering, University of Limerick, Limerick, Ireland Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Health Research Institute, University of Limeric, Limerick, Ireland
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Introduction Joaquim Miguel Oliveira, Hajer Radhouani, and Rui L. Reis
Contents 1 Context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Abstract
Microbial polysaccharides are a renewable resource that has gained increasing interest for biomedical applications due to their outstanding properties. The abundance of these polysaccharides allows their sustainability and provides monetary value for new functional biomaterials. Furthermore, the increasing worldwide demand for active substances offering several health benefits could be the future source of innovation for microbial polysaccharides. The latest developments involving polysaccharides of microbial origin and their biomedical applications are overviewed herein. Keywords
Microbial · Polysaccharides · Biomedical applications
J. M. Oliveira (*) · H. Radhouani · R. L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected]; [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_1
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Context
Nature comprises a wide diversity of microorganisms, and in consequence it is predictable that microorganisms produce new materials with different structures and properties. This unlimited structural diversity results in an extensive range of applications for microbial polysaccharides that can be obtained from microorganisms such as bacteria, yeast, fungi, and microalgae (Smelcerovic et al. 2008). Polysaccharides, in many forms, play an essential function in all living organisms for storage and supply of energy and/or protection and structural integrity of cells (Shanmugam and Abirami 2019). Microbial polysaccharides, one of the most diverse families of biopolymers, are high molecular weight polymers being able to reach until several millions of Dalton, sharing a substantial component of the cellular carbohydrates found in and surrounding microbial cells (Delattre et al. 2007). Thus, microbial polysaccharides can serve as renewable resources and are now widely used in different industry sectors, principally due to their amazing rheological properties, where they are used as coagulants, binders, gelling agents, lubricants, film formers, emulsifiers, thickeners, and stabilizers, among others (Jindal and Singh Khattar 2018). In the last few years, polysaccharide-based materials have been receiving an increasing interest in the pharmaceutical and materials engineering fields because of their biocompatibility, bio-absorbability, versatility, and nontoxic nature, among others (Dave 2016). In fact, xanthan, xylinan, gellan, curdlan, dextran, pullulan, scleroglucan, schizophyllan, and alginate are, among others, common microbial polysaccharides in current use (Ahmad et al. 2015; Lei and Edmund 2019). These polysaccharides can also offer not only the advantages of a well-controlled production process with consistent and reproducible yield that can be continued throughout the year but also constant structural, chemical, and physical characteristics (Giavasis 2013). Commonly, microbial polysaccharides are being used in food industries (Giavasis 2013; Ramalingam et al. 2014; Jindal and Singh Khattar 2018; Nešić et al. 2020) but recently other successfully developed microbial polysaccharides have found their applications in cosmetic, pharmaceutical, and medical fields (Morris and Harding 2009; Ahmad et al. 2015). In fact, there are significant advances focused on the use of polysaccharides of microbial origin for biomedical applications including but not limited to drug delivery (Miao et al. 2018), imaging (Moscovici 2015), wound healing and dressing (Azuma et al. 2015; Zhu et al. 2019), surgery (Gupta et al. 2019), and tissue engineering (Delattre et al. 2007; Silva et al. 2017). Developments in the applications of polysaccharides from microorganisms are thoroughly related to the capability of the scientific community to entirely understand the complexity of these biopolymers. Hence, translating this knowledge to practical applications is necessary to fully characterize their structural, biological, and physicochemical properties. Herein, the “Polysaccharides of Microbial Origin: Biomedical Applications” book presents the microbial polysaccharides status, from their classifications to their wide range of biomedical applications. The book is presented in six major sections, conceptualized in Fig. 1.
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Fig. 1 Polysaccharides of microbial origin in biomedical applications
Section 1 provides an overview of some of the major bacterial polysaccharides such as cellulose, dextran, levan, gums, curdlan, bacterial alginate, bacterial hyaluronic acid, kefiran, and cyanobacterial polysaccharides, among others. Essentially, they have been identified both in Gram-positive and Gram-negative bacteria, having in general an exocellular location (Delattre et al. 2007). Section 2 comprises the main fungal and microalgal polysaccharides which include chitin, chitosan, pullulan, scleroglucan, and schizophyllan, among others, with successful examples of their biomedical exploitation. Moreover, new bioactive polysaccharides from marine bacteria are highlighted in this section as a valid alternative to traditional polysaccharides. Section 3 describes the most recent isolation, extraction, purification, and production processes used to obtain microbial polysaccharides of high purity and yields, together with an economic efficiency. These production processes are currently a matter of intense research, and appropriate techniques need to be implemented to enhance process optimization. There are several methods, described in this book, that are used to isolate and purify microbial polysaccharides; the main criterion to select a certain method is to maintain the intrinsic properties of these polymers unchanged during the whole process. Section 4 presents a comprehensive understanding on the structural, physicochemical, and biological properties of microbial polysaccharides that could guide researchers in the development of target therapeutic products meeting desired characteristics and profiles. Furthermore, this section provides a number of approaches that can be followed to modify these polymers in order to enhance
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their functional and technological properties through either physical or chemical cross-linking reactions. One of the greatest biotechnological approaches is the transfer of specific gene constructs by several methods (Ahmed 2003). In this section, genetic engineering technologies are described to increase the production yield of biopolymers in a short span of time. Genetically modified organisms are continually being explored, and these techniques certainly offer promising results as well as pose both technical and scientific challenges. Sections 5 and 6 provide a review on the pharmaceutical and tissue engineering applications of microbial polysaccharides according to current and outstanding research works in biomedical science. The probable future trends for the utilization of these bioactive compounds in biomedical fields are also discussed. It has been previously shown that these polysaccharides have huge potential owing to their unique rheological properties, and great antitumor, antioxidant, anti-inflammatory, anticoagulant, antiallergic, and antidiabetic properties, among others (Ahmad et al. 2015; Jenab et al. 2020). Promising applications of these polysaccharides have been reported in different therapeutic fields such as cancer, diabetes, vaccines, wound healing, and surgery, among many others (Hasnain and Nayak 2019). During the last decades, the use of microbial polysaccharides, in tissue engineering and regenerative medicine, has noticeably changed from a simple three-dimensional scaffold for cell and drug incorporation, to be used as new functional biomaterials with excellent physicochemical and biological properties. Due to their unique properties, microbial polysaccharides are considered potential candidates for natural product drug discovery and for the delivery of new derived products such as genes, drugs, vaccines, peptides, and proteins, particularly in form of matrix tablets, microspheres, nanoparticles, hydrogels, and capsules (Tiwari et al. 2012), resulting in a powerful synergism of treatment options for biomedical applications. The development of controlled drug delivery systems opens a new opportunity in the biomedical use of these biopolymers being their vast area of application supported by the multifarious series of derivatives available whose valuable properties can be controlled. The potential of these bioactive polysaccharides is being constantly studied, and a new generation of therapeutics will be accomplished by combining their biological activity with natural biodiversity. Nonetheless, the growing worldwide demand for active ingredients providing health benefits will expectedly act as a springboard for future innovation regarding microbial polysaccharides.
References Ahmad NH, Mustafa S, Man YBC. Microbial polysaccharides and their modification approaches: a review. Int J Food Prop. 2015;18:332–47. Ahmed FE. Genetically modified probiotics in foods. Trends Biotechnol. 2003;21:491–7. Azuma K, Izumi R, Osaki T, et al. Chitin, chitosan, and its derivatives for wound healing: old and new materials. J Funct Biomater. 2015. https://doi.org/10.3390/jfb6010104. Dave SR. Microbial exopolysaccharide – an inevitable product for living beings and environment. J Bacteriol Mycol Open Access. 2016. https://doi.org/10.15406/jbmoa.2016.02.00034.
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Delattre C, Laroche C, Michaud P. Production of microbial and fungal polysaccharides. In: Advances in fermentation technology. New Delhi: Asiatech Publisher Inc.; 2007. p. 483–522. Giavasis I. Production of microbial polysaccharides for use in food. In: Microbial production of food ingredients, enzymes and nutraceuticals. Oxford: Woodhead Publishing; 2013. Gupta RC, Lall R, Srivastava A, Sinha A. Hyaluronic acid: molecular mechanisms and therapeutic trajectory. Front Vet Sci. 2019. https://doi.org/10.3389/fvets.2019.00192. Hasnain MS, Nayak AK. Natural polysaccharides in drug delivery and biomedical applications. San Diego: Elsevier Science & Technology; 2019. Jenab A, Roghanian R, Emtiazi G. Bacterial natural compounds with anti-inflammatory and immunomodulatory properties (mini review). Drug Des Devel Ther. 2020;14:3787–801. Jindal N, Singh Khattar J. Microbial polysaccharides in food industry. In: Biopolymers for food design. London: Academic; 2018. Lei S, Edmund TF. Polysaccharides, microbial. In: Encyclopedia of microbiology. San Diego: Academic; 2019. Miao T, Wang J, Zeng Y, et al. Polysaccharide-based controlled release systems for therapeutics delivery and tissue engineering: from bench to bedside. Adv Sci. 2018;5:1700513. Morris G, Harding S. Polysaccharides, microbial. In: Encyclopedia of microbiology. Academic Press, London, UK. 2009;482–494. Moscovici M. Present and future medical applications of microbial exopolysaccharides. Front Microbiol. 2015;6:1012. Nešić A, Cabrera-Barjas G, Dimitrijević-Branković S, et al. Prospect of polysaccharide-based materials as advanced food packaging. Molecules. 2020;25:135. Ramalingam C, Priya J, Mundra S. Applications of microbial polysaccharides in food industry. Int J Pharm Sci Rev Res. 2014;27:322–4. Shanmugam M, Abirami RG. Microbial polysaccharides – chemistry and applications. J Biol Act Prod Nat. 2019. https://doi.org/10.1080/22311866.2019.1571944. Silva SS, Fernandes EM, Pina S, et al. Natural-origin materials for tissue engineering and regenerative medicine Simone. In Comprehensive Biomaterial II. Ducheyne P, Healy KE, Hutmacher DE., Grainger DW, Kirkpatrick CJ (Eds), Elsevier, Amsterdam, Netherlands. 2017;228–252. Smelcerovic A, Knezevic-Jugovic Z, Petronijevic Z. Microbial polysaccharides and their derivatives as current and prospective pharmaceuticals. Curr Pharm Des. 2008. https://doi.org/10. 2174/138161208786404254. Tiwari G, Tiwari R, Bannerjee S, et al. Drug delivery systems: an updated review. Int J Pharm Investig. 2012. https://doi.org/10.4103/2230-973x.96920. Zhu T, Mao J, Cheng Y, et al. Recent progress of polysaccharide-based hydrogel interfaces for wound healing and tissue engineering. Adv Mater Interfaces. 2019.
Part I Bacterial Polysaccharides
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Glucans Cesar A. Tischer
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Glucans and the Immune System: Firsts Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Basis of the Immune Response to Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Dectin-1 Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Glucan as Immunoadjuvant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Final Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Glucans are a group of polysaccharides that are isolated from plants and microorganisms that have interesting specificities on physicochemical properties that make each one useful polymers for food and other industrial applications, but that regard some key resemblance, the β-1➔3-glucopyranose presence and the modulation of the immune system. Versatile, accept a wide range of conjugable groups, and its being receiving more and more attention as investigations reveals the mechanisms and the relation between structure and function that move specific events in response to antigens. This chapter review a short period of time, mostly the last 3 years on the research of this immunomodulators polysaccharides as therapeutic agents and vaccine adjuvants. Keywords
Exopolysaccharide · Immune system · Vaccine adjuvant · Dectin-1 · Structural motif C. A. Tischer (*) Biotechnology and Glicoconjugate Laboratory, Department of Biochemistry and Biotechnology, State University of Londrina – UEL, Londrina, Brazil e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_2
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Introduction
Linear glucans are produced by a variety of organisms as plants, bacteria, and fungus but cannot be found on mammals. Great reviews about structure, production, industrial uses of glucans were written that give us a wide spectrum of potentialities of these biopolymers: • A critical review on production and industrial applications of beta-glucans (Zhu et al. 2016) • Bacterial glucans: production, properties, and applications (Xu and Zhang 2016) • A critical review on the impacts of β-glucans on gut microbiota and human health (Jayachandran et al. 2018) • A concise review on the molecular structure and function relationship of β-glucan (Du et al. 2019) • Effect of the modifications on the physicochemical and biological properties of β-glucan – a critical review (Yuan et al. 2020) • Cell wall glucans of fungi. A review (Ruiz-Herrera and Ortiz-Castellanos 2019) • β-glucan, a dietary fiber in effective prevention of lifestyle diseases – an insight (Maheshwari et al. 2019) • Fermentative production of beta-glucan: properties and potential applications (Philippini et al. 2019) • Industrial production and applications of α/β linear and branched glucans (Venkatachalam et al. 2020a) The attention that it has been receiving is an indicator of the value of these materials, how versatile on its production as renewable, secure to use, and liable to be chemically modified. The uses to tune the rheological behavior of aqueous systems, on the scope of the science of colloids (Dickinson 2006), are interesting with regard to its applications for the food industry but also in health. Many interesting properties as curdlan, i.e., that once heated in an aqueous suspension can form two types of gels depending on the heating temperature, one of which is a high-set thermal nonreversible gel (80 C) (Yan et al. 2020), that can be a tunable excipient for antibacterial formulations (Tong et al. 2020), or dextran that generates fluids with Newtonian characteristics and it is used as anticoagulant and blood volume expander by the pharmaceutical industry (Schött et al. 2018). These interesting properties are by themselves a huge field of exploration and are given by its tridimensional arrangement, but most of time the iteration with other molecules and particularly with proteins (Zielke et al. 2018). Nilson and cols. (Zielke et al. 2019) summarize data that correlate the serine, threonine, and tyrosine phosphorylation to the hydrogen bond that aggregates proteins to phosphorylated β-glucans, and that these interactions are due by not only charge distribution but through specific amino acids. Dectin-1 is a membrane protein that activate immune responses depending on oligomerization to act in response to β-glucans; Dectin-1 will be detailed later in this
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chapter. Dulal et al. observed that if tryptophan, histidine, tyrosine, respectively, W221, H223, and Y228 lacks on Dectin-1, the dimers and trimers does not aggregate and no immune response happened even in the presence of the polysaccharide (Dulal et al. 2018). The structural motif arginylglycylaspartic acid (RGD), a tripeptide constituted of the arginine-glycine-aspartic acid sequence related as key to aggregation between polysaccharides and proteins like fibrinogen (Alipour et al. 2020; Tchobanian et al. 2019), also show that play a role on the linkage of β-glucans with β-1,3-glucan binding proteins (LGBPs). The LGBPs are present on the surface of the hemocytes and is involved in the pattern recognition mechanism in invertebrates (Huang and Ren 2020), that recognizes the pathogen cell wall and act against these PAMPs (pathogen-associated molecular patterns: β-glucan, laminarin, lipopolysaccharide, lipotechoic acid, peptidoglycan). Sivakamavalli et al. found that changing Asp in RGD motif results in complete loss of binding of Pm-LGBP of Asian tiger shrimp (Penaeus monodon) to βG and pathogen recognition introducing a specific mutation D134K, a central area of the sugarbinding (βG) site (Sivakamavalli et al. 2019). (Fig. 1).
Fig. 1 β-glucan and laminarin are pathogen-associated molecular patterns, PAMPs, that are recognized by immune system and led the interactions with specific amino acids. Due to the change of Asp (mutant D134K), laminarin and glucan could not interact with protein of LGBP. (From Sivakamavalli et al. 2019, with permission)
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Glucans and the Immune System: Firsts Experiments
Immune system discerns what is and what is not proper, and clean tissues from cells or molecules potentially deleterious. This system also recognizes abnormal cells engendered inside the tissues. Molecules that are recognized by the immune system are considered antigens, Ag. It was near to 1980s when the firsts experiments shown that glucans plays a role on immune system, stimulating the cell and humoral immunity. The β-(1➔3) glucan from the Saccharomyces cerevisiae cell wall received attention and its influence on lymphoreticular system resulting in a hypertrophy of the major reticuloendothelial organs and intense macrophage proliferation was studied (Di Luzio and Williams 1978), as well induced septicemia has regressed (Di Luzio and Williams 1979). At this time, a variety of glucans was tested. Reynolds et al. show that S. cerevisiae glucan alter the course of several lethal experimental infections in mice and rats (Reynolds et al. 1980). They inoculate Rift Valley fever virus and Venezuelan equine encephalomyelitis (VEE) virus on the animals 0.5 g to 2 mg/100 g, via intraperitoneal or inhalation. The uses against a variety of pathogens with the immune system tools knew at those time, for example, stimulating the cell-mediated and humoral immunity against Plasmodium berghei showing that 1 mg/mouse of β-1,3 glucan provides complete protection through development of a well-defined cellular and humoral immunity (Kumar and Ahmad 1985).
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Basis of the Immune Response to Polysaccharides
The pattern recognition receptors (PRRs) induces immune defense on virtually in all live kingdoms, from plants, invertebrates, and vertebrates, recognizing molecular structures, the pathogen-associated molecular patterns or PAMPs (Wang et al. 2018). Some microbial carbohydrates are PAMPs that could induce adaptative and the training immunity that depends on receptors at the functional cells surface (Netea et al. 2020). The C-type lectins are proteins that binds to carbohydrate at the “C-type carbohydrate recognition domain” (CTLD), a compact region, and the major β-glucan receptors in mammals is Dectin-1, encoded by CLEC7A (Adachi et al. 2004). The T helper cells Th1, Th17, cytotoxic T lymphocyte responds to carbohydrates as β-glucan, mannan, and monophosphoryl lipid A (MPLA) (Lang and Huang 2020). The Th1 cells, Th17 have the Dectin-1 as receptor, and the integrin CR3 (complement receptor 3, also called CD11b/CD18) is the main response activator for macrophages (Brown et al. 2002; Li et al. 2019). The concise Brown and Gordon article hold out a glimpse of what expect about Dectin-1 and opens an entirely field of studies that covers from glucan structure,
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phenotypic biochemical changes, and physiological interactions (Brown and Gordon 2001). Dectin-1 receptor plays an important role for training immunity, that develops long-term memory in response to some PAMPs, as bacilli Calmette-Guerin (BCG) (Arts et al. 2016) and β-Glucans, developing nonspecific cross protection (Cheng et al. 2014). The trained immunity led to the new field of trained immunity-based vaccines (TIbV), defined as “vaccine formulations that induce training in innate immune cells” (Sánchez-Ramón et al. 2018). Metabolic changes are the key to phenotypic reprogramming inducing immune response, driving to the Warburg effect (DeBerardinis and Chandel 2020), from oxidative phosphorylation to aerobic glycolysis. Under β-glucan PAMPs exposure, Dectin-1 primes serine/threonine protein kinase AKT, mechanistic target of rapamycin (mTOR), and hypoxia-inducible factor 1α (HIF1α), that increases glucose consumption and higher the lactate production (Mulder et al. 2019; Schmitz et al. 2008). These changes affects glutaminolysis and cholesterol synthesis with epigenetic rewiring in monocytes and macrophages inducing the innate immunity (Sánchez-Ramón et al. 2018).
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Dectin-1 Receptor
Dectin-1 is a 28-kDa type II membrane protein, expressed on a variety of cells as monocytes, macrophages, neutrophils, Kupffer cells, Langerhans cells, CD1c+DC, pDC, CD141+DC, B cells, basophils, and eosinophils (Tone et al. 2019). The mast cells release some of its immuno-regulatory and anti-inflammatory molecules in dependence of Dectin-1 (Żelechowska et al. 2020). Is a C-type lectin, it means that have carbohydrate binding properties, within a compact protein region with a unique structural fold that became known as the C-type carbohydrate recognition domain (CTLD) (Weis and Drickamer 1996). More than 1000 C-type lectin receptors, 17 groups, affects immunity against pathogens and autoimmune diseases, cancer, also homeostasis and physiological regulation (Brown et al. 2018). The Dectin-1 is one on the cluster with seven structurally related receptors (group V), all with a single carbohydrate recognition domain, encoded in human genome (Tone et al. 2019). The three parts of Dectin-1 are (Effendi et al. 2020; Goodridge et al. 2012): • Single extracellular C-type lectin-like domain (CTLD) • A transmembrane region • A cytoplasmic tail that contains a single tyrosine-based activation motif Analytical tools such as x-ray diffraction, solid state cross polarized-magical angle spinning nuclear magnetic resonance (13C CP-MAS NMR), 1H-NMR titration and saturation transfer difference (STD)-NMR, diffusion ordered spectroscopy (DOSY)-NMR help to define the role of interaction groove where the glucan like molecules could link to the CTLD. The higher lengths of the β-glucan chains
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apparently increases the recognition, that could change the arrangement from unstructured to helically, or triple-helically complexes, to the activation of the innate immune response by mouse Dectin-1 (Hanashima et al. 2014). The exclusion chromatography and multi-angle light scattering experiments shown that induces the oligomerization of the Dectin-1 receptor leading to an active tetramer (Dulal et al. 2018). Studies with mutant proteins varying key amino acids shown that not only the presence of the hydrophobic Trp221/His223 groove CTLD is fundamental to accommodate a β-glucan chain, but also interaction loci are present and acts cooperatively to elicit a response in order to promote the tetramer aggregation. The Förster resonance energy transfer (FRET) measurements carried out by Anaya et al. revealed that molecular aggregation of Dectin-1 occurs dependently upon glucan higher order structure, forming very small (90 C, to kill microbial cells, reduce the broth viscosity, facilitate mixing during precipitation, and inactivate the enzymes that can modify the polymer in the following steps (Prajapati et al. 2013; Liu et al. 2017a). After removal of the cells by filtration or centrifugation, precipitation of the polymer in cell-free filtrate or supernatant occurs often by adding alcohols (ethanol, isopropanol, and methanol) and acetone (Palaniraj and Jayaraman 2011; Giavasis 2013). Extraction solution containing the precipitated polysaccharide still may have impurities, so additional steps include removal of proteins (deproteinization) and pigments (decoloration). Sewage and trichloroacetic acid methods cause protein denaturation, while hydrogen peroxide, activated carbon, dialysis, and macroporous resin techniques are useful for the removal of colored substances (Liu et al. 2017a, 2020). After this cleaning of impurities, the process of purification of the EPS fraction commonly encompasses membrane separation methods, such as microfiltration and ultrafiltration, which promote the separation of the polymer based on molecular weights through synthetic membranes with different pore sizes (Ruas-Madiedo and De Los Reyes-Gavilán 2005; Liu et al. 2020). Final purification stage involves the use of chromatographic columns, such as ion exchange and gel filtration chromatography, promoting separation of the polymers based on charge and molecular weight, respectively (Liu et al. 2017a).
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Antimicrobial Properties
Antimicrobial agents present in antibiotics, food preservatives, and disinfectants can cause inhibition of growth or death of microorganisms. However, microbial resistance is a reality, and these compounds can no longer cause metabolic and/or physiological alterations in the pathogenic microorganisms (Abushaheen et al. 2020). Moreover, the US Centers for Disease Control and Prevention (CDC) have estimated that antibiotic-resistant bacteria are responsible for causing about more than two million illnesses and over 23,000 deaths per year (Cao et al. 2020a). Thus, natural products may have effects against pathogenic microorganism strains and even action on those resistant to synthetic antimicrobials. Various microorganisms can produce chemicals with antimicrobial activity, such as protein or peptide compounds called bacteriocins and reuterin produced from glycerol by some strains of Lactobacillus reuteri (Gyawali and Ibrahim 2014). In addition, polysaccharides from different natural sources can have antipathogenic action (Albuquerque et al. 2020), such as the gums. For example, an EPS characterized as branched heteropolysaccharide constituted of galactoglucan and levan, produced by Lactococcus lactis, exerted a wide inhibitory activity against different species of bacteria and the fungus C. albicans (Nehal et al. 2019). Levan is a neutral homopolysaccharide constituted of d-fructo-furanosyl residues joined by β-2,6 linkages in core with lateral branches by β-2,1 linkages, and for its biosynthesis, the enzyme levansucrase is necessary, which is also called sucrose 6-fructosyltransferase (Srikanth et al. 2015a). Antimicrobial activity of levans produced by different microorganisms has been reported. Three levan compounds showed action against various foodborne pathogenic bacteria; these β-2,6-fructan compounds were the following: high-molecular-weight levan with molecular weight (MW) of 3 106 Da, and low-molecular-weight levan (MW ¼ 5 104 Da) synthesized by the levansucrase from Zymomonas mobilis and difructose dianhydride IV (DFA IV) produced by a levan fructotransferase from Arthrobacter ureafaciens (Byun et al. 2014). An exopolysaccharide produced by Bacillus tequilensis-GM was identified as levan, and this polysaccharide was able to inhibit biofilm formation and disrupt preformed biofilms from E. coli, S. aureus, and Enterococcus faecalis (Abid et al. 2019). Antiviral action has been reported for levan produced by different Bacillus sp. strains, which showed antiviral effect against respiratory virus (HPAI and H5N1) and enteric virus (adenovirus type 40) (Esawy et al. 2011). Dextran is an exopolysaccharide α-D-glucan, mainly composed of α-1,6-glycosidic linkage and in lower proportion of α-1,2; α-1,3; and α-1,4 branched linkages, obtained by different lactic acid-producing bacteria (Sajna et al. 2015) that can act against microorganisms. A dextran extracted from Leuconostoc pseudomesenteroides, for example, had an inhibitory activity against Escherichia coli and Staphylococcus aureus (Ye et al. 2019). However, some dextrans may not have any antimicrobial action as soon as they are isolated and may need to undergo chemical changes in their structure in order to exhibit this action (McCarthy et al. 2019). For example, amphiphilic dextran esters were obtained by the reaction between this polysaccharide and different substituted 1,2,3-triazoles-4-carboxylates. Antimicrobial analyzes by discdiffusion assay revealed that the presence of chemical groups -CH3 and -OCH3 as
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Fig. 1 Schematic representation of the mechanism of action from a cationic amphiphilic polysaccharide on the microorganism membrane surface
substituent groups on para position of aromatic ring had action against some microorganisms, such as S. aureus and C. albicans (Stanciu et al. 2020). Moreover, an approach that has been employed to make gums with antimicrobial activity is to make them cationic and amphiphilic. As shown in Fig. 1, death induced by these polysaccharides occurs since the positively charged layer can interact by electrostatic adsorption with the negative surface of the cell membrane followed by the weakening of the membrane by the interaction of a hydrophobic chain in the nonpolar tails of the membrane (Pan et al. 2019). Cationic amphiphilic polymers were obtained from dextran backbone containing hydrophobic alkyl chain and quaternary ammonium groups. These polymers had antimicrobial action against different species of bacteria and fungi (Tuchilus et al. 2017). A similar approach was carried out with the neutral polysaccharide curdlan, a linear β-1,3-glucan produced by Agrobacterium biovar, which upon receiving quaternary ammonium groups generated an amphiphilic polyelectrolyte with antibacterial action (against E. coli and P. aeruginosa) and antifungal (in assays with C. albicans) (Popescu et al. 2019). Xanthan gum is an anionic polysaccharide secreted by Xanthomonas campestris composed of a β-1,4-D-glucose backbone and side chains of D-mannose and D-glucuronic acid (Elella et al. 2020). The action against pathogenic microorganisms of xanthan has been attributed when this polysaccharide was grafted with poly (N-vinyl imidazole). The imidazole ring has cationic nature and confers antibacterial properties since it is able to interact electrostatically and hydrophobically with the cytoplasmic membrane; the penetration of this ring in the bacterial cell can cause its binding and destabilization of the bacterial DNA (Elella et al. 2017). Although this carbohydrate has a negative charge, xanthan-oligosaccharide exerted antibacterial action against S. aureus probably brought by its low molecular weight and hydroxyl groups in its structure; this oligosaccharide was also able to affect the cell membrane permeability and inhibit the biofilm formation of this microorganism (Wang et al. 2020).
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Antioxidant Action
Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are compounds that have one or more unpaired electrons and can participate in several pathological processes (Oliveira et al. 2018). However, natural
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compounds, such as polysaccharides from bacteria and fungi, can combat these oxidizing agents (Wang et al. 2013). A levan produced by Acetobacter xylinum had its antioxidant activity revealed by the ability to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Srikanth et al. 2015b); this happens when the unpaired electron of nitrogen in DPPH receives a hydrogen atom from antioxidant compound (Kedare and Singh 2011). Furthermore, the antioxidant mechanism of gums can also be characterized through scavenging assays of other free radicals besides DPPH. Another levan obtained from Bacillus megaterium was able to scavenge the DPPH, superoxide anion, hydroxyl, and 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals in a dose-dependent manner (Pei et al. 2020). Ability of gums, such as dextran, to reduce free radicals can be affected by their monosaccharide composition, concentration, content of carboxy and hydroxy groups, and hydrogen or electron donation capacity. The ion chelation assay can also be used to evaluate the antioxidant action of these polysaccharides since the presence of -Oand -OH- groups in dextran may be able to chelate Fe2+ (Du et al. 2018). It is important to evaluate the antioxidant activity in different assays since the singular experimental chemical conditions may indicate that the gum may have action in microenvironments of different chemical composition inside the cell. For example, beside testing the ability to reduce free radicals, the capacity to donate electrons from dextran was also determined by total antioxidant capacity assay, which assesses the property of reduction of the Mo+6 to Mo+5 ions and the reducing power test that estimates the potential to reduce potassium ferricyanide (Soeiro et al. 2016). Xanthan gum may also be able to fight oxidizing agents due to its pyruvate, acetyl, and glucuronic acid groups; the addition of the furfural radical in this polysaccharide increased the hydroxyl radical scavenging (Kang et al. 2019). A paradoxical factor can be attributed to the molecular weight of bioactive polysaccharides, since these macromolecules with high molecular weight can find barriers to penetrate the cell and exert their pharmacological action; nevertheless, their very low molecular weight can suppress its bioactivity (Li et al. 2016). Therefore, the antioxidant property of gums should also be evaluated when there is a reduction in their structure. Thus, a low-molecular-weight xanthan obtained by the biodegradation of this gum by the fungus Chaetomium globosum was able to exert scavenging effects on different free radicals due to the increase of carboxyl and hydroxyl groups that occurred by decrease in molecular weight (Hu et al. 2019). In other approaches, xanthan-derived oligosaccharides obtained by hydrolysis of xanthan in alkaline conditions also exhibited antioxidant activity (Xiong et al. 2013; Wu et al. 2013). Gellan gum is an anionic polysaccharide composed of repeating units of glucose, glucuronic acid, and rhamnose purified from Sphingomonas elodea. There are reports of low antioxidant activity for native gellan, which possibly stems from strong intermolecular and intramolecular hydrogen bond in their structure. However, carboxylated derivatives of gellan with different uronic acid content did improve the activity of scavenging radicals (Redouan et al. 2011). Another way to improve the antioxidant performance of gellan gum is by forming
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composite films based on lignin, a biopolymer consisting of aromatic groups, beside hydroxyl and methoxy functional groups that can donate hydrogens to free radicals (Rukmanikrishnan et al. 2020). Nonconventional EPS has been purified from bacteria and fungi, and its antioxidant capacity has been evaluated through different assays, mainly by the reduction of free radicals, as shown in Table 1. It is also noted in the Table that even due to the diversity in the monomeric composition of these polysaccharides, concentrations below 10 mg/mL can have the maximum efficiency of combating oxidizing agents. EPS with different saccharide compositions can be obtained from the same source, as can be seen from Weissella cibaria; however, they can have antioxidant effects in similar concentrations. Furthermore, it is observed that the genera Leuconostoc and Lactobacillus can be important suppliers of antioxidant EPS.
5
Anticancer Activity
An abnormal mass of tissues originated by abnormal growth or division of cells is called a neoplasm or tumor. The tumor can be classified as benign or malignant. When the tumor does not extensively invade the healthy surrounding tissues or spread to other parts of the body, it is classified as benign, whereas a malignant tumor is an uncontrolled growth of cells, and it can become progressively invasive. In this case, the term cancer refers specially to this malignant tumor (Hanahan and Weinberg 2011). Cancer could be considered the most aggressive disease worldwide despite the advancing technology and intensive researches about therapy and cure; cancer still ranks among the first cause of death in the world (Yildiz and Karatas 2018). Conventional cancer therapies, such as surgery, chemotherapy, and radiotherapy, present important limitations, such as poor prognosis and negative side effects (Bao et al. 2013). In view of this, researches of the last 20 years focused on the use of natural products in order to circumvent the toxic effects of anticancer agents. The question of identifying natural polymers with potential anticancer activity has contributed to carbohydrates from various sources being investigated. Some mechanisms for the antitumor effects of polysaccharides have been identified via in vitro cell line studies and in vivo protocols using animal models: NO pathway, cell cycle arrest (related to G0, G1, G2, and S cell cycle phases), depolarization of the mitochondrial membrane, and immunomodulation. Some polysaccharides act through multiple pathways, while others do not have the mechanism of action well defined (Albuquerque et al. 2020). In order to understand the role of polysaccharides as antitumor agents, it is important to explain the hallmarks of cancer; they comprise six biological capabilities acquired during the multistep development of human tumors, which occur under an organizing principle for rationalizing the complexities of neoplastic disease. The hallmarks include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (Hanahan and Weinberg 2011).
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Table 1 Exopolysaccharides with antioxidant activity
EPS source Weissella cibaria
Weissella confusa
Monosaccharide composition Glucose and rhamnose
Mannose, glucose, galactose, arabinose, xylose, and rhamnose Mannose
Leuconostoc pseudomesenteroides
Glucose
Leuconostoc mesenteroides
Fructose
Lactobacillus plantarum
Mannose, glucose, and galactose
Lactobacillus acidophilus
Glucose, galactose, and maltose Galactose, glucose, and mannose
Lactobacillus helveticus
Microbacterium aurantiacum
Glucuronic acid, glucose,
Mechanism of antioxidant action Capacity to reduce Fe3+/ferricyanide complex and by scavenging effect on superoxide anion, DPPH, and hydroxyl radicals Reducing power and scavenging ability on hydroxyl, DPPH, and superoxide anion radicals Capacity to reduce Fe3+/ferricyanide complex and by scavenging effect on superoxide anion, DPPH, and hydroxyl radicals Scavenging ability on hydroxyl, DPPH, and ABTS radicals Scavenging ability on hydroxyl radical Ferric reducing power, scavenging capacity on DPPH and ABTS radicals Scavenging ability on DPPH radical Metal ion-chelating activity and scavenging ability on DPPH, hydroxyl, and superoxide anion radicals Reducing power and scavenging ability on
Concentration with higher activity (mg/mL) 4
References AdesuluDahunsi et al. (2018)
5
Zhu et al. (2018a)
1.25
Lakra et al. (2020)
5
Farinazzo et al. (2020)
6
Taylan et al. (2019) Bomfim et al. (2020)
8
0.45
4
0.75–3.5
Abedfar et al. (2020) Xiao et al. (2020)
Sran et al. (2019) (continued)
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Table 1 (continued)
EPS source
Monosaccharide composition
Mechanism of antioxidant action
mannose, and fucose
hydroxyl, DPPH, and superoxide anion radicals Ferric reducing power and scavenging ability on hydroxyl, DPPH, and superoxide anion radicals Inhibiting the lipid peroxidation, scavenging ability on DPPH and superoxide anion radicals Inhibiting the lipid peroxidation, scavenging ability on DPPH and superoxide anion radicals Reducing power, inhibiting the lipid peroxidation, and scavenging ability on DPPH and superoxide anion radicals Reducing power and scavenging ability on hydroxyl radical
Bacillus sp.
Galactose, glucose, and mannose
Aspergillus sp.
Mannose and galactose
Oidiodendron truncatum
Glucose, mannose, and galactose
Lasiodiplodia sp.
Glucose and mannose
Fusarium equiseti
Mannose, glucose, fucose, rhamnose, xylose, and arabinose Glucose, mannose, arabinose, and galactose
Chaetomium sp.
Scavenging ability on hydroxyl and DPPH radicals
Concentration with higher activity (mg/mL)
References
7
Hu et al. (2019)
10
Chen et al. (2011)
4
Guo et al. (2013)
0.2
Kumar et al. (2018)
3
Prathyusha et al. (2018)
10
Zhang et al. (2017)
DPPH: 2,2-diphenyl-1-picrylhydrazyl. ABTS: 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
The progression of the aberrant proliferative cell cycle and the resistance of the cell death are hallmarks closely related to the activation of proto-oncogenes and/or inactivation of tumor suppressor genes. Regulators of cell cycle phase transitions are important targets for cancer treatment to reduce clonogenic capacity of tumor cells (Mastbergen et al. 2000). The two tumor suppressor genes encode the proteins RB
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(retinoblastoma associated) and TP53; they operate as central control points within two key complementary cellular regulatory circuits that govern the decisions of cells to proliferate or, alternatively, activate programs of apoptosis and senescence (Hanahan and Weinberg 2011). In relation to programmed cell death, the mitochondrial apoptotic pathway is one of the major routes of apoptosis, and mitochondria play a crucial role in processes of apoptosis signal. In this pathway, the key event is associated with the loss of the mitochondrial membrane potential, which allows the release of proapoptotic factors, such as cytochrome c, Smac/DIABLO, Omi/Htra2, endonuclease G, and apoptosis-inducing factor (Yu et al. 2016). Still above the response of the apoptotic stimuli, regulatory proteins of the Bcl-2 family, which contains pro- and antiapoptotic members, control the trigger that transmits signals between regulators and effectors. The archetype, Bcl-2, and their relatives Bcl-xL, Bcl-w, Mcl-1, and A1 are apoptosis inhibitors, acting most of the time by suppressing two proapoptotic triggering proteins: Bax and Bak. These are proteins of the mitochondrial outer membrane and, when out of the Bcl-2 inhibition action, can disrupt the integrity of the outer mitochondrial membrane, causing the release of proapoptotic signaling proteins. The most important proapoptotic protein is the cytochrome c, whose release is associated to the activation of a cascade of caspases that act via their proteolytic activities to induce the multiple cellular changes associated with the apoptotic program. In addition, Bax and Bak share domains of proteins, termed BH3 motifs, with the antiapoptotic Bcl-2-like proteins, thus mediating various physical interactions (Hanahan and Weinberg 2011). Up to this point, it was possible to define the hallmarks of cancer as acquired functional capabilities that allow cancer cells to survive, proliferate, and disseminate; these are the functions essentially acquired by different tumor types via distinct mechanisms and at various times during the course of multistep tumorigenesis. Modern knowledge about enabling characteristics and emerging hallmarks depicts other distinct attributes of cancer cells, being two of them particularly compelling. The first involves major reprogramming of cellular energy metabolism in order to support continuous cell growth and proliferation, thus replacing the metabolic program that operates in most normal tissues and fuels the physiological operations of the associated cells. The second one deals with the evasion by cancer cells from attack and elimination by immune cells, a capability that highlights the dichotomous roles of an immune system that both antagonizes and enhances tumor development and progression (Hanahan and Weinberg 2011). It is possible to predict that the most important bioactivities displayed by natural polysaccharides acting as immunoceuticals in clinical cancer therapies should be associated with their immunostimulant and antitumor effects. Most polysaccharides use specific receptors, for example, toll-like receptors 2 and 4, on the macrophage cell surface, and stimulate the cells via the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. NF-κB is a protein complex of inducible transcription factors responsible to regulate the expression of many genes encoding enzymes, chemokines, cytokines, and adhesion molecules involved in innate and adaptive immunity, antiapoptosis, inflammation, and proliferation. The MAPK family is a serine-threonine kinase that plays important roles in
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the regulation of cellular activities such as gene expression, cell proliferation, differentiation, development, and apoptosis (Chaisuwan et al. 2020). A comprehensive explanation of the main antitumor mechanisms displayed when microbial bioactive exopolysaccharides stimulate macrophages is shown in Fig. 2. Thereafter, the anticancer activity is demonstrated in various cancer cell lines through different mechanisms. Sarcoma is a nonepithelial tumor currently treated by chemotherapy, which is often characterized by serious side effects. Antineoplastic agents from natural sources can be suggested as the new generation of products from sustainable materials that can combine both ecological and health aspects. Therefore, exopolysaccharides from secure natural sources, for instance, Bacillus, might be a good alternative to synthetic antineoplastic drugs. CPS from B. velezensis SN-1, isolated in Da-jiang, China, was made and extensively characterized, in addition to being evaluated by antioxidant and antitumor activities against hepatocellular carcinoma (HepG-2). The antineoplastic analysis showed that the exopolysaccharide displayed significant antitumor activity toward HepG-2 tumor cells, which suggests the CPS generated by B. velezensis SN-1 as a natural antitumor drug (Cao et al. 2020b). Still in view of the genus Bacillus, the endophytic bacterium, MD-b1, isolated from the medicinal plant Ophiopogon japonicas and identified as the B. amyloliquefaciens
Fig. 2 A comprehensive explanation of the main antitumor mechanisms displayed by microbial exopolysaccharides. Polysaccharides bind to specific receptors on the macrophage cell surface and then stimulate important vias: the nuclear factor kappa B (NF-KB) and the mitogen-activated protein kinase (MAPK) signaling pathway. They are able to block the tumorigenesis through signals that translocate to the nucleus and activate transcription factors that regulate the gene expression of important proteins. Activated proteins, such as cytokines and enzymes, could initiate different mechanisms of action, including the NO pathway, modification of the cell cycle, depolarization of the mitochondrial membrane, and modulation of the immune system. Some polysaccharides act through more than one of the abovementioned mechanisms
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sp., produced EPSs with significant therapeutic activities against gastric carcinoma cell lines (MC-4 and SGC-7901). It is important to mention that this was the first study investigating the endophytic microorganism isolated from O. japonicas and also the first discovery of antitumor activity for EPSs derived from the genus Bacillus (Chen et al. 2013). Other Bacillus strains, in this case the B. circulans isolated from the slimy layer of coconut, produced appreciable amounts of an exopolysaccharide composed of glucose, mannose, and galactose that showed bioactivities including antioxidant, antiinflammatory, and antitumor activities. The strong antioxidant activity exhibited by this EPS forecasts its ability to serve as an antitumor compound against cultures of VERO cells and HepG-2 and Hep-2 cancer cell lines. The results clearly indicate that the EPS from B. circulans can serve as a potential candidate for the development of medical formulations (Vidhyalakshmi et al. 2016). Another study used seven mud samples (named as BS) collected from a gas station in Giza governorates, Egypt; the samples were located at the discharge of water from station that loaded with large quantities of gasoline spilled on the ground. The molecular identification using 16S rRNA gene fragments of the isolate BS4 belonged to the genus Bacillus with 99% identity as Bacillus mycoides strain ATCC 6426. Then, different concentrations of EPSs produced by B. mycoides strain BS4 were evaluated against normal cell baby hamster kidney (BHK) and tumor cells (HepG-2 and colorectal adenocarcinoma cells, Caco-2). The results provided a promising microbial BS4-derived EPS with significant therapeutic antitumor activities against hepatic and colon cancer; however, authors highlighted the importance of further works to study the relationship between the structure and the EPS function (Farag et al. 2020). Among the wide range of bacterial species able to produce EPSs, lactic acid bacteria (LAB) are a major group of species whose compositions, structure, and properties have been extensively studied. They usually have low cytotoxicity and side effects and may serve as an efficient alternative to the synthetic antitumor agents (Rahbar Saadat et al. 2019). For example, EPSs extracted from Lactobacillus casei 01 exerted antiproliferation effect on human colon cancer cell, HT-29 (Liu et al. 2011). The EPS isolated from L. helveticus MB2-1 was characterized and demonstrated to be composed of glucose, mannose, galactose, rhamnose, and arabinose. Preliminary in vitro tests revealed that EPS significantly inhibited the proliferation of HepG-2, BGC-823, and especially HT-29 cancer cells (Li et al. 2015). Also, the EPS of L. plantarum 70810 showed a significant antitumor activity on HepG-2, BGC-823, and HT-29 cancer cells (Wang et al. 2014). The EPSs from L. acidophilus have shown the antitumor properties against colon cancer cell lines, HCT15 and CaCo2 (Deepak et al. 2016). Colon cancer affects many organs and tissues and could be considered the most common type of cancer. Studies have shown that EPSs produced by probiotic strains can be effective against this disease. Probiotics are living microorganisms, preponderantly lactobacilli and bifidobacterial, or living microbial food ingredients that provide useful effects to consumers when applied in sufficient amounts. Some labs produce EPS, whose health benefits are related to their biological activities; in this case, EPSs might contribute to human health as prebiotics, i.e., nondigestible food
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ingredients/components/supplements that confer health benefits to the host upon specific stimulatory modulation of selected populations (Ruas-Madiedo et al. 2002; Yildiz and Karatas 2018). Leukemia, another important type of cancer and commonly known as cancer of the blood, is a malignant disease of the blood cell-forming tissue. There are important findings suggesting the use of EPSs as potential adjuvant chemotherapeutic and chemopreventive agents, also as effective drugs against human leukemia. For example, the anticancer activity of the EPS from Antarctic bacterium Pseudoalteromonas sp. S-5 was tested in human leukemia K562 cells. The EPS significantly inhibited the proliferation of K562 cells by morphological characteristics of apoptosis, such as condensation of chromatin and the formation of apoptotic bodies. EPS induced collapse of mitochondrial membrane potential and activation of caspase-9, suggesting that the intrinsic apoptotic signaling pathway was involved in apoptosis induced by the K562-treated cells. In addition, the ratio of Bax/Bcl-2 increased, and the calcium signal was associated with the cytotoxicity of the EPS against K562 cells (Chen et al. 2015). An EPS was isolated from the fermentation broth of Trichoderma pseudokoningii, and its anticancer activities on human leukemia K562 cells were studied. The results demonstrated that EPS significantly inhibited K562 cell proliferation in a time- and concentration-dependent manner, and this was mainly caused by apoptosis. Meanwhile, the dissipation of mitochondrial membrane potential, increased production of ROS, enhancement of the concentration of intracellular calcium, upregulation of Bax and p53 mRNA, and downregulation of Bcl-2 mRNA were detected, characteristics primarily involved in the mitochondrial pathways (Huang et al. 2012). Other study found that the halophilic bacterium Halomonas stenophila, strain B100, produced a heteropolysaccharide that, when oversulfated, exerted antitumor activity on T cell lines deriving from acute lymphoblastic leukemia. B100S was the first bacterial EPS that has been demonstrated to exert a potent and selective proapoptotic effect on T leukemia cells (Ruiz-Ruiz et al. 2011). In what concerns the group of eukaryotic microorganisms that have been reported to produce polysaccharides, fungi are demonstrated to produce EPSs with interesting bioactivities. For instance, an exopolysaccharide (EPS1-1) composed of glucose, mannose, galactose, and fructose was purified from the fermentation broth of the filamentous fungus Rhizopus nigricans and suggested as an antitumor drug against human colorectal carcinoma. EPS1-1 demonstrated significant inhibition of the proliferation of human colorectal carcinoma HCT-116 cells in vitro, in addition to induced S phase cell cycle arrest and increased sub-G0/G1 population, which could be considered a hallmark of apoptosis. It was also demonstrated that the mitochondrial pathway was involved in the EPS1-induced apoptosis since the polymer caused dissipation of mitochondrial membrane potential, accumulation of reactive oxygen species, up-regulation of Bax and p53 mRNA expression, and down-regulation of Bcl-2 mRNA expression (Yu et al. 2016). The anticancer activity of the polysaccharide extracted from the Auricularia polytricha fungus (APPs) was evaluated toward A549 human lung cancer cells, and its underlying mechanisms were investigated. APPs significantly inhibited the
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proliferation and DNA synthesis of A549 cells in a concentration-dependent manner. Also, the polysaccharide induced apoptosis in A549 cells by arresting cell cycle progression at the G0/G1 phase and significantly increased the expression of cyclindependent kinase (CDK) inhibitors p53 and p21, whereas the expression of cyclin A, cyclin D, and CDK2 was decreased by treatment with the polymer. The apoptotic induction in APPs-treated A549 cells could also be associated with the release of cytochrome c from mitochondria to cytosol, which in turn resulted in the activation of caspases-9 and -3, and the cleavage of poly (ADP-ribose) polymerase. Furthermore, the inhibitory effect of APPs on the growth in BALB/c-nu nude mice bearing A549 cells was also proven (Yu et al. 2014). Some bioactivities, including the antitumor activity, of the EPS extracted from an endophytic fungus of Crocus sativus L. (saffron), a scarce plant used in Chinese traditional herbal medicine, was examined for the first time. EPS exhibited remarkable cytotoxicity against K562, A549, HL-60, and human cervical cancer (HeLa) cells in a dose-dependent manner (Wen et al. 2018). The antitumor activity of the EPS extracted from other endophytic fungi, in this case the JY25, Chaetomium sp., isolated from the leaves of the Chinese medicinal plant Gynostemma pentaphylla, was evaluated against A549 cells and demonstrated good inhibitory effects (Zhang et al. 2017). A neutral water-soluble polysaccharide (HPA), mainly composed of mannose, was isolated from the marine fungus Hansfordia sinuosae, and its antitumor effect in vitro was tested. HPA showed a remarkable inhibitory effect on human cervical carcinoma HeLa cells and human breast carcinoma MCF-7 cells. Furthermore, for HeLa cells, HPA was able to increase intracellular ROS levels, induce cell apoptosis, decrease mitochondrial membrane potential, and elevate the expression of caspase3; all these findings are significant for suggesting HPA as a potential antitumor agent (Li et al. 2018). Another neutral polysaccharide (LGPS-1) had its anticancer efficacy tested; it was isolated from Lentinus giganteus, an edible mushroom with high protein content. The antitumor activity of LGPS-1 was assessed using HepG-2 cells and showed an inhibition proliferation rate of the carcinoma cells due to induced apoptosis through intrinsic mitochondrial apoptosis and PI3K/Akt signaling pathways (Tian et al. 2016). The strain Neopestalotiopsis sp. SKE15 was isolated and used for the production of an EPS tested against three cell lines: HeLa, human stomach cancer AGS gastric carcinoma, and human breast cancer MDA-MB-231. The EPS treatment (at the dose of 100 μg/mL) inhibited both the HeLa cells and breast cancer cells proliferation in the rate of around 61% and 56%, respectively, indicating the performance of the EPS as promising therapeutic agent since it was safe and active at low concentrations (Fooladi et al. 2019). The antiproliferative and apoptosis-inducing activities of an acidic polysaccharides fraction from Pholiota dinghuensis Bi mycelium (PDP-3) against human breast cancer MCF-7 cells were investigated. Interesting results displayed a significant prevention of cell growth toward MCF-7 cells by the p38/MAPK signal transduction pathway, which was the potential mechanism of PDP-3 in regulating both cell proliferation and apoptosis. The protein expressions of MCF-7 cells were
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upregulated p21 and downregulated cyclin D1, CDK4 and PCNA, upregulated Bax and downregulated Bcl-2, and caspase-9 and caspase-3. In MCF-7 cells treated with PDP-3, downregulated TRAF2, and upregulated ASK1, phosphorylated of p38 and p53 were also found (Gan et al. 2015). A crude polysaccharide extract obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz-cp) was able to inhibit the growth of cancer cells by inducing apoptosis preferentially in transformed cells. The main mechanism was reported to be the increase of intracellular calcium concentration and the activation of P38 to generate ROS via NADPH oxidase. The apoptosis was caspase dependent and occurred via a mitochondrial pathway (Kim et al. 2014). Two polysaccharides (termed PLPS-1 and PLPS-2) were isolated from mycelia of cultured Phellinus linteus, structurally characterized and submitted to in vitro antitumor assays. The monosaccharide composition, the molar ratio, and the carbohydrate components of their side branches were different, thus suggesting that the distinct antitumor activity between the two PLPS evidently occurred due to their structural alterations. In vitro antitumor assays for PLPS-1 displayed strong antiproliferative effect against S-180 sarcoma cells through apoptosis, whereas PLPS-2 had no such effect. Thus, PLPS-1 was considered a potential anticancer agent (Mei et al. 2015). The action of microbial polysaccharides on malignant cells was mainly demonstrated via immunomodulation and apoptosis. The anticancer activity was reported in various cancer cell lines, such as hepatocellular carcinoma, colorectal adenocarcinoma, human colon cancer cells, human leukemia cells, human lung cancer cells, human cervical cancer cells, and human breast carcinoma, the growth of the cells being directly suppressed by the polysaccharides. In addition, different anticancer efficacies might be involved with the molecular weight, composition, structural conformation, and charge characteristics of the polysaccharides.
6
Healing Capacity
The wound-healing process is a tissue survival mechanism initiated after an injury and roughly involves four integrated stages, including hemostasis, inflammation, proliferation, and remodeling; it is the guarantee that damaged or destroyed tissues are removed and the breach in tissue integrity is restored. The wound repair is performed spontaneously or by a connective tissue repair, both of them being independent of how the injury has been sustained, or whether the injury is minor, major, or confined to soft tissue trauma or is a surgical wound (Beldon 2010). Figure 3 presents the report for the normal acute physiological wound-healing process. In the course of a normal wound-healing process, immune-inflammatory cells appear transiently and then disappear. The innate immune system is involved in cleaning dead cells and the cellular debris; the adaptive immunity, which is accomplished by inflammatory cells such as macrophages, neutrophils, and myeloid progenitors, is engaged in the wound healing itself. The abovementioned immune cells
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Fig. 3 Normal wound-healing process and particular characteristics of its four orchestrated phases
are one of the major sources of the angiogenic, epithelial, and stromal growth factors and matrix-remodeling enzymes that are needed for wound healing; similarly, these factors are also needed to support neoplastic progression. In addition, lymphocytes B and T may facilitate the recruitment, activation, and persistence of both wound healing and tumor-promoting immune cells (Hanahan and Weinberg 2011). Given the last information, one could ask what is the main relationship between the wound-healing process and the development of cancer? This motivating question was already proposed by Schäfer and Werner (2008). In their review article, they highlighted some similarities between tumor generation and wound healing, including the abovementioned immune factors. They also pointed crucial differences, such as the altered metabolism, impaired differentiation capacity, and invasive growth of malignant tumors. In view of the sophisticated repair process, the reactions are synergic and ordered, contributing to a wound repair without interruption, for example, how it occurs in acute wounds. Differently, chronic or inert wounds are those interrupted by an orderly process for some reason, infected with microorganisms, or unable to heal because of a potential disease (Xiang et al. 2020). Defects in wound repair are considered a severe health problem and frequently affect aged patients, diabetics, immunosuppressed, and individuals submitted to chemotherapy or radiotherapy. They often develop painful, nonhealing ulcers. Neoplasia is a particularly dangerous complication of nonhealing ulcers, and the development of cancer in fibrotic tissue is a usual event. This fact was reported by Rudolf Virchow in 1863, who postulated
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that chronic irritation and previous injuries are a precondition for tumorigenesis (Schäfer and Werner 2008). Some important factors allow wounds to become one of the leading causes of death worldwide, for example, the high cost for wound care and the strong correlation between chronic wounds and increasing age. In view of this, there is a growing pressure for the development of advanced wound care that reaches the soaring demands; then, wound dressings are developed with the aim to protect the wound bed and promote skin regeneration to accelerate wound healing (Xiang et al. 2020). When choosing a wound dressing, many factors must be considered, including the stage of the current wound, the frequency of dressing replacement, the cost of the treatment, and associated drugs (such as antibiotics and painkillers). Characteristics of nontoxicity, biocompatibility, viscosity, and adequate mechanical properties must be considered when choosing an ideal wound dressing; it is essential that it could absorb the exuded tissue fluid, allow the exchange gas in time, prevent bacterial infection, and maintain a humid environment around the wound interface to allow the cells to adhere and proliferate properly, thereby promoting the wound healing (Xiang et al. 2020). The majority of the currently available wound dressings are either passive or interactive materials such as hydrogels, hydrocolloids, and films that provide a controlled environment for the site of healing. However, there are some of them whose ability to initiate the cellular responses is deficient, and hence they require the necessity of combining with other molecules that can stimulate and trigger target cell responses crucial to the wound-healing process (Mayet et al. 2014). Biopolymers able to interact with innate cells and promote wound repair have an advantage over the existing synthetic dressings, being an advantageous alternative for the current wound care sector. Polysaccharides are the major class of biomolecules with ideal physical, chemical, mechanical, and biological properties for wound dressings. Besides the mentioned properties of polysaccharides, they can be molded in different scaffolds that are crucial in developing functional biomaterials (Sahana and Rekha 2020). In particular, polysaccharides from microbial sources have emerged as economical and sustainable biomaterials for this field due to their fast and high-yielding production, and the excellent biocompatibility with human tissues (Ng et al. 2020). It is important to mention that the wound repair might be analyzed besides the antioxidant activity of the wound dressing. It is well known that, when the tissue is injured, short-term process of inflammation occurs because of the release of inflammatory mediators and ROS by the macrophages; thus, some steps of the wound healing process are impaired or delayed, retarding the repair. In view of this, the inhibition of ROS production is an important point to guarantee the proliferative phase of repair by fibroblasts (Trabelsi et al. 2017). The majority of works reporting microbial polysaccharides as wound-healing agents were developed using hydrogels scaffolds. For a better understanding, it is important to explain that hydrogels are artificial biomaterial scaffolds able to offer a much-favored 3D microenvironment for regenerative medicine. Exopolysaccharidebased hydrogels have been proposed as efficient dressings given their excellent water-reserving ability, and biocompatibility, i.e., because they do not interact
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biologically with human tissues, a critical limitation hampering their translation into paradigmatic scaffolds for tissue engineering (Ng et al. 2020). For example, a hydrogel composed of dextran-hyaluronic acid (Dex-HA) enriched with sanguinarine (SA) incorporated in the gelatin microsphere (GMs) was successfully evaluated in a rat full-thickness burn infection model. The wound-healing effects and antibacterial properties were analyzed; overall, the hydrogel exhibited the best healing effect when analyzed by in vivo wound healing; additionally, the inflammation reaction was lower than the other groups, and the epithelization, collagen deposition, and uniform distribution were faster when compared with the others. The results suggested that the SA/GMs/Dex-HA hydrogel provides a potential way for infected burn treatment with high-quality and efficient scar inhibition (Zhu et al. 2018b). A highly stretchable hydrogel composed of hyaluronan and sodium alginate was suggested as a matrix that could favor the anchorage of keratinocytes and thus enhance its cell carrier role for tissue regeneration (Murphy et al. 2012). A customized dextran-based was used for clinical translation and its regenerative response mechanisms in wound healing were determined. The hydrogel alone, with no additional growth factors, cytokines, or cells, was developed to treat thirddegree burn wounds on mice. Results demonstrated that the treatment promoted remarkable neovascularization and skin regeneration, which was suggested as a lead to treatments for dermal wounds (Sun et al. 2011). More recently, another hydrogel based on dextran demonstrated an accelerated healing mechanism, this time in a third-degree porcine burn model. The results suggested that the hydrogel may substantially improve healing quality and reduce skin grafting incidents, thus serving as a base for clinical studies to improve the care of severe burn injury patients (Shen et al. 2015). A thermo-reversible hydrogel composed of xanthan gum-konjac glucomannan (at different concentrations and ratios) was produced and characterized. The blends were considered hydrophilic and suitable for their future application as wound dressings. The ability to provide a moist local wound environment that absorbs excess exudate and promotes a proper wound healing was evidenced. Besides, the hydrogels also possessed adequate biological properties for supporting cell adhesion, migration, and proliferation, thereby promoting the cell secretion of extracellular matrix components to accelerate the granulation process (Alves et al. 2020). The wound-healing effects of the EPS produced by Lactobacillus sp.Ca6 (EPS-Ca6) were reported using an excision wound model in rats, which demonstrated that the gel of EPS-Ca6 accelerated significantly the wound-healing activity when compared to the nontreated group, and a total closure was achieved after 14 days of wound induction. Furthermore, histological examination of biopsies showed fully re-epithelialized wound with a complete epidermal regeneration. The antibacterial and antioxidant activities of this EPS were also evaluated through different assays, the results being compatible with a potential antioxidant activity (Trabelsi et al. 2017). The in vivo wound-healing performance, the in vitro antioxidant activity, and the rheological characterization of the EPS produced by Pseudomonas stutzeri AS22 (EPS22) were investigated. EPS22 showed good chelating ability and a
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pseudoplastic behavior, high elasticity, good mechanical strength, and stability with high water-absorption ability at 0.5% concentration, thus being considered a gel. The application of the PES22 gel was tested on dermal full-thickness excision wounds in a rat model every 2 days and enhanced significantly wound-healing activity and total closure (Maalej et al. 2014). An EPS produced and purified from Streptococcus zooepidemicus MTCC 3523 was identified as hyaluronic acid (HA) and showed significant wound-healing activity in a rat model. It is important to mention that HA is the major component of the extracellular matrix and directly contributes to wound healing through its role in repair and increasing strength of tissues. According to the authors, the obtained results of wound healing by treatment of HA were encouraging and in perfect agreement with the abovementioned HA information (Patil et al. 2011). Several bacteria from marine niche are adapted to synthesize EPSs with unique chemical compositions. When compared to polysaccharides sourced from plants and animals, they present advantages such as the ease of downstream processing, higher yield, purity, and lower cost (Moscovici 2015). An EPS isolated from a marine bacteria Alteromonas sp. PRIM-28 was used for EPS production. The polysaccharide, namely EPS-A28, was extensively characterized and tested for its bioactivities using in vitro models. EPS-A28, reported as an anionic heteropolysaccharide, showed biocompatibility and induced proliferation and migration of dermal fibroblasts and keratinocytes. In addition, EPS-A28 was able to increase the S-phase of cell cycle and to induce nitric oxide and arginase synthesis in macrophages. The results suggested that EPS-A28 could be potentially used as a multifunctional bioactive polymer in wound care (Sahana and Rekha 2019). Another marine bacterium, this time identified as Pantoea sp. YU16-S3 (EPS-S3), was evaluated as wound dressing by studying the key-molecular mechanisms in vitro and in vivo. The results demonstrated that EPS-S3 activated macrophages, facilitated cell migration in fibroblasts, and accelerated the phases of the cell cycle. In vivo experiments in rats showed the re-epithelialization of injured tissue with increased expression of growth factors in EPS-treatment. EPS-63 biosynthesized by the marine bacterium was a potential biomolecule for cutaneous wound-healing applications (Sahana and Rekha 2020). Considering that correct moisture levels are required for efficient recovery times in wound-healing processes, bacterial cellulose presents a high-water holding ability, which allows for the wound site to have the ideal moisture conditions. Due to the network of its nanofibers, scaffolds of bacterial cellulose (BC) might prevent infection by creating a physical barrier, thus preventing bacteria colonization into the wound (Ahmed et al. 2020). BC possesses an exceptional water vapor permeability which can be hugely beneficial in wound dressings due to the multitude of hydroxyl groups (Fu et al. 2013). Other publications highlighted BC suitable as a skin substitute material or a temporary wound treatment, for example, the ones who reported BC similar to skin (Lee and Park 2017) and able to maintain an optimal moisture content for the proliferation and regular function of epidermal cells and fibroblasts in a threedimensional culture model (Xu et al. 2016).
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Microbial polysaccharides with multifunctional activities targeted to the distinct phases of wound healing, i.e., hemostasis, inflammation, proliferation, and remodeling, have great promise as a single treatment to the complex physiological process of healing. EPS-based hydrogels have been considered the most ideal scaffold for the production of wound dressings since they display a threedimensional structure that mimics the native extracellular matrix of the injured tissue; additionally, their high-water content is essential in conferring a moist environment at the wound site, thus aiding the wound repair.
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Drug Delivery
Conventional methods to deliver drugs as oral and injection often present limitations in a particular therapy related to high dosages and repeated administration that can result in lower patient compliance, severe side effects, and toxicity. Other drug delivery alternatives including transdermal, transmucosal, ocular, and pulmonary pathways have been developed using biological polymeric films, hydrogels, micro-/ nanoparticles, beads, and tablets (Fig. 4) to address controlled drug release to cells and tissues over time and in space. These delivery routes can be more efficient,
Fig. 4 Examples of microbial gums biobased formulations for controlled drug delivery
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promoting an increase in the patient compliance, and reduction in the required dosage and side effects (Shirsath and Goswami 2019). Materials based on microbial gums have some functional properties, biocompatibility and biosafety required for the delivery of various drugs, which can be metabolized by the intestinal microflora and enzymes. However, gums in their native form have showed some disadvantages that include pH-dependent swelling/solubility, alteration in viscosity degree on storage, and uncontrolled rates of hydration that can interfere in their use as drug delivery (Rana et al. 2011). Thus, gums have been modified to minimize these limitations and improve their drug release properties, through grafting with polymers, cross-linking, and derivatization of functional groups, among others (Rana et al. 2015; Bhatia 2016). Xanthan gum is an important exopolysaccharide produced by Xanthomonas campestris bacteria with unique rheological properties for pharmaceutical formulations. The use of xanthan gum in various drug delivery systems has been reported highlighting this potential to control drug release due to its gelling property and ability to maintain the drug within the gel (Verma et al. 2017). A study investigated the application of a xanthan-based hydrogel to drug delivery for colon treatment. Xanthan gum was hydrolyzed and grafted with acrilamide using microwave irradiation. The grafting process has been used to alter its swelling and drug release properties (Rana et al. 2011). Morphological and thermal analysis showed that this modified-xanthan gum became somewhat smoother and more heat tolerant than nonmodified xanthan. The grafted xanthan was bound to a triamcinolone drug and showed a reduction in its swelling in media similar to the gastro-enteric and colonic systems, as well as a controlled drug release and noncytotoxicity, suggesting an efficient and safe drug delivery system for human use (Anjum et al. 2015). Xanthan gum hydrogels were also developed using xanthan gum/polyvinylpyrrolidone polymer cross-linked with acrylic acid for controlled delivery of 5-fluorouracil at colon-specific site. Cross-linking is an alternative to reduce the swelling degree of the gums, since the hydrophilic swell of gums can result in the leaking out of the entrapped drug prior to arrival at its specific site (Rana et al. 2011). Drug release studies revealed the controlled release pattern of 5-fluorouracil at colon-specific site for prolonged time period, and nontoxicity (Anwar et al. 2020). Another study has presented a cross-linked injectable hydrogel based on aldehyde-modified xanthan and carboxymethyl-modified chitosan for drug delivery. This hydrogel demonstrated self-healing, biocompatibility, biodegradability, and stable drug release within 10 h after injection in liquids. Controlled drug release was also observed, especially in tissues with abundant excretion and exudation. When loading a vascular endothelial growth factor in rats, the hydrogel allowed an interaction between this biomaterial and the animal, accelerating the angiogenesis and reconstruction of the abdominal wall tissue (Huang et al. 2018). Chitosan/ xanthan gum-based hydrogels were prepared as drug carrier system cross-linked for controlled acyclovir delivery. Acyclovir is an antiviral drug used against herpes simplex virus infections. The characterization analyses revealed that acyclovir was efficiently encapsulated into hydrogels, and the drug release behavior was pH dependent (Malik et al. 2020).
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Cationic polymeric nanoparticles based on gums as dextrans have showed great potential for drug delivery (Nguyen and O’Rear 2017). Biocompatible cross-linked chitosan-dextran sulfate nanoparticles have been synthesized for ocular controlled release of ciprofloxacin antibiotic drug, in order to increase the bioavailability compared to conventional eye drops. The therapeutic value of nanoparticles was evaluated in simulated tear fluid at pH 7.4. Ocular irritancy was evaluated and revealed that chitosan-dextran sulfate nanoparticles did not induce any vascular response, being nonirritant to the ocular surface. They also showed monotonous controlled-release for duration of 21 h. Antimicrobial assays done against ophthalmic microbes showed good results of minimum inhibitory concentration and minimum bactericidal concentration, confirming the drug efficacy (Chavan et al. 2017). Thus, chitosan-dextran sulfate nanoparticles can be considered a ciprofloxacin vehicle nonirritant that promotes prolonged time of drug availability, offering efficient therapeutic value. The use of dextran-sulfate as drug delivery platform for drug-coated balloons was also investigated. Drug-coated balloons are a promising alternative for peripheral artery disease treatment, but a significant drug loss from the balloon surface is observed during balloon tracking, reducing the therapeutic dose required at the diseased site (Kaule et al. 2015). To minimize this limitation, dextran sulfate films were prepared to carry an antiproliferative drug paclitaxel, and the drug release was investigated. Studies revealed that 10–20% of the drug loaded was lost during the time period of balloon tracking (1 min), allowing that a clinically relevant dose of drug be released during the typical time period of balloon inflation and treatment (from 1 min to 4 min). These data suggest the potential of dextran-sulfate films for controlled drug delivery from balloons (Lamichhane et al. 2016). Magnetic nanoparticles were synthesized with cationic dextran conjugated with the oligoamine spermine to improve hydrophilic capecitabine antineoplastic delivery to negatively charged cancerous cells. Dextran-spermine nanoparticles showed an attractive sustained release behavior up to 56% after 24 h at neutral pH, and a burst release up to 98% after 3 h in acidic pH. The in vitro cytotoxicity of the drug-loaded nanoparticles was evaluated in glioblastoma U87MG cells and indicated promising theranostic effect when compared to free capecitabine, showing the need of more studies for glioblastoma-therapeutic application (Ghadiri et al. 2017). Other approach using dextran consisted in cross-linked poly(ethylene glycol)-graft-dextran nanoparticles for reduction and pH dual response drug delivery into cancer cells. These nanoparticles were loaded with the antineoplastic drug doxorubicin and were efficiently internalized into cancer cells, when the drug was released in response to acidic pH or reduction environment. In addition, they showed good biocompatibility and significant inhibition of cancer cells proliferation, suggesting their potential for safe cancer therapeutics (Lian et al. 2017). Curdlan-based nanoparticles were also developed for biocompatible and safe therapeutic nucleic acid delivery. iRGD peptide-conjugated 6-amino-6-deoxy curdlan nanoparticles loading short interfering RNA (siRNA) were designed for delivery to integrin-expressing cancer cells. Curdlan was chemically modified in order to bind to siRNA and specifically enter integrin-expressing HepG2 cancer
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cells. The siRNA was effectively carried and released in the cytoplasm. Moreover, it induced significant inhibition of the polo-like kinase-1 (Plk1) gene, related to mitotic events and frequently over-expressed in human cancers (Ganbold et al. 2019). It suggests that iRGD peptide-functionalized curdlan nanoparticles for siRNA delivery are a promising therapeutic strategy for cancer. Other study investigated the efficiency of multiparticulate regioselective curdlan gum-based drug delivery system of moxifloxacin hydrochloride, an antibiotic absorved in the anterior part of the gastrointestinal tract, and therefore with a narrow therapeutic absorption window in this tract. Moxifloxacin beads were prepared via cross-linking of calcium, sodium alginate, and curdlan gum, and their release behavior of floating in gastric fluid and controlled release were investigated in vitro. More than 90% drug release was observed in about 10 h, and the beads remained buoyant more than 12 h. The kinetics modulation was controlled by the extent of cross-linked formation and bead sizes. These data suggest the potential of curdlan-based moxifloxacin drug delivery system for regioselective release and to improve bioavailability and therapy (Gurav and Sayyad 2019a). Curdlan gum was also used for design gastroretentive tablets of lamotrigine as regioselective drug delivery system. Lamotrigine is an antiepileptic agent exclusively absorbed from the upper region of gastrointestinal tract, and its unregulated plasma concentrations induce toxic effects, which can be prevented by a controlled gastroretentive drug delivery system. The lamotrigine tablets based on curdlan showed significant floating ability to release the drug regioselectively in stomach and proximal part of small intestine, and a floating time of at least 12 h (Gurav and Sayyad 2019b). Pullulan is another microbial gum that has been also reported as an efficient matrix for fast oral dissolving drug delivery applications due to its bioadhesive, mechanical strength and good properties of film formability (Prajapati et al. 2018). A pullulan film-loading enalapril maleate, an antihypertensive agent, was developed and exhibited good physicochemical properties, no interaction between drug and polymer, and high dissolution rate, favoring this use for mucoadhesive buccal drug delivery (Gherman et al. 2016). An electrically responsive poly(acrylamide)graft-pullulan copolymer was synthesized and evaluated under electric stimulation for transdermal drug delivery of rivastigmine tartarate through skin. When electric stimulation was applied, the drug diffusion rate was significantly enhanced in comparison with no electric stimulation condition. Analyses of skin histopathology revealed a reversible alteration in skin structure when it was submitted to electric stimulation, being an efficient biomaterial for transdermal drug delivery (Patil et al. 2020). Levan has been applied as a capping agent for gold and silver salt solutions in order to obtain polymer-capped nanoparticles (Sathiyanarayanan et al. 2017). Levan-capped silver nanoparticles showed bactericide effect against Escherichia coli and Bacillus subtilis. These nanoparticles were also introduced in an alginate gel in order to create a silver release system with bactericidal activity. The system reduced the bacterial survival up to 20% in 5–10 h depending on the bacteria genera, being useful for drug delivery applications, such as wound dressing (GonzálezGarcinuño et al. 2019).
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β-glucan has also been used for the delivery of drug molecules (Zhang et al. 2018). Particles based on β-glucan were prepared to contain a payload of rifabutin, an antituberculosis drug, for macrophage-targeted delivery. β-glucan particles were extracted from yeast cells by acidic and alkaline extraction and were either spray dried or lyophilized, followed by rifabutin loading and alginate sealing. Spray-dried β-glucan particles showed great results of thermal stability, drug entrapment, and release. The formulation was also phagocytosed by macrophages within 5 min of exposure, being a promise for targeted drug delivery to macrophage in the tuberculosis therapeutics (Upadhyay et al. 2017). Gellan gum is another microbially derived polysaccharide that has been applied in drug delivery formulations (Cardoso et al. 2017). A mucoadhesive in situ gel based on gellan gum and hydroxylpropyl methyl cellulose was developed for nasal delivery of the antiasthmatic drug salbutamol sulfate. In situ gel formulations are attractive for nasal applications due to their low viscosity into the nasal cavity, prolonged contact time, and continuous drug releasing (Gu et al. 2020). In this study, the drug release was found to be approximately 98% in 11 h. Viscosity and pH range were compatibles for nasal administration, and no histopathological damage was observed during ex vivo permeation studies. In this context, the in situ gel of gellan gum may be an efficient vehicle for nasal drug delivery (Salunke and Patil 2016). Other preparation of in situ gel of gellan gun was formulated for ocular delivery of brinzolamide, an antiglaucoma drug, in order to improve its therapeutic efficacy. In vitro drug release showed controlled and prolonged effect and less irritation degree than brinzolamide solutions after administration (Sun and Zhou 2018). A low-acyl gellan macrobeads-carrying meloxicam was evaluated for oral delivery in the prophylaxis of colorectal cancer. Nonalteration on meloxicam structure was observed during production of the beads. Swelling and release studies showed a pH-dependent behavior of the meloxicam release from gellan gum-based beads in vitro, allowing the application for specific delivery in the distal parts of the gastrointestinal tract (Osmałek et al. 2017). The structural properties of microbial gums make them promising materials for new drug delivery formulations for prevention and treatment of different diseases with more specificity and efficiency.
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Challenges in Microbial Gums
There is a great diversity of molecular structures for microbial gums; however, a few have been commercialized due to limitations related to production, extraction, and purification, including substrate availability, high costs, and cultivation challenges. With growing interest in microbial gums, research focused on approaches to solve the issues and challenges in their obtaining and applications. Many studies are taking place to isolate new strains, to use low-cost substrates, optimizing the process conditions involved in the extraction and purifications (Barcelos et al. 2019). The improvement of curdlan production from Agrobacterium sp. ATCC 31749 was evaluated with the addition of juice of discarded bottom part of green asparagus
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spear on culture medium. An increase of Agrobacterium sp. ATCC 31749 cell mass and enhancement of the curdlan production yield were observed, as well as a reduction in the cost of production and environmental waste resulting from the large-scaled discarded bottom parts of green asparagus spear (Anane et al. 2017). The high cost of the gellan gum production from Sphingomonas paucimobilis may limit its commercialization. A study showed the use of corn steep liquor as a costeffective nutrient source in culture medium to improve gellan gum yields, interesting for industrial production (Huang et al. 2020). A promise for increasing purity and high quality, these polysaccharides are metabolic engineering, which can manipulate the genes involved in encoding of the catalytic enzymes of the reactions in the pathway, or by altering a regulatory pathway (Shanmugam and Abirami 2019). This approach has been attempted for the production of some microbial gums, such as xanthan gum. Large amounts of ethanol are required to extract xanthan gum from Xanthomonas campestris fermentation broth, as well as to remove xanthomonadin impurities. In order to reduce the amount of ethanol and the production cost, a xanthomonadin-deficient strain of X. campestris was constructed by inserting the Vitreoscilla globin gene into the region of the pigA gene, resulting in the reduction of xanthomonadin synthesis. The insertion also induced an increase in the metabolism of X. campestris, allowing that the product ion of xanthan gum reaches wild-type levels. Thus, the engineered bacteria promote a reduction in the use of ethanol and the downstream-processing cost in xanthan gum production (Dai et al. 2019). This same strategy has also showed an enhancement of welan gum production in Sphingomonas sp., being of great importance for industrial applications (Liu et al. 2017b). Moreover, microbial gums have been modified via physical and chemical reactions to improve their functional properties for biomedical applications, such as antimicrobial agents, antitumor drugs, and polymeric matrices to produce hydrogels, coatings, and nanoparticles useful for controlled drug delivery, tissue engineering, and wound dressings (Ahmad et al. 2015; Shanmugam and Abirami 2019). For example, gellan gum oxidized with hydrogen peroxide showed an antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Aspergillus niger, growing with the oxidation level, having a potent application as antimicrobial coating material in the food industry (Lu et al. 2019). Other study developed a formulation of xanthan gum and polyvinylpyrrolidone polymer chemically crosslinked with acrylic acid monomer to fabricate pH-sensitive hydrogels for nontoxic and controlled drug delivery (Anwar et al. 2020). Thus, the scale of production, purity, and quality of microbial gums may be improved, contributing for industrial demands and commercialization.
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Conclusions
Gums of microorganisms are polysaccharides capable of forming viscous dispersions or gels in water, and many of these biopolymers have already been well characterized, structurally and biologically. EPSs can be produced on a large scale
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by microorganisms applying SSF and SmF methods, which use solid and liquid substrates, respectively. Optimization of this production can still be achieved through changes in parameters in the fermentation processes. Microbial gums, such as dextran and xanthan, had their antimicrobial action improved when they became cationic and amphiphilic, capable of interacting with the negative surface and hydrophobic tails of microbial cell membranes to cause its death. Antioxidant potential of EPSs is another biological property that these biopolymers can have and is generally determined by assays involving the scavenging of free radicals. It is very meaningful to review different sources of microbial polysaccharides which possess splendid activities in health, such as the activity of inducing apoptosis in oncotherapy and adjuvant therapy, and the ability to hasten the wound-healing process. Microbial gums-based delivery formulations are promising candidates for more specific, efficient, and controlled release of drug compounds, allowing the direct treatment of the cause of the disease. Despite challenges, progresses in the use of microbial gums have been registered through understanding the complexity and biomedical applications of these polysaccharides to shape and improve their various biological, physical, and chemical properties. Acknowledgments Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) is recognized for fellowships (LCBBC and MTSC) and grants. This work was also supported by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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The Promise and Challenge of Microbial Alginate Production: A Product with Novel Applications Wael Sabra
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Biosynthesis of Alginates and Their Biological Functions in Producing Bacteria . . . . . . . . . 2.1 Postpolymerization Modification of Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Biological Role of Alginate Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Industrial Production of Microbial Alginate: Challenges and Opportunities . . . . . . . . . . . . . . . 3.1 Technical Challenges in Alginate Production from A. vinelandii . . . . . . . . . . . . . . . . . . . . . 3.2 Nonpathogenic Strains of Pseudomonas as Promising Alginate Producers . . . . . . . . . . 4 Applications of Alginate in Food, Pharmaceutical, and Biomedical Sectors . . . . . . . . . . . . . . . 4.1 Alginate Applications in Food Sectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Pharmaceutical and Biomedical Applications of Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Alginate is a polysaccharide that has various established applications in food, textile, and pharmaceutical industries for its viscosifying and gelling properties. In the last two decades, and owing to their biocompatibility, and nontoxicity as well as versatility in view of modifications, several novel applications have been emerged that can be the base of novel future markets. Currently, seaweeds are the largest source for alginate production, and despite the variations in quality and quantity due to changes of climate and sources, most of the commercially produced alginates are still based on brown seaweeds. Nevertheless, alginate can also be biotechnologically produced by species of two families of heterotrophic bacteria, namely Pseudomonas and Azotobacter. Efforts
W. Sabra (*) Faculty of Life Sciences, Rhine-Waal University of Applied Sciences, Kleve, Germany e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_5
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have been made in the past to produce alginate polymers from these bacteria; however, its production never left the lab scale. Herein, the biological function of microbial alginate and the current process aspects with regard to its industrial production as well as the bottlenecks that still exist are highlighted. Based on their unique biochemical and biophysical characteristics and the ability to upgrade microbial alginate polymers through in vitro modifications, the achievements for alginate polymers of the last 10 years in food, pharmaceutical, and biomedical applications are discussed. Keywords
Azotobacter vinelandii · Pseudomonas · Tissue engineering · Scale-up challenges · Microaerobic culture · Alginate applications
1
Introduction
Alginate is a linear polysaccharide composed of (1–4)-β-D mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G). The distribution of the acids residues along the chain is nonrandom and involves relatively long sequences of each uronic acid arranged in a block-wise pattern (Fig. 1). The extent and composition of these sequences together with the relative molecular mass of the alginate chain determine the gel-forming ability and the physicochemical properties of the polymer. In the presence of divalent cations, such as calcium, alginate gels can be formed due to ionic cross-linking of G-blocks with some contribution from the MG-blocks by certain divalent cations which form bridges between L-guluronic acid residues on adjacent chains (Fig. 1). Alginate occurs as the main cell wall constituent of marine algae and comprises up to 50% of the dry weight. The different species of the brown seaweeds such as Laminaria hyperborea, Macrocystis pyrifera, and Ascophyllum nodosum are the main current source of all commercial alginate materials. Seaweeds are collected from coastal waters or cultivated seaweeds farms, dried, and crushed, and the polysaccharide is extracted and purified by several ion exchange operations. Although the quantities of brown seaweed exceed the global current demand, there is actually a lack of this raw material because they exist in remote areas where transport costs are excessive. In addition, alginates from seaweeds are subjected to product variations because of seasonal variations and growth conditions. It can even vary within the plant itself, with the frond (leaves), stipe (stem), and holdfast (anchor) showing progressively more poly-G. Indeed, algal alginates with predefined properties cannot be easily obtained from a particular algal species, thus limiting their use in the pharmaceutical and chemical industries. In food and beverage, paper, textile and printing, and various biomaterials industries, algal alginates are used as viscosifier, thickener, stabilizer, and gelling agent. In fact, there is a huge market for alginate with an estimated annual worldwide production of around 45,000 metric tons (Stanisci et al. 2020). Some of the manufacturers of sodium alginate include FMC Corporation; Cargill, Inc.; Qingdao Lanneret; and KIMICA and DANISCO.
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Fig. 1 Block structure of alginate (a) and its crosslinking with calcium cations in an eggbox model (b, c)
The price of alginate varies according to the source and quality, between 2 US$ and 40 US$/kg, and may reach more than 40,000 US$/kg for purified alginate for pharmaceutical applications (Pacheco-Leyva et al. 2016). Alginate can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum, and several species of Pseudomonas (Aarstad et al. 2019; Hay et al. 2013; Lotfy et al. 2018; Rehm and Valla 1997; Sabra et al. 2002). Unlike seaweed alginate, bacterial alginate production can be easily tailored to obtain molecules with different characteristics and unique compositions and, therefore, has the advantage that it may potentially be sold at higher prices, and this may open new markets for this polymer (Ertesvag 2015; Ertesvag et al. 2017; Gawin et al. 2020; Maleki et al. 2017). In fact, bacterial alginates are of potential commercial interest due to the fast development of pharmaceutical applications of this polymer as well as the discovery of its unique immunological properties (Aarstad et al. 2019). Certainly, the advances in biotechnological techniques have aroused the interest of industrial investors in developing a microbially optimized production process through controlling the growth conditions in a bioreactor while tailoring the production of biologically active high-value compounds.
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Biosynthesis of Alginates and Their Biological Functions in Producing Bacteria
A. vinelandii is a diazotrophically strict aerobic bacterium that synthesizes two polymers during vegetative growth, alginate, and the intracellular polyesters – polyhydroxybutyrate (PHB) (Fig. 2) (Castillo et al. 2013; Sabra et al. 1999, 2001; Sabra and Zeng 2009). All of the steps involved in the conversion of central sugar metabolites into the alginate precursor in A. vinelandii have been previously identified and characterized (Flores et al. 2015; Pacheco-Leyva et al. 2016). Pyruvate is
Fig. 2 Simplified metabolic pathway for the biosynthesis of alginate and poly-ß-hydroxybutyrate in Azotobacter vinelandii. The multiprotein complex involved in alginate polymerization and transport across the cell membrane is also shown. (Refer to Rehm and Moradali (2018), Urtuvia et al. (2017) for details about the protein-protein interactions and the complexity of alginate polymerization and transport)
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first formed from the available C6 sugar through the Entner-Douderoff pathway, which is then channeled to the tricarboxylic acid (TCA) cycle, while oxaloacetate from the TCA cycle is converted to fructose-6-phosphate via gluconeogenesis. The formation of guanosine di-phosphate (GDP)-mannuronic acid, the active precursor for alginate synthesis, from fructose 6 phosphate requires the action of several cytosolic enzymatic steps (Fig. 2). Phosphomannose isomerase (AlgA) catalyzes the conversion of fructose-6-phosphate to mannose-6-phosphate, which is directly converted to its isomer form, mannose-1-phosphate, by phosphomanno-mutase (AlgC). The activated mannose-1-phosphate is converted to GDP-mannose with the hydrolysis of GTP by the GDP-mannose pyrophosphorylase (GMP) activity of AlgA. The GMP activity of this enzyme favors the reverse reaction, but AlgD (GDP-mannose-dehydrogenase) constantly converts GDP-mannose to GDP-mannuronic acid, and the reaction is shifted toward GDP-mannuronic acid and alginate production. This AlgD-catalyzed reaction is essentially irreversible and provides the direct precursor for polymerization, GDP-mannuronic acid. The high intracellular levels of GDP-mannose indicate that this AlgD-catalyzed step is a limiting step and was shown to represent the metabolic bottleneck in alginateoverproducing strains of P. aeruginosa (Rehm 2009). Polymerization is done by membrane-bound glycosyltransferases which catalyze the transfer of an activated sugar moiety onto a receptor molecule while forming a glycosidic bond. The polymannuronate polysaccharide resulting from polymerization and then translocation to the A. vinelandii periplasm is composed of M-residues, which can then be further modified during its passage across the periplasm.
2.1
Postpolymerization Modification of Alginate
A unique feature for alginate biosynthesis is its synthesis in the periplasm as a homopolymeric strand that contains solely mannuronic acid residues. During its passage across the periplasm, several alginate-modifying enzymes work in a coordinated action to create alginate polymers of different properties at different environmental conditions. This postpolymerization modification of the alginate strands represents the critical steps needed to create several polymers with unique characteristics like gel-strength, chain stiffness, water binding capacity, viscosity, immunogenicity, and biocompatibility (Ertesvag 2015). In A. vinelandii, only one periplasmic epimerase, which incorporates single G residues into the alginate during secretion of the polymer, was found (AlgG). Moreover, a family of seven calciumdependent-mannuronan C5 epimerases (AlgE1–AlgE7) modifies the polymannuronate and produces specific epimerization patterns while introducing guluronic acid residue either as monomers or in blocks in the alginate strands. Recently, Aarstadt et al. (2019) showed the importance of the AlgE1 to form extralong G-blocks, when acting on poly-M, and concluded that A. vinelandii alginates can form gels with mechanical properties similar to the desired seaweed alginates and of comparable compositions, which strengthens the possibilities of commercial alginate production from microbial sources.
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The action of the different epimerases on the polymannuronate strand depends on the acetylation pattern by O-acetylases. Acetylation can occur at the hydroxyl groups of either the C2 or C3 position of the mannuronic acid residue. Only nonacetylated M-residues can be epimerized to G-residues by epimerases, while O-acetylated M-residues are protected from epimerization. Finally, the length of the alginate strand is determined by the activity of different alginate lyases. A multiprotein complex that is anchored to the membrane is responsible for (1) the polymerization (forming poly M); (2) protecting the nascent alginate against lyase activity, epimerization, and acetylation; and finally (3) completing the translocation of the modified alginate strands across the outer membrane and secretion to the external milieu (Flores et al. 2015; Hay et al. 2014; Rehm and Moradali 2018). Both alginate lyases and C-5-epimerases can be used in vitro to tailor alginates for specific purposes. Accordingly, the biochemical properties of these enzymes have been extensively characterized to understand the production mechanism (Aarstad et al. 2019; Castillo et al. 2013; Ertesvag 2015; Gawin et al. 2020; Stanisci et al. 2020), especially due to their potential biotechnological applications (Aarstad et al. 2013). Interestingly, most of these polymer-modifying enzymes in A. vinelandii were reported to be affected by the oxygen concentrations in culturing media (Castillo et al. 2013, 2020; Pacheco-Leyva et al. 2016). The molecular mechanism involved in alginate production linked to the availability of oxygen in A. vinelandii was recently discussed by Pacheco-Leyva et al. (2016).
2.2
Biological Role of Alginate Production
The role of exopolysaccharides (EPS) in nature has not been clearly established and is probably diverse and complex. It has been suggested that EPS may protect against desiccation, phagocytosis and phage attack, participate in uptake of metal ions, function as adhesive agents and stabilize membrane structure against the harsh external environment, or act as ATP sinks, or be involved in interactions between plants and bacteria. Alginates in phaeophyta are believed to act as a structureforming component providing mechanical strength and flexibility. This explains the difference in alginate compositions in different algae or in different regions of the same alga. As an alternative source of algal alginate and for the many advantages of bacterial alginates, several studies have been performed for the production of alginate by different strains of A. vinelandii and Pseudomonas spp. (Aarstad et al. 2019). A. vinelandii is distinguished from the rest of the family Azoto bacteriaceae by the presence of a characteristic life cycle which includes the formation of a spherically dormant cyst at carbon-limited conditions (Galindo et al. 2007). For the successful formation of the cyst, both PHB and alginate are essential. Alginate production is, therefore, a mandatory requirement for cyst formation in A. vinelandii and helps to protect the cells from desiccation, mechanical stress, and represents a diffusion barrier against heavy metal contamination. The alginate capsule was also shown to control the intracellular redox status of Azotobacter cells grown under N2-fixing conditions (Sabra et al. 2000).
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Diazotrophic-grown Azotobacter cells are able to grow under fully aerated conditions, despite the fact that the nitrogenase enzyme complex, which catalyzes the reduction of N2 to ammonia, is highly sensitive to oxygen. Therefore, the growth of N2-fixing A. vinelandii was shown to favor microaerobic condition (Sabra and Zeng 2009). Nevertheless, at high-oxygen concentrations, the bacteria have developed many mechanisms to protect their oxygen-sensitive nitrogenase. A. vinelandii was shown to create their microaerobic conditions by controlling the alginate capsules formation, and by varying their permeability properties through the actions of the different epimerases (Figs. 2 and 3). In a phosphate-limited continuous culture, it was shown that the specific rate of oxygen consumption (qO2) of the cells increased
Fig. 3 Thin-section electron micrograph of diazotrophically growing A. vinelandii cells in phosphate-limited chemostat cultures at low- (2.5% of air saturation) and high- (20%) dissolved oxygen concentrations (pO2, “a”), and the diffusion barrier exerted by a compact alginate capsule against oxygen at higher pO2 to protect nitrogenase (“b”). At a constant intracellular pO2 value suitable for nitrogenase, the difference in cell wall permeability as a function of extracellular pO2 values is shown in “c”
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as the dissolved oxygen tension (pO2) was elevated from 1% to 5% air saturation (Sabra et al. 2000). The qO2 remained, however, essentially constant when the pO2 was controlled above 5%. These results cannot be interpreted in terms of the “respiratory protection” concept. This is in agreement with several studies that indicate that respiratory protection is not the prevailing mechanism for nitrogenase protection above a certain oxygen concentration (Oelze 2000). Indeed, at constant qO2 values, while increasing the pO2 values, the bacteria could keep the intracellular oxygen concentrations (CO2, int), at a microaerobic or merely anaerobic range suitable for nitrogenase, if the permeability “P” of the cell wall to oxygen decreases sharply, as can be derived from (1). qO2 ¼ qO2, max ¼ P pO2,ext CO2,int constant
ð1Þ
It was also observed that both the molecular weight and the guluronic acid content of the alginate formed in phosphate limited chemostat cultures monotonically increased with pO2. The transmission electron microscopy of negatively stained A. vinelandii showed that cells at lower and higher pO2 formed capsules with significant differences in the thickness and compactness of the polysaccharide (Fig. 3). Such an alginate capsule was proposed to function as diffusion barrier against oxygen for the O2-sensitive nitrogenase enzyme complex (Sabra et al. 2000) (Fig. 3). The fact that alginate production in A. vinelandii is dependent on oxygen concentration has attracted many scientists to understand the link between oxygen availability and alginate production at a molecular level (Aarstad et al. 2019; Castillo et al. 2013; Gawin et al. 2020; Pacheco-Leyva et al. 2016; Urtuvia et al. 2017). Interestingly, earlier studies also showed an oxygen-dependent alginate biosynthesis with P. aeruginosa (Bayer et al. 1990; Leitao and Sa-Correia 1997). Concerning the biological role of alginate for the pathogenic bacteria P. aeruginosa, the causative agent of cystic fibrosis, it serves to protect the bacteria from adversity in its surrounding and also enhances adhesion to solid surfaces. Alginate is considered as a virulence factor, and as a result, biofilm develops which is advantageous to the survival of the bacterium in the lung (Hay et al. 2014). P. aeruginosa produces also alginate lyase which cleaves the polymer into short oligosaccharides resulting in increased detachment of the bacteria away from the surface, allowing them to spread and colonize new sites (Ertesvag 2015).
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Industrial Production of Microbial Alginate: Challenges and Opportunities
3.1
Technical Challenges in Alginate Production from A. vinelandii
In aerobic industrial processes, limitation of oxygen during growth is a major technical problem. Increasing the oxygen transfer rate through increasing the agitation speed and/or the aeration rate is normally done. On the one hand, the stirrer
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provides enough force to disperse the gas bubbles and increase their residence time in the medium. On the other hand, it provides homogeneity and mixing. Alginate production like many polysaccharides causes a drastic increase in viscosity. Moreover, alginate solution shows a non-Newtonian fluid behavior, in which the viscosity is shear rate dependent (Pandit et al. 2019). Whereas in lab-scale bioreactors turbulent flow condition is feasible by increased agitation, the high viscosities of polysaccharide fermentation broths make fully turbulent flow conditions impossible, or extremely difficult to achieve at large sale. During the growth of A. vinelandii in a bioreactor at microaerobic conditions, the rheological behavior of the broth changes from an “almost” Newtonian to a non-Newtonian behavior depending on the concentration of the polymer formed. In Newtonian fluids, it is manageable using reasonable techniques to relate operating variables (power number of the impeller, external power from the agitator, liquid density, impeller agitation speed, and impeller diameter) to power consumption and, hence, to the degree of agitation which proportionally affects the oxygen transfer rate. Hence an accurate control of the dissolved oxygen tension at an optimal value for polymer production can be achieved at the beginning of the fermentation. However, during alginate production, the broth behavior becomes non-Newtonian, and the apparent viscosities, which are not uniform within the fermentation vessel even at the same polymer concentrations, become extremely challenging for the process control. In such a solution, liquids-mixing intensity drops abruptly with distance from an impeller blade and, therefore, the apparent viscosities decrease at regions near the stirrer, while stagnant zones with higher apparent viscosities become near the vessel wall (Fig. 4). This rheological behavior of the alginate broth affects the oxygen transfer rates (kLa), the mixing characteristics of the fermentation, the power requirements, and scaling up. As a consequence, oxygen and nutrient limitations will mostly dominate near the bioreactor wall, while
Fig. 4 Heterogeneities in viscous fermentation and challenges for the formation of alginate in large-scale bioreactors by Azotobacter vinelandii
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oxygen and nutrient enrichments are to be found near the impeller zone. The insufficient mixing and mass transfer limitations with more gradient zones will escalate in industrial scale bioreactors (Fig. 4). Because of such heterogeneities, the reproducibilities in different scale bioreactors are challenging. Diaz- Barrera et al. (Diaz-Barrera et al. 2014) compared alginate production by A. vinelandii in two bioreactors with different scales (3 L and 14 L). While the two reactors had similar maximum oxygen transfer rates (OTRmax ¼ 19 mmol L1 h1) and produced the same alginate concentrations, the molecular weight of alginate obtained in the 3 L bioreactor was 1250 kDa compared to 590 kDa in the 14 L stirred fermenter (Diaz-Barrera et al. 2014). Several studies have demonstrated that the G/M ratio, the molecular weight, and acetylation degree of alginates produced by A. vinelandii can be modified by the dissolved oxygen concentrations. In fact, a narrow range of dissolved oxygen tension, within the microaerobic range, is needed for the optimal alginate production with a defined quality (Fig. 4) (Castillo et al. 2020; Garcia et al. 2020; PachecoLeyva et al. 2016; Sabra et al. 1999, 2000, 2001; Sabra and Zeng 2009). Since the control of the dissolved oxygen tension (pO2) at such a microaerobic range in a viscous pseudoplastic alginate broth is challenging, alternative process parameters (OTR, RQ) to monitor polymer production were investigated. Nevertheless, they have had limited success and were difficult to apply (Garcia et al. 2020; Sabra et al. 1999). Outside this narrow range for alginate production in A. vinelandii, the cell either exhibits a high-respiratory activity and hence wasting the C- source in respiration, or diverts the C-source to PHB synthesis at oxygen-limited conditions. In fact, this explains the complications associated with the biotechnological alginate production using A. vinelandii, and the additional risk accompanied by the process transfer to larger scales. It should be emphasized here that for other viable industrial polysaccharides processes like xanthan production by Xanthomonas compestris or gellan by Sphingomonas sp., such narrow ranges of pO2 for the polymer production are lacking (Lobas et al. 1994; Peters et al. 1989). Indeed, the deep understanding of bioreactor characteristics at different scales facilitates the successful development of any industrial processes. For the establishment of successful alginate fermentation processes using a productive A. vinelandii strain, a combination of strain improvements and process improvements should work in an integrated approach. While several studies on strain improvements and tailormade alginate production by different mutants were intensively carried out in lab-scale bioreactors, process improvements in different bioreactors and scaling up studies are largely missing in the literatures. In fact, more fundamental and engineering studies are desired that deal with lowering the oxygen sensitivity of alginate production (Fig. 4), novel bioreactors, and bioreaction techniques for large-scale and long-term operation.
3.2
Nonpathogenic Strains of Pseudomonas as Promising Alginate Producers
The knowledge about biosynthesis of alginates was first gained in P. aeruginosa, which has been widely studied as a model organism for alginate production and biofilm formation in the lung, especially in patients suffering from the genetic
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disease called cystic fibrosis (Rehm and Moradali 2018; Sabra et al. 2014). Although most studies have focused on clinical assays using P. aeruginosa, nonpathogenic species of the genus Pseudomonas were also studied for alginate production, and strains of P. mendocina, P. syringae, and P. fluorescens were shown to produce alginate under several growth conditions (Chang et al. 2007; Fan et al. 2019, 2020; Guo et al. 2012; Müller and Alegre 2006). P. fluorescens, a nonpathogenic plantcommensal strain was intensively investigated for alginate production by Ertestvag and her colleagues. While wild-type strains do not naturally produce alginate under laboratory conditions, the authors reported stable alginate production using mutant strains of P. fluorescens and reached up to 17 g/L alginate at a productivity of 0.35 gL1 h1 and a yield of 40% based on carbon (Gimmestad et al. 2014; Maleki et al. 2016). Unlike A. vinelandii, alginate production in Pseudomonas was constant within a wide range of pO2 values. Such a difference is not surprising, especially because of the lack of respiratory protection in Pseudomonas. Nevertheless, alginates synthesized from pseudomonads lack the polyguluronic block structures normally found in A. vinelandii and in algae. It is well known that solutions of alginate polymers with the same molecular weights result in more viscous broth as the G/M ratio increases. While the low G/M ratio alginate polymers are undesirable in many industrial applications, their production process in bioreactors is easier to control with less high-viscosity-related upscaling problems. Similar to A. vinelandii, the factors controlling alginate production and structure in P. fluorescens are well known, and different strains with different polymer characteristics can be obtained (Maleki et al. 2016, 2017). Gimmestad et al. (2003) produced a high-molecular weight polymannuronate polymer by using an epimerase negative strain of P. fluorescens. Such a polymer can be tailored for the production of particular alginate types for specialized applications by the action of different epimerases (Fig. 5). In fact, the in vitro use of different enzymes makes it possible to produce perfectly tailored alginate sequences not found in nature and thus improving polysaccharide performances for several applications. Recently, Gawin et al. (Gawin et al. 2020) used epimerase enzyme from A. chroococcum to introduce long G- blocks in the alginate strands. In general, alginates with a G-content of over 60% are highly desired for some medical and pharmaceutical applications. At the moment, such alginates with high G/M ratio can be extracted from the stem of Laminaria hyperborean which contains up to 70% G-content. However, the limited supply of such high G/M content alginates is currently limiting such applications. Interestingly, the alginate produced by the in vitro epimerization using the AlgE1 enzyme form A. chroococcum was reported to produce higher G-block polymers with a high degree of epimerization (up to 87%) (Gawin et al. 2020). This in vitro enzymatic approach to composition tailoring will definitely open the door for upgrading of various alginate samples into more valuable polymers. Indeed, engineering more robust industrial strains to produce alginate at industrial scales is the future goal, especially due to the growing interest in synthetic biology, which has great potential for enhancing wide range of industrial applications. Beside the quality of alginate in terms of molecular weight and the G/M ratio, the stable production of the polymer in large-scale bioreactors should be taken as a critical factor for evaluating the alginate-producing bacterium. The use of a microbial host,
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Fig. 5 Alginate production form brown seaweed are currently used in some established industries (shown in gray arrows). However, the seasonal variations, the sea current, the water temperature, and processing method are all factors that affect important structural features of the algal alginate produced, and hence heterogeneous qualities of alginate polymer are produced from brown algae. The advantage of using controlled growth conditions in bioreactors for bacterial alginate production, followed by the biocatalytic steps and valorizing alginate by the action of different modifying enzymes, will result in the formation of different high-value-tailored alginate products. This will open a new market for novel pharmaceutical and biomedical industries (shown in blue arrows)
whether the soil bacteria A. vinelandii or a nonpathogenic Pseudomonas strain, for alginate and/or high-value polymers production is clearly entering a new phase right now. The winner of such a race will definitely emerge in the next few years and will depend on developing a production process at the lowest possible cost. Designing new-generation bioprocesses that depend on engineering process-compatible microorganisms is the new roadmap. To achieve these goals, innovative fermentation process design that can reduce downstream-processing costs significantly, designing integrated systems with more fundamental knowledge about the control
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mechanisms, and process dynamics in large bioreactor are needed. Only by developing cost-efficient processes through integration of fermentation in large-scale and downstream processing, the microbial production of alginates can fulfill their potentials as biotechnologically produced high-value chemicals. On this journey, chemical engineers, metabolic/genetic engineers, system biologists, and microbial physiologists will have to work all together in an integrated cooperative approach.
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Applications of Alginate in Food, Pharmaceutical, and Biomedical Sectors
From the standpoint of the commercial use of alginate, three different criteria are important: • Alginate’s ability to form viscous solutions in aqueous media, which is in turn directly affected by the molecular weight, the number of M or G residues, and the solution strength. It is well known that the viscosity increases as the stiffness of the constituent chain blocks increases in the order MG7 from glucuronans degradation (Da Costa et al. 2001). On the same way, two cellouronate lyases were isolated from the bacterial strain Brevundimonas sp. SH203. The CUL-I and CUL-II enzymes synergistically perform, with respectively an endo- and exolytic mode, the depolymerization of the cellouronate, a (1,4)-β-D-glucuronan obtained by TEMPO-mediated oxidation from regenerated cellulose (Konno et al. 2006, 2008). Among fungi, the Trichoderma sp.GL2 strain was identified as producing an extracellular glucuronan lyase (TrGL), when cultivated on minimal medium as sole source of carbon (Delattre et al. 2006). The purified enzyme of 27 kDa, requiring
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Table 2 Glucuronan lyases (GL) enzymes characterized in various species PL Family PL5
GL activitiy mode Exo
PL14
Organism/Strain Stenotrophomonas maltophilia K279a, mutant H208F Catenulispora acidiphila DSM 44,928 Chlorovirus CVK2
Endo
PL20
Trichoderma reesei
Endo
PL31
Saccharophagus degradans 2–40 Streptomyces hygroscopicus
Endo
PL38
Brevundimonas sp. SH203
ND
Sinorhizobium meliloti M5N1CS
Endo and exo Endo
PL7
ND
Protein identification number GenBank: SNW08053.1 GenBank: ACU70527.1 DDBJ accession number 044791 GenBank: BAG80639.1 GenBank: ABD82242.1 GenBank: AGF62897.1 GenBank: GAW41138.1 ND
References Mac Donald et al. (2016) Helbert et al. (2019) Sugimoto et al. (2004) Konno et al. (2009a) Helbert et al. (2019)
Kikuchi et al. (2020) Da Costa et al. (2001)
ND Non-determinated
bivalents ions (Ca2+, Mg2+, Li2+, Mn2+) as cofactors for its enzyme activity, was shown functionally active on all glucuronans, with a variable efficiency depending to the acetylation rate of the substrate, and also on ulvan, a glucuronorhamnoxyloglucan extracted from Ulva lactuca (Delattre et al. 2006). The production of glucuronan lyase by virus was also reported, notably with the example of the chlorovirus CVK2 expressing the vAL-1 enzyme, in part involved in the cell wall degradation of the Chlorella host cells (Sugimoto et al. 2000, 2004). The identification of the genes encoding glucuronan lyases in some organisms has considerably contributed to acquire knowledge of structural features and families classification of these enzymes. Based on amino acids sequence similarities, the glucuronan lyases have been distributed within six families of PLs: PL5, PL7, PL14 (vAL-1), PL20 (TrGL), PL31, and PL38 (CUL-1) (CAZy database, http:// www.cazy.org) (Sugimoto et al. 2004; Konno et al. 2009a; Helbert et al. 2019; Kikuchi et al. 2020). Although the amino acids sequences may be highly divergent between the different families, the catalytic domains adopt a classical β-jelly roll-type structural scaffold, except for PL5 having an α/α-barrel organization. Interestingly, several studies underlined some convergences between the glucuronan lyases with the alginate lyases that may suggest a relatively close evolutionary process of these enzymes. For example, the alignment of β-jelly roll structures of the glucuronan lyase TrGL and PL7-A1 alginate lyase revealed the overlap of secondary features and the conservation of some amino acids catalytic residues (Konno et al. 2009b). In addition, the mutation (H208F) of an alginate lyase (PL5) from the Stenotrophomonas maltophilia K279a (Smlt2602) conferred the acquirement of a significant exolytic poly-β-D-glucuronic acid activity by the mutant strain (Mac Donald et al. 2016).
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Currently, the genomic and metagenomic data related to polysaccharide lyases allowed to identify and list at CAZy server many PLs for several organisms, which remained unexplored functionally. The contribution of research in activity and structural definition of “non classified” enzymes should lead to discover new PLs, among which glucuronan lyases, and could contribute to a better understanding of the large diversity of theses enzymes.
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Succinoglycans
3.1
Sources and Structures of Microbial Succinoglycans
Succinoglycan is a high-molecular-weight (>1 106 Da) heterogeneous polysaccharide with a branched repeating unit consisting of seven (1,3), (1,4), (1,6), and (1,4; 1,6)-linked β-D-glucoses and one (1,3)-linked β-D-galactose residues, associated to succinic and pyruvic acids adducts (Reinhold et al. 1994). Its basic structure is shown in Fig. 4. It is produced by bacteria belonging to Agrobacterium, Sinorhizobium, Rhizobium, Pseudomonas, and Alcaligenes genera when carbohydrates are used as carbon source. These bacteria often produce other EPSs. In several rhizobia an acetate substitution is also present on the main chain but not in agrobacteria. Pyruvate is always present in a stochiometric ratio whereas succinyl and acetyl ratios depend on culture conditions and bacterial species. The backbone of succinoglycan is composed of a tetrasaccharididic β-(1,4/3)-Dgalactoglucan. The side chain is linked to it by a β-(1,6) glycosidic linkage to the terminal glucose unit and has the following structure: [β-(1,3)-D-GLC-β-(1,3)-DGLC-β-(1,6)-D-GLC-β-(1,6)-D-GLC]. The terminal glucose of the side chain contains the pyruvate acetal O4 and O6 linked whereas the other β-(1,3)-linked D-GLC of the side chain is O6 connected with the succinate. The O-acetyl is present on the main chain of some suvccinoglycans O6 linked to a Glc unit. This structure was fully elucidated using methylation analysis,1H/13C NMR spectroscopy but also electrospray ionization and collision-induced dissociation and tandem mass spectrometry after methylation and deuteriomethylation of the octamer. 1H NMR spectroscopy gave access to to chemical shifts of CH3 protons of pyruvate and acetyl groups but also to methylene protons from succinyl one. 13C NMR revealed the chemical shift
Fig. 4 Chemical structure of succinoglycan
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of CH3 carbon of pyruvate (25.7 ppm), CH2 carbons of succinate (32.4 and 31.2 ppm), and acetyl carbon (102.2 ppm). Contrary to glucuronan, the biosynthesis pathway of succinoglycan is well known as it was abundantly studied in Sinorhizobium meliloti in 1990s. It consists of the assembly of nucleotide sugars (UDP-glucose formed from glucose-1-phosphate by a phosphoglucomutase being the precursor) in an octasaccharide repeating unit and the addition of acetate, succinate, and pyruvate before its polymerization and exportation. A large exo/exs cluster located on megaplasmid pSymB is responsible of the succinoglycan biosynthesis (Halder et al. 2017). Succinoglycan is the first EPSs identified as a molecule signal for numerous Rhizobium and Sinorhizobium invasions of leguminous nodules. The biological role of succinoglycan for other producers such as Pseudomonas or Agrobacterium is unknown but seems to be not implicated in interactions with plants. The production of succinoglycan has been abundantly documented in regards to its potential as texturing agent. Classical strategies of industrial microbiology have been applied to its production, including fed batch, as well as continuous and batch fermentations (Stredansky 2002). As observed with other production of soluble microbial EPSs, the rheology of broths limited the mixing of culture media but also the heat and gaz transfer with a negative impact on succinoglycan yields. Indeed oxygen availability in the reactor depend mostly on agitation and unlimited oxygen cultures giving the higher succinoglycan yields. This problem has been successfully overcame at the laboratory scale using solid-state fermentation (Stredansky and Conti 1999). The culture media for succinoglycan production in reactor by strains of Agrobacterium, Pseudomonas, or Rhizobium are not complex and with high C/N substrates ratio. They include a salt buffer, a low level of other mineral such as Mg2+ source, nitrogen, and carbohydrate sources. The nitrogen sources in industrial production are nitrates or ammonium salts even if other sources such as yeast extract, lysine, or glutamate are used. The carbohydrate sources are mainly sucrose even if glucose, maltose, and polyols such as mannitol at concentrations between 1% and 5% (w/v) give good yields. The pH has to be maintained around 7 and the optimal temperature of culture is 30 C. Batch processes are the more commonly used to produce succioglycan as they are much either to implement in industry with cheap substrates. They give also the higher final concentration in a time of culture of 3–4 days. Other processes such as continuous or fed batch ones have been also published but not really developed for large-scales productions. Indeed, even if continuous or fed batch processes have major advantages (productivity, for example), they have some disadvantages such as stability of strains, risk of contamination, and control of the dilution rate. Whatever the fermentation process, the extraction procedure is commun to other microbial hydrocolloids. Firstly the biomass is extracted from the broths using speraration technics such as filtration or centrifugation. The cell-free broths are then concentrated (or not) before to be dried to obtain low-grade succinoglycan or can be precipitated using polar solvent and mainly ethanol or isopropanol befor to be collected and dried leading to high-grade succinoglycan.
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3.2
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Enzymes Acting on Succinoglycan
The identification of the succinoglycan degrading enzymes was impaired by the relatively complex structure of this polysaccharide and its resistance to several β-Dglucanases (Amemura et al. 1974). The first successful approach was performed by the screening of the microorganisms able to grow on medium containing succinoglycan as the sole source of carbon, and thus likely producing succinoglycan depolymerizing enzymes (Oyaizu et al. 1982). Such an approach allowed the isolation and purification of an extracellular β-D-glucanase of 180 kDa (succinoglycan depolymerase) from the Flavobacterium sp. M64 strain (Amemura et al. 1974). The succinoglycan depolymerase, whose production is induced by the presence of succinoglycan or desuccinylated succinoglycan (D-SG) as sole carbon source, was reported to depolymerize both D-SG and succinoglycan to yield a polymer with a degree of polymerization, firstly estimated to 12 and finally considered as 8 (Amemura et al. 1974; Hisamatsu et al. 1978). The high specificity of the enzyme was clearly demonstrated for succinoglycan and D-SG, when no depolymerization activity was observed for others oligo- and polysaccharides, containing β-(1,3), β-(1,4) or β-(1,6) glycosidic bonds (Amemura et al. 1974). Due to its specificity, the succinoglycan depolymerase was used to elucidate the structure of polysaccharides related to succinoglycan, and has notably led to define octasaccharide as repeating units forming the polymer chain of polysaccharides elaborated by Rhizobium, Alacaligenes, and Agrobacterium, (Reinhold et al. 1994). In parallel to the action of the succinoglycan depolymerase, an intracellular endo(1,6)-β-D-glucanase was isolated from Flavobacterium M64, and was shown to be involved in the hydrolyzing of desuccinylated octasaccharide to two tetrasaccharides, one composed of D-glucose and pyruvic acid in a molar ratio 4:1 and the other composed of D-glucose and D-galactose in a molar ratio 3:1 (Abe et al. 1980). The combination of the succinoglycan depolymerase and endo-(1,6)-β-Dglucanase enzymes on different succinoglycans from diverse microorganisms has led to the same tetrasaccharide hydrolysis products, confirming the synergistic action of these enzymes (Fig. 5) (Abe et al. 1980; Reinhold et al. 1994). The ability for some microorganisms to regulate the size of their exopolysaccharides has suggested the hypothesis of the production of endogene glycanases acting on the depolymerization of these polymers. Rhizobium meliloti was described to produce in culture both a high-molecular-weight (HMG) succinoglycan (Wang et al. 1999) and low-molecular-weight (LMG) forms, including monomers, trimers, and tetramers of the octasaccharide-repeating unit (Battisti et al. 1992; Leigh and Lee 1988). Two endo-(1,3)-(1,4)-β-glycanases, ExoK and ExsH, were identified functionally active for the cleavage of succinoglycan from R.meliloti, only during the ongoing synthesis of succinoglycan by bacteria, but not efficient on succinoglycan present in cell-free medium (Glucksmann et al. 1993; York and Walker 1998). The physiologically role of ExoK and ExsH, specifically acting on nascent succinoglycan is clearly different from the succinoglycan depolymerase from Flavobacterium.
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Fig. 5 Action of glucanases from Flavobacterium in the hydrolysis of succinoglycan. SD succinoglycan depolymerase, EGase Endo-(1,6)-β-D-glucanase, Acl Acetyl, Sul Succinyl, Pyl Pyruvyl, О Glucose, ♦ Galactose
In conclusion, succinoglycan is a relatively complex structure which requires specific enzymes, remaining still little known, to ensure the degradation of this polysaccharide.
4
Rheology of Glucuronan and Succinoglycan
One of the main advantages of microbial extracellular polysaccharides over plant and seaweed polysaccharides is the possibility to induce constant chemical and physical properties (Manivasagan and Kim 2014) providing desirable and stable characteristics for high-value technological applications. Many authors have underlined the theoretical and practical value of the knowledges related to the rheological behavior of polysaccharides aqueous solutions through relations between viscoelastic flow properties, the chain structural conformation, and other physicochemical properties crucial for establishing potential applications area for these polymers. Microbial glucuronan and succinoglycan are water-soluble biopolymers at pH close to neutral and at room temperature and could have a significant commercial value related to interesting and specific features such as viscous, thickening, and gelling property. However, only a few studies detailed the rheological behavior of these polymer solutions. The main factors that affect the viscoelastic properties are classified as relative to the polymer-solvent system (as the polymer concentration, size and shape, the polydispersity index, the nature and the concentration of the solvent cations, and the interactions between polymer chains and between polymer and solvent) and relative to the operating parameters (shear rate, temperature, etc.). Heyraud et al. (1993) reported about the thickening properties of low concentrated (1 g.L1) solution of partially acetylated (16% of O-acetyl groups (w/w))
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extracellular (1,4)-β-D-glucuronan produced by fermentation by a mutant strain of Rhizobium meliloti M5Nl. At high concentrations, the polymer produced a thermoreversible gel in monovalent cation media and a thermally stable gel in presence of divalent cation (as Ca2+) for temperatures lower than 100 C (Heyraud et al. 1993). The gel strength increased when the degree of acetylation of the polymer decreased. The same authors proposed an empirical relationship between the intrinsic viscosity and the molecular weight close to models proposed for sodium alginate. The acetylation degree of glucuronan also influences the polymer solubility as high acetyl content promotes interchain associations and aggregate formation. Hence, Elboutachfaiti et al. (2011) suggested that the solubility is higher for less acetylated exocellular β-(1,4)-D-glucuronan from Rhizobium meliloti M5N1CS strain. Navarini et al. (1992) studied the viscoelastic properties of a mixture of succinoglycan and galactoglucan produced by Rhizobium meliloti YE-2 grown under different osmolarity conditions. The proportion of succinoglycan increases at high osmolarity (0.4 and 0.6 M) till 85% and as this polymer adopts an ordered conformation in solution, the mixture forms a “weak gel” instead of an entangled system as at low osmolarity (0.0 and 0.2 M) galactoglucan is dominant. The viscoelastic behavior of polymer mixture recovered from the culture media of 0.4 M osmolarity is similar to that obtained for xanthan/guar mixture and the shear-rate-dependent behavior is shear thinning for all osmolarities. Succinoglycan produced by Pseudomonas sp. NCIB 11592 exhibits reversible pseudoplastic behavior in dilute and semi-dilute solutions (Gravanis et al. 1990). Viscosity is sensitive to the ionic strength since a decrease was observed when ionic strength increases. This is explained by the worm-like chain behavior of the polymer as a result of electrostatic interactions when cations are present in the solvent. The overlap parameter C*[η] for succinoglycan solubilized in 0.1 M NaCl was estimated at 1.3. In comparision to xanthan, the succinoglycan exhibites a sharper conformational transition with a greater decrease in the viscosity explained by the greater flexibility of the chain conformation. Simsek et al. (2009) correlate physical, chemical, and rheological properties of succinoglycans of different molecular weights produced by four different strains of Sinorhizobium meliloti strains. The rheograms show typical shear thinning behavior following a power law model for all samples and a flow index between 0.61 and 0.79. Oscillatory dynamic tests have shown a more elastic behavior at high frequencies and various degrees of entanglement of these molecules. A low abundance of succinyl group favors the elasticity and viscosity of succinoglycans. Andhare et al. (2017) proved that succinoglycan produced by Rhizobium radiobacter strain CAS has a high industrial significance due to its higher viscosity at the same concentration (1% aqueous solution) regarding widespread commercial biopolymers such as xanthan, guar gum, and sodium alginate. The rheological analysis shows that the polymer solution exhibits high stability at several thermal cycles, high temperatures, large pH range, and wide electrolytes concentrations (mono- and divalent cations) and presents succinoglycan as a great rheological modifier/stabilizer in food and pharmaceutical industries.
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Conclusions
The developments of succinoglycan and glucuronan applications is clearly limited by the market competition of other microbial and above all terrestrial plants or seaweeds polysaccharides. Indeed, these two anionic and water-soluble rhizobacterial EPSs with varied biological and physicochemical properties should be more investigated for their commercialization and for their costs of production. Process dévelopments to increase the yield of production and metabolic engineering could be two levers to increase their marketization. Process developments should focus on improvement of fermentation techniques with the aim to a better oxygen diffusion in the culture medium and a limited foam formation in the reactor. The oxygen and nutrients diffusion being currently the main limiting factor implements the mixing of systems. Moreover, the extraction and purification processes avoiding high water and/or alcohol consumptions could be also a part of the production process to implement. However the metabolic engineering of the production strains is probably the largest potential of innovation for the production of these two polysaccharides. The metabolic engineering should deregulate the biosyntheis pathways of these two EPs but also to inactivate polysaccharide hydrolases and polysaccharide lyases expressed by the producing strains probably to regulate the molecular weights of these biopolymers. For that a better understanding of biosyntheis pathway of glucuronan should be of first importance. Moreover, majority of patents focusing on succinoglycan and glucuronan applications are older than 15 years and will have shortly no more patented protections.
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Cyanobacterial Extracellular Polymeric Substances (EPS) Rita Mota, Carlos Flores, and Paula Tamagnini
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Cyanobacterial Extracellular Polymeric Substances (EPS) and Their Biological Roles . . . 3 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 First Steps and Modification Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Assembly and Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Regulatory Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Optimization of Production and Isolation Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Tailoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Biotechnological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Water Treatment and Soil Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Food, Personal Care, and Other Industries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Biomedical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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R. Mota i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal e-mail: [email protected] C. Flores i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Centre for Urological Biology, Department of Renal Medicine, University College London, London, UK e-mail: c.fl[email protected] P. Tamagnini (*) i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_11
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7 Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Abstract
Cyanobacteria are a very particular group of photoautotrophic prokaryotic organisms with major ecological significance in the carbon and nitrogen cycles. Most cyanobacterial strains produce extracellular polymeric substances (EPS), mainly heteropolysaccharides that can remain attached to cell surface or be released into the extracellular environment (RPS). These EPS play prominent roles on cell protection and biofilms/colonies formation, and have distinct features compared to other bacterial EPS. Here, we review the cyanobacterial EPS biological roles, what is known/can be inferred about their biosynthetic pathways, and describe their particularly complex composition/structure. In addition, we address how to optimize the production and yield, and how to tailor the polymers either by genetic manipulation or by chemical modification. At last, already implemented applications as well as promising ones are discussed foreseeing the commercialization of cyanobacterial EPS-based products. Keywords
Cyanobacteria · Extracellular polymeric substances (EPS) · EPS applications · EPS biosynthesis · EPS characteristics
1
Introduction
Cyanobacteria are an ancient group of gram-negative prokaryotes with the ability to perform oxygenic photosynthesis, and many strains can also fix atmospheric nitrogen, making these organisms prominent players in the carbon and nitrogen cycles. Moreover, cyanobacteria are highly diverse morphologically, comprising species that are unicellular, form multicellular filaments, or grow in colonies. Some filamentous strains undergo cell differentiation, forming heterocysts, specialized cells involved in nitrogen fixation, and akinetes, resting cells that are able to endure environmental stress. Some have also the ability to produce hormogonia, small chains with different size, shape, and motility compared to the parent filament, essential for short-distance dispersal and host infection. The cyanobacteria long evolutionary history, autophototrophy, and diazotrophy are regarded as the main reasons for their success in modern habitats, allowing these microorganisms to thrive from fresh to salt water, soils, and extreme environments. Additionally, cyanobacteria managed to adapt their living styles according to the metabolic necessities, being able to live as planktonic free-living cells, in biofilms, aggregates, and complex mats, or even in symbiosis with a wide range of eukaryotic hosts (providing them combined nitrogen and/or carbon). Most of the cyanobacterial strains are also able to produce extracellular polymeric substances (EPS) that can remain attached to the cell surface or be released into the surrounding environment – RPS (Rossi and De Philippis 2016). The development
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and biotechnological application of added value products based on cyanobacterial EPS is increasingly attractive, since there is a constant implementation of restrictive regulation in the synthetic polymer industry to promote environmental preservation. This led to an increase in the global market demand for products with a lower ecological footprint (“green materials”), even considering the production costs, downstream processing, and some batch inconsistency in quantity/quality of the final product (Singh et al. 2019). The natural polymers are derived from renewable resources and by definition, they present several competitive advantages compared to the recalcitrant petroleum/oil-based products, such as biodegradability, eco-friendliness, and often biocompatibility. Furthermore, production of bacterial EPS at the industrial scale presents important advantages compared to the production of biopolymers from other natural sources: (i) they are generally actively secreted by the cells, being easy to extract and usually do not need a challenging purification process reducing the production costs; (ii) are not ruled by the stringent environmental measures applied in the production of polymers using animals or plants (e.g., ethics and animal protection or deforestation preventive measures); (iii) the strains used have usually fast growth rates accelerating the production process; and (iv) in some cases, the polymers and/or the producing strain can be easily functionalized/ engineered to obtain a product with the desired properties and/or enhanced performance. Additionally, EPS production by cyanobacteria brings also two other major advantages, even compared to the production of bacterial EPS that are currently being commercially exploited: (i) cyanobacteria have extremely simple nutritional requirements, since their growth and EPS production do not depend on the addition of expensive EPS precursors or substrates, and (ii) the cyanobacterial EPS are more complex, which make them more versatile and with a broader spectrum of potential application fields, mainly in high-value market niches, such as cosmetic, pharma, and biomedicine (Singh et al. 2019). However, despite all the already available data, the cultivation, extraction, and purification of these polymers is not yet implemented at industrial level allowing the control of fundamental requirements for clinical applications such as purity, stability, and safety. In addition, a detailed knowledge on the cyanobacterial EPS biosynthetic pathways is still required in order to enhance bespoke production of target EPS, and to engineer structural and compositional variants tailored for a given application. Therefore, the main aim of this chapter is to provide the state of the art on cyanobacterial EPS biosynthesis, characteristics, and putative biotechnological applications, seeking to contribute to the implementation of cyanobacterial polymers or products based on cyanobacteria polymers on the market.
2
Cyanobacterial Extracellular Polymeric Substances (EPS) and Their Biological Roles
Cyanobacteria can produce EPS, mainly composed by complex heteropolysaccharides (but also containing proteins, nucleic acids, and lipids) that can remain attached to the cell surface as capsular polysaccharides (CPS), or be released into the surrounding environment (RPS). According to their thickness and
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consistency, the EPS attached to the cell surface can be designated as sheaths (usually a thin, dense layer loosely covering cells or groups of cells); capsules (a thick and structurally coherent layer tightly associated with the cell surface); or slimes (a mucilaginous material dispersed around the cells but not reflecting their distinct shape) (Fig. 1) (Rossi and De Philippis 2016). The RPS biosynthetic process seems to diverge from the synthesis of CPS, and this hypothesis is mainly supported by differences in their composition, namely the presence of different monosaccharidic residues and sulfur content, Synechocystis sp. PCC 6803, and some of its EPS-related mutants (Pereira et al. 2019a). The cyanobacterial EPS can have distinct biological functions, depending on their structure and complexity and, thus, their physicochemical properties. The most acclaimed EPS role is cell protection, since they constitute a physicochemical barrier surrounding the cell that protects it against several environmental and stress factors, such as desiccation, heat, ultraviolet radiation, high salts concentration, predation, infection, and other external agents (e.g., antibiotics and toxic metal cations) (Kehr and Dittmann 2015). EPS can also contribute to the concentration/sequestration of nutrients, namely of essential metal cations and minerals, in particular in environments
Fig. 1 Light micrographs highlighting extracellular polymeric substances (EPS) from several cyanobacteria stained with Alcian blue. (a) Cyanothece sp. VI 22, (b) Calenema singularis LEGE 06188, (c) Geminobacterium atlanticum LEGE 07459, and (d) Cyanothece sp. CCY 0110. cp capsule, sht sheath, sl slime, RPS released polysaccharides. (Images b and c were kindly provided by Ângela Brito. Scale bars: 15 μm)
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where the availability is limited, e.g., marine. Metabolic imbalances can also represent a challenge for cyanobacteria, and EPS can serve as a sink for excess of energy if the carbon/nitrogen balance is shifted toward excess carbon (Pereira et al. 2019b). Moreover, it is widely known that the establishment of symbiosis between cyanobacterial symbionts and their hosts is mediated by glycans and lectins (Kehr and Dittmann 2015), and that EPS play a role in the gliding motility mechanism of hormogonia by slime extrusion through junctional pore complexes (Khayatan et al. 2015). Moreover, EPS can be involved in biofilm formation, having a major role in the mats complex microbial community structure. Furthermore, EPS can mediate cell adhesion to surfaces by a cohesive three-dimensional polymer network that allows cell-cell communication (either as vehicles of compounds or as quorum sensing molecules) and retention of extracellular enzymes (Rossi and De Philippis 2016). Likewise, they can contribute to cell aggregation and adhesion to solid surfaces and particles leading to the formation of biological soil crusts (Rossi et al. 2018). The contribution of cyanobacterial EPS to the establishment of colonies is also evident, and in the case of Microcystis they are involved in the initial steps of the formation of cell aggregates that can lead to the formation of toxic mucilaginous blooms with serious environmental and ecological hazard (Kehr and Dittmann 2015).
3
Biosynthesis
The intricate machinery underlying bacterial EPS biosynthesis have been extensively studied over the last two decades, mainly in pathogenic gram-negative and lactic acid gram-positive bacteria (Low and Howell 2018; Schmid 2018). In general, the biosynthetic process relies on three major steps (Fig. 2): (i) in the cytoplasm, energy-rich metabolites, mainly from glycolysis, react with monosaccharides to form nucleotide sugar precursors; (ii) glycosyltransferases catalyze the formation of glycosidic bonds by transferring sugar residues from the nucleotide sugars onto acceptor molecules (e.g., lipid carrier at the plasma membrane or other sugars) defining the composition of the polysaccharide backbone; and (iii) proteins in the cell envelope continue the assembly and/or polymerization and export of the polymer (participating in the control of both EPS composition and chain length). The last steps are quite conserved and mainly occur via three different pathways, the so-called Wzy-, ABC transporter- or synthase-dependent pathways (see Sect. 3.2).
3.1
First Steps and Modification Enzymes
The group of enzymes working in the first steps of bacterial EPS biosynthesis is highly diverse and heterogeneous, varying significantly between strains. Furthermore, the majority of these proteins are not exclusively dedicated to EPS production, being important players in the general (carbohydrate) metabolism. After the synthesis of nucleotide sugar precursors, carbohydrate-active enzymes (well known as CAZymes) with functional domains that degrade, modify, or create glycosidic bonds
Fig. 2 Schematic representation of the main bacterial EPS biosynthetic pathways. (I) Nucleotide sugar precursors are formed in the cytoplasm; (II) the monosaccharides start to be assembled onto a lipid carrier by glycosyltransferases (GT) and modified by enzymes such as epimerases and other carbohydratemodifying enzymes (ME); (III) the polymerization of the repeating units and export of the polymer through the cell envelope may occur by one of the three main mechanisms (Wzy-, ABC-, or synthase-dependent). In the synthase-dependent pathway, a lipid carrier may not be present, and the synthase accumulates the functions of GT and transporter. IM inner membrane, PG peptidoglycan, OM outer membrane, TPR tetratricopeptide repeat (TPR)-containing scaffold lipoprotein, CR c-di-GMP receptor
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start the assembly and modification of the polymer or its repeating units (Lombard et al. 2014). Although a wide range of these enzymes have been identified in cyanobacteria, the majority is not biochemically characterized, being difficult to associate them specifically to EPS biosynthesis (Lombard et al. 2014). The most studied group of CAZymes in the context of cyanobacterial EPS production are the glycosyltransferases (GTs). They have been mainly identified in filamentous strains as players involved in the production of the protective heterocyst envelope polysaccharide (HEP) layer, and in hormogonia contributing to their EPS-mediated gliding motility. For example, in Anabaena sp. PCC 7120, nine GTs encoded in a gene cluster, so-called “HEP island” (alr2825-alr2841) (Huang et al. 2005), and other three outside this cluster, alr3698 (hepB), alr3699 (hepE), and all4160 (Wang et al. 2007), were shown to be essential for the HEP layer biosynthesis. In addition, other genes in the “HEP island” encode relevant proteins that act on the modification of the EPS, such as epimerases (alr2827 and alr2830) and an oxireductase (alr2831) (Huang et al. 2005). The importance of 13 GTs-encoding genes for the production/secretion of hormogonia EPS was also reported for Nostoc punctiforme (Zuniga et al. 2020; Risser and Meeks 2013). These genes are either in EPS-related gene clusters (hpsA-K, hpsL-P, hpsS-U) or isolated, such as hpsQ and hpsR, and their deletion frequently leads to the loss of gliding motility by this strain (Zuniga et al. 2020; Risser and Meeks 2013). In unicellular strains, such as Synechoccocus species, GTs have been studied in the context of cellulose production, which can accumulate between the outer and inner membranes or be exported via synthase-dependent pathway (Zhao et al. 2015). These enzymes seem to be ancestral to the GTs involved in the plants cell wall formation. In Synechocystis sp. PCC 6803, five GTs encoded in the gene cluster sll1722-sll1726 were also shown to be involved in EPS production (Foster et al. 2009). Moreover, Fisher et al. (2013) associated the gene cluster slr0977-sll0575 to EPS biosynthesis, with genes encoding a GT (slr0983, rfbF) and carbohydrate-modifying enzymes, such as the epimerase (slr0985, rfbB) and the dehydratase (slr0984, rfbG). They also showed that the methyltransferase Slr1610 participates in Synechocystis EPS production, through the formation of carbohydrate-methylated residues that are most probably recognition sites for the end of the polymerization and/or EPS export. Moreover, another modification enzyme, the polysaccharide monooxygenase Sll1783 (glycoside hydrolase family), was shown to be involved in EPS degradation and uptake in this strain (Miranda et al. 2017). In addition, recent studies suggest that a wide range of carbohydrate-modifying enzymes might be involved in cyanobacterial EPS production, such as enzymes responsible for the addition of methyl, acetyl, pyruvyl, and/or sulfate groups (Rossi and De Philippis 2016), as well as extracellular enzymes responsible for the reutilization of EPS (Stuart et al. 2016).
3.2
Assembly and Export
In spite of the diversity of EPS producers and of the produced polymers, the proteins involved in the last steps of EPS assembly and export are relatively well conserved
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among bacteria. This is a challenging process for the bacterium, since these large and usual hydrophilic molecules have to cross the cell envelope without compromising the critical barrier properties. In general, the EPS export follows one of the three main mechanisms (recently reviewed in Schmid 2018): the Wzy-, the ABC transporter-, or the synthase-dependent pathways (Fig. 2). The genes encoding proteins involved in these pathways are usually localized in large gene clusters in both gram-positive and gram-negative bacteria, nearby genes encoding sugar-activating/modifying enzymes, and glycosyltransferases. In the Wzy-dependent pathway, oligosaccharide repeating units linked to the lipid carrier are flipped across the plasma membrane by Wzx. Then, Wzy continues the polymerization task at the periplasmic face of the membrane. Both enzymes cooperate in order to control the polymer chain length. Subsequently, the translocation process of the polymer through the cell envelope is performed by the Wza/Wzc complex, which forms a transmembrane channel. Wzc also works as copolymerization unit in collaboration with Wzy, being implicated in the chain length control, and its activity is regulated by phosphorylation/dephosphorylation cycles, usually by a Wzb phosphatase. In the ABC transporter-dependent pathway, the polysaccharide is fully polymerized at the cytoplasmic face of the plasma membrane. The KpsC/KpsS, KpsF, and KpsU are usually involved in the synthesis of the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) linker or its activated donor that connect the polysaccharide to a terminal lipid in the membrane. After that, the polymer is translocated across the plasma membrane by an ABC transporter complex formed by the KpsM and KpsT subunits. Finally, the export of the polymer is accomplished by the activity of the transmembrane complex KpsE/KpsD. KpsE and KpsD may form a transenvelope system analogous to Wzc and Wza, and they belong to the same protein families, the polysaccharide copolymerase (PCP) and the outer membrane polysaccharide export protein (OPX), respectively. Regarding the molecular machinery involved in synthase-dependent pathways, it may differ significantly depending on the polymer produced (Low and Howell 2018). In general, a polysaccharide synthase embedded in the plasma membrane (e.g., Alg8 or BcsA) is responsible for the simultaneous polymerization and export of the polysaccharide. This process occurs in the presence or absence of a lipid acceptor molecule and it is controlled by the secondary messenger c-di-GMP. Meanwhile, other proteins in the periplasm may modify the polymer (e.g., AlgI, AlgF, and AlgG) or even degrade polymer surplus (e.g., AlgL or BscZ). The polymer is then guided and protected from degradation by a periplasmic tetratricopeptide repeat (TPR)-containing scaffold lipoprotein through a β-barrel porin, which will export it. This step can be performed by two independent proteins (e.g., AlgK and AlgE) or a single protein that combines both functions (e.g., BcsC). In cyanobacteria, a phylum-wide analysis revealed homologs from the three aforementioned pathways among the 124 genomes analyzed, but often not the complete set defining one pathway (Pereira et al. 2015). In general, multiple gene copies were found in cyanobacterial strains with higher morphological complexity and larger genomes, while strains with reduced genomes, such as Synechococcus and Prochlorococcus, seem to have lost most of the EPS-related genes. This study
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suggested that EPS production in cyanobacteria is much more complex compared to other bacteria, probably with more players involved, which may cooperate, cross talk, or display redundant functions. Recently, the involvement of some of these genes/proteins in EPS production has been elucidated, mainly using Synechocystis sp. PCC 6803 knockout mutants in homologs from the Wzy-dependent pathway: sll1581 (wza), sll0923 (wzc), and sll0328 (wzb) (Pereira et al. 2019a; Jittawuttipoka et al. 2013), and the ABC-dependent pathway: slr0977 (kpsM), slr0982 (kpsT), sll0574 (kpsM), and sll0575 (kpsT) (Fisher et al. 2013). In general, these knockout mutants produce a lower amount of EPS that also vary in composition, compared to the polymer produced by the wild type. Moreover, and in agreement to what was previously observed for other bacteria, slr0328 (wzb) encodes a phosphatase envisaged to be important for the regulation of Sll0923 (Wzc) activity, and consequently EPS export, but it might also interact with many other targets such as proteins involved in photosynthesis (Pereira et al. 2019a). In addition, slr1875 (exoD) has also been associated to EPS assembly/export in Synechocystis, being a homolog of the exoD involved in the production of rhizobia EPS (Jittawuttipoka et al. 2013). However, it is still unclear whether ExoD cooperates with the conventional EPS assembly/export pathways. In filamentous strains, mainly in Anabaena sp. PCC 7120, a few proteins responsible for the assembly/export of the HEP layer have been described, such as homologs from KpsT (Alr2835, HepA), Wzc (All1711, HepP), and Wzb (Alr5068) (Huang et al. 2005; Herrero et al. 2016). Furthermore, for the assembly and secretion of hormogonia EPS, two distinct mechanisms were proposed, either involving homologs to proteins of the Wzy-dependent pathway (Zuniga et al. 2020), or of the type IV secretion system (pili secretion) in cooperation with the well-known junctional pore complex (JPC) (Risser and Meeks 2013). In filamentous cyanobacteria, JPC components are encoded in the universally conserved hormogonia polysaccharide (hps) cluster, and homologs to these genes/proteins are rarely found in unicellular strains (Khayatan et al. 2015).
3.3
Regulatory Mechanisms
Up to now, very few cyanobacterial regulatory elements were undoubtedly associated to EPS production, and mainly in Anabaena sp. PCC 7120 and Nostoc punctiforme. In these filamentous cyanobacteria, the global nitrogen-control transcription factor NtcA was shown to be a key player in the regulation of the transport of glycosides, which has an indirect influence in the distribution of the building blocks for EPS biosynthesis (Herrero et al. 2016). Another global regulator, the alternative sigma factor SigJ was specifically associated with the heterocysts HEP layer formation, controlling the expression of genes involved in the first steps of EPS production (e.g., genes encoding glycosyltransferases), as well as in EPS assembly/ export (Gonzalez et al. 2019). In addition, a two-component system involving the response regulator DevR (Huang et al. 2005), histidine kinases, such as HepK and HepN, and a serine/threonine kinase (HepS) (Fan et al. 2006) were also implicated in
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the control of the synthesis of the HEP layer. Other crucial regulators for heterocysts differentiation and patterning also directly or indirectly influence the production of EPS by these cells, such as the Het and Pat regulators, sigma factors, and the noncoding RNA, NsiR1 (Herrero et al. 2016). For the production of hormogonia EPS, a few regulatory mechanisms have also been described, mainly for Nostoc punctiforme, such as the Hmp (hormogonium motility and polysaccharide) signal transduction system (Risser and Meeks 2013), and the partner-switching regulatory system involving the HmpU (phosphatase), the HmpV (with a STAS domain), and the HmpW (kinase) (Riley et al. 2018). These systems are controlled by a hierarchical sigma factor cascade, being primarily dependent on SigJ, and to a lesser extent on SigF, which interferes only with the regulation of the hormogonia polysaccharide (hps) gene cluster and the type IV secretion system (Gonzalez et al. 2019). This study also suggested that there is an overlap in the components involved in the synthesis of the HEP layer and hormogonia EPS. Moreover, and in agreement with the results from Gonzalez et al. (2019), the alternative group 3 sigma factor SigF was shown to influence several secretion pathways in Synechocystis sp. PCC 6803, including the production/export of EPS and the type IV secretion system (Flores et al. 2019a). The ΔsigF also exhibits cell envelope alterations, and a three- to fourfold increase in RPS production compared to the wild type. The secondary messengers, cAMP and c-di-GMP, might also be involved in regulatory mechanisms of cyanobacterial EPS production, since they were already associated to other processes intrinsically related, such as cell motility, biofilm formation, and heterocyst differentiation (Agostoni and Montgomery 2014). In addition, the regulation by noncoding RNAs (ncRNAS) might also be important, similarly to what occurs in the control of the production of other cyanobacterial polysaccharidic polymers, such as glycogen (de Porcellinis et al. 2016). Most importantly, the abovementioned secondary messengers and several ncRNAS were also shown to be essential for EPS production in a wide range of bacteria (O’Toole and Wong 2016). Overall, the knowledge about the regulatory network behind cyanobacterial EPS production is still very scarce, being the full picture far from elucidated.
4
Optimization of Production and Isolation Procedures
The use of cyanobacterial EPS for biotechnological applications depends on the knowledge of the physiological conditions that enhance the productivity, the desired characteristics (e.g., monosaccharidic composition, structure, and molecular mass), and consequently the functional properties of the polymer. This task is particularly difficult since there is a lack of systematic studies, and the influence of each factor is strongly strain dependent and, often, related to the cell growth (Mota et al. 2013; Pereira et al. 2009). In some strains, the EPS are produced under virtually all physiological conditions (possibly constitutively), while in others this depends on specific nutritional conditions, growth phase, or as a stress response (Delattre et al. 2016). For obvious reasons, and in contrast to their bacterial counterparts, light
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(intensity and quality) is most probably the main factor influencing cyanobacterial EPS production (Fig. 3). For some strains, the culture exposure to continuous light or high-light intensities enhances EPS production (Mota et al. 2013). In other cases, specific light wavelengths stimulate production, e.g., UV radiation for Nostoc punctiforme (Soule et al. 2016) or red light for Nostoc flagelliforme, which also change significantly the monosaccharide composition of their CPS and RPS (Han et al. 2015). Light may also cause temperature fluctuations (e.g., due to heat generation), resulting in alterations on EPS productivity. Although only a few studies addressed the impact of temperature on cyanobacterial EPS production (and often in combination with changes in light intensity), it seems that the production tends to be higher under slightly higher temperatures than the ones used for optimal cell growth. However, in other cases, no effect or even slight reductions in EPS production were also detected (Pereira et al. 2009). Moreover, higher temperatures compared to the ones used for optimal cell growth also altered the monosaccharide composition of the CPS bulk from a biofilm formed by the cyanobacterium Synechocystis and the algae Chlorococcum (Di Pippo et al. 2012). Nutrient availability (namely carbon, nitrogen, phosphate, and sulfate) and its cell ratio (e.g., C:N) are also very important for cyanobacterial EPS production, thus selecting the most appropriate culture medium is vital (Rossi and De Philippis 2016). The presence of a combined nitrogen source, such as nitrate, ammonium, or urea, usually results in increased EPS production, probably by requiring less energy than nitrogen fixation. However, nitrogen starvation may also lead to higher production due to the increase of C:N
Fig. 3 Key parameters influencing the production of cyanobacterial extracellular polymeric substances (EPS)
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ratio, and the availability of different nitrogen sources may affect EPS composition and/or cyanobacterial productivity. Furthermore, it is known that under low carbon dioxide mass-loading rates the conversion of CO2 is mainly to biomass, while under high CO2 mass-loading rates, the main routes are toward the synthesis of EPS and volatile organic compounds (Pereira et al. 2019b). Nutrient limitation/starvation, as depletion of phosphorous and sulfate sources, trace metals (e.g., calcium, magnesium, iron, and potassium), or vitamins (e.g., cyanocobalamin), is a common strategy used to trigger EPS synthesis and accumulation, however this can lead to cell growth impairment (Delattre et al. 2016). Therefore, this strategy is only used for batch culture systems, consisting in an initial growth phase with nutrients supply and then a production phase under nutrient limitation. Aeration can also be important for EPS production, by promoting a physical separation of cell bound EPS or simply a better stirring of the culture improving light penetration and nutrient availability (Pereira et al. 2009). In addition, increased salinity is a common stimulator of carbohydrate synthesis, mainly for compatible solutes production, but also for EPS production, and may also contribute for the alteration of their monosaccharide composition (Pereira et al. 2009). Nowadays, most large-scale production of cyanobacteria occurs in outdoor open ponds that are relatively inexpensive, but several aspects, such as temperature, light distribution, and microbial contamination, are difficult to control. These raceway ponds are used worldwide to grow the most commercially exploited cyanobacterium Arthrospira platensis (Spirulina), where microbial contamination can be overcome through highly selective operating conditions, namely high pH (Takenaka and Yamaguchi 2014). However, other commercially interesting strains do not tolerate high pH, temperature, and salt concentration. Therefore, a variety of closed photobioreactor configurations have been developed to suit the particular characteristics of cyanobacteria, including flat panels reactors (vertical or alveolar), tubular reactors (horizontal or helical), cascade reactors, or bubble columns. The selection of the appropriate system should be based on several factors, such as the strain characteristics, the costs of land, energy, nutrients and labor required, the availability of water, the suitability of climate, and the specification of the final product (Takenaka and Yamaguchi 2014). In particular, the cultivation of EPS producers may result in some drawbacks, namely higher grazing susceptibility and slow growth due to redirection of nutrients and energy for EPS production. Furthermore, in an industrial context, it may lead to some problems of cultivation and cell harvesting, due to cell adhesion to the bioreactors and/or the impairment of culture mixing/aeration. Therefore, to overcome these obstacles, and after lab-scale optimization, several issues should be taken into consideration for each strain, such as the inoculum quality (age and concentration), the photobioreactor design, and the downstream processing (Pierre et al. 2019). Following the optimization of the conditions to increase the EPS production, it is necessary to develop economic, effective, and environmentally friendly protocols for the isolation and purification of the polymers, depending on their cellular location. To detach the EPS from the cell surface it is necessary to apply physical methods, such as sonication, heating or cation exchange resin, and/or chemical methods, such
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as the use of ethylenediaminetetraacetic acid (EDTA), formaldehyde, sodium hydroxide (NaOH), or glutaraldehyde as detaching agents (Pereira et al. 2019b). However, it is essential to cause minimal cell lysis and disruption in the EPS structure. The EPS are then separated from the cells by centrifugation or filtration, and the supernatant/filtrate is precipitated using an absolute alcohol, such as ethanol, methanol or isopropanol, or acetone (two–three volumes for one volume of supernatant/filtrate) (Flores and Tamagnini 2019). However, the polarity of the alcohol and the temperature of precipitation can influence the EPS yield and coprecipitate impurities (Delattre et al. 2016). Dialysis and membrane techniques can be used to remove the impurities, such as salts from the culture medium and nucleic acids (Flores and Tamagnini 2019). To increase the purification degree of EPS fractions it might be necessary to remove nonpolysaccharidic molecules, as proteins, pigments, or salts, with trichloroacetic acid treatment, tangential ultrafiltration, or selective alcoholic precipitation (Delattre et al. 2016). Finally, the EPS should be dried, usually by spray-, freeze-, or oven-drying. It should also be taken into account that the extraction protocol used can affect the quality and amount of EPS extracted, particularly the harsh procedures generally applied for removing EPS attached to the cells (Rossi and De Philippis 2016). Moreover, all the downstream processing need to be improved and optimized for high-efficient industrial scale-up to avoid expensive and time-consuming drawbacks, e.g., contamination, membrane clogging, and loss of yield of the final product. Therefore, alternative strategies for EPS extraction and purification are being proposed, such as nanofiltration using antifouling nanocomposite membranes, and ultrasound- and microwave-assisted extraction, but it is still necessary to consider the advantages and disadvantages for each specific strain/ product (Delattre et al. 2016).
5
Composition and Structure
Cyanobacterial EPS consist of repeating units built from monosaccharides resulting in molecules several hundred kDa in size, with a molecular mass that can surpass 2 MDa (Pereira et al. 2009). These EPS possess a large number of different monosaccharides (up to 13), which is a unique feature, since bacterial EPS frequently contain up to four monosaccharide building blocks. Therefore, the cyanobacterial polymers have several repeating units and branching points, resulting in a complex structure that allow a wide range of possible conformations. Glucose is usually predominant, but they can also contain other hexoses (mannose, galactose, and fructose), deoxyhexoses (fucose and rhamnose), and pentoses (arabinose, ribose, and xylose), as well as acidic sugars (glucuronic and galacturonic acid) and amino sugars (glucosamine, galactosamine, and their N-acetyl derivatives) (Fig. 4). The presence of uronic acids (in ~90% of cyanobacterial EPS) results in polymers rich in negatively charged groups, but is rarely observed in the EPS of other gram-negative bacteria (Pereira et al. 2019b). Another unusual feature in bacterial EPS, but common among EPS from algae and some archaea, is the presence of sulfate groups (up to 20% of cyanobacterial EPS dry weight). Moreover, peptides and noncarbohydrate constituents (e.g., phosphate,
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Fig. 4 Abundance of a given monosaccharide in cyanobacterial polymers. *Depending on the methodology, monomers in low amount or rare in cyanobacterial polymers might be underestimated. (Data from 145 cyanobacterial strains and adapted from Rossi and De Philippis (2016), Delattre et al. (2016), Flores et al. (2019a), and Mota et al. (2020))
pyruvate, lactate, acetate, and glycerol) have been reported. The presence of unusual EPS sugars, such as methyl, acetyl, and amino sugars, are expected to contribute to cyanobacterial polymers’ biological activity, most probably involved in cell recognition, adhesion, and protection. The peptides, enriched in glycine, alanine, valine, leucine, isoleucine, and phenylalanine, but also with aspartic and glutamic acids in some cases, contribute to the stabilization of the cyanobacterial EPS through the creation of hydrogen bonds. Despite the complexity usually observed, there are some particular cases of cyanobacterial homopolymers, namely Microcystis wesenbergii RPS constituted by uronic acids only and Cyanothece sp. 113 EPS constituted by D-glucose only (Pereira et al. 2019b). Cyanobacterial EPS can have an amphiphilic behavior, due to the presence of different polysaccharidic fractions with hydrophilic and/or hydrophobic properties, determining the rheological behavior and jellification ability of the polymer. The hydrophobic fractions (ester-linked acetyl groups, peptidic residues, and deoxyhexoses) enhance the capacity of cell adhesion and the polymers emulsifying capacity, while the hydrophilic ones (sulfate groups, uronic acids, and ketal-linked pyruvyl groups) confer a strong anionic charge and sticky behavior to the polymer, being involved in the entrapment of minerals, nutrients, and water (Rossi and De Philippis 2016). The particular features and the complexity of the cyanobacterial EPS hinder the solving of their structures (reviewed in Pereira et al. 2009), but at the same time
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make these polymers more versatile and particularly attractive for specific industrial and biotechnological application (see Sect. 6). To determine the EPS structure, it is required the identification of type, sequence, and branching patterns of monosaccharides, and the anomeric configuration and isomer position of each glycosidic bond. A previously depolymerization into oligo- or monosaccharides is usually necessary, most often by acidic hydrolysis or enzymatic cleavage, due to their complex structure and conformation (Delattre et al. 2016). The content of neutral carbohydrates and other constituents of the EPS as uronic acids, proteins, and sulfate groups are usually quantified by colorimetric assays. Other techniques are commonly used to analyze physicochemical properties of EPS, such as scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), thermogravimetry (TGA), differential scanning calorimetry (DSC), and rheology (Xiao and Zheng 2016). Moreover, the carbohydrate residues can be further analyzed qualitatively and quantitatively using several methods: (i) spectroscopic, such as Fourier transformed infrared (FTIR) to detect the functional groups, and X-ray photoelectron spectroscopy (XPS) to study the elemental composition; (ii) chromatographic, such as gas (GC), liquid (LC), and ion-exchange chromatography (IEC) to determine the monosaccharidic composition, and size exclusion chromatography (SEC) to analyze the molecular mass; and (iii) spectrometric techniques, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), to elucidate the branching patterns and molecular structure (Delattre et al. 2016). Since many different techniques can be used for the analysis of the EPS monosaccharidic composition and structure, the comparison of the results available in the literature is not straightforward.
5.1
Tailoring
The manipulation of cyanobacterial EPS has two main goals: the cost-effective maximization of productivity/polymer yields and the generation of tailor-made polymer variants with desirable properties. Recently, strategies to obtain designed polymers based on cyanobacterial EPS were proposed/reviewed by Pereira et al. (2019b). These authors also discuss approaches to improve the amount of polymers produced through metabolic engineering, and relevant advances for modification of EPS to generate biomaterials, such as hydrogels, coatings, or scaffolds, e.g., through chemical modification or combination of EPS with plasticizers. Regarding the increase of cyanobacterial polymer yields, other procedures have also been shown to be very promising, such as the manipulation of global regulators (e.g., sigma factors) that are involved in the control of carbon metabolism and/or secretion mechanisms (Flores et al. 2019a), and the heterologous expression of genes involved in EPS biosynthesis. This strategy is still in an early stage regarding cyanobacteria, but successful examples using Synechococcus sp. PCC 7002 were reported like the overexpression of six genes related to the synthase-dependent pathway of Gluconacetobacter xylinus that resulted in a high-yield production of extracellular type-I cellulose (Zhao et al. 2015), and the expression of two hyaluronic
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acid (HA) synthetases from the gram-negative Pasteurella multocida and the grampositive Streptococcus equisimilis that allowed the production of ~80 mg/L of HA by this unicellular cyanobacterial strain (Zhang et al. 2019). Preliminary evidences also showed that the manipulation of genes encoding carbohydrate-modifying enzymes, e.g., methyltransferases (Fisher et al. 2013) or proteins involved in EPS assembly/export (Fisher et al. 2013; Pereira et al. 2019a), might be used to tailor cyanobacterial EPS length, molecular mass (MM), and composition. In agreement, Wzy and Wzx proteins are widely considered as preferential targets for the incorporation of desired sugars during EPS polymerization, due to their broad specificity observed among different bacterial genus (Schmid 2018). Concerning the generation of tailor-made cyanobacterial EPS, many physiochemical modifications have been increasingly used to alter the isolated polymers, such as trifluoroacetic acid hydrolysis to reduce polymers’ MM (Flamm and Blaschek 2014a), peptide removal by trichloroacetic acid precipitation (Delattre et al. 2016), or oversulfation with dicyclocarbodiimide-sulfuric acid (Lee et al. 2007). For desulfation, solvolysis remains the most common method used, but this method can have several side effects, such as polymer degradation, and reduction of polymer MM and yield (Flamm and Blaschek 2014a; Lee et al. 2007). Alternative, silylating agents have been used to efficiently desulfate cyanobacterial EPS (up to ~98% of desulfation), although often accompanied by modifications in the linkage pattern of some monosaccharides (Flamm and Blaschek 2014b). Other strategies to achieve higher polymer yields and/or tailor-made EPS are still largely unexplored using cyanobacteria, such as protein engineering regarding enzymes involved in EPS biosynthesis. In this context, several approaches have been efficiently used in heterotrophic bacteria, such as the shuffling of glycosyltransferases domains, the alteration of active sites of proteins involved in carbohydrate modification (e.g., epimerases) and in the assembly/export pathways, and the use of platforms with immobilized and designed enzymes for EPS production/ modification (Schmid 2018).
6
Biotechnological Applications
As cyanobacteria have been emerging as source for the development of novel, versatile, and tailor-made “green materials” based on EPS, their application broadens to different fields such as wastewater treatment and soil conditioning, food, cosmetic, textile and painting industries, biomedicine, tissue engineering, and pharmaceutics (Singh et al. 2019). To promote the commercial use of cyanobacterial EPS/EPS-based products, several patents have been filled, regarding the polymers/ polymer products, their production, extraction, and/or downstream processing (Table 1). Interestingly, the same polymer frequently shows the potential to be used in completely distinct fields. For example, the EPS from Nostoc flagelliforme are promising emulsifying and flocculating agents that could be used in cosmetics and food industries (Han et al. 2014), but these have also potent antiviral activity that will allow its exploitation in biomedicine (Kanekiyo et al. 2007).
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Table 1 Patents related to cyanobacterial polysaccharides published in the last 15 years Patent numbera WO2006078284A3
US7205284B2
WO2007044439A2
Microbial exopolymers useful for water demineralization
US8350024B2
Sugar derivatives and application of same Production of polysaccharides in mixotrophic mode using Nostoc Cosmetic compositions comprising exopolysaccharides derived from microbial mats, and use thereof Cyanobacterium extracellular polymer, compositions, and uses thereof Uses of cyanobacterium extracellular polymer, compositions, coated surfaces, or articles
WO2013136029A1
US20180256627A1
WO2018042378A1
WO2019171344A1
a
Title Methods and compositions related to antiviral therapy using algae and cyanobacteria Potent immunostimulants from microalgae
Strains Spirulina
Inventors (year) Teas J (2006)
Aphanizomenon flos-aquae, Spirulina platensis Oscillatoria, Lyngbya, Schizothrix, Chroococcus, Calothrix Aphanothece sacrum Nostoc
Pasco DS, Pugh ND, Elsohly M, Ross S, ElSohly NM (2007)
Phormidium, Scytonema, Schizothrix, Chlorococcales
Loing E, Briatte S, Vayssier C, Beaulieu M, Dionne P, Richert L, Moppert X (2018) Mota R, Tamagnini P, Gales L, Leite JP, Pereira SB (2018)
Cyanothece sp. CCY 0110
Cyanothece sp. CCY 0110
Perry TD (2007)
Kaneko M, Kaneko T (2013) Romari K, Godart F, Calleja P (2013)
Mota R, Tamagnini P, Costa F, Martins MCL (2019)
Includes US patents and applications under the Patent Cooperation Treaty (PCT)
6.1
Water Treatment and Soil Conditioning
The use of EPS-producing cyanobacteria/isolated EPS in metal removal has been the subject of several studies in the last decade (Table 2), with some reports presenting insights on the mechanisms/functional groups involved in this process (De Philippis et al. 2011; Mota et al. 2016). Metal bioremediation by microorganisms comprises two processes: bioaccumulation, an active process that results in intracellular metal accumulation mediated by cell metabolism, and biosorption, a passive process generally involving several complex physicochemical mechanisms, such as ion exchange, complexation, and adsorption (De Philippis et al. 2011). Biosorption has been suggested to be the main mechanism by which the positively charged metal ions are removed from water solutions, being this due to the presence of a large
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Table 2 Examples of cyanobacterial EPS and their applications in water treatment and soil conditioning Cyanobacteria Water treatment Cyanothece sp. CCY 0110 Oscillatoria limnetica; Anabaena spiroides Soil conditioning Leptolyngbya ohadii
Polymers
Applications
References
Cyanoflan (RPS) Mucilage
Heavy metal bioremediation Heavy metal bioremediation
Mota et al. (2016) Tien (2002)
EPS
Mugnai et al. (2018) Chen et al. (2009) Colica et al. (2014) Tamaru et al. (2005) Chamizo et al. (2020) Chamizo et al. (2020)
Microcoleus vaginatus
EPS
Bare sandy substrates stabilization UV-B radiation protection
Microcoleus vaginatus; Scytonema javanium Nostoc commune
EPS
Water retention
EPS
Phormidium ambiguum
EPS
Desiccation and freezing protection Soil physical stability
Scytonema javanicum
EPS
Soil fertility
number of negatively charged groups on the EPS attached to the cell and/or on the RPS, such as carboxyl, hydroxyl, phosphoryl, sulfhydryl, and amino functional groups. Various culture fractions of Cyanothece sp. CCY 0110 (living cells plus RPS, dead cells plus RPS, or isolated RPS only) were tested for their affinity toward different metal ions in aqueous solutions. The results obtained revealed that the RPS played the main role in the removal process due to the organic functional groups available, mainly carboxyl and hydroxyl (Mota et al. 2016). However, up to now, no industrial-scale process has been fully developed since the process is much more complex than under laboratory-controlled conditions due to, e.g., the variety of the chemicals present in wastewaters. Moreover, the implementation of a new process involves always significant upfront costs, usually considered too high for the treatment of wastewaters containing low valuable metals. Nonetheless, these disadvantages could, in the future, be counterbalanced with the revenue derived from the recovery of commercially valuable heavy metals, a process that is not efficient using the traditional physicochemical methods (Colica and De Philippis 2014). Cyanobacteria have also been described as useful agents in the remediation and amelioration of soils, by contributing with organic matter and improving soil biological activity, since they are pioneers in the formation of biocrusts – complex communities composed by different proportions of cyanobacteria, microalgae, microfungi, lichens, and bryophyte – that are important for the establishment and growth of vascular plants (Rossi et al. 2018). In the biocrusts, the EPS matrix play a significant role in sediment cohesion, resistance to erosion, moisture maintenance, immobilization of nutrients, protection from external harmful factors, and enhancement of soil fertility. The inoculation of soil with EPS-producing cyanobacteria, such
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as Microcoleus and Nostoc spp. (Table 2), improves the aggregation of the top coat, and increases the water retention capacity and the regeneration of the ecosystems through the in situ fixation of nitrogen and carbon (Colica and De Philippis 2014). Moreover, some cyanobacterial strains, in particular from soil and extremophilic habitats, can excrete a brown pigment deposited in EPS (particularly in the sheath), named scytonemin, which can protect the cells against long-wavelength ultraviolet A (UVA) radiation. Likewise, mycosporine-like amino acids (MAAs) are natural UV sunscreen compounds that can be found in the sheath of some cyanobacterial strains (Chen et al. 2009). Furthermore, the inoculation of EPS-producing cyanobacteria in desert areas promotes the formation of soil crusts, stabilizing sand dunes and driving ecological succession, which has been already successfully applied in large hyper arid areas in China (Colica et al. 2014). Lab-scale studies also revealed that the use of EPS-producing cyanobacteria in burned soils can decrease soil hydrophobicity and increase surface penetration resistance, resulting in higher stabilization of postfire soils (Chamizo et al. 2020). Fixing dunes to fight desertification and prevent sandstorms as well as recovering and stabilizing unconsolidated soils due to fires or floods are examples of the valuable use of EPS-producing cyanobacteria.
6.2
Food, Personal Care, and Other Industries
The first studied features of cyanobacterial EPS envisaging industrial exploitation were the high viscosity of their aqueous solutions, and the capabilities to stabilize emulsions and to form gels with good tensile strength (reviewed by Colica and De Philippis 2014). This interesting rheological behavior is mostly determined by the complex composition, structure, and high molecular mass (MM) of the cyanobacterial EPS. The most promising feature is their ability to stabilize the flow properties of aqueous solutions against changes in temperature, ionic strength, and pH. In addition, the amphiphilic character contributes to their possible use in the stabilization of emulsions, as bioflocculants and/or viscosifiers (Table 3). The pseudoplastic behavior usually observed for aqueous solutions of these polymers is important for the food industry, in order to provide good sensory qualities, flavor release, and suspending properties in the production of, e.g., cake mixtures, salad dressings, sauces, puddings, and dairy products, as well as in the cosmetic industry, for the production of lotions, creams, and gels. These polymers can also be used to disperse hydrocarbons or oils in water or food preparations, in the secondary recovery of petroleum, or as a stain remover. This is the case of emulcyan, an extracellular sulfated heteropolysaccharide containing fatty acids and peptides, produced by the filamentous cyanobacterium Phormidium J-1, which is capable of forming emulsions of hydrocarbons and/or oils in water and that can act as a bioflocculant, clarifying the water column in natural turbid-water habitats (Fattom and Shilo 1985). Due to other natural features, namely moisturizer, photoprotector, antioxidant, and anti-inflammatory properties, the application of cyanobacterial EPS is also being studied in the field of cosmetics, cosmeceuticals (i.e., cosmetic products with active
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Table 3 Examples of cyanobacterial EPS and their applications in food and personal care industries Cyanobacteria Aphanothece sacrum Cyanothece sp. CCY 0110 Lyngbya stagina Nostoc commune Nostoc flagelliforme Nostoc punctiforme Phormidium J-1
Polymers Sacran (EPS) Cyanoflan (RPS) EPS
Applications Moisture retention Emulsification Viscosifier
Sheath
Moisture absorption and retention, Antioxidant, Photo-protection (MAAs)
EPS
Flocculation and emulsification
Sheath
Photo-protection (scytonemin)
Emulcyan (RPS)
Flocculation and emulsification
References Okajima et al. (2008) Mota et al. (2020) Jindal et al. (2013) Li et al. (2011), Nazifi et al. (2015) Han et al. (2014) Soule et al. (2016) Fattom and Shilo (1985)
ingredients resulting in a pharmaceutical therapeutic benefit), and nutraceuticals (i.e., nutritional supplements) (Pierre et al. 2019). For instance, the EPS produced by the edible Nostoc commune have strong moisture absorption and retention capacities (Table 3), greater than urea and chitosan, having a high potential to be used as humectants in moisturizers without the need to be combined with undesirable occlusive agents (Li et al. 2011). In addition, these EPS have antioxidant activities since they are capable of scavenging both superoxide anions and hydroxyl radicals in a dose-dependent manner, and they can increase antioxidant enzymes activity, and decrease the content of lipid peroxidation. Other example is the moisture retention capacity of Sacran, a giant anionic polysaccharide extracted from the cyanobacterium Aphanothece sacrum, which is tenfold higher than hyaluronic acid (Okajima et al. 2008). It has been suggested that the moisture retention capacity of cyanobacterial EPS is due to strong interactions between water molecules and the hydrophilic hydroxyl groups of the polysaccharides (Derikvand et al. 2016). The photo-protective compounds that often appear within the EPS (scytonemin and MAAs) have also great potential to be used as ingredients in sunscreens due to their inherent good solubility, photostability, and effective UVA absorption properties (Derikvand et al. 2016). These properties can be beneficial for several personal care products beyond the traditional skin protection and suntan lotions, including daytime facial moisturizers, lipsticks, and makeup. Despite some cyanobacterial EPS exhibit similar or even better properties than the commercially available microbial EPS, such as xanthan gum and chitosan (Li et al. 2011; Mota et al. 2020), it is still difficult to compete to the well-established products in the market. Therefore, only polymers with novel and unique properties may be capable to overcome this challenge. Nonetheless, the interest in cyanobacterial-based products by the cosmetic industry is evident since several of them can be found at the European Commission Database for information on
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cosmetic substances and ingredients (CosIng), such as Aphanothece sacrum “exopolysaccharides” with stated functions of absorbent, emulsion stabilizing, film forming, and viscosity controlling.
6.3
Biomedical
A wide range of potential biomedical applications have been described for cyanobacterial EPS, which make them very attractive for the development of innovative bioproducts, such as drugs, nanocarriers, coatings, or scaffolds. The majority of the envisaged applications are based on the biological activity of the cyanobacterial EPS, e.g., antioxidant, immunostimulatory, cytotoxic, pro-inflammatory, antiviral, and antimicrobial (Table 4). These activities mostly result from a complex interaction of several structural features, including the sugar residue composition, molecular mass, sulfation level, distribution of sulfate groups along the polysaccharide backbone, and stereochemistry (Pereira et al. 2019b). The high sulfate content is one of the most promising cyanobacterial EPS features in the biomedical context, having been associated to different bioactivities, such as antiviral (Reichert et al. 2017), antioxidant (Parwani et al. 2014), anticoagulant (Flamm and Blaschek 2014a), and antimicrobial (Challouf et al. 2011). Nonetheless, only in a few cases, the bioactivities exhibited were undoubtedly associated with the EPS features, and usually this association is inferred based on other compositionally similar bioactive polymers from other bacterial sources or eukaryotic organisms (mainly macroalgae). At the top of the list of the most studied bioactive EPS are those produced by the filamentous edible cyanobacterium Arthrospira platensis, well known as Spirulina, for which several patents were filled. At least nine biological activities, e.g., antiviral and immunostimulatory, are well described for different types of EPS from A. platensis (Table 4). However, at the industrial level, EPS form A. platensis that are still considered as a co-product from the extraction of high-value compounds (e.g., phycocyanin pigment used as natural colorant) (Pierre et al. 2019). In contrast, the antitumor activity of cyanobacterial EPS is one of the least studied bioactivities; however, recent works demonstrated that completely distinct cyanobacterial EPS have strong antiproliferative effects and/or efficiently trigger apoptosis in different tumor cell lines (Flores et al. 2019b; Li et al. 2018). For example, the polymer produced by a Synechocystis ΔsigF EPS-overproducing mutant decreases the viability of melanoma, thyroid, and ovary carcinoma cells by inducing high levels of apoptosis, through p53 and caspase-3 activation (Flores et al. 2019b), and the nostoglycan isolated from Nostoc sphaeroides colonies suppresses the proliferation of several types of tumor cell lines due to its antioxidant properties and induces apoptosis in human lung adenocarcinoma A549 cells via caspase-dependent pathway (Li et al. 2018). However, other cyanobacterial EPS with no apparent biological activity can also be interesting for the pharmaceutical or biomedical field, for example, the RPS produced by the unicellular strain Cyanothece sp. CCY 0110 that showed to be a promising vehicle for topical and oral administration of therapeutic macromolecules
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Table 4 Examples of cyanobacterial EPS and their biological activities and/or applications in biomedicine Cyanobacteria Anabaena variabilis; A. anomala; A. oryzae; Tolypothrix tenuis Aphanothece sacrum Arthrospira platensis (Spirulina)
Polymers EPS
Activities/applications Hemostatic blood clotting agent, wound dressing/ healing, antibacterial, antioxidant
References Parwani et al. (2014)
Sacran (EPS)
Anti-inflammatory, hydrogellike scaffold Immunostimulatory, pro-inflammatory Antiviral, tissue remodeling and migration, stimulation of anticoagulant proteoglycan secretion, antithrombin, antiatherogenic, antitumor Antimicrobial (bactericidal, bacteriostatic), antioxidant, bacterial anti-adhesive, antiviral Controlled drug delivery, anti-adhesive coating Inhibition of human complement, biomaterial for coatings/membranes, capping agent of silver nanoparticles (bactericidal) Antitussive, bronchodilator, immunomodulatory Antifungal film
Ngatu et al. (2012), Okajima et al. (2008) Løbner et al. (2008)
Immulina (EPS) Spirulan (EPS)
EPS
Cyanothece sp. CCY 0110 Nostoc commune
Cyanoflan (RPS) EPS
Nostoc sp.
EPS
Nostoc sp.; Synechocystis sp. Nostoc flagelliforme Nostoc sphaeroides Nostoc microscopicum Synechocystis aquatilis Synechocystis sp. PCC 6803
EPS Nostoflan (EPS) Nostoglycan (EPS) EPS EPS RPS
Antiviral, Antitumor Antitumor, Antioxidant Bactericidal, Antibiofilm Inhibition of human complement, Anticoagulant Antitumor
Kaji et al. (2002), Lee et al. (2007), Mader et al. (2016), Mishima et al. (1998) Reichert et al. (2017), Challouf et al. (2011)
Estevinho et al. (2019), Costa et al. (2020) Morsy et al. (2014), Brull et al. (2000)
Uhliariková et al. (2020) Morales-Jiménez et al. (2020) Yue et al. (2011), Kanekiyo et al. (2007) Li et al. (2018) Ramachandran et al. (2019) Flamm and Blaschek (2014a) Flores et al. (2019b)
(Estevinho et al. 2019). This polymer was also used to produce an anti-adhesive coating capable of efficiently preventing the adhesion of relevant etiological agents, aiming at developing an antibiotic-free alternative to fight catheter-associated urinary tract infections (Costa et al. 2020). The lack of knowledge about the mode of action and the production mechanisms of cyanobacterial EPS hinders the approval of these polymers as qualified
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commodity materials by the policy agencies worldwide. In addition, this approval process is even more restrictive regarding products with biological activity and for biomedical use. So far, only Arthrospira (namely A. platensis and A. maxima biomass/extracts) successfully accomplished the high-quality criteria to be commercialized worldwide as dietary supplement and cosmetic product. Another significant milestone was reached using A. platensis carbohydrate-based molecules, such as a peptidoglycan complex that is being tested as anticancer drug in patients with advanced pancreatic cancer (Khan et al. 2019). This molecule reached the phase I in December 2016, but the results of this study are still not currently available. Overall, although very promising in vitro results have been obtained testing cyanobacterial EPS, more comprehensive studies are needed to confirm these results in vivo, as well as to fully understand the mechanisms and the EPS features associated with their bioactivity.
7
Conclusions and Future Perspectives
The complexity of the chemical composition and structure, rheological behavior, and diversity of bioactivities of the cyanobacterial EPS results in an almost endless list of biotechnological applications. In addition, with the expected growth of the universal current “green trend,” the development of novel natural solutions with better performance and positive cost-benefit is a must. However, despite the recent advances on the knowledge on cyanobacterial EPS biosynthetic pathways, the identification of key components and the elucidation of their regulatory networks is mandatory to control EPS production/EPS characteristics. Enduring efforts to improve the photobioreactors in terms of design, operation, and scaleup, together with modern genetic/ protein engineering approaches, can significantly improve the economics and feasibility of cyanobacteria/cyanobacterial EPS industrial production. In addition, costeffective methodologies covering the entire product development, including biomass production, product processing and purification, and packaging and marketing, still need to be further addressed to make cyanobacteria/cyanobacterial extracellular polymers economically attractive. Acknowledgments This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028779 (PTDC/BIA-MIC/28779/2017).
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Glycosaminoglycans Hajer Radhouani, Susana Correia, Cristiana Gonc¸alves, Rui L. Reis, and Joaquim Miguel Oliveira
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Structural Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Heparin (HEP) and Heparan Sulfate (HPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Chondroitin Sulfate (CS) and Dermatan Sulfate (DS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Keratan Sulfate (KS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Hyaluronan/Hyaluronic Acid (HA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Industrial Production of GAGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Bacterial Production of GAGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Bacterial Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Bacterial Chondroitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Bacterial Heparosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Properties and Clinical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Chondroitin Sulfate and Dermatan Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Heparin and Heparan Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Keratan Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Conclusions and Future Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Glycosaminoglycans (GAGs), also known as mucopolysaccharides, are linear anionic compounds composed of repeating disaccharide units and classified into four groups: heparin and heparan sulfates, chondroitin sulfate and dermatan H. Radhouani (*) · S. Correia · C. Gonçalves · R. L. Reis · J. M. Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_12
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sulfate, keratan sulfate, and hyaluronan/hyaluronic acid. These large complex carbohydrate molecules are present in every mammalian tissue where they are known to bind and regulate a broad range of proteins involved in a myriad of physiological and pathological processes. GAGs are found in (in)vertebrate animals, implying a conserved function in the animal kingdom. Though, today there is an increasing number of examples of GAGs of microbial origin. There are concerns such as environmental impact, presence of undesirable animal products, and contamination risks that have necessitated alternate sources for industrial GAG production. The GAGs produced by microorganisms (microbial GAGs) are renewable resources and meet current market demands. Besides, these microbial GAGs are less complex and lack some modifications usually observed in animal GAGs. In fact, certain bacteria such as Escherichia coli, Pasteurella multocida, and Streptococcus have the necessary enzyme machinery to produce simple, nonsulfated GAGs, such as hyaluronan, heparosan, and chondroitin, among many more. Due to the recent expansion of GAG demand, a summary of the molecular structures, biosynthesis, physiologic functions, and clinical applications of the four primary groups of GAGs, and also a brief description of the microbial production of GAGs, is of particular interest. Keywords
Bacteria · Glycosaminoglycans · Microbial production · Biological properties · Pharmaceutical applications
1
Introduction
Glycosaminoglycans (GAGs) are long linear anionic polysaccharide compounds composed of repeating disaccharide monomers that are present in animal tissues (Casale and Crane 2019). GAGs are constituted of combined sulfated or nonsulfated uronic acids (glucuronic acid or iduronic acid) and amino sugars (glucosamine or galactosamine). From the combination of different uronic acids and amino sugars, four main groups of GAGs can be distinguished: heparin (HEP) and heparan sulfates (HPS), chondroitin sulfate (CS) and dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan or hyaluronic acid (HA) (Neves et al. 2020). These heteropolysaccharides can occur with different sulfation degrees and patterns that affect their biological function, with exception of HA, the only nonsulfated GAG (Soares da Costa et al. 2017). Sulfated GAGs are synthesized by specific enzymes in the Golgi apparatus of the cell, whereas hyaluronan (HA) is synthesized by transmembrane proteins called HA synthases. While HA is not linked to a protein and produced from its reducing end, sulfated GAGs are synthesized from the nonreducing end as side chains attached to a protein that forms proteoglycans (PGs) (Fig. 1) (Köwitsch et al. 2018). Their functions within the body are widespread and determined principally by their molecular structure. These polysaccharides are present in the extracellular matrix (ECM) of all animal cell surfaces and some are known to bind a number of
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Fig. 1 Structure of an aggrecan-type complex proteoglycan
different proteins, including cytokines, growth factors, chemokines, enzymes, and adhesion molecules (Jackson et al. 1991; Gandhi and Mancera 2008). The function of GAGs was believed to be limited to structural scaffolding and cell hydration; however, evidence now suggests that GAGs play a crucial role in cell signaling, which attends to modulate a vast amount of biochemical processes (Linhardt and Toida 2004). Some of these processes include regulation of cell growth and proliferation, promotion of cell adhesion, anticoagulation, and wound repair, among others (Casale and Crane 2019). Over the past few decades, GAGs are being used extensively in the pharmaceutical industry because of their multiple biological properties. Likewise, the therapeutic potential of GAGs and their mimetics for the treatment of many diseases, including thrombosis, thrombophlebitis, cancer, inflammation, infection, wound healing, lung diseases, Alzheimer’s disease, etc., are being actively investigated (Weitz and Harenberg 2017; Pomin and Mulloy 2018; Morla 2019; Neves et al. 2020). Usually, the production of GAGs is obtained from mammalian tissues mainly generated in slaughterhouses: rooster combs, cartilage (tracheas and nasal from bovine and swine), and umbilical cords. However, as a consequence of concerns due to bovine spongiform encephalopathy (BSE) and undesired contaminations causing possible allergic inflammation responses, the extraction of GAGs from microorganisms has received an increasing attention. The microbial production of GAGs can generate best yields with higher concentrations at lower costs and with more efficiency (Mishra and Pavelko 2006; Vázquez et al. 2013; Delbarre-Ladrat et al. 2014). The main microbial glycosaminoglycans are those produced by Group C Streptococcus (GCS), Escherichia coli K5, E. coli K4, and Pasteurella multocida. In fact, these bacteria have the necessary enzyme machinery to produce simple, nonsulfated GAGs, such as hyaluronan, heparosan, and chondroitin. One of the most important commercial sources of hyaluronan polysaccharides for surgical, ophthalmic, and viscoelastic applications is GCS (Nwodo et al. 2012; Cimini et al. 2018).
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Herein, the current knowledge of the four primary groups of GAGs regarding their structure, biosynthesis, properties, microbial production, and clinical applications are overviewed.
2
Structural Features
Glycosaminoglycans are structurally complex molecules, with varying lengths, backbone sugars, and modifications. GAGs are linear and highly negatively charged polysaccharides due to the presence of uronic acid residues and sulfate groups. These biopolymers are composed of repeating disaccharide subunits, which are made of an amino sugar and a hexuronic acid, that have molecular weights of roughly 10–100 kDa. These building blocks consist of variously N-substituted glucose- or galactosamine linked to glucuronic or iduronic acid (Almer et al. 2018; Morla 2019). These disaccharides can suffer modifications at multiple positions by epimerization, sulfation, and/or acetylation, resulting in the different major categories of GAGs (HEP/HPS, CS/DS, KS, and HA) (Gandhi and Mancera 2008). Except for HA, all GAGs are negatively charged due to the presence of varying amounts of sulfate and carboxyl groups within their structure. Thus, HA is the only GAG that is not sulfated and, hence, is the least negatively charged GAG, while HEP is the most negatively charged (Pomin and Mulloy 2018). The molecular structure of each of the major categories appears below.
2.1
Heparin (HEP) and Heparan Sulfate (HPS)
Heparin and heparan sulfate (Fig. 2) contain repeating disaccharide units of N-acetylglucosamine and hexuronic acid residues. The hexuronic acid residue glucuronic acid is identified in heparan sulfate, while iduronic acid is present in heparin (Casale and Crane 2019). The major repeating disaccharide sequence of heparin (75–90%) is [!4)-2-O-sulfo-α-L-ido-pyranosyluronic acid (IdoAp) (1!4)2-deoxy-2-N-,6-O-sulfo-α-D-glucopyranosylsamine (GlcNp) (1!], while minor sequences contain β-D-glucopyranosyluronic acid (GlcAp) residues, a reduced content of sulfo groups as well as N-acetylation. Regarding HPS, this polysaccharide has a similar structure with heparin, primarily containing nonsulfated disaccharides [!4) β -D-GlcAp (1!4)-α-D-GlcNpAc (1!] and monosulfated disaccharides, such as [!4) β -D-GlcAp (1!4)-6-sulfo-α-D-N-GlcNpAc]. HEP and HPS are composed of sulfated linear polysaccharide combinations, with a molecular weight (MW) ranging from 5000 to 40,000 and a MW average from 10,000 to 25,000 kDa (Zhang et al. 2010). HPS polysaccharides are abundant components of the cell surface and extracellular matrix of all multicellular animals, whereas HEP is present within most cells and contains higher levels of sulfo groups and more iduronic acid. Heparin is usually extracted from animal organs, predominantly porcine intestinal mucosa (Meneghetti et al. 2015).
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L-iduronate-2-sulfate N-sulfo-GlcNAc-6-sulfate Fig. 2 Heparin and Heparan sulfate structures they are composed of L-iduronate (IdoA) or D-glucuronate (GlcA) and N-sulfo-D-glucosamine-6-sulfate
2.2
Chondroitin Sulfate (CS) and Dermatan Sulfate (DS)
Chondroitin sulfate (Fig. 3A) and dermatan sulfate (also known as chondroitin sulfate-B, Fig. 3B) are similar in their structural composition to HPS. Their disaccharide repeat consists of N-acetylgalactosamine and hexuronic acids – iduronic acid in CS and glucuronic acid in DS (Raman et al. 2005). The CS/DS family of GAGs is composed by alternating 1!3, 1!4 linked 2-amino, 2-deoxy-α-D-galactopyranose (GalNpAc) and uronic acid (β-D-GlcAp in CS and α-L-IdoAp in DS) residue. Moreover, CS-GAGs also have domain structures similar to HPS-GAGs. The molecular weights of CS/DS range from 2 to 50 kDa. Furthermore, CS and DS can be identified on the cell surface and in the extracellular matrix like HPS (Suflita et al. 2015; Casale and Crane 2019).
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Keratan Sulfate (KS)
Keratan sulfate (Fig. 4) contains a disaccharide repeat composed of galactose and N-acetylglucosamine; the sulfation patterns may be present on either unit, although with increased frequency on N- acetylglucosamine. As stated before, KS is the only sulfated GAG that is not connected by a tetrasaccharide linker compound to the PG protein core (Prydz 2015). KS has two distinct forms, originally designated as KS-I and KS-II based on differences between cornea and cartilage KS. However, a third type of KS linkage (mannose-O-Ser) has been identified that has been named KS-III. KS is comprised primarily of 6-O-sulfo-GlcNpAc and galactose (Gal) (which may
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(A)
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O N
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L-iduronate (IdoA)
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Fig. 3 Chondroitin sulfate (A) and Dermatan sulfate (B) structures
contain 6-O-sulfo groups). KS has been known in an extensive range of tissues, such as animal cornea, cartilage, bone, neural cells, dermis, ovarian zona pellucida, and even mammal uterine lining (Funderburgh 2000).
2.4
Hyaluronan/Hyaluronic Acid (HA)
Hyaluronan (Fig. 5) is an anionic linear polysaccharide composed of disaccharide units consisting of ([!3)-D-GlcNpAc(1!4)-β-D-GlcAp(1!]) (Moscovici 2015). This homocopolymer has the simplest structure of all GAGs, consisting of sequentially bound glucuronic acid and N-acetylglucosamine residues, without the need for
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O
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Fig. 4 Keratan sulfate structure
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OH
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HO
O
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HO
O N
OH O
CH3
D-glucuronate (GlcA)
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Fig. 5 Hyaluronan structure
additional functional group sulfation in the Golgi apparatus as other GAGs (Ghiselli 2017). This unsulfated GAG is characterized by a very high MW of up to 2000 kDa. Hyaluronan is present in the vitreous humor, cartilage tissue, and synovial fluid (Tamer 2013). The presence of hyaluronan outside of vertebrates has been identified only in a mollusk (Yamada et al. 2011).
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Biosynthesis
Glycoscience faces many challenges due, mainly, to the nontemplate-driven nature of GAG’s biosynthesis and their structural complexity (Lopez Aguilar et al. 2017). The biosynthesis process involves complex reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. The carbon backbone
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of the GAG chain may not be modified (e.g., hyaluronan) or may suffer modifications at different regions through sulfation, de-acetylation, and/or epimerization (e.g., heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate) (Datta et al. 2019). One of the critical modifications is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, being this reaction demonstrated to be crucial for animal development (Raedts et al. 2013). In eukaryotes, the process of GAG biosynthesis takes place in the endoplasmic reticulum and the Golgi apparatus, with the synthesis of five uridine diphosphate (UDP) derived activated sugars (UDP-glucuronic acid, UDP-N-acetylglucosamine, UDP-xylose, UDP-galactose, and UDP-N-acetylgalactosamine) (Fernández and Warren 1998). These sugars are then secreted through an antiporter transmembrane transporter from the cytoplasm to the Golgi apparatus for further modification (Ghiselli 2017). An exception is HA, which is exclusively synthesized at the plasma membrane by a membrane-bound HA-synthase. Instead of undergoing modification and sulfation in the Golgi apparatus, the HA precursor sugars UDP-glucuronic acid and UDP-N-acetylglucosamine are transferred from the cytoplasm to the plasma membrane for further processing without sulfation resulting in HA production (Raedts et al. 2013; Casale and Crane 2019). To apply their biological function, GAGs are further processed by a series of modification steps such as sulfation of functional groups by the action of the sulfate donor compound 3’-phosphoadenosine-5’-phosphosulfate (PAPS). It is important to highlight that the accessibility of PAPS for GAG’s sulfation will disturb the biosynthetic rate of production of the sulfated GAGs (Ghiselli 2017). Moreover, these sulfated GAG chains which were synthesized in the Golgi apparatus are added to proteoglycan. Regarding the GAGs heparin/heparan sulfate, chondroitin sulfate, and dermatan sulfate, the tethering process happens through a serine amino acid residue present on the protein core that relates to a common tetrasaccharide linker between the GAG and PG. Moreover, the only sulfated GAG that is not related to a PG protein core by this process is keratan sulfate and is instead linked by several other compounds depending on the subtype of keratan sulfate (I, II, and III) (Prydz 2015).
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Industrial Production of GAGs
Nowadays, the commercial production of GAGs is commonly based on three origin categories (1) industrial production from animals, (2) industrial production using microbial cells, and (3) industrial production using eukaryotes (Datta et al. 2019). Most of the commercially available GAGs have so far been extracted and purified from mammalian tissues, such as heparin from bovine and porcine mast cells that are found in intestine and lung tissues, chondroitin from animal (bovine, chicken and porcine) cartilage, dermatan sulfate from mucosal tissue and bovine and porcine intestine or porcine skin, and hyaluronan from rooster combs, umbilical cords, synovial fluids, skin or vitreous humor (Datta et al. 2019). This often highlights health concern issues on viruses and prions, as well as some lower vertebrates that are potentially endangered. The tissue extraction method has
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many disadvantages such as insufficient raw materials, nonuniform products, waste liquid pollution, and potential pathogenic factors (Jin et al. 2019). Recently, in order to avoid health issues and wider ecosystem damages derived from the extensive uses of mammalian and fishery wastes as substrate, diverse investigations on the industrial production of GAGs from microorganisms have been described (Vázquez et al. 2013; Datta et al. 2019). It is important to point out that prokaryotic GAGs are usually less complex than the eukaryotic ones due to an absence of chemical processes such as sulfation modification (Raedts et al. 2011).
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Bacterial Production of GAGs
Certain bacteria have the capacity to synthetize simple, nonsulfated GAGs, such as chondroitin, heparosan, and hyaluronan. These bacterial GAGs, known as capsular polysaccharides, are commonly produced by Streptococcus sp., Escherichia coli K5, E. coli K4, and Pasteurella multocida (Fallacara et al. 2018; Datta et al. 2019). These polysaccharides represent amazing substitutes for GAGs of animal origin (Nwodo et al. 2012). Some examples of the most common bacterial GAGs from serotypes of Streptococcus, E. coli and P. multocida, among others, are described in the following sections.
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Bacterial Hyaluronan
Hyaluronan production through bacterial fermentation started in the 1960s, but its production on industrial scale was firstly achieved by Shiseido in the 1980s (Liu et al. 2011). It is important to highlight that bacterial fermentation produces hyaluronan with high molecular weight and purity, but the risk of some contamination still exists such as bacterial endotoxins, proteins, nucleic acids, and heavy metals (Hussain et al. 2017). On the other hand, bacterial hyaluronan production has a great advantage since bacteria can be metabolically and/or physiologically improved to produce with high molecular weight and purity. Nowadays, there is a rising interest on using microorganisms such as pathogenic streptococci or safe recombinant hosts for bacterial hyaluronan production since they have the necessary hyaluronan synthase (Boeriu et al. 2013). Recently, the most significant alternative has been the development of bacterial hyaluronan production by the gram-positive Streptococcus sp. and gram-negative bacteria P. multocida, which can naturally produce hyaluronan (Schiraldi et al. 2010; Cimini et al. 2018). Group C streptococci (S. equi subsp. equi and S. equi subsp. zooepidemicus) have been shown to produce hyaluronan with a higher quality and quantity than Group A streptococci or pathogenic bacterium P. multocida. Microbial fermentation of S. zooepidemicus is considered as one the most proficient and widespread processes of hyaluronan production. More recently, a recombinant S. thermophilus was
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developed with the ability to produce 1.2 g.L1 hyaluronan with a molecular weight similar to the wild type strain (Izawa et al. 2011; Zakeri et al. 2017). Research on hyaluronan production optimization through bacterial fermentation attended yield improvement, molecular weight increase, and also polymer polydispersity reduction, parameters which are fundamental for hyaluronan applications. The main characteristic was to regulate the molecular weight through several parameters such as optimized process conditions, improved bacteria, or even using metabolic engineering (Fallacara et al. 2018). Recent research in alternative systems for sustainable production of hyaluronan proposed new microbial strains such as E. coli, Bacillus sp., Agrobacterium sp., and Lactobacillus lactis. Hence, nowadays researchers have an interest to develop new strains like Pichia pastoris (Komagataella pastoris), Lactic acid bacteria (Lactococcus lactis), Bacillus subtilis and Corynebacterium glutamicum, and other safe strains to recreate the production pathway for HA synthesis (Vázquez et al. 2013; Datta et al. 2019).
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Bacterial Chondroitin
Another example of a GAG-like polysaccharide produced by bacterial strains is chondroitin; the microbial synthesis of this biomolecule is, now, one of the most potential alternatives to animal sources of chondroitin/CS. Pathogenic bacteria such as E. coli K4 and P. multocida type F are known to produce a capsular polysaccharide (CPS) that possessed a disaccharide repeat like unsulfated chondroitin and thus can be used as a starting biopolymer for further chemo/enzymatic modifications to CS with the desired sulfation degree and pattern (Deangelis et al. 2013; Cimini et al. 2018). The capsules of P. multocida type A, D, and F could be extracted upon treatment with different glycosaminoglycan hydrolases. These CPS are constituted of hyaluronan, N-acetylheparosan (heparosan or unsulfated, unepimerized heparin), and unsulfated chondroitin, respectively (DeAngelis and White 2004). In the beginning, P. multocida, an important gram-negative pathogenic bacterium, was one of the preferred bacteria to produce chondroitin; however, its cholera pathogenicity has impeded and reduced its interest (Naccari et al. 2009; Vázquez et al. 2013). Another member of the Pasteurellaceae family, Avibacterium paragallinarum genotype A, was also described to produce a chondroitin capsule. It is important to point out that a fragment of its genome draft sequence revealed high homology to the capsule gene clusters of P. multocida type F and E. coli K4 (Wang et al. 2018).
5.3
Bacterial Heparosan
Heparosan is biosynthesized as a polysaccharide capsule in bacteria including E. coli K5 and P. multocida type D, being the common precursor molecule of both heparin and heparan sulfate which have similar backbone structures to the commercial heparin.
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These bacterial polysaccharides have been synthesized by E. coli K5 in the nonsulfated forms and in the sulfated forms by E. coli K4. Moreover, heparan sulfates, produced by P. multocida, are characterized by higher molecular weights (200 and 300 kDa) than those produced by E. coli strains (DeAngelis and White 2004). Contrary to animal sources, no postpolymerization modifications occur on the K5 heparosan molecule, which makes this K5 polysaccharide a valuable substrate to investigate the enzymes in heparin biosynthesis and a potential precursor for heparin chemo-enzymatic synthesis (Vann et al. 1981; Raedts et al. 2011). Recent research demonstrated that the extracted heparosan from microbial sources could be modified chemoenzymatically in order to produce the anticoagulant heparin (Bhaskar et al. 2015). Besides, other study demonstrated that mammalian cells that naturally produce heparan sulfate can be metabolically modified for heparin production. The recovery and modification of these polysaccharides into biologically active GAG-like polysaccharides, such as heparin or heparan sulfate, will serve as an important substitute for animal derived GAGs (Baik et al. 2012; Datta et al. 2019).
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Properties and Clinical Applications
Glycosaminoglycans, as one main part of the glycocalyx, are involved in a myriad of biological and therapeutic functions, as well as in several diseases. Certain GAGs are known to bind and regulate a number of different proteins, such as growth factors, cytokines, morphogens, chemokines, enzymes, and cell adhesion molecules (Gandhi and Mancera 2008; Mende et al. 2016). Additionally, GAGs play a key role in cell signaling and development, axonal growth, anticoagulation, angiogenesis, tumor progression, and metastasis. Uncontrolled progenitor cell proliferation leads to malignant tissue transformation and cancer (Morla 2019). GAG bioactivity is conferred by intrinsic structural features such as sugar compositions, glycosidic bonds, and sulfation patterns and degrees. Since GAGs are engaged in a variety of biological processes, their use in drug development has been for long interesting in the pharmaceutical industry (Table 1), and more recently in tissue engineering, as shown in Fig. 6 (Lane et al. 2017; Köwitsch et al. 2018; Morla 2019).
6.1
Hyaluronan
The most abundant nonsulfated GAG, hyaluronan, has numerous important functional roles, such as signaling activity during embryonic morphogenesis, pulmonary and vascular diseases, and wound healing. HA also acts in the lubrication of synovial joints and joint movement; it has been described to function as space filler, wetting agent, flow barrier within the synovium, protector of cartilage surfaces and has undergone clinical testing as a means of relieving arthritis (Ondresik et al. 2017). Moreover, the best-known physicochemical property of HA is its aptitude to form hydrogels. This property allows HA to be used as a vehicle to make specific hydrogel formulations for regenerative medicine. HA-based products used to be applied in
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Table 1 Summary of the most common GAGs (DeAngelis 2002; Gandhi and Mancera 2008; Raedts et al. 2011; Köwitsch et al. 2018) Main disaccharide -4)GlcAβ(1-4) GlcNAc-α(1-
Sulfation pattern GlcA(IdoA) at C2, GlcNAc (NS) at C6, C3
Molecular weight 10–70 kDa
HEP
-4)IdoAα(1-4) GlcNS-α(1-
Like heparan sulfate but heavier
5–40 kDa
CS
-4)GlcAβ(1-3) GalNAc-β(1-
GalNAc at C4/C6 and GlcA at C2
5–50 kDa
Cartilage, tendons, ligaments, bone, heart valves
DS
-4)IdoAα(1-3) GalNAc-β(1-
GalNAc at C4/C6 and IdoA at C2
15–40 kDa
KS
-3)Galβ(1-4) GlcNAc-β(1-
Gal and GlcNAc at C6
4–19 kDa
Skin, blood vessels, heart valves, tendons, lungs Cornea, bone, cartilage
HA
-4)GlcAβ(1-3) GlcNAc-β(1-
Unsulfated polysaccharide
4–8000 kDa
GAGs HPS
(In)vertebrate sources Component of cell surface membranes, ECM Component of intracellular granules of mast cells lining the arteries of the lungs, liver, skin
Synovial fluid, vitreous humor, ECM of loose connective tissue
Potential therapeutic application Antiinflammatory, antitumor Anticoagulant/ antithrombotic, anti-inflammatory, wound healing (OA), antitumor, promoter of cell growth and differentiation in tissue engineering, hydration therapies, and protective function on cosmetic industry Antiinflammatory, wound healing (OA), antitumor, promoter of cell growth, and differentiation in tissue engineering Anticoagulant/ antithrombotic
Antiadhesive, active ingredient in eye drops Antiinflammatory, antitumor, wound healing, promoter of cell growth and differentiation in tissue engineering, lubricating agent of synovial joint fluid, hydration therapies, and wrinkle prevention on cosmetic industry
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Fig. 6 GAGs and derivatives in some tissue engineering applications
cosmetics to, firm, soften and smooth skin by dint of its hydrating properties and inherent regenerative; HA is an important functional component of the extracellular matrix (ECM) of skin and has shown clinical utility in preventing formation of postsurgical adhesions (Zhu et al. 2017). Due to its relative ease to be extracted and modified and its capability to form 3D structures, HA has become the most used GAG in medical product development (Fallacara et al. 2018; Pomin and Mulloy 2018).
6.2
Chondroitin Sulfate and Dermatan Sulfate
The dermatan molecule has residues of L-iduronic acid, which is commonly considered as the principal difference between chondroitin sulfate and dermatan sulfate. CS is essential in cell–cell interaction and communication, and DS reveals a potential antithrombotic effect. These two GAGs also have remarkable functions in growth factor signaling, neurite outgrowth, cell division, morphogenesis, inflammation, and infection (Hayes and Melrose 2018).
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Chondroitin sulfate has an anticoagulant activity and so can be used also as a natural alternative for heparin in several pharmaceutical industries and also as a dietary supplement to treat arthritis pain and inflammation, and as hydrogels for cartilage tissue engineering (De Jesus Raposo et al. 2015). In fact, this polysaccharide can be used as an alternative treatment in cases of osteoarthritis, and occasionally even osteoporosis, because of its essential roles in cartilage and other connective tissues. The use of CS for arthritis treatment is usually related to the use of glucosamine, another key constituent of cartilage tissues (Schiraldi et al. 2010). On the other hand, dermatan sulfate is used as a stabilizer for growth factors and cytokines. This biomolecule demonstrated a significant anticoagulant property. The anticoagulant activity of dermatan sulfate inhibits thrombin, having no effect on the clotting cascade and in platelet function. With its anticoagulant and antithrombotic activity, DS could be considered a good alternative for heparin, one of the most popular and widely used anticoagulants (Cardoso et al. 2016). Moreover, DS is thought to be involved in wound repair, in cell differentiation, morphogenesis, and migration, which is why this substance is used for cosmetic and clinical applications (Sobczak et al. 2018).
6.3
Heparin and Heparan Sulfate
Heparin is an invaluable drug for the treatment of coagulation and thrombotic disorders and is listed as one of the World Health Organizations essential drugs. It has been widely used as an anticoagulant for more than 80 years and is characterized by several biological activities with potential clinical applications, such as antiinflammatory, angiogenic and antiangiogenic, anticancer and antiviral activities. It has effects on lipoprotein lipase, on smooth muscle proliferation, and on inhibition of complement activation, among others (Zhang et al. 2010). Furthermore, due to its high charge density, HEP is also important in cell–cell interactions involving adhesion proteins, cell–cell communication involving chemokines, and cell signaling involving growth factors (Suflita et al. 2015). When crosslinked (physically and chemically), heparin hydrogels have been developed for a variety of clinical applications including the encapsulation of fibroblasts, differentiation of stem cells, and liver tissue engineering applications (Roberts and Martens 2016). Because of the presence of sulfated groups in the HPS molecule, the polysaccharide is capable to bind to numerous proteins and thus regulates biological processes such as coagulation. Moreover, HPS can bind to several polypeptides like growth factors as well as cellular receptor complexes (Cheng et al. 2017). Recently, it has been used in the development of some new antineoplastic drugs along with new drug delivery systems and also in diagnostics (Weissmann et al. 2016).
6.4
Keratan Sulfate
Keratan sulfate is one of the principal functional components in cornea, particularly in the maintenance of corneal transparency, and it has antiadhesive properties. This
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polysaccharide can be used as an active ingredient in eye drops for the treatment of certain visual dysfunctions. This biomolecule has been detected in several neural and epithelial tissues, in which KS expression responds to wound healing, physiological variations, and embryonic development. It has been revealed that KS has functional roles in cellular recognition of protein ligands, axonal guidance, cell motility, in embryo implantation, and in decreasing the immune response in diseases like osteoarthritis in cartilage (Caterson and Melrose 2018).
7
Conclusions and Future Trends
Glycosaminoglycans, with their exceptional properties and diverse applications, have proved to be remarkable materials for medical, pharmaceutical, and cosmetic applications. Most of GAGs are extracted from animal sources to produce active biopolymers to use in several applications. However, GAG-producing microorganisms are renewable resources and could meet current market demands, but remain largely unexplored. Microbial GAG production has the additional and important advantage that bacteria can be physiologically and/or metabolically improved for better biopolymer production. Thus, there is an urgent need to develop industrialscale microbial production processes of GAGs, with new microorganisms that could be further engineered. Genome sequencing and bioinformatics analysis could be used to discover novel GAG-producing bacteria and/or optimize microbial GAG production. The availability of new genome sequences, together with advances in metabolic engineering and analytical tools, could be applied to evaluate the correlation between cell metabolism and new biologically active GAG compounds. Further investigations on designing new metabolic engineering approaches will also be needed to construct microbial cells that will produce biopolymers with desired structures for a certain application. Acknowledgments H. Radhouani and C. Gonçalves were supported by the Foundation for Science and Technology (FCT) from Portugal, with references CEECIND/00111/2017 and SFRH/BPD/94277/2013, respectively. J. M. Oliveira thanks the FCT for the fund provided under the Investigador FCT 2015 (IF/01285/2015) program. S. Correia and this work were funded by the R&D Project KOAT – Kefiran exopolysaccharide: Promising biopolymer for use in regenerative medicine and tissue engineering, with reference PTDC/BTMMAT/29760/2017 (POCI-01-0145FEDER-029760), financed by FCT and co-financed by FEDER and POCI.
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Part II Fungal and Microalgal Polysaccharides
Botryosphaeran
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An Unusual Exocellular Fungal (1!3)(1!6)-β-D-Glucan with Notable Biomedical Applications Robert F. H. Dekker and Aneli M. Barbosa-Dekker
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Chemical Structure of Botryosphaeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 A Family of Botryosphaerans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Sulfonylation of Botryosphaeran: Anticoagulant and Antiviral Activities . . . . . . . . . . 3 Antimutagenicity and Chemopreventive Effect of Botryosphaeran . . . . . . . . . . . . . . . . . . . . . . . 4 Anti-Obesogenic Activity of Botryosphaeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Hypoglycemia and Hypocholesterolemia Exhibited by Botryosphaeran . . . . . . . . . . . . . . . . . . 5.1 Improving the Diabetic Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Improving the Dyslipidemic Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Anticancer Activity of Botryosphaeran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Botryosphaeran [(1!3)(1!6)-β-D-glucan] is an exopolysaccharide (EPS) produced by the ascomycete Botryosphaeria rhodina MAMB-05 when cultivated on glucose medium. Botryosphaeran has an unusual chemical structure in the sense that one of its two appendage residues (gentiobiose) attached to the (1!3)-linked backbone chain is rare, the other, glucose, is common to most β-glucans of this type. A family of botryosphaerans is produced on different carbohydrate substrates. Botryosphaeran is neither mutagenic nor genotoxic, and presents antioxidant, anticlastogenic, hypoglycemic, hypocholesterolemic, and hypotriglyceridemic R. F. H. Dekker (*) Universidade Tecnológica Federal do Paraná (UTFPR), Programa de Pós-Graduação em Engenharia Ambiental, Câmpus Londrina, Londrina, Paraná, Brazil A. M. Barbosa-Dekker Beta-Glucan Produtos Farmoquímicos - EIRELI, Lote 24A, Bloco Zircônia, Universidade Tecnológica Federal do Paraná, Avenida João Miguel Caram, Londrina, Paraná, Brazil © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_63
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activities and reduced the development of Walker-256 bearing tumors and cachexia syndrome in rats. Botryosphaeran exhibited antiproliferation of cancer cells that is associated with cell cycle arrest, apoptosis, necrosis, and oxidative stress in breast carcinoma MCF-7 cells, and cell cycle arrest in human T-lymphocyte tumorigenic cells. Botryosphaeran is commercially used in formulated cosmetic products to promote skin health and treat skin conditions. Keywords
Family of botryosphaerans · Sulfonated botryosphaeran · Antimutagenicity · Anti-obesogenicity · Hypoglycemia · Dyslipidemia · Breast cancer MCF-7 · Walker 256 tumor
1
Introduction
The discovery of botryosphaeran (an exopolysaccharide) evolved from a fungal isolate collected from a colony of fungi growing on a stem canker of a eucalyptus tree. The isolate was purified to an axenic culture, designated MAMB-05, morphologically characterized as a species of the genus Botryosphaeria (taxa Botryosphaeriaceae) (Barbosa et al. 1996), and molecularly identified to the species level as Botryosphaeria rhodina (Saldanha et al. 2007). Cultivation of isolate MAMB-05 on glucose media by submerged fermentation resulted in increased viscosity of the culture fluid suggesting the presence of an exocellular biopolymer. On treating the cell-free fermentation broth with ethanol, a white precipitate resulted, which when isolated and subjected to total acid hydrolysis, yielded glucose as the only hydrolysis product, indicating that the biopolymer was an exopolysaccharide constituted of D-glucose residues; i.e., a D-glucan (Dekker and Barbosa 2001). After isolation, purification, and chemical structural characterization, the exopolysaccharide was identified as a (1!3)(1!6)-β-D-glucan, and named botryosphaeran (Barbosa et al. 2003). The polysaccharide group of β-D-glucans consist chiefly of a backbone chain comprising β-(1!3)-linked D-glucose residues containing appendages comprising D-glucose units through β-(1!6)-linkages (Stone 2009). The chemistry and biology of the (1!3)-β-D-glucans have been widely studied and have attracted much interest because of their unique structure-biological function relationships (Stone and Clarke 1992; Bacic et al. 2009). Besides being produced exocellularly, the (1!3)(1!6)-linked β-D-glucans also participate as structural elements in the cell wall of fungi including yeasts, where they are found in a matrix along with other components including proteins and other polysaccharides: chitin, α- and β-glucans of various glycosidic linkages, and mannans (Gow et al. 2017). The β-glucans vary in fine physical structure, molecular size, degree of branching, and conformation (random coil, single and triple helices), properties that confer a crucial role on their biological properties (Leung et al. 2006; Wang et al. 2017). The
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β-glucans possess strong antioxidant activity through scavenging of free radicals thereby avoiding cellular damage by reactive oxygen species (ROS) that trigger oxidative stress (Wang et al. 2013, 2016). The (1!3)-β-D-glucans possess biological response modifying activities such as immunostimulation and immunomodulation (Bohn and BeMiller 1995), and are well recognized in effectively treating human disease conditions that includes obesity, dyslipidemia, diabetes, inflammation, cardiovascular diseases, and cancers (Chen and Raymond 2008; Vetvicka and Vetvickova 2012; Wouk et al. 2021). The biological activities of the β-glucans are manifested through internal cellsignaling events on binding to the carbohydrate-binding domain on surface receptors (Dectin-1, Toll-like Receptors (TLR), Complement Receptor CR-3) of immune cells, where they are recognized as pathogen-associated molecular patterns, and initiate the innate and adaptive immune response of the host resulting in the systemic activation of the whole immune system. This triggers the secretion of cytokines (signaling proteins) and chemokines (small chemo-attractant cytokines) that influence cellular functions.
2
Chemical Structure of Botryosphaeran
The chemical structure of the botryosphaeran (designated EPSGLC) from Botryosphaeria rhodina MAMB-05 cultivated on glucose was determined through methylation analysis, Smith degradation, Fourier-Transform Infra-Red (FT-IR), and 13 C Nuclear Magnetic Resonance (NMR) spectroscopy and provided precise details on the sugar moiety, nature and configuration of the glycosidic linkages, and the polysaccharide’s appendages (Barbosa et al. 2003). Methylation and acid hydrolysis of the permethylated polysaccharide revealed the presence of 2,3,4,6-tetra-O-methyl-glucose (I, nonreducing terminal unit), 2,4,6-tri-O-methyl-glucose (II, 3-O-substituted glucosyl residue), 2,3,4-tri-O-methyl-glucose (III, 6-O-substituted glucosyl residue), and 2,4-diO-methyl-glucose (IV, 3,6-di-O-substituted unit) as the main methylated sugar derivatives (molar proportions 26:36:16:22, respectively). Methylation analysis demonstrated that botryosphaeran is a glucan consisting of a (1!3)-linked glucose backbone substituted with approximately 22% branch points on C-6 of the glucose moiety. Periodate oxidation and Smith degradation revealed side chains consisting of both single glucose residues, and short-chained (1!6)-β-linked glucosyl disaccharides. FT-IR spectroscopy confirmed β-anomeric carbons in botryosphaeran and (1!3)-di-O-substituted glucose residues. 13C NMR spectral analysis on purified botryosphaeran and the periodate-oxidized polysaccharide revealed that all the carbon lines were well resolved, and chemical shifts were congruent with literature values. 13C NMR analysis revealed no peaks resonating around δ 100.0 ppm, typical of α-configuration of anomeric carbons. Peaks resonating between δ 103.3 and 102.9 ppm provided clear evidence that only β-anomeric carbons were present. The structural investigation concluded that botryosphaeran was a (1!3)-β-Dglucan with (1!6)-β-linked single glucose and gentiobiose appendages attached at random along the backbone chain at a frequency of one branch point (glucose or
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Fig. 1 The chemical structure of botryosphaeran – (1!3)(1!6)-β-D-glucan – an exopolysaccharide produced by the ascomycete Botryosphaeria rhodina MAMB-05 when cultivated on glucose medium
gentiobiose) to every five glucose residues (Barbosa et al. 2003). The proportion of glucose-to-gentiobiose along the backbone chain was 6:16 (Fonseca et al. 2011). The structure of botryosphaeran (EPSGLC) is shown in Fig. 1.
2.1
A Family of Botryosphaerans
When Botryosphaeria rhodina was cultivated on other carbohydrate substrates (galactose, mannose, fructose; lactose, sucrose), the fungus produced exopolysaccharides, which after isolation and acid hydrolysis produced only glucose; i.e., they were glucans. FT-IR spectroscopy of the exopolysaccharides presented confirmation they all possessed a (1!3)-β-glucan backbone, thus constituting a family of botryosphaerans (Steluti et al. 2004). Although the carbohydrate sources did not change the chemical structure of the backbone chain of botryosphaeran, they strongly influenced the degree of branching by glucose and gentiobiose on the polysaccharide chain. When Botryosphaeria rhodina was grown on either fructose or sucrose, exopolysaccharides designated EPSFRU and EPSSUC, respectively, resulted (Corradi da Silva et al. 2005). Methylation analysis of EPSFRU and EPSSUC revealed they both constituted a typical botryosphaeran structure comprising a backbone chain of β-(1!3)-linked glucose units with side chains of a single glucose residue, and also a short-chained (1!6)-β-linked glucosyl disaccharide (gentiobiose), in keeping with the chemical structure of botryosphaeran (EPSGLC). EPSFRU was more branched (31%) than botryosphaerans, EPSGLC and EPSSUC, having a branch point to every three glucose units along the backbone chain. Additionally, the relatively low amount of the tri-O-methyl derivative (II, 3-Osubstituted glucosyl residue) of EPSFRU by comparison to the tetra-O-methyl (I) and the di-O-methyl (IV) derivatives presented evidence of a highly branched structure. Furthermore, the low value of the tri-O-methyl (III) derivative suggested
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that the backbone chain of EPSFRU consisted essentially of consecutive (1!3)linked D-glucose residues with side branching on C-6. FT-IR and 13C NMR spectra of EPSFRU and EPSSUC revealed very similar data to that obtained for EPSGLC, the only difference being in the intensity of the 13C NMR signals of EPSFRU. The most important difference was the higher amount of the 6-Osubstituted derivative in EPSFRU compared to EPSSUC. This evidence was confirmed by the intensity of the signal at δ 70.1 and correlated to glucose residues with substitution on C-6 with glucose or gentiobiose residues, in accord with the methylation results. The degree of branching for EPSSUC (21%) (Corradi da Silva et al. 2005) was similar to that of EPSGLC (22%) (Barbosa et al. 2003). The proportion of glucose to gentiobiose along the backbone chain for EPSFRU was 16:15, and for EPSSUC, 12:9 (Fonseca et al. 2011). Our studies on botryosphaeran indicated that the extent of substitution of repetitive branching residues could be modified through the source of carbohydrate during fungal growth. Manipulation of the nutrient media, however, did not change the structure or molecular constitution of the backbone chain of the botryosphaerans. All three botryosphaerans exist in a triple helix conformation (Giese et al. 2008), which strongly influences biological activity (Yoshitomi et al. 2005), and all exhibited free-radical scavenging properties and antioxidant activities (Giese et al. 2015).
2.2
Sulfonylation of Botryosphaeran: Anticoagulant and Antiviral Activities
Certain physical structural features of (1!3)-β-glucans (e.g., molecular weight, degree of polymerization, chemical nature of the side-branch residues, and degree of ramification as well as conformation of the constituent chains in solution) can lead to solubility problems in aqueous solutions (Wang et al. 2017). The problem of water solubility can be resolved by adding appropriate functional groups (e.g., sulfonyl) to hydroxyl positions along the polysaccharide chain (Kagimura et al. 2015). Sulfonylation of polysaccharides introduces a charged group (anionic sulfonate) that improves water-solubility, but also changes the biological functions of the modified polysaccharide, e.g., mimicking heparin activity, i.e., anticoagulation (Jiao et al. 2011). Sulfonated polysaccharides, naturally derived from various plant sources including algae and microorganisms, and those chemically derived by sulfonylation reactions, are well known to present anticoagulant activity. A blood clot is initiated with the activation of platelets that is followed by a cascade of coagulation events catalyzed by enzymes (serine proteases) leading to the formation of thrombin, which acts in the conversion of fibrinogen to fibrin to form a stable cross-linked clot. Heparin has the property of an anticoagulant, and interferes in this process preventing blood clotting, and acts by inhibiting antithrombin of the intrinsic pathway of the coagulation cascade. The biological mechanism of sulfonated polysaccharides occurs by the potentiation of plasma cofactors that are natural inhibitors of the coagulation cascade of events (Melo et al. 2004).
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Anticoagulation activity of a sulfonated polysaccharide can be determined by specific assays monitoring clot formation of blood plasma, and measures the clotting times taken for the formation of activated partial thromboplastin (APTT; intrinsic phase of coagulation), thrombin (TT; conversion of plasmatic fibrinogen to fibrin), and prothrombin (PT; extrinsic phase of coagulation). Heparin is used as the reference standard. The coagulation time in the last stage of the cascade is the conversion of fibrinogen to fibrin by thrombin. Botryosphaerans EPSGLC and EPSFRU sulfonylated using chlorosulfonic acid in formamide solvent readily became water-soluble. The sulfonated botryosphaerans showed an absorption peak at 259 nm in the ultraviolet (UV) spectrum, which was due to the introduced sulfonate group, while FT-IR spectral analysis supported evidence of the introduced sulfonate groups in both botryosphaerans (Mendes et al. 2009; Brandi et al. 2011). 13C NMR spectral analysis of the sulfonated polysaccharides showed that the C-6 signals shifted to around δ 68.0 compared to the parent botryosphaeran (δ 60.7–61.1) indicating that most of the hydroxyl-free C-6 on glucose was substituted by the introduced sulfonate groups. The primary hydroxyl groups on position C-6 is more accessible for sulfonation than the secondary hydroxyl groups, which may be sterically hindered when introducing sulfonate groups. The presence of the β-(1!6)-glucosidic linkages in the side chains may also sterically reduce the number of primary hydroxyl groups available for sulfonation. Brandi et al. (2011) presented evidence that sulfonate substitution on botryosphaeran was absent from hydroxyls at C-2 and C-4, presumably due to steric hindrance. When the sulfonated botryosphaerans were assayed in blood clotting tests, they exhibited anticoagulation activity compared to the parent, unmodified polysaccharide (Mendes et al. 2009; Brandi et al. 2011). The APTT and TT tests of highly branched sulfonated botryosphaeran (S-EPSFRU) indicated significant in vitro anticoagulant activity that was both dose- and degree of sulfonation-dependent. The prolongation of APTT in the presence of S-EPSFRU indicated inhibition of the intrinsic pathway of coagulation, while the extension of the TT time indicated inhibition of the conversion of fibrinogen into fibrin (Brandi et al. 2011). The anticoagulant action of S-EPSFRU is most likely related to the activation of antithrombin that leads to the inhibition of thrombin formation, as the times of clotting were severely prolonged. Un-sulfonated botryosphaeran did not inhibit any of the three coagulation tests, hence has no anticoagulant activity (Mendes et al. 2009; Brandi et al. 2011). Besides anticoagulation activity, sulfonated polysaccharides are also well known for antiviral activity by inhibiting infection of viruses (Chen and Huang 2018), and do this by interacting with the positively charged motifs on glycoproteins of the virus responsible for binding to cell surface receptors on the human host cell. Sulfonylation of botryosphaeran (EPSSUC) exhibited strong antiviral activity against the enveloped viruses Dengue and Herpes simplex (Sacchelli et al. 2019). The parent unmodified botryosphaeran moderately inhibited acyclovir-sensitive (HSV-KOS) and acyclovir-resistant (HS-VAR) strains of Herpes simplex infection in Vero cells (monkey kidney epithelial cells), and demonstrated that native
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botryosphaeran exhibited antiherpetic activity. Sulfonated botryosphaerans of low (0.4) and high (1.1) degrees of substitution (DS) potently inhibited infection against HSV-KOS and HSV-AR. Likewise, dengue viral (DENV-3) infection of Vero cells was weakly inhibited by the parent botryosphaeran, but inhibition was remarkably stronger for the two sulfonated botryosphaerans. Apparently, the presence of sulfonate groups on the polysaccharide chain, and the DS, were important characteristics of antiviral activity of botryosphaeran (Sacchelli et al. 2019).
3
Antimutagenicity and Chemopreventive Effect of Botryosphaeran
New compounds with potential nutraceutical and biomedical applications need first to be screened for mutagenic and genotoxic activities, as this property assesses cellular damage on the host. There are a number of tests considered biomarkers for assessing mutagenic and genotoxic damage. These can be conveniently performed on animal models and mammalian cell-lines, and include: cell viability and cell proliferation – MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann 1983), micronucleus test (Hayashi 2016), comet assay (Tice et al. 2000), and the Ames test – uses bacteria (Salmonella typhimurium strains) instead of animals (Maron and Ames 1983). Genotoxicity can be assessed by the cytokinesis-block micronucleus assay (Fenech 1993). Another important characteristic in assessing a new compound is whether it can exert a chemoprotective effect to reduce mutations induced by common chemotherapeutic drugs to treat cancers (e.g., cyclophosphamide, doxorubicin, methyl methanesulfonate, bleomycin), or mutagens (benzo[a]pyrene). These drugs can be used experimentally as cytostatic agents to induce cellular damage in micronucleus formation in bone marrow and peripheral blood cells of model lab animals. Botryosphaeran was demonstrated to be non-mutagenic, nor cytotoxic or genotoxic, and exhibited strong antimutagenic activity against the in vivo DNA-damaging effect of cyclophosphamide, reducing the clastogenic (aneugenic) effect of cyclophosphamide-induced micronucleus formation in bone marrow and peripheral blood cells in Swiss mice (Miranda et al. 2008). In a further study using a higher dose of botryosphaeran (30 mg/kg body weight (b.w.)/day/15 days), SilvaSena et al. (2018) supported the findings of Miranda et al. (2008), and confirmed that botryosphaeran was not mutagenic nor genotoxic in mice (young and adult of both genders). Botryosphaeran, furthermore, exhibited strong antimutagenic activity being able to reduce the number of micronucleated cells formed by clastogenic cyclophosphamide. Similar studies with mammalian cell-lines: Chinese hamster lung fibroblasts (V79), rat hepatocarcinoma cells (HTC), and cultured normal and tumor (leukemic) human T lymphocytes also confirmed that botryosphaeran was neither mutagenic nor genotoxic, and exhibited chemoprotective effects against the clastogenic effects of doxorubicin and methyl methanesulfonate (Malini et al. 2016), and the mutagen benzo[a]pyrene (Kerche-Silva et al. 2017).
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The findings that botryosphaeran did not present clastogenic or aneugenic damage in mice, nor in several mammalian cell-lines, indicated that it was safe for human use, and thus possessed GRAS status (Generally Recommended As Safe). An interesting feature of botryosphaeran was its strong chemoprotective effect against DNA damage and cell-death induced by chemotherapeutic drugs. This property, and its immunopotentiation function, could find applications of botryosphaeran as a coadjuvant in vaccines, and in treating cancers to lessen the side effects of therapeutic drugs.
4
Anti-Obesogenic Activity of Botryosphaeran
Obesity, an overweight condition prevalent worldwide, is based upon a body mass index (BMI) exceeding 30 kg/m2, and is a strong risk factor for metabolic dysfunction, diabetes, dyslipidemia, and hepatic steatosis that can lead to cardiovascular diseases and cancer. Obesity occurs when there is an energy imbalance with caloric intake far exceeding caloric expenditure. Obesity is characterized by an excess of white adipose tissue stored in the body as an energy source. The incidence of obesity along with diabetes, dyslipidemia, hepatic steatosis, and their associated comorbidities is a worldwide health problem increasing at an alarming rate. Fungal (1!3)-β-glucans are known to reduce the risk of obesity and diabetes, and are effective in treating human hyperglycemia, hyperlipidemia, and hypercholesterolemia that pose cardiovascular risks (Chen and Raymond 2008); Wouk et al. 2021). Studies have revealed that β-glucans can reduce body weight through weight control and metabolism (Rahmani et al. 2019). Silva et al. (2018) induced obesity in rats by feeding a high-fat and high-sugar diet over an 8-week period, and observed symptoms of glucose intolerance, insulin resistance, dyslipidemia, and hepatic steatosis. Treatment with botryosphaeran (12 mg/kg. body weight (b.w.)/day over 15 days) attenuated obesity (reduced weight gains and adipose tissue mass), hepatic steatosis (reduced total lipids, triglycerides, and cholesterol), dyslipidemia (reduced the levels of triglycerides and very-low density lipoprotein (VLDL), and increased high density lipoprotein (HDL) levels), and improved glucose intolerance and insulin sensitivity, as well as increasing AMP-activated protein-kinase (AMPK) and Forkhead transcription factor, FOXO3a, activities in adipose tissue (Silva et al. 2018). AMPK plays a key role in metabolism, especially in cellular energy homeostasis. AMPK regulates several metabolic enzymes and transcription factors including the FOXO forkhead transcription factors, and signaling pathways in liver, adipose tissue, skeletal muscle, and heart that contribute to cellular metabolism of glucose and lipids (Kahn et al. 2005). FOXO is involved in regulating energy metabolism (Calnan and Brunet 2008). Obese rats bearing Walker-256 tumors presented accumulation of adipose tissue mass, reduced muscle mass, glucose intolerance, insulin resistance, hyperglycemia, anemia, leukocytosis, and thrombocytopenia. Botryosphaeran corrected insulin resistance and hyperglycemia, modulated cholesterol levels, and increased total
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leukocyte and lymphocyte counts. Obesity increased tumor development and cachexia syndrome (prognostic in cancer patients, which is characterized by weight loss, anorexia, asthenia, and anemia), and was significantly higher for the obese group than the nonobese group of rats. Treatment with botryosphaeran (12 mg/kg. b. w./day/15 days) attenuated these parameters. The immune response is thought to contribute to lowering the development of tumor, and botryosphaeran effectively attenuated tumor growth (Comiran et al. 2021). In non-obese rats bearing Walker-256 tumor, treatment with a higher dose of botryosphaeran (30 mg/kg. b.w./day/15 days) maintained glycemia within the normal limits, and ameliorated the hypertriglyceridemic condition. Botryosphaeran modulated the levels of glucose, triglycerides and HDL-cholesterol, corrected macrocytic anemia (condition of enlarged red blood cells with low hemoglobin content), and significantly reduced tumor development and cancer cachexia syndrome by increasing apoptosis (ordered cascade of enzymatic events leading to cell death) of the tumor cells through increasing bax expression in the rats (Geraldelli et al. 2020a). Bax, known as the bcl-2-like protein 4, is a member of the Bcl-2 gene family that acts as a regulator of cell death by either inhibiting or inducing apoptosis. In obese rats with Walker-256 bearing tumor, botryosphaeran treatment at the same dose (30 mg/kg b.w./day for 15 days) significantly decreased tumor growth and the intensity of the neoplastic cachexia syndrome, and corrected the macrocytic anemia condition. The mechanism is thought to be associated with the reduction of visceral adipose tissue, the improvement of insulin sensitivity, and increased activity of FOXO3a observed in the tumor tissue (Geraldelli et al. 2020b). From the aforementioned studies on botryosphaeran, we conclude that this unusual fungal (1!3)(1!6)-β-D-glucan presented anti-obesogenic activity beneficial in the obese condition that may be associated with other comorbidities, such as dyslipidemia, insulin resistance, and cancer.
5
Hypoglycemia and Hypocholesterolemia Exhibited by Botryosphaeran
5.1
Improving the Diabetic Condition
Diabetes, a debilitating chronic metabolic disorder, is characterized by hyperglycemia (high glucose levels) due to defects in insulin secretion and insulin action. Epidemiological studies have presented evidence that diabetes is linked to an increased risk of cancer and cardiovascular diseases with the prevailing factor being the high glucose levels. The diabetic condition is manifested by insulin resistance and insufficient insulin secreted by the endocrine system (β cells) of the pancreas. Hyperglycemia also results in the generation of reactive oxygen/nitrogen species that severely impact on the diabetic condition. Management of the diabetic condition focuses primarily on controlling hyperglycemia through administering insulin or drugs like metformin, depending upon the type of diabetes mellitus; type 1 (genetic, severe deficiencies of insulin), and type 2 (late-onset and prediabetic
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condition manifested by glucose intolerance). Managing diabetes is essential in order to decrease, or avoid, other conditions that include cardiovascular-related diseases and cancer, among others. Cereals and mushrooms contain β-glucans that effectively lower blood glucose levels through dietary changes that are helpful for diabetics. Such products display antiglycemic properties and reduce the condition of diabetes, and the risk of developing diabetes, without experiencing undesirable side effects common to drugs used to treat the diabetic condition. Diabetes can be induced experimentally in lab animal models through injection of diabetogenic agents such as alloxan (class of pyrimidones) and streptozotocin (alkylating antineoplastic agent). They selectively cause injury to the insulinsecreting β cells of the pancreas that leads to type-1 diabetes. Treatment of streptozotocin-induced diabetic male rats with botryosphaeran (12 mg/kg. b.w./day) lowered blood glucose levels by 52% over a 15-day period, and was accompanied by a reduced intake of ration and an increase in the median body weight gain, as well as in the efficiency of food conversion in the diabetic animals. Botryosphaeran was furthermore observed to exert a protective effect by reducing the symptoms of cachexia in the type-1 diabetic animals (Miranda-Nantes et al. 2011). In related studies, obese rats presented glucose intolerance because of the obesogenic condition, and treatment with botryosphaeran (12 mg/kg. b.w./day) over 15 days was effective in decreasing obesity (reduced feed intake, weight gain, and periepididymal and mesenteric adipose tissue weights), and improved glucose intolerance (reduced plasma glucose levels, 26%) and insulin sensitivity in the obese animals (Silva et a. 2018). In another related study using 18-month old male knockout LDLr/ mice genetically predispositioned to hyperlipidemia, treatment with botryosphaeran (30 mg/kg b.w./day) over 15 days improved glucose intolerance reducing the plasma glucose levels (36%) (Silva-Sena et al. (2018).
5.2
Improving the Dyslipidemic Condition
Dyslipidemia is often related to obesity, and is the condition of abnormally elevated levels of cholesterol and lipids in the circulating blood system, and is the primary cause of cardiovascular diseases such as atherosclerosis, cardiac infarction, and stroke (ischemic and hemorrhagic) (Wouk et al. 2021). Prevention and management of this condition is crucial to decrease the alarming number of morbidities and mortalities related to heart diseases. Dyslipidemia is associated with hypertriglyceridemia and hypercholesterolemia, and its prevention decreases fat accumulation and manages obesity. The control of cholesterol can be successfully targeted by statins (lipid-lowering drugs) that block metabolic pathways leading to the synthesis of cholesterol. Statins, although effective in reducing cardiovascular diseases, also possess adverse side effects (Goulomb and Evans 2008). A class of natural products with GRAS status that lower cholesterol without experiencing side effects are the (1!3)-β-D-glucans
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(Sima et al. 2018). They have been demonstrated to be efficacious in treating hypercholesterolemic activity (Chen and Raymond 2008). The β-glucans are categorized in the polysaccharide group that constitutes “dietary fibers” and remain undigested in their passage through the human gastrointestinal tract. Fiber is characterized by its physical properties that feature their soluble or insoluble nature. The β-glucans as fibers present health benefits that have been reported to include reduction of bowel transit time thus preventing constipation and reducing the risk of colorectal cancer. Besides lowering cholesterol and regulating glucose blood levels in the respective management of dyslipidemia and diabetes, they also promote the growth of beneficial gut microflora (gut microbiota) producing short-chain fatty acids that influence metabolism in the host (Brennan and Cleary 2005). Botryosphaeran treatment (12 mg/kg. b.w./day) of rats preconditioned to hyperlipidemia and fed a high-fat diet over 15 days reduced the total plasma cholesterol and LDL-cholesterol (bad cholesterol) levels by 18.6% and 27%, respectively (Miranda-Nantes et al. 2011). In a related but independent study, male knockout LDLr/ mice developed elevated plasma cholesterol levels and atherosclerosis on feeding a high-fat diet. Treatment with botryosphaeran (30 mg/kg. b.w./day) over 15 days attenuated the lipidic profiles reducing them by 84%, and decreased LDL-cholesterol by 86%. Additionally, the deposition of lipids in the aorta decreased by ~33% in the mice group tested compared with a control, thus lowering the cardiovascular risk of atherosclerosis (Silva-Sena et al. 2018). Botryosphaeran can therefore serve as a promising agent to treat cardiovascular-related diseases with potential for use as a nutraceutical to treat these metabolic conditions. In a further related study from our research group, Silva et al. (2018) demonstrated that obese rats induced on a high-fat and high-sugar diet showed significant increases in weight gain and adipose tissue mass, and presented hepatic steatosis and dyslipidemia. Treatment of the obese rats with botryosphaeran (12 mg/kg b.w.) over 15 days attenuated obesity significantly reducing feed intake, weight gain, deposition of periepididymal and mesenteric fat, reduced VLDL-cholesterol, and raised HDL-cholesterol (good cholesterol) levels. Furthermore, botryosphaeran corrected dyslipidemia by reducing the content of total lipids, triglycerides, and cholesterol in the obese animals. Botryosphaeran modulated the expression of AMPK and FOXO3a proteins in adipose tissue of the obese rats.
6
Anticancer Activity of Botryosphaeran
Cancer, a multifactorial disease, is one of the principal causes of deaths in the world and is increasing globally. Cancer treated by chemotherapy and radiotherapy bears the hallmark of debilitating side effects that can contribute to cancer mortality. In targeting cancers, it’s important to develop appropriate treatments with minimal side effects. In this respect, β-glucans of the β-(1!3)-linked kind are recognized as possessing anticancer activity, and bear no side effects (Vetvicka and Vetvickova 2012). Their anticancer effects are mediated directly on cancer cells via cytotoxicity,
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and indirectly through immuno-modulation and metabolism (Chan et al. 2009; Batbayar et al. 2012). Botryosphaeran mediated cell-signaling pathways in human breast carcinoma MCF-7 cells leading to suppression of tumorigenesis, decreased proliferation of cancer cells, and promoted apoptosis, necrosis, and oxidative stress. The antiproliferative effect of botryosphaeran was mediated by the actions of AMPK and FOXO3a (Queiroz et al. 2015). AMPK activates the regulation of cancer cell proliferation and is important in inhibiting cellular growth and promoting apoptosis in cancer cells (Shackelford and Shaw 2009). AMPK can activate FOXO3a and consequently promotes cell cycle arrest, apoptosis, and tumor suppression (Brunet et al. 1999; Dijkers et al. 2000). Activated FOXO3a induces the transcription of certain target genes involved in cell-cycle arrest, cell-death, and tumor suppression, and this effect was induced by botryosphaeran in MCF-7 cells (Queiroz et al. 2015), and in Walker-256 tumorbearing obese rats (Geraldelli et al. 2020b). In MCF-7 cells, botryosphaeran increased the intracellular expression of mRNA’s of p53 and p27 (involved in cellcycle arrest) and bax (apoptosis regulator), and cleaved caspase-3 proteins involved in apoptotic pathways, confirming this occurred via FOXO3a activity (Queiroz et al. 2015). Malini et al. (2015) reported cell-cycle arrest by botryosphaeran in human tumorigenic (Jurkat) T-lymphocytes. Botryosphaeran increased oxidative stress in breast cancer MCF-7 cells, and this effect was associated with increased generation of ROS with consequent apoptosis and necrosis. Oxidative stress is the imbalance between the production of reactive oxygen species (superoxide anion radical, hydroxyl radical, hydrogen peroxide, and organic hydroperoxides), and the endogenous protective actions of enzymes (superoxide dismutase, catalase, among others) and non-enzymatic antioxidants (natural products that includes plant polyphenols, vitamins C and E, and also β-glucans, such as botryosphaeran; Giese et al. 2015). In the presence of the pro-oxidant hydrogen peroxide, botryosphaeran increased ROS levels in the MCF-7 cells above that generated by hydrogen peroxide alone, indicating that oxidative stress induced by botryosphaeran was responsible for cancer cell death (Queiroz et al. 2015). Oxidative stress generated by ROS can promote cell apoptosis, and consequently reduce cell viability and stimulate AMPK activity (Queiroz et al. 2014). In Walker-256 tumor-bearing rats (non-obese), botryosphaeran (30 mg/kg b.w. over 15 days) significantly reduced tumor development and cancer cachexia syndrome through increasing apoptosis of tumor cells via bax protein expression (Geraldelli et al. 2020a). In related work with obese rats bearing the Walker-256 tumor, botryosphaeran decreased tumor growth and the intensity of the neoplastic cachexia syndrome, and increased FOXO3a activity (Geraldelli et al. 2020b). Botryosphaeran acted in a concentration-dependent manner decreasing tumor growth and cachexia syndrome in both obese and non-obese rats bearing Walker256 tumors (Comiran et al. 2021). The relative differences in tumor development between non-obese and obese male rats bearing Walker-256 tumors, and following treatment with botryosphaeran
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Fig. 2 The relative sizes of Walker-256 bearing tumors in non-obese (CT) and obese (OT) male rats, and after 15 days of treatment with botryosphaeran (CTB and OTB)
(30 mg/kg. b.w./day) for 15 days, is shown in Fig. 2, which indicates a notable reduction in the size and weight of the tumors before and after treatment with botryosphaeran.
7
Conclusion and Future Perspectives
As aforementioned, botryosphaeran manifests many biological activities that find applications as alternative and complementary medicines. The uniqueness of this fungal β-glucan activity we believe is influenced by the chemistry of the side-chain residues, especially the di-glucoside unit, gentiobiose, in synergy with glucose, as well as the degree of branching of substituents along the backbone chain. Our research continues to unravel new knowledge on botryosphaeran that leads to further applications. Three commercial cosmetic products have evolved from ~20 years of studies on botryosphaeran. They include a formulated cosmetic facial crème, body lotion, and a serum all containing β-glucan that promote skin health and treat skin conditions. Unpublished data has revealed that botryosphaeran promotes healing of skin wounds that leads to re-epithelialization of the wound area, and the synthesis of collagen fibers. Additionally, botryosphaeran possesses antinociceptive activity (removal of pain sensation), an important factor in treating wound injuries. In wider applications, botryosphaeran and its chemical derivative, carboxymethyl-botryosphaeran, have been used to construct novel electrochemical
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sensors and biosensors as platform materials assembled on simple electrodes to rapidly and quantitatively measure various drug components, e.g., dopamine and spironolactone (Coelho et al. 2019), hydroquinone (Mattos et al. 2019), desloratadine (Salamanca-Neto et al. 2020), dopamine and paracetamol (Eisele et al. 2019), and quercetin (Gomes et al. 2020). Acknowledgments The authors are grateful to Danielle Geraldelli and Dr. Eveline Queiroz (Núcleo de Pesquisa e Apoio Didático em Saúde, Instituto de Ciências da Saúde, Universidade Federal de Mato Grosso, Sinop-MT, Brazil) for the provision of photographs of rats bearing the Walker-256 tumors.
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Chitin and Its Derivatives Applications in Biomedical Devices
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Simone S. Silva, J. M. Gomes, L. C. Rodrigues, and Rui L. Reis
Contents 1 2 3 4 5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chitin Extraction and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chitin and Chitosan Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chitin and Chitosan-Based Matrices: Processability and Chemical Modification . . . . . . . . . Biomedical Applications of Chitin and its Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Skin Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Bone Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Cartilage Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Chitin and chitosan are well-known natural polymers that have been considered a sustainable feedstock for the production of high-value biomaterials. In particular, the wide availability of the main sources of chitin, e.g., squid pens, shrimp, crab shells, insects, and fungal cell walls, is associated to different methodologies for its extraction, namely, classical methods based on strong acids, alkali, and enzymes, or alternative ones like the use of ionic liquids and/or natural deep eutectic solvents. The latter method offers opportunities to extract
S. S. Silva (*) · J. M. Gomes · L. C. Rodrigues · R. L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_13
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and process chitin directly from the raw materials, preserving the chitin qualities. The global market of chitin is continuously increasing across different fields, namely, the textile industry, packaging, technology, and biomedical field. In the biomedical field, many chitin and chitosan-based matrices, namely, membranes, hydrogels, nano/microfibers, and scaffolds have been produced using various methodologies such as blending chitin and chitosan with other natural/synthetic polymers (such as alginate, gelatin, polycaprolactone), metals and/or ceramics (such as HAp, SiO2, TiO2, ZrO2). Besides, the functional groups of chitin and chitosan can be modified for the development of a broad range of derivatives with a wide range of applications. The achieved architectures have the suitable physicochemical and biological performance for applications in wound repair, bone regeneration, cartilage repair, and infectious diseases. In this chapter is performed an overview of the recent research on the extraction, production, and applications of chitin and chitosan-based matrices envisaging their biomedical potential. Keywords
Chitin · Chitosan · Marine residues · Biomaterials · Biomedical applications Abbreviations
(H-HTCC)-NS/MS [Amim]Br [Amim]Cl [Bmim]Cl [Bmim]OAc [Emim]OAc AIBN Bio-IL BMSC C-CBM CDI CMCS COP DA DD DES ECM EGCG ESCs GAG Hap ILs
N-(2-hydroxypropyl)-3-trimethyl chitosan 1-allyl-3-methylimidazolium bromide 1-allyl-3-methylimidazolium chloride 1-butyl-3-methylimidazolium chloride 1-butyl-3-methylimidazolium acetate 1-ethyl-3-methylimidazolium acetate 2,20 -azobisisobutyronitrile Biocompatible ionic liquids Bone marrow-derived stem cells Collagen—chitin biomimetic membrane 1,1-carbonyldiimidazole Carboxymethyl chitosan sulfate Collagen peptide Degree of acetylation Degree of deacetylation Deep eutectic solvent Extracellular matrix Epigallocatechin gallate Epidermal stem cells Glycosaminoglycans Hydroxyapatite Ionic liquids
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MSCs MTGase MW NACOSs NS/MS PCL PEI SF TEOS TGF-β
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Mesenchymal stem cells Microbial transglutaminase Molecular weight N-acetyl chitooligosaccharides Nano/microspheres Polycrapolactone Polyethylenimine Silk fibroin Tetraethylorthosilicate Transforming growth factor- β
Introduction
Chitin is a naturally occurring polymer, and it has even been reported to be one of the most abundant biomolecules on earth, with an estimated annual production of 1011–1014 tons. Chitin is present in a variety of biomass, such as squid pens, shrimp, crab shells, insects, and fungal cell walls (Bastiaens et al. 2019). The wide availability of the raw material used to obtain chitin and its derivatives associated with various facets of the global market of chitin that appears across different fields, namely, textile industry, packaging, biotechnology, and biomedical field, suggests that the market potential for chitin is rising. In fact, according to different key players, it is expected to boost the growth of the global market for chitin during the forecast period. The global Chitin market size is predicted to gain a CAGR of 3.8% in the forecast period of 2020–2025. It will be expected to reach USD 59 million by 2025, from the actuals USD 50 million (Chitin Market Size 2020). Despite the above trends, the lack of solubility of chitin in water and organic solvents is challenging, and different strategies involving ionic liquids (ILs) (Silva et al. 2017; Silva et al. 2013a; Silva et al. 2013b; Silva et al. 2011; Silva et al. 2020) and deep eutectic solvents (DES) (Vicente et al. 2020; Sharma et al. 2013) have been explored to help to unlock this limitation. Special emphasis is placed on methods conducted with the use of ILs, defined as salts in the liquid state at ambient temperature (Anthony et al. 2002), which allows not only the extraction in higher product yields as compared to traditional reactions but also its transformation in high-value medical devices (Silva et al. 2013a; Silva et al. 2013b; Silva et al. 2011; Shamshina 2019). Then, the solvating power of ILs on the extraction of chitin from shellfish waste represents a total byproduct use concept with practical implications in crustacean processing industries (Patrick et al. 2013). Another strategy to explore chitin is mainly focused on the use of chitosan (CHT), a partially deacetylated derivative of chitin that is soluble in aqueous acetic acid and, therefore, easier to process. Both chitin and chitosan have been fabricated as
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2D-/3D-based matrices, namely, membranes, scaffolds, micro/nanofibers, and hydrogels (Tao et al. 2020; Muzzarelli 2009; Kumar et al. 2010). These architectures have appropriate features such as large surface area, mechanical properties, and biocompatibility needed for tissue regeneration and sustained release of bioactive compounds (Muzzarelli 2009). The feasibility of the use of those matrices as wound dressings for skin repair, drug delivery systems, bone repair, cartilage regeneration, and chronic disease treatment have been investigated intensively (Silva et al. 2013b; Tao et al. 2020; Muzzarelli 2009; Kumar et al. 2010; Saravanan et al. 2016). However, it sometimes implies its physical or chemical modification and/or blending with other polymers (either natural or synthetic) to reach adequate features for its biomedical application. This chapter addresses an overview of the fundamental features of the extraction from different sources of chitin, its matrices, and potential tissue engineering approaches (Fig. 1).
2
Chitin Extraction and Structure
Chitin is one of the most abundant polysaccharides on earth, existing widely as one of the main components of the cell walls of fungi and algae, exoskeletons of arthropods such as crustaceans (e.g., shrimps and crabs ranging from 15–30%), and insects (up to 60% in special parts such as the flexible portions), radulas of mollusks, and the beaks of cephalopods, providing structural integrity and protection to those animals (Muzzarelli 2009; Muzzarelli 2011). Chitin may be extracted according to chemical and biological extraction methodologies, through deproteinization and demineralization steps (Fig. 2), possessing both strategies advantages and drawbacks (El Knidri et al. 2018; Arnold et al. 2020). The chemical approach is the most commonly used for industrial and commercial proposes due to the reduced extraction time, productivity, and practicality. However, it requires the use of strong acids and bases with the intent to, respectively, dissolve calcium carbonates and proteins, which is associated with many disadvantages, as high costs, reduced purity, and particularly in the environmental field through effluent generation (El Knidri et al. 2018). Decolorization and purification as additional steps are often required to remove pigments and obtain pure and colorless chitin, which is carried out using acetone, sodium hypochlorite, hydrogen peroxide, or potassium permanganate (Pighinelli 2019). To overcome the disadvantageous hazardousness and to achieve a higher quality final product, a more time-consuming biological approach is being employed using proteases from bacteria and lactic acid-producing bacteria to ensure the demineralization and deproteinization steps (El Knidri et al. 2018; Arnold et al. 2020; Tan et al. 2020). The proteolytic enzymes ensure protein hydrolysis through activation of the low pH of the medium, with the advantage of allowing the recovery of enzymes, proteins, and pigments for further application or reuse. Moreover, into fermentative and enzymatic processes, digestive and microbial enzymes able to consume organic material are produced, potentiating as well the
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Fig. 1 Overview of the sources, matrices, and tissue engineering applications of chitin
Fig. 2 Steps sequence of chitin extraction from marine wastes
production of hydrolyzed proteins. To increase the effectiveness of the extraction process, a methodology resulting from the combination of the chemical process with biological methods, with the application of microorganisms, is also being explored (Pighinelli 2019). This biotechnological approach presents a new sustainable vision with new quality parameters, offering a more suitable biomaterial, mainly to be applied to health solutions (Pighinelli 2019). Chitin is a long and regular chain structure made up of β (1 ! 4)-linked primary units of N-acetyl D-glucosamine (monomer units of 2-acetamido- 2-deoxy-b-d-glucose) (Silva et al. 2020; Younes and Rinaudo 2015; Pillai et al. 2009) (Fig. 2). Chitin, depending on the nature of the H-bond, can assume three crystalline polymorphic forms designated as α, β, and γ chitin; however, the existence of γ chitin is a controversy (Silva et al. 2020). α- and β-chitins present stable antiparallel and metastable parallel chain alignments being mostly present in different animal species, respectively, α-chitin in arthropods and crustaceans (major sources) and β-chitin in
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marine diatoms and squid pens (minor sources) (Younes and Rinaudo 2015; Pillai et al. 2009). The arrangement of the γ-chitin is more complex being composed of three chitin chains; two are parallel, and the third one is arranged in an antiparallel fashion (Silva et al. 2020). The existence of different arrangements leads to differences in the packing and polarities, which consequently promote the existence of distinct physicochemical properties. The strength of the intermolecular forces in β-chitin is much weaker than in α-chitin, β-chitin being observed to be more soluble and reactive (Silva et al. 2020; Hirayama et al. 2018), and α-chitin the most stable form of chitin. Chitin is found to occur as ordered crystalline microfibrous material embedded in a six-stranded protein helix (Qiu and Wang 2017), with reduced reactivity due to the strong intra- and inter-molecular interactions, particularly the H-bonds established between the C ¼ O and NH groups of the adjacent polymeric chains, which make its processability difficult and are the major obstacle to its dissolution, being insoluble in common solvents. However, it can be dissolved in concentrated acids, as hydrochloric, sulfuric, phosphoric, dichloro- and trichloroacetic or formic acids and in hexafluoroisopropanol or hexafluoroacetone, N, N-dimethylacetamide mixture of DMA and LiCl, lithium thiocyanate, CaBr2H2O saturated methanol, hexafluoroisopropanol alcohol, hexafluoracetone, and N-methyl-2-pyrrolidone and some hot and concentrated neutral salts (El Knidri et al. 2018). Although enabling chitin dissolution, many of those solvents are toxic, corrosive, harmful, or scarcely degradable. Moreover, the most environmentally friendly approach is the possibility to dissolve chitin using ILs (Silva et al. 2011, 2013a, b, 2017). Recent studies described that ILs not only dissolve raw chitin but also isolate it from crustaceans’ biomass, making possible the production of chitin fibers or nanofibrous mats via one-pot protocol (Shamshina 2019; Barber et al. 2013). Many of those works suggest that chitin dissolution requires an anion with an increased number of hydrogen bond donors and acceptors (more basic anion), as acetate, able to interact with the chitin H-bonds, destroying them, and leading to chitin crystal dissolution (Uto et al. 2018). Chitosan, the most important derivative of chitin, is obtained by partial deacetylation of chitin (Daraghmeh et al. 2011); however, during the process also depolymerization reaction occurs, promoting changes into chitosan molecular weight (Younes and Rinaudo 2015). In chitin, the ratio of 2-acetamido-2-deoxy-dglucopyranose to 2-amino-2-deoxy-d-glucopyranose structural units, commonly named as degree of acetylation (DA), is typically 0.90 indicating the presence of about 5–15% of amino groups due to deacylation that might occur during chitin extraction (Pillai et al. 2009). On its glucose ring, chitin has acetamido groups that can undergo incomplete hydrolysis into primary amine groups, N-deacetylation of chitin, which leads to the formation of chitosan that can be easily dissolved in aqueous acidic solutions, which makes it suitable for various applications. Similarly to chitin extraction, its deacetylation may also occur according to chemical or enzymatic processes, the chemical one being mostly used for commercial purposes due to its reduced cost and higher efficiency (Younes and Rinaudo 2015). Chemical deacetylation of chitin may likewise involve the use of acids or alkali’s solutions; however, due to the increased susceptibility of glycosidic bonds to acids, the resource to alkali deacetylation is most frequently employed (Hajji et al. 2014) producing an insoluble residue of chitosan deacetylated up to 85–99%. The
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deacetylation reaction may occur under homogeneous or heterogeneous conditions, which may influence the distribution of N-acetyl-D-glucosamine and D-glucosamine residues with some blockwise acetyl group distribution along polymeric chains, consequently promoting a variation of the solubility and degree of aggregation of chitosan leading to changes in their average characteristics (Younes and Rinaudo 2015). According to Younes et al. and its reported fractional factorial design and mathematical model, seven factors may influence deacetylation reaction extension; however, in different extension (Younes et al. 2014). The study revealed a significant influence of the temperature, of the nature of the alkali reagent, of the number of successive baths, of the reaction time, and of the alkali concentration. In contrast, a reduced influence was attributed to the atmospheric conditions (nitrogen or air) and the use of reducing agents (e.g., NaBH4) (Younes and Rinaudo 2015; Younes et al. 2014). Despite its efficacy, chemical deacetylation also has disadvantages: energy consumption, and waste of concentrated alkaline solution, contributing to an increase in environmental pollution (Younes and Rinaudo 2015). The resource to chitin deacetylases for the conversion of chitin to chitosan offers the opportunity of producing well-defined chitosan-based on a controlled, nondegradable products (Younes and Rinaudo 2015). However, the deacetylation based on enzymes is not very effective (5%) may also show inhibition effect on pullulan synthesis. Nitrogen Source The ammonium ion (NH4+) has substantial part in pullulan synthesis and diminution of nitrogen is considered as an indication for pullulan production. It is reported that ammonium ions may apply its impact as an influencer of enzyme activity and
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Fig. 3 Factors that influence the effective pullulan synthesis
controller of carbon flow. The surplus nitrogen source to the process does not enhance the exopolysaccharide yield but it can increase the biomass growth. With higher nitrogen concentration at the initial stages, the yield of pullulan is decreased as the enzyme activity is noticed at the later pullulan fermentation stages. As per the reports, 10:1 C/N ratio is the utmost favorable state for synthesis of exopolysaccharide. In many studies, the combined source of ammonium sulfate and yeast extract had been used for pullulan production. Apart from simpler nitrogen forms, multifaceted nitrogen sources were also used as substitute and this in fact augment synthesis of pullulan. Supplements Along with carbon and nitrogen sources in the fermentation broth, various supplements, viz., mineral salts, inducers, inhibitors, precursors, etc., should be monitored
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for improved pullulan synthesis in submerged fermentation. The variety of sources (C and N) and also various supplements play a significant role in microbial production of pullulan. Uracil, a predecessor for UDP-glucose, has substantial part on biopolymer and biomass synthesis, and uridine phosphorylase (UPase) activity. By the UPase activity and addition of uracil (5 mM), the rate of pullulan production increased to 49.07 g/L. Other supplements, viz., olive oil and tween 80 were also used for enhancing the biopolymer yield. The biopolymer yield was extremely enriched by adding tween 80 in the medium. The influence of various iodoacetic acid concentrations was assessed to evaluate the function of key enzymes that are involved in biopolymer production. Few studies portrayed the effect of soya bean oil as nitrogen source for production of biopolymer.
3.4.2 Fermentation Type Numerous studies evaluated the effect of fermentation type, i.e., batch, fed-batch, and continuous for the increased efficiency of pullulan production. The difficulty of suppression effect caused by higher substrate concentrations could be evaded by sporadic supply of limiting substrate to the medium. A study was conducted to evaluate the sucrose suppression effect on pullulan production under fed-batch mode and observed the production of 58 g/L exopolysaccharide. However, the fed-batch mode showed increased productivity till certain time and did not show substantial yield upsurge after adding sucrose. Moreover, the fed-batch mode showed little decrease in pullulan concentration after 7 days of cultivation. Several studies depicted the usage of continuous mode for the production of pullulan. Reports suggested the increased exopolysaccharide production relatively for a long time without any problems. But, the dilution rate was extremely low at the continuous mode of operation. Rate of dilution is a critical parameter that influences biopolymer synthesis in chemostat. Literature reports show that the utilization of chemostat system enhanced the pullulan productivity even at low dilution rates. The continuous processes of fermentation in combination with enhanced cell biomass are feasible for long-term production. 3.4.3 Bioreactor Configuration and Operation The various factors that highly influence the polymer synthesis in submerged fermentation include the broth composition and behavior at different agitation speeds, sturdy air provision, and low shear rate, etc., thereby providing optimal conditions for the growth of microbes. All the above-mentioned parameters can be controlled in the bioreactor. Hence, bioreactor design and operation play a significant role in enhanced efficiency of pullulan production. The advancement of novel and innovative fermentation reactors will aid in high productivity. Different bioreactors, viz., reciprocating plate bioreactor, were developed to suit the fermentation process and yield good productivity of pullulan. Reactor configuration, viz., biofilm and suspended culture also affect the performance of biological system and regulate the process. The carriers for biofilm configuration had been widely utilized to immobilize the strain. In spite of numerous advantages with biofilm configuration, the
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formation of substrate agglomeration and other factors like low void volume, aeration rate, etc., affected the metabolite production.
3.4.4 Fermentation Time The amount of pullulan produced and its yield varies with fermentation time. Reports suggest that the fermentation time for achieving maximum yield of pullulan changes with respect to different operating conditions and microbial cultures. Hence, the optimal time for production of high pullulan yields varies from 48 h to 5.36 days depending much on the microbial cultures and operational parameters (Sugumaran and Ponnusami 2014). 3.4.5 Microbial Culture Another important factor that affects the pullulan productivity is the type of microbial culture. As reported in the previous literature, A. pullulans is the highest pullulanproducing strain ever reported among wild strains. Apart from A. pullulans, there are other strains also that have the ability to synthesize pullulan. Aureobasidium melanogenum TN1-2 strain isolated from natural honey was used to synthesize pullulan. The low productivity obtained with A. pullulans when maltose was used as carbon source was overcome by using the mutant strain. The mutant strains aided in performing reactions in large scale with optimal conditions. Other mutant strains aided in producing high molecular weight pullulan, increasing the cell growth along with reduction in melanin pigmentation. Coculturing of pullulan-producing strain, A. pullulans SH 8646, and an insulin degradation strain, Kluyveromyces fragile ATCC 52466 provide the carbon source for A. pullulans by the hydrolyzation of inulin by the enzyme inulase synthesized by K. fragile. The efficiency of fermentation (FE) indicates that the presently used mutant strains of A. pullulans are nearly indistinguishable with regard to their polymer synthesizing activity (Mishra et al. 2018). 3.4.6 Oxygen Intensity The provision of air to the pullulan production is one of the critical parameters as the microbe does not grow or produce pullulan under anaerobic conditions. Concentrated air provision during fermentation leads to enhanced pullulan production as pullulan-producing cells accumulate in the medium. As viscosity of medium upsurge swiftly, the oxygen demand intensifies as polymer elaborates during submerged fermentation. Hence, optimum DO (dissolved oxygen) concentration and agitation speed are required for obtaining maximum yield of pullulan. The drawbacks associated with intense aeration during pullulan production include lower molecular weight polymer as the vital enzyme for defining the molecular weight, α-amylase, will be altered and also formation of excess foam due to high viscous nature of medium with pullulan. Another report suggested the enhancement in pullulan productivity at low constant dissolved oxygen as it has decreased shear rate. The increase in partial air pressure improves the oxygen rate of transfer to the microorganism thereby synthesizing higher exopolysaccharide.
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3.4.7 pH The pH of the fermentation medium not only influences the yield of pullulan but also affects the cultivation time, metabolite molecular weight, and morphology of the A. pullulans. The ideal pH will increase the action of the enzyme, UDPG pyrophosphorylase. As per the reports, the pullulan productivity increases with the pH from 2.5 to 5.5 but thereafter decreases. The pullulan production is suppressed by the lower pH conditions, but the amount of insoluble glucan production is elevated. The optimal pH of 5.5 to 7.5 varies with respect to strain and the mutated strains of A. pullulans showed good productivity at pH 4 and 5.9 at different conditions. At near-neutral pH conditions, the microbes produce high molecular weight (MW > 2,000,000) pullulan. The ideal pH values for biopolymer synthesis are varied due to the diverse operating conditions and microorganisms used. 3.4.8 Temperature Like pH, the temperature also varies with respect to the strain used for pullulan production and it is one of the most influential parameters for biopolymer synthesis. The ideal temperature varies from 25 to 37 C with respect to different strains. Studies reported the isolation of strain that tolerates high temperature (42 C) and that which does not produce melanin. The synthesis of pullulan is evaluated by using two-stage temperatures for higher cell growth and pullulan synthesis. The lower temperature of 26 C reinforced the unicellular biomass growth of A. pullulans.
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Characterization of Pullulan
4.1
Structural Characterization
The cationic groups in pullulan can be determined by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (pNMR). Centered on the absorption of infrared (IR) energy by unique functionality groups/linkages in the sample with a specific wave number, the structure of the compound could be predicted. Chemical bonding of biopolymers, stretching and functional groups, can be easily identified by FTIR spectroscopy. The peaks at 856 cm1 and 1156 cm1 are due to α- glycosidic linkage between individual glycoside residues and D-glucose in pyronose form. The absorption peak intensity was recorded at 931 cm1 and 764 cm1 confirming the presence of α-1,4 glycosidic linkage and α-1,6 glycosidic linkage, respectively. The hydroxyl proton present in the samples exhibit the signal between 4.5 and 5.6 ppm using proton NMR spectra. In the purified sample, the pyranose is identified by the absence of signals ranging from 82 to 88 ppm. The presence of anomeric carbon α-1,6 is observed on the peak at 100.1 ppm. One-dimensional proton NMR spectra of the pullulan exhibits signals integrated for the total of 23 protons. The signals arrived in the downfield region between 3 and 5 ppm infers proton carrying carbon atoms attached to an electronegative atom, which is a characteristic chemical environment of a carbohydrate moiety. Total integration for 23 protons infers that the repeating unit of the carbohydrate polymer contains three
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monosaccharide units. In order to characterize and identify the composition of the pullulan, the pullulanase hydrolysis study has been also performed (Mishra and Varjani 2019). Generally, pullulanase hydrolyses α-1,6 glycosidic linkage of branched chain of pullulan and releases the reducing sugars with one –OH free group in each case. The extent of hydrolysis percentage (%) was calculated as follows. Hydrolysis ð%Þ ¼
4.2
Amount of reducing sugar released after the hydrolysis 100 Amount of pullulan
Molecular Weight
The molecular weight of pullulan is greatly affected by the initial pH of the medium, the form of strain used, the quality and composition of substrate used, the composition of medium, and the harvesting time. Weight-average molecular weight and number-average molecular weight can be determined by size exclusion chromatography coupled to multi-angle laser light scattering (MALLS). The molecular weight depends upon the various parameters like (a) composition of substrate (b) initial pH (c) types of microbial strain (d) composition of the media, and (e) time of fermentation. The average molecular weight of pullulan is ranged from 362 to 480 kDa. The molecular weight is ranged from 100 to 200 kDa. The molecular weight of pullulan is higher that biosynthesized from agro wastes than that of synthetic medium. The polydispersity index (ratio of weight-average molecular weight and number average molecular weight) for pullulan ranges from 2.1 to 4.1. The variation in polydispersity index depends upon variation in metabolic pathway and cell morphology (Singh et al. 2009). The medium composition like nitrogen source, carbon source, phosphate, and NaCl affect the molecular weight during fermentative production of pullulan. The molecular weight of the pullulan was found to be varied depending upon the concentration of soybean pomace in a study. In a similar way, ammonium ions play a major role in the production of high molecular weight pullulan as compared to nitrate ions. In many cases, the molecular weight of pullulan has been decreased in the due course of fermentation time due to coproduction of pullulanase and α-amylase that degrades the maltotriose subunits present in the pullulan.
4.3
Thermal and Mechanical Properties
Thermal property is an important parameter for exopolysaccharides, which is analyzed by Thermogravimetric Analysis-Differential scanning calorimetry (TGA-DSC). It specifies the decomposition point for pullulan with respect to temperature. Singh and Saini (2008) had observed that the decomposition for pullulan was found to be in the range of 250–280 C. The decomposition property of pullulan also depends upon the substrate that is used for it biosynthesis and the type of strain used for this. It was found to be 245 C and 296.72 C, respectively, that was synthesized by A. pullulans by utilizing Asian Palm kernel and Cassava
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bagasse, respectively (Sugumaran and Ponnusami 2017). The molecular weight, compositions, and structure also affect the decomposition behavior of pullulan. In many cases, the substitution of any groups (-H or –OH) of pullulan with other groups also changes the thermal property due to intermolecular interaction. Incorporating nanofibrillated cellulosic substances to pullulan also enhances its decomposition point to 370 C. The mechanical property of pullulan is the essential factor to be considered for its applications in pharmaceuticals and food industries. The molecular weight also affects its mechanical properties and tensile strength. The tensile strength can be modulated by the substitution of any groups. For example, substitution with nanocellulosic substances enhances the tensile strength where with acetylated pullulan (pullulan acetate) was found to possess lower tensile strength. As per the X-ray diffraction study, pullulan is amorphous in nature. Here, the intensity level at the 2-theta value of 20 to 30 denoted the amorphous nature of the pullulan. It was also found that, there was no shifting of the band found in the X-ray power diffractometer graph.
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Surface Derivatization Approaches for Pullulan
5.1
Polyionic Derivatives
Polyionic derivatives are formed due to mixing of two oppositely charged particles. When charged molecules of opposite natures are mixed in the aqueous solution, nanoparticles can be created. The size, charge, and effectiveness of the nanoparticles depend upon amount of polyelectrolyte relatively. The polyelectrolytic effect of chemically modified pullulan based upon its charges has been illustrated in Fig. 4. There are various ionizable groups like carboxylate, sulfate, and trialkyl ammonium that regulate the viscosity of pullulan solution at different levels. The ionic strength of the ionizing groups and formed polyelectrolyte can control the viscosity of pullulan solution (Spatareanu et al. 2014). The derivatives of pullulan can also be complexed with oppositely charged polymeric groups to prepare a hybrid polyelectrolyte membrane (Jafari et al. 2019). Generally, anionic modifications (sulfation or carboxylation) can create negatively charged pullulan, while cationic modification (Diethyl amino ethyl or polyethylenimine) creates positively charged pullulan. Cationic derivatives of pullulan serve as the vector for delivering DNA and specific genes. Cationic derivative of pullulan act as the immunotherapeutic agents in cancer treatment due to their more interaction with the tumor cells. Scientists report that cationic pullulan-nanoparticles can act as anticancer molecules for multidrugresistant malignant cells even without application of drugs. Here, these nanoparticles irritate the cell membrane through electrostatic stress. When pullulan is modified with sulfide groups, drug delivery can be made easily by disulfide linkages formed through reduction. Attachment of cysteine to the derived pullulan-PEI has enhanced the uptake of plasmid DNA and the drug (doxorubicin) into the cancer cells.
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Fig. 4 Schematic representation to illustrate the derivatization of pullulan through cationic and anionic substitution
5.2
Cross-Linking
Pullulan is highly hydrophilic and due to this, it creates an obstacle for certain application. Cross-linking method can be useful to address this problem. In most of the cases, with regard to pullulan film the cross-linking could enhance the swelling, permeability, water absorption, and tensile strength. Pullulan-based hydrogels can be formed by the application of cross-linking processes. Researchers had developed a cross-linkedpullulan/Gelatin gel by alkyne-azide cycloaddition that exhibited unique mechanical properties (Highly stable; Not melted with heating up to 50 C) (Han and Lv 2019). This can be possible by stabilized configuration of -OCO-, -OCONH-, and -OCOO- linkages in the cross-linked pullulan as illustrated by Fig. 5. There are various reports dealing with the cross-linking of pullulan using commercially available crosslinkers. The properties of the final cross-linked product depend upon the concentration of the pullulan and the cross-linker. Various studies have been performed in order to optimize the concentration of cross-linkers. Sodium trimeta phosphate used as crosslinker with pullulan has been studied extensively with different concentrations. Enhancing the concentration of photo initiator in the reaction, cross-linking density also increased in pullulan. However, retardation of the reaction was observed in higher concentration of photo initiator (Tiwari and Bahadur 2019).
5.3
Hydrophobic Modification
Pullulan can be modified hydrophobically in the form of highly ordered selfassembled structure in aqueous system to impart plasticity for developing functional
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Fig. 5 Schematic representation for creating cross-linked pullulan with gelatin and gellan
biomaterial for various applications. Based upon the chemical composition and block length, different forms of nanoaggregates like vesicles, micelles, and wormlike micelles are formed .These kinds of formulations have been used for target drug delivery system for both small molecules and large molecules. Special types of recognition factors have been tagged with this formulation in order to target them to specific set of cells. As explained by Wang et al. (Wang et al. 2014), due to steric hindrance, aggregation number of these derivatives of pullulan is relatively low. Pullulan was modified with reacting 1-ethyl-3-(3 dimethyl amino propyl) carbodiimide hydrochloride and 4- dimethyl amino pyridine with the application of α-tocopheryl succinate. Applications of cholesterol to convert hydrophilic pullulan to hydrophobic have been described in so many literatures. The schematic representation of the formation of hydrophobic pullulan with cholesterol has been illustrated in Fig. 6. The hydrophobic domains of cholesterol groups form nanogels of size 20–30 nm with hydrophilic outer shell formed by pullulan (Fig. 6). As per the published report, these nanogels were used as carrier for enzymes, vaccines, drugs, molecular chaperon, and therapeutic proteins (Fujioka-Kobayashi et al. 2012). The surfactants play a major role in association and dissociation of these gels. It was reported that sodium dodecyl sulfate (SDS) helps in the self-aggregation of modified pullulan. A transition from macroscopic gel-to-sol occurred as excess SDS is added. Addition of more SDS triggers the solubilization of hydrophobic groups resulting in reduction of viscosity of the solution.
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Fig. 6 Schematic representation for developing hydrophobic pullulan through application of cholesterol
5.4
Grafting
The performance and range of applications of polysaccharides, specifically involving melt processing methods can be improved by grafting process. This can be carried out by “grafting from” and “grafting onto” strategies, which produce a macromolecular architecture with multiple side chains (molecular brushes) and a linear backbone. The former involves attachment of pre-synthesized polymers containing complementary functional groups. Despite its simplicity, it cannot produce dense polymer brushes, as the attachment of the incoming chains is hindered by the steric congestion of the chains previously attached to the side. A substantial decrease in the response rate was identified as a result of the increase in side chain conversion. On the other hand, “grafting from” requires the covalent attachment of a monomer over the polymer backbone and its subsequent polymerization as a side chain (Lepoittevin et al. 2019). The monomer acts as an active center and initiates chain growth via free radical or redox-initiated polymerization (Fig. 7). The efficiency of the grafting can be determined by initiator density, which is comparatively higher in “grafting from” procedure due to its small size of initiator molecule. Molecular weight and weight distribution of the product can be
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Fig. 7 Schematic representation for making grafted pullulan-PLGA with application of poly (D, L-lactide-co-glycolide) (Note: DMSO Dimethyle sulfoxide, DCC N,N0 -Dicyclohexylcarbodiimide, DMAP 4-Dimethylaminopyridine)
controlled by varying the reaction conditions. Knowing the total number and size of graft polymers through characterization could be difficult but they act as good multiphase polymer systems. Graft polymers are widely used in drug delivery, tissue engineering, and biosensing. The solubility in solvents such as methanol and dimethyl sulfoxide can be expanded by grafting of polyethylene glycol (PEG) on pullulan. On other hand, grafting of poly (methyl acrylate) residues decreases its hydrophilicity. However, there is limited amount of acrylate grafting application in biomedical due to its nonbiodegradability under physiological conditions. Since the grafting of poly (N-isopropylacrylamide) (pNIPAM) undergoes sharp phase transition at physiological conditions the intensive tests are done to introduce temperature sensitivity in polysaccharides. Poly-N-isopropylacrylamide (pNIPAM)-grafted pullulan displayed a reversible phase transition at about 32 C thereby producing nanoaggregates in aqueous system. Nanoparticles showed a high loading efficiency for indomethacin due to strengthened hydrogen bonding and hydrophobic interactions between the drug and pNIPAM grafts. The dissociation of hydrogen bonds at high temperatures enabled the controlled drug release (Constantin et al. 2017). Fundueanu and coworkers reported the attachment of thermo- and pH-sensitive units onto pulllulan microspheres. pNIPAM-co-acrylamide grafts were inserted for thermo-responsive units. Remaining –OH groups were linked with pH-sensitive
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groups (succinic anhydride). The sharp volume phase transition was maintained by the microspheres at low values of exchange capacities at both below and above pKa of carboxylic acid. Akiyoshi’s group integrated the self-assemblage of collagen hybridizing peptide (CHP) with thermo-responsive behavior of pNIPAM to develop botryoidal clusters of CHP/pNIPAM nanogels. They have observed that the solution concentration and relative amount of components determines the nanogel morphology. It revealed two distinct volume transitions over 20–55 C temperature window. A decrease in hydrodynamic radii from 93 to 57 nm as the temperature increased from 20 to 35 C, followed by a sharp increase from 57 nm to 90 nm at 55 C. Authors attributed the phase change of CHP-pNIPAM nanogels to its botryoidal assembly, which is different from the assembly of block copolymers that possess distinct temperature-sensitive blocks. Individual units in this structure were held together by cholesteryl cross-linking points (Morimoto et al. 2007).
6
Recent Applications and Uses in Biomedical and Pharmaceutical Fields
6.1
Pharmaceutical Formulations
Pullulan, often available in powdered form, can also be manufactured in thin film format. These films have many applications in the health-care industries, pharma® ceutical, and food. Listerine mouth freshener is the most common and commercially effective product in pullulan films. Pullulan can be used in pharmaceuticals, such as in pill and capsule coatings, including formulations for sustained release (Dubey 2018). Ulmus davidiana var. Japonica (UD) has historically been used to treat various types of illnesses, including skin wounds and inflammation, with Korean medicine. Compared to pullulan-only gel film, UD gel films showed good thermal stability and better mechanical properties with outstanding swelling performance and favorable skin adhesiveness. The latest method mixes the essential ingredients in current vaccines with a sugar gel where they remain viable for 8 weeks or more, even at high temperatures. The method produces light, effective, and compact doses that, according to the researchers, would be perfect for shipping Ebola vaccine to affected African regions, for example. Consequently, framework only adds marginal costs to the preparation of a vaccine and eliminates almost all transportation costs, which can account for 80% of the revenue of inoculation. Mixing the vaccines and sugars (pullulan and trehalose) is almost as simple as pouring cream and sugar into coffee, the researchers conclude. At McMaster, the storage device was developed by chemical engineers who had already proven its utility in other applications, such as an edible coating that can improve the shelf life of fruit and vegetables. To adapt the technology to the vaccines, the engineers partnered with colleagues from health sciences across campus who specialize in virology and immunology. The new vaccine storage method suspends the active components of the vaccine in a thin, single-dose jar filled with a sugar-gel mixture that dries into the vaccine for sealing. Clinicians then
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reconstitute and administer the vaccine with water as they usually would to patients. The researchers have proved the viable approach of using two experimental vaccines such as herpes simplex virus and influenza virus, inoculate and research the exposure of viruses to mice since the immune response of mice is similar to that of humans. The US Food and Drug Administration has already licensed the products in the storage medium, thereby simplifying the marketing route (Leung et al. 2019). Based on a mixture of trehalose/pullulan, protein-loaded orodispersible films (ODFs) were prepared by air-and freeze-drying. Depending on the outstanding trehalose-stabilizing protein capacity and pullulan film-forming capability, these two carbohydrates were chosen. Three model proteins were mounted onto ODFs. Ovalbumin was used to analyze the impact of protein incorporation on mechanical properties, time of disintegration, uniformity of weight, and thickness of ODF. The stability of the proteins has been tested by lysozyme and β-galactosidase. The loading of ovalbumin did not have a noticeable effect on the mechanical properties of freeze-dried ODFs, while the introduction of ovalbumin into air-dried ODFs resulted in a large decrease in tensile strength. Lysozyme stability was not affected by the trehalose/pullulan ratio, whereas β-galactosidase stability improved with increased trehalose/pullulan ratios. Likewise, for process stability over airdrying, freeze-drying proved to be advantageous, while for storage stability, the opposite was observed. In addition, ODFs based on trehalose/pullulan are promising for potential protein delivery via the oral cavity from a technical point of view (Tian et al. 2018). The inclusion of therapeutic proteins in a sugar glass matrix is known to increase protein stability, but when exposed to elevated relative humidity (RH), safety is sometimes lost. It was hypothesized that the use of disaccharide and polysaccharide binary glasses may be beneficial for the stability of proteins, particularly under these conditions. Different amounts of polysaccharide pullulan were then used in glasses of freezed-dried trehalose. When subjected to elevated RH, the presence of pullulan above 50% weight in these homogeneous blends prevented trehalose crystallization. The β-galactosidase protein model incorporated into pullulan/trehalose blends tested for storage stability for up to 4 weeks showed superior pure trehalose activity at 30 C/0 percent RH, while pullulan/trehalose blends showed the highest stability at 30 C/56 percent RH. Therefore, binary pullulan and trehalose glasses can provide excellent protein stability under storage conditions, such as high RH and high temperatures, which can occur in activity (Teekamp et al. 2017). Hussain et al. (2017), have synthesized using the 1,10-carbonyldiimidazole (CDI) reagent activating green carboxylic acid. In order to prepare aspirin-imidazolide at room temperature for 24 h, the aspirin was first reacted with CDI, which reacted in situ with predissolved pullulan, and the reaction was then followed by nitrogen at 80 C for 24 h. Using 1H NMR spectroscopy, the degree of aspirin substitution on pullulan (DS 0.32–0.40) was determined. The elevated charge and purity of the covalent content have been verified by spectroscopic techniques. Thermal research has shown that the new macromolecular prodrugs (MPDs) of aspirin are thermally more stable than pure aspirin. As analyzed by transmission electron microscopy (TEM), the amphiphilic pullulan-aspirin conjugates self-assembled in nanoparticles
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at a solvent interface within the range of 500–680 nm without further structural changes. This novel pullulan-aspirin can theoretically be safe stomach prodrugs combined with masked functional group COOH.
6.2
Tissue Engineering
In medical applications, biomimetic hydrogels are structurally and mechanically identical to the extracellular matrix (ECM) and have several possibilities. Nevertheless, new methods of controlling hydrogel feature resolution, biofunctionalization, and mechanical properties are required to create synthetic microenvironments that mimic the cell growth and differentiation effects of natural tissue niches. As a celladhesive, hydrogel possesses three-dimensional (3D) photo-printing in size ranging from macro- to micro-scale dimensions, and tunable mechanical properties. This could produce multiscale printed scaffolds that are found to be inert and biologically compatible with cell adhesive proteins for cell adhesion. Using natural polysaccharide pullulan, cross-linked with sodium trimetaphosphate (STMP) and combined with collagen, a three-dimensional (3D) scaffold was manufactured to build a polymeric network. Collagen has been derived from the skin of unexplored puffer fish (Lagocephalus inermis). The cross-link took place at alkaline pH at room temperature to give translucent, transparent, and soft hydrogels. Swelling tests showed remarkable properties of water absorption with a swelling ratio of up to 320%, an ideal feature for the hydrogel for a moist wound healing environment. SEM research revealed that there was a closely interconnected porous structure of the collagen blended pullulan hydrogels. On NIH3T3 fibroblast cell lines, the MTT assay showed that the prepared hydrogels were 100% biocompatible with increased cell adhesion and proliferation. The hydrogels promoting angiogenesis in the chick chorioallantoic membrane were studied in the Chick Chorioallantoic Membrane (CAM) assay. The highly porous hydrogels of collagen-pullulan, a promising biomaterial for wound healing applications, have been successfully developed with substantial biological output in vitro and in vivo (Iswariya et al. 2016). In tissue engineering and regenerative medicine, a multifunctional biomaterial with the ability to bind to hard tissues, such as bones and teeth, is required for medical and dental applications. Phosphorylated-pullulan (PPL) is able to bind hydroxyapatite to the bones and teeth. PPL has been used for bone engineering as a novel biocompatible material. First, an in vitro assessment of the mechanical properties of PPL showed that composites of both PPL and PPL-Linked-β-tricalcium phosphate (β-TCP) have higher shear bond strength than existing clinical use products, including polymethylmethacrylate cement (PMMA) and α-tricalcium phosphate cement (α-TCP), Biopex-R. In addition, the composite compressive strength of PPL/β-TCP was substantially higher than Biopex-R. Next, PPL/β-TCP in vivo composite osteoconductivity was investigated in a murine intramedullary injection model. Bone formation, which was even more visible at 8 weeks, was observed 5 weeks after injection of PPL/β-TCP composite; and now no bone
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formation was found after injection of PPL alone. Later, PPL/β-TCP was composed of an ulnar rabbit bone defect model, and bone formation similar to that induced by Biopex-R was observed. At 4 weeks, PPL/β-TCP composite implantation induced new bone growth, which was shockingly obvious at 8 weeks. By comparison, after 8 weeks, Biopex-R remained isolated from the surrounding bone. In a model of pig vertebral bone defects, PPL/β-TCP composite-treated defects were almost entirely replaced by new bones, while PPL alone failed to induce bone formation. Taken together, these results demonstrate that PPL/β-TCP composites can be useful in bone engineering (Takahata et al. 2015). Pullulan is a biologically compatible and efficient cytoadhesive medium for mesenchymal stem cell (MSC) tissue engraftment. Prolonged pullulan exposure has no detrimental effect on the morphology, viability, and differentiation capability of the cells. In the fibrillated surface of articular osteoarthritic cartilage, pullulan greatly increases the survival of MSCs. In the expression of a lectin transmembrane complex of type Dectin-2 C, pullulan induces upregulation. A pamidronate-pullulan conjugate has been shown to be involved in hydroxyapatite binding and accumulating in regenerating bone tissue (Liu et al. 2012).
6.3
Targeted Drug Delivery
Solid dispersion (SD) technique is the innovative method used to improve the solubility of BCS (Biopharmaceutical Classification System) Class II drugs. Throughout their solid state the drug was molecularly distributed in the SDs in a hydrophilic polymer. The major challenge in the development of the dosage form was the drug’s poor aqueous solubility. In this study the technique of solid dispersion was used to improve solubility. Pioglitazone is an oral antidiabetic medication, which is used in the treatment of type II diabetes mellitus and is used as a model drug for solubility enhancement testing. The SDs were prepared using the kneading method, solvent evaporation method, and combining method of solvent evaporation with kneading process. The solid dispersions are formulated using various drug ratios: polymer (1:1, 1:1.5 and 1:2). In fact, the results obtained show that the solubility decreases as the polymer concentration decreases. It was concluded from this that natural hydrophilic polymer is important for the improvement of the solubility of Biopharmaceutical Classification System (BCS) class II drug (Kulkarni et al. 2019).
6.4
Gene Delivery
Doxorubicin (DOX) is an important antitumor agent, which is commonly used. However, its therapeutic use is limited due to its side effects, including anti-apoptotic defense of cancer cells caused by DOX-induced autophagy and deleterious effects on normal tissues. In order to enhance the anticancer effect of DOX by blocking the autophagy mechanism mediated by the Beclin1 protein, a new folate (FA)-decorated
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amphiphilic bifunctional pullulan-based copolymer (named FPDP) was developed as an effective nano-carrier for the co-delivery of Beclin1’s DOX and short hairpin RNA, a pivotal autophageal gene. In the FPDP molecules, pullulan was modified for the formation of micelles with lipophilic desoxycholic acid, the introduced branched polyethylenimine (PEI) of low molecular weight (1 kDa) was used to deliver shBeclin1, and folate (FA) was used as the target group for the tumor. FPDP micelles have an average diameter of 161.9 nm, sufficient storage stability, good biocompatibility, excellent DOX and shBeclin1 loading capacities, and a sustained drug release profile. Moreover, in vivo studies demonstrated superior antitumor efficacy compared to non-FR-targeted PDP and free DOX micelles of tumor-targeted FPDP/ DOX/shBeclin1. These findings showed that Beclin1 DOX and shRNA co-delivery with FPDP micelles has the potential to overcome DOX shortcomings in clinical cancer therapy (Chen et al. 2018). Using oxidized pullulan (oxPL) and a disulfide-containing poly(β-amino) ester (ssPBAE) for the effective delivery of genes and chemotherapeutic agents via polymer degradation that responds to intracellular pH and redox tumor status, a dual-responsive nanoparticles system was created. By means of the pullulan oxidation process, OxPL containing excess aldehyde groups was obtained. With diethylenetriamine, ssPBAE was modified and then grafted onto oxPL by Schiff’s base reaction. Via the acid-cleavable hydrazone bond, DOX was bonded to ssPBAEoxPL, resulting in ssPBAEoxPL-DOX with a DOX content of about 3.6%. SsPBAEoxPL-DOX has shown in vitro hepatoma targeting properties and good condensing capacity of fluorescein-labeled oligoDNA (FAM-DNA) and plasmid DNA (pDNA) genes to some degree. There were non-sphere shapes and fairly uniform sizes of ssPBAE-oxPL-DOX/pDNA nanoparticles. The in vitro releases of DOX and pDNA from ssPBAEoxPL-DOX/pDNA nanoparticles showed strong pH-and redoxresponsive properties. The nanoparticles of ssPBAE-oxPL-DOX/pDNA effectively inhibited cell proliferation in HepG2 hepatoma cells, and induced S-phase cell apoptosis and cell cycle arrest. Furthermore, ssPBAE-oxPL-DOX/FAM-DNA nanoparticles effectively delivered DOX and FAM-DNA to the nucleus and cytoplasm in HepG2 cells, respectively, and also showed distinct hepatoma targeting properties after injection in HepG2 tumor-bearing mice. In short, this innovative dualresponsive nanoparticle device has shown tremendous potential for combination gene therapy and chemotherapy for hepatoma (Wang et al. 2016). A modern type of anticancer therapy has emerged as a combination treatment of drugs and therapeutic genes. A new amphiphilic bifunctional pullulan derivative (known as PSP) consisting of stearic acid and low molecular weight (1 kDa) branched polyethylenimine was prepared and characterized in this study as a nanocarrier for potential cancer therapies for drug and gene co-delivery. The drug loading content and encapsulation efficacy of PSP nanomicelles for DOX, an antitumor drug, was approximately 5.10% and 56.07%, respectively, and the continued release of DOX in PSP nanomicelles. The in vitro IC50 of PSP/DOX nanomicelles was significantly lower than the free DOX against MCF-7 cells. In addition, PSP nanomicelles effectively condensed DNA in gene transfection experiments to form compact structures and induced comparable rates of GFP gene expression to
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Lipo2000 at N/P ¼ 10. Compared with single DOX or p53 delivery, co-delivery of DOX and therapy gene p53 using PSP micelles showed higher cytotoxicity and induced higher apoptosis rates of tumor cells in vitro. Furthermore, PSP showed low cytotoxicity and good blood compatibility in the MTT and hemolysis assays. All in all, PSP nanomicelles have tremendous potential to provide hydrophobic anticancer drugs and therapeutic genes for enhanced cancer treatment at the same time (Chen et al. 2015). A combination of excellent protection and adequate performance is what is desperately required in the development of a gene carrier network. For successful genetic photodynamic therapy, a straightforward and flexible synthesis of a cationic guanidine-decorated dendronized pullulan (OGG3P) was studied here. With a weight ratio of 2, OGG3P has been able to block DNA mobility. Nonetheless, G3P missing guanidine residues could not block DNA migration until disclosure of guanidation could facilitate DNA condensation at a weight ratio of 15. A zeta potential plateau (~ + 23 mV) of OGG3P complexes through unique guanidinium– phosphate interactions suggested that pullulan nonionic hydrophilic hydroxyl groups neutralize excessive detrimental cationic loads. Compared with that of native G3P in Hela and HEK293 T cells, there was no clear cytotoxicity and hemolysis, and also transfection efficiency improvement with respect to OGG3P. More precisely, the uptake efficiency between OGG3P and G3P complexes in Hela cells was found not to be significantly different. However, guanidation induced changes in the uptake pathway and contributed to the macropinocytosis pathway, which can be an important factor in improving transfection efficiency. OGG3P complexes achieved substantial improvements in the expression of KillerRed protein and in the development of ROS under irradiation following the introduction of a therapeutic pKillerRedmem plasmid. Suppression of proliferation of the cancer cells caused by ROS was also reported. This study shows the possible development of dendronized pullulan decorated with guanidine as a robust nonviral gene carrier specifically designed to carry therapeutic genes (Zhou et al. 2018). Magnetic nanoparticles have been used as efficient vehicles for the targeted delivery of therapeutic agents that can be controlled by the concentration and distribution of externally guided magnets to a particular part of the body. The functionalization, characterization, and synthesis of pullulan-spermine(PS) magnetic nanoparticles for medical applications is the subject of this study. Thermal decomposition of goethite (FeOOH) produced magnetite nanopowder in oleic acid and 1-octadecene; pullulan-spermine was deposited on the magnetite nanoparticles in the form of pullulan-spermine clusters. EGFP-p53 plasmid was loaded to transfer functional iron oleate into cells. The nanocomplexes have been tested for the efficacy of encapsulation and drug processing. FTIR studies demonstrated the existence of oleic acid and 1-octadecene in their nanopowder on oleate and authenticated the spermine–pullulan interaction. In the pullulan spermine-coated ironoleate (PSCFO) spectrum, the characteristic PS bands indicated that PS covered the surface of their onoleate particles. Magnetic tests found that the magnetic saturation of the PSCFO was lower compared to those on oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity studies performed using U87 cells as
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glioblastoma cells found a survival rate of 92% at 50 mg/ml of plasmid-carrying PSCFO with an IC50 value of 189 mg/ml (Eslaminejad et al. 2016).
7
Future Perspectives
Despite a large number of valuable applications, the major constraint prevailing on the use of pullulan is its cost, which is three times higher than the price of other polysaccharides such as Dextran and Xanthan. Previous studies have addressed the melanin by-product in pullulan synthesis but its cost (25–30 USD/Kg) is even a greater problem. Engineering innovations or improved production strains, particularly with reduced melanin production, could be beneficial to improve the economics of the production, thereby opening new avenues for pullulan utilization. Pullulan does not have antimicrobial activity. The clear understanding of mechanism of pullulan biosynthesis is required to enhance the product quality and also to its study in Metabolic Engineering and Molecular Editing. The biochemistry of pullulan can answer the major down streaming and production problems. A comprehensive review of pullulan production in relation to molecular properties, upstream genetic regulations, and downstream processes, including new bioreactor design, cultivation parameters, and applications, is still to be explored. Through further chemical modifications, pullulan can be a potential source in various industries as a novel bioactive derivative. In the last two decades, the main demand for pullulan has been in food applications. With the innovative technologies of modification and cultivation skills, the development of modified pullulan derivatives with distinctive material properties and pullulan with a particular molecular weight distribution can be achieved. As a result, an increase in the number of studies on pullulan applications in medical, environmental, and pharmaceuticals has been reported. The demand for pullulan is increasing in cancer therapy with its related studies. The high bioactivity with some cytotoxic molecules is exhibited by the modified pullulan and it has known to form the complexes of those molecules. The gradual release of cytotoxic molecules is facilitated by accumulation of these conjugates at target sites. In many medical cosmetic industries other synthetic materials which produce CO2 are replaced with pullulan as it does not have any adverse effects. It is useful to assess them for a more intensive use of this polymer applications related to personal care and cosmetics, not only as a revolutionary active ingredient, but also as a safe compound for biodegradable materials and packaging. There appears to be a robust demand for antiaging cosmetics. The personal care and cosmetic products should be packed in biodegradable containers in such a way they do not affect the environment. This opens a broad scope of using pullulan exopolysaccharide in dermatological and cosmetic sector to produce smart products. The other emerging market is in biomedical engineering as pullulan has strong absorption ability. Safer innovative methods are being discovered for biosynthesis of pullulan. Numerous methods have been developed for the use of pullulan in drug delivery like in subcellular targeting, stimulus-responsive drug delivery systems, and
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nanoplatforms. Pullulan derivative nanoparticles or gels have wide applications in drug delivery material and gene transfer in pharma and food industries. Various notable developments are happening with pullulan in stem cell therapy, imaging, cancer cell targeting, etc. Considering these aspects, there is potential future for pullulan in health care sector for the betterment of human race. The surface modification of pullulan can be done to widen its application. Pullulan’s metallic stability is greatly decreased due to its hydrophilic nature. Pullulan hydrogels have non-fouling properties and are less studied in bone tissue culture applications. The future research can be oriented to provide surface support for cell adhesion and proliferation through osteogenesis in bone tissue culture applications.
8
Conclusion
The main advantage of polysaccharides produced from the microorganisms over the plant or algae is that, microorganism exhibits higher growth rate than plants and algae; moreover, they are more amenable to the manipulation and adjustment of the conditions for the enhancement of the growth and production. The progress of the researches in the area of fungal polysaccharides are also very less. The optimal conditions of process parameters for pullulan production are still debating.
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Scleroglucan and Schizophyllan Microbial Polysaccharides of Functional Importance
14
Hafiz Ubaid ur Rahman, Waqas Asghar, and Nauman Khalid
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Structural Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Physicochemical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Properties of Scleroglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Properties of Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Production and Isolation of Scleroglucan and Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Production and Isolation of Scleroglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Production and Isolation of Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Applications for Scleroglucan and Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Applications in the Food Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Biomedical/Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusion/Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Microbial exopolysaccharides, particularly scleroglucan and schizophyllan, have shown outstanding potential for use in food and medical/pharmaceutical industries. Scleroglucan and schizophyllan are obtained from fungal sources and their exclusive physico-chemical attributes offer a wide range of applications as stabilizers, gelling agents, thickeners, carriers of nutraceuticals, drugs and other bioactive compounds, prebiotics, immunostimulants, immunomodulators, antimicrobial and antiviral agents, hypoglycemic and hypocholesterolemic substances, and excipients. Additionally, several potential applications of these exopolysaccharides are yet to be explored for the development of functional foods and commercial pharmaceuticals. This chapter provides a comprehensive overview about the structural description, sources, physicochemical properties, H. U. u. Rahman · W. Asghar · N. Khalid (*) School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_16
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production, isolation, and recovery of scleroglucan and schizophyllan. Moreover, potential applications of these exopolysaccharides for the development of functional foods, edible coatings and delivery systems for drugs, nutraceuticals, and bioactive substances have been discussed in detail. Furthermore, various healthpromoting aspects of scleroglucan and schizophyllan in controlling lifestylerelated disorders (hyperglycemia and hypercholesterolemia) have also been conversed in this treatise. Keywords
Microbial exopolysaccharides · Scleroglucan · Schizophyllan · Biomedical applications · Functional foods
1
Introduction
Polysaccharides have been extensively distributed in the nature and are being obtained by all organisms of animal, plant, and microbial origin. These functional molecules have been associated with various biological functionalities such as cell wall formation (cellulose), energy storage (starch), and cellular communications (glycosaminoglycans) (Ates 2015). Polysaccharides are complex macromolecules having very high molecular weights reaching up to millions of Daltons. Generally, polysaccharides are classified as homo- and hetero-polymers, contain either anionic (uronic acid) or neutral sugars (pentoses and hexoses), and sometimes are linked with nonsugar components as conjugated molecules (Jindal and Khattar 2018). Mostly, polysaccharides of industrial importance are obtained from plants or seaweeds; however, microbial polysaccharides have also been emerged as quite useful biopolymers due to their unique physico-chemical attributes. In addition, polysaccharides obtained from microbial origin have also been used as alternatives to natural gums such as carrageenan and Gum Arabic (Milani and Maleki 2012; Shit and Shah 2014). Some key advantages of using microbial polysaccharides over plant-based or synthetic polysaccharides are discussed below: • Production of microbial polysaccharides is not influenced by regional or climatic factors. • Recovery of microbial polysaccharides is a quite easy and quick process with special reference to optimization of the process and rate of production. • Production of polysaccharides with required properties is easy through genetic modifications of microorganisms. • The materials used as growth media for microorganisms are simple and safe. • Production of microbial polysaccharides is quite cheap and environment friendly as food industrial wastes can also be used as microbial growth medium (Ladrat et al. 2014). Microbial polysaccharides are generally classified as intracellular and extracellular polysaccharides; however, extracellular polysaccharides have been used more
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extensively in the food processing industries as compared to the intracellular microbial polysaccharides (Patel et al. 2010). The extracellular polysaccharides of microbial origin are either attached on the cellular surfaces as sheath/capsules or can be secreted by microbial cells as slime (Madhuri and Prabhakar 2014). The polysaccharides have different structural characteristics depending upon their mode of cellular production. For example, the sheath exists in the form of an electrondense, thin layer surrounding the microbial cells, or colonies, whereas the capsules are present in the form of a slimy and relatively thick layer on the cell surface having sharp outlines containing exudate particles. Additionally, the slime is released into the medium in the form of water-soluble materials (Li et al. 2001). The water-soluble microbial polysaccharides (slime) can be easily extracted/isolated from the medium as compared to the sheaths or capsules, therefore, have more attraction for the processors as compared to those microbial polysaccharides which remain attached on the cellular surfaces. Microorganisms produce a wide range of polysaccharides. The list of polysaccharides obtained from various microorganisms along with their industrial applications is provided in Table 1. Accordingly, the present chapter is mainly focused on the physico-chemical properties, structural attributes, production, identification, isolation, characterization, and industrial applications of scleroglucan and schizophyllan with special reference to food and medicinal importance.
2
Structural Description
The main features associated with the primary structure of polysaccharides include presence of various sugars, phosphate or sulfate containing substitute groups, specific sequence of sugars, size of furanose or pyranose rings, α or β configurations, types of glycosidic linkages, and absolute configuration (d or l). Polysaccharides have different secondary structures, exhibiting a specific set of helix parameters. For instance, type-A helix has a ribbon-like structure which is a distinctive property of structural polysaccharides. These polysaccharides have β-(1,4) linkage and strong hydrogen bonding. Storage polysaccharides have type-B helical structure with less compactness and higher number of residues (8) per turn. The polysaccharides containing type-C helix have coil-like flexible structure which is formed by joining several monomers with β-(1,2) linkages (Venugopal 2011). The two most common examples of exopolysaccharides of fungal origin are scleroglucan and schizophyllan. Both of these compounds are the examples of beta-glucans and these are also regarded as neutral exopolysaccharides. Scleroglucan is mainly produced by Sclerotium rolfsii, (Survase et al. 2007b), whereas schizophyllan is naturally produced by Schizophyllum commune (Zhang et al. 2013a). These fungal exopolysaccharides contain a branched backbone of β-(1!3)-D-glucopyranose molecule with β-(1!6)-D-glucopyranose residue located at every third glucose unit of the structure (Moscovici 2015). The structural configuration of both exopolysaccharides is quite similar; however, scleroglucan has
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Table 1 Examples of microbial polysaccharides and their potential applications Example of polysaccharide Glucan Gellan Xanthan
Microbial source Saccharomyces cerevisiae Pseudomonas elodea Xanthomonas campestris
Levan
Alcaligenes viscosus
Pullulan
Aureobasidium pullulans Azotobacter and Pseudomonas Alcaligenes faecalis
Alginate Curdlan Emulsan Dextran Welan
Acetan Kefiran Scleroglucan
Schizophyllan
Acinetobacter calcoaceticus Leuconostoc mesenteroides Alcaligenes spp.
Acetobacter xylinum Lactobacillus hilgardii Sclerotium, Sclerotinia, and Corticium Schizophyllum commune
Industrial applications Food thickener Gelling agent in jams, jellies, glazes, frostings, and icings Oil recovery, pharmaceutical applications, cosmetics industry, gelling agent for ice creams, cheese spreads, and puddings Functional biopolymer in food and pharmaceutical industries, food additive, skin moisturizer Food additive to provide texture, inhibits fungal growth in foods Stabilizer, thickening agent, gelling agent in dairy products, sauces, jams, and soups Texture modifier in foods, viscosity improvement in foods, gelling agent Stabilizer, emulsifying agent Viscosity improvement in ice creams Thickening agent in beverages, salad dressings, jellies and dairy products, medical and pharmaceutical industry Gelling agent in confectionary products Improves viscosity of foods Gelling agent, pharmaceutical industries, medical uses
Thickening agent, food preservative, pharmaceutical and medical applications
more intense branching as compared to schizophyllan with a glucose residue at every two β-1, 3-glucosyl units. Scleroglucan is a homopolysaccharide having branched structure and it only gives D-glucose on hydrolysis. This polysaccharide mainly possesses a chain of 1!3-linked units of ß-D-glucopyranosyl. The chemical structure of scleroglucan was established through periodate oxidation and hydrolysis using selective glucanase enzymes. Hydrolysis of scleroglucan yields gentiobiose (one mole) for two moles of glucose, which affirmed the ratio of 1!3 and 1!6 glycosidic linkages. Additionally, methylation analysis is helpful in quantitative and qualitative determination of various types of glyosidic bonds present in the structure. Nuclear magnetic resonance (NMR) spectral analysis also confirmed the regular polymer structure corresponding to various types of glucose units. Moreover, light scattering technique has also been used for determining the molecular weights of scleroglucan. Dissolved chains of scleroglucan possess a rod-shaped triple helix structural conformation with
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side groups of D-glucoside units which mainly prevent the aggregation of helices and provide structural integrity to the scleroglucan molecule (Brigand 2012). Schizophyllan is a neutral exopolysaccharide produced by submerged culture of Schizophyllum commune (Zhang et al. 2013b). Studies involved in the enzymatic degradation have documented that the repeating unit of schizophyllan is comprised of three β-(1-3)-linked residues of D-glucopyranose residues having one β-(1-6) linkage. NMR spectral analysis also confirmed the structural configuration of schizophyllan with a regioselective cleavage of β-1,3 chain with β-l,6 side chains. The triple helix structural conformation of schizophyllan describes that the molecule has a rigid chain with rod-like structure having largest chain length (LpE200 nm). It has also been investigated that the rigid structure of schizophyllan is converted to a slightly flexible structure when the molecular weight exceeds 500 kDa. The triple helix is synthesized by convolution of three individual helices with β-(1-6) D-glucose side chains and the helix is stabilized by hydrogen bonding. Higher concentrations of schizophyllan develop a liquid crystalline phase (Zhang et al. 2013b). The mentioned structural properties are responsible for diverse applications of these microbial exopolysaccharides in food and medicinal industries.
3
Physicochemical Aspects
Microbial exopolysaccharides are ubiquitous in nature and composed of repeating units of sugar moieties attached to a lipid carrier. Functionality and properties of exopolysaccharides mainly depend on ecological niche of microorganisms and structural characteristics of polysaccharides. Additionally, the commercial applications of exopolysaccharides of microbial origin also depend on their physicochemical properties and mainly include food processing industries, pharmaceutical applications, detoxification, petrochemical industries, bioremediation, biotechnology, paint industry, etc. (Mishra and Jha 2013). This chapter is mainly focused on the two important exopolysaccharides of microbial origin: scleroglucan and schizophyllan. Hence, the physicochemical properties are also directed towards these two biomolecules. Gels produced from scleroglucan have vast stability as these gels can survive in the temperature, pH, and salt concentration ranges of 0–120 C, 1–12.2, and 0–20%, respectively (Zhang et al. 2013b). These properties make scleroglucan a good candidate for various applications in food, pharmaceutical, and medical industries (Smelcerovic et al. 2008). Schizophyllan is a water-soluble and nonionic exopolysaccharide having wide applications such as thickening agent and food preservative in food industries, petroleum recovery, thickening substance in cosmetic creams and lotions, and pharmaceutical and medical applications. Some important properties of both exopolysaccharides have been discussed here.
3.1
Properties of Scleroglucan
Scleroglucan can be easily dissolved in cold and hot water. Addition of alkalis or acids does not impose significant effect on the properties of scleroglucan solution
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over a pH range of 2.5–12. Moreover, the viscosity of the solution is not influenced by ionic species. The solution of scleroglucan is thermostable and possesses pseudoplastic behavior at higher concentrations. Scleroglucan also has significant emulsifying potential which makes it useful as stabilizer in food and cosmetic industries (Brigand 2012; Zhang et al. 2013a). The important characteristics of scleroglucan have been further elaborated as follows.
3.1.1 Properties of Scleroglucan Aqueous Solutions Powdered or granular form of scleroglucan has the ability to disperse in water at all temperatures. However, scleroglucan powder must be added to the water slowly with high speed mechanical stirring to prevent lumping. Additionally, the rate of dissolution or dispersion can be increased by pretreatment of powder with alcohol. Likewise, the required maximum viscosity of the solution can be achieved in a shorter period of time by strong agitation while preparing the solution. Alternatively, the viscosity can be attained by keeping the solution for 24 h or heating the solution up to 90 C followed by cooling. The pH range is also important to get desired viscous properties of the solution. The ideal pH range for viscosity development is 6.5–9. Studies have also shown that concentrated solutions have faster rates of viscosity development as compared to the diluted solutions (Brigand 2012). 3.1.2 Rheological Properties of Scleroglucan Due to the high viscosity, scleroglucan solutions have high shear-thinning properties which provide good suspending characteristics to the solutions. The concentrated solutions of scleroglucan behave like pseudoplastic materials and have high practical yield. For example, 0.5% scleroglucan solution has the yield value of about 50 dynes/cm2, while 1% solution contains a yield value of 150–200 dynes/cm2. This high yield value along with shear-thinning characteristics of scleroglucan solution gives good pourability and suspending power to the solution. Additionally, high yield value of scleroglucan solutions is useful in stabilizing the emulsions. For instance, 0.25% solution containing equal parts of scleroglucan and a vegetable oil develops highly stable emulsion. The viscous properties of scleroglucan solutions are not affected over a wide temperature range and remain constant between 15 and 90 C. At lower temperature values, the scleroglucan solutions develop thermo-reversible gels. Furthermore, viscosity of scleroglucan solutions is not significantly affected at low pH values but pH value above 12.5 results in decreasing the viscosity due to the transition of triple helical structure to random coil-like structure. Scleroglucan is also quite stable in acidic medium and forms gels due to the breakdown of some glycosidic bonds and aggregation of triple helix. Moreover, at high electrolyte concentrations, scleroglucan solution flocculates and forms gels. Likewise, high alkaline conditions also result in the precipitation and formation of gels. 3.1.3 Physiological Properties of Scleroglucan Bioefficacy studies based on animal modeling have described that intake of scleroglucan is not associated with any kind of toxicity, tissue pathology, or blood
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abnormalities. Likewise, skin and eye tests did not exhibit any significant adverse effects on the skin or sensitization. Studies have also revealed that dietary administration of scleroglucan to the chicks is helpful in reducing the cholesterolemia and increasing the excretion of cholesterol and lipids. Additionally, scleroglucan also displays considerable antitumor capability.
3.2
Properties of Schizophyllan
3.2.1 Properties of Dilute Schizophyllan Solutions Schizophyllan is fairly soluble in water and it dissolves in water as a triple helix with rod-like structure, whereas it is dispersed in the basic solutions like dimethyl sulphoxide solution in the form of single coil. It has also been studied that schizophyllan is completely dissolved in water at lower salt concentrations (0.01 M NaOH) without disturbing the helical structure and provides stability to the microgels. Dilute solutions of schizophyllan also show conformational transitions due to temperature variations. The conformational transitions at higher temperature are thermo-irreversible and mainly involve the dissociation of complex triple helical structure into random coils. Contrarily, the low-temperature conformational transitions are thermo-reversible and are associated with the dissociation of triple helices of schizophyllan aggregates or intramolecular conversion between type I and type II triple helices (Zhang et al. 2013b). 3.2.2 Behavior of Concentrated Schizophyllan Solutions Generally, the shear rate at which the viscosity of a solution starts decreasing is lowered with increased concentration of the solution. It is reported that the dependence of molecular weight on the shear rate of schizophyllan solutions also becomes weaker with increased flexibility resulting due to the higher solution concentrations. However, the solutions containing rod-like structure of schizophyllan in the solution have stronger dependence on the molecular weight of the compounds as compared to the solutions containing flexible polymers of schizophyllan. Additionally, the behavior of the solutions is also changed as the concentration of schizophyllan is changed. For instance, at higher concentrations, schizophyllan produces liquid crystals with varied dependence of the viscosity on the concentration for different types of solutions. It is observed that at lower concentrations (2.33%), the solution remains isotropic in nature at ambient temperature, whereas at higher concentration (14.34%), the solution of schizophyllan shows cholesteric mesophase. It has also been investigated that the optical rotatory dispersion (ORD) of the concentrated schizophyllan solution is drastically changed upon cooling the solution to 5 C due to the transition of isotropic phase to the cholesteric phase (Zhang et al. 2013b). 3.2.3 Gelation Behavior of Schizophyllan Schizophyllan produces weak gels at lower temperatures, that is, below 6 C (Zhang et al. 2013b). However, the addition of several compounds such as sorbitol or borax develops relatively stronger gels. Addition of borax provides strength to the
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schizophyllan gels by making complexes between borate ions and hydroxyl groups of schizophyllan side chains. The rate of gel formation is directly related to the concentration of borax, salt, and schizophyllan in the solution, but inversely related to the temperature. In contrast to the schizophyllan-borate system, the gelation behavior of schizophyllan is quite different in case of sorbitol addition. It has been reported that addition of sorbitol leads to the reduced water mobility resulting in the aggregation of schizophyllan molecules to develop a three-dimensional network of gel. It has also been proposed that triple helical structure of schizophyllan is broken down in the presence of sorbitol and the broken chains are then re-associated through hydrogen bonding to form gel networks. Moreover, it has also been noticed that the temperature of the system is increased due to the increased concentration of the sorbitol. Another gelation mechanism of schizophyllan-sorbitol system involves the conformational transition of schizophyllan chains from type II to type I helix which increases the stiffness and diameter of the helices and reduces the mobility of water and schizophyllan molecules (Zhang et al. 2013b) resulting in chain enlargement and formation of strong gel network. These properties of the schizophyllan gels are helpful in developing the schizophyllan-protein co-gels and widening the applications of schizophyllan as gel metrices in food and pharmaceutical industries.
4
Production and Isolation of Scleroglucan and Schizophyllan
For the production microbial polysaccharides, the most extensively practiced method is fermentation due to its financial aspects and feasibility. Hence, optimization of the fermentation process for the production of exopolysaccharides of microbial origin is of prime importance and the cost of the production process can be significantly reduced by controlling several parameters such as temperature, pH, rate of agitation, concentration of oxygen, and compositional properties of culture medium. Additionally, the microbial strains used for the production of polysaccharides also influence their properties such as structural conformation, rheological and physicochemical attributes, and monomer composition (Lembre et al. 2012). Therefore, control of fermentation parameters and careful selection of feedstock and microbial strains are important to achieve the polysaccharides with required specifications.
4.1
Production and Isolation of Scleroglucan
Various species of Sclerotium such as Sclerotium glucanicum, S. delphinii, and S. rolfsii are involved in the extracellular production of scleroglucan (Farina et al. 2001; Survase et al. 2007). Sclerotium species are filamentous fungi and heterotrophic in nature. These fungi are also regarded as plant pathogens due to the oxalic acid producing ability which facilitates plant cell lysis. These fungal species possess several types of enzymes such as cellulases, arabinose, galactosidase, phosphatidase,
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exo-galactanase, exo-mannase, and poly-galacturanase, etc. Sclerotium species contain light-colored mycelia, or black to brown-colored sclerotia which are resistant to biochemical and chemical degradation, but do not have the ability to sporulate. In liquid media, these organisms produce pellets having central capsules with extended hyphae. On solid media, aerial hyphae are produced and arranged in the mycelia. Sclerotium species produce scleroglucan which assists these organisms in the attachment to the surface of plants and also provides protection against harsh environmental conditions. Additionally, scleroglucan is also used by these organisms as energy source due to the activity of hydrolytic enzymes which degrade the biopolymer into glucose molecules. The optimum growth temperature for Sclerotium species is reported as 30 C. Additionally, these organisms can tolerate lower sucrose (0.15–1.17 M) and salt (0.86 M) concentrations; however, higher concentrations cause growth inhibition (Farina et al. 1996).
4.1.1 Biosynthesis of Scleroglucan The generic pathway for biosynthesis of scleroglucan involves three prime steps which include (a) uptake of the substrate, (b) intracellular production of polysaccharide, and (c) extrusion of the produced polysaccharide from the fungal cell. During the biosynthetic production of scleroglucan, glucose is firstly transferred to the cells by hexokinase enzyme and then phosphorylated by two enzymes, namely, phosphor-glucomutase (PGM) and phosphor-glucoisomerase (PGI). Afterwards, UDP-G (uridine diphosphate glucose) is produced due to the action of pyrophosphorylase (UGP) enzyme. UDP-G starts polymerization by reacting with the lipid carrier. The proposed schematics of the biosynthetic pathway of scleroglucan are illustrated in Fig. 1. Glucose Hexokinase Glucose-6-phosphate PGM Glucose-1-phosphate PGI UMP
UDP-G
Lipid pyrophosphateglucose
Lipid phosphate
UDP-glucose UDP-glucose
Pi
Lipid pyrophosphate
(Lipid pyrophosphateglucose)n
(Lipid pyrophosphateglucose)2 UDP
Fig. 1 Biosynthetic pathway of scleroglucan production
UDP
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4.1.2 Fermentation Conditions for Scleroglucan Production Generally, several factors influence the production of scleroglucan. These factors include preparation of inoculum, type of growth medium, environmental conditions, and byproduct formation. Additionally, the production of desired polysaccharide can also be enhanced by controlling the activities of undesirable enzymes and modifying the genetic determinants. The composition of growth medium is highly important for effective utilization of substrate by the microbial enzymes and transformation into the final product. Normally, the media with high carbon to nutrient (limiting nutrient) ratio is recommended for efficient production of polysaccharides. High yield fermentation processes generally convert 60–80% of consumed carbon source into crude biopolymer. The main nutrients needed by fungal cells include carbon, nitrogen, oxygen, phosphorus, potassium, sulfur, and magnesium. It has also been reported that addition of several precursor molecules improves the rate of polysaccharide synthesis by providing metabolic force. In general, nucleotide phosphate sugars improve the metabolic flux of exopolysaccharide production. Incorporating amino acids (L-lysine) and sugar nucleotides can be used as metabolic precursors for the production scleroglucan (Survase et al. 2007a). Among various environmental factors, temperature is of prime importance to carry out the microbial activities smoothly. The optimum temperature for growth of culture is 28 C (Wang and McNeil 1995a), whereas for the production of scleroglucan is 20–37 C. It is also observed that the growth of culture is reduced below 28 C due to excessive formation of oxalic acid which ultimately influences the yield of scleroglucan in an adverse way. Similarly, pH range also imposes significant effect on the growth culture as well as on scleroglucan production rate. The optimum pH value for effective fungal growth is 3.5, while production rate of scleroglucan is enhanced at pH value of 4.5. It is also documented that fermentative production of scleroglucan under these pH conditions results in 10% mitigation of byproduct formation (Wang and McNeil 1995b). Oxygen concentration also plays an important role in the growth of aerobic microorganisms. Studies have affirmed that high dissolved oxygen facilitates the growth and metabolic activities of Sclerotium species which promotes the reduction of carbon and formation of scleroglucan. Moreover, it has also been investigated that higher rate of aeration and agitation (600 rpm) facilitates the fungal growth by improved dissolution of oxygen and nutrient mixing; however, reducing the rate of agitation at later stage of fermentation enhances the scleroglucan synthesis because higher agitation can induce molecular degradation of the produced polysaccharide. The main byproduct during scleroglucan production is oxalic acid which in undesirable because oxalates block the biosynthetic pathway of scleroglucan production and need another purification step for its separation. Hence, pH adjustment is important to reduce the production of oxalic acid during fermentation. Generally, low pH values reportedly enhance the activity of oxalate decarboxylase enzyme which breaks down oxalate into carbon dioxide and formate and prevents the blockage of scleroglucan formation pathway.
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4.1.3 Downstream Processing for Recovery of Scleroglucan Optimizing all the fermentation conditions is not enough to ensure the good yield of scleroglucan, but another important step for obtaining high yield of scleroglucan is recovery of the product. The selection of suitable recovery method for exopolysaccharides mainly depends on several factors including properties of the organisms used for the production of exopolysaccharide and desired purity level of the product. Usually, the crude product is obtained by completely drying the whole fermentation broth. The actual product/polysaccharide can be separated by filtration or differential centrifugation. Additionally, drum drying or spray drying methods can be used for water and solvent removal. Sometimes, use of electrolytes is helpful in the precipitation of the polysaccharides by neutralization of the charges (Taurhesia and McNeil 1994). In general, three different methods have been reported for the recovery of scleroglucan produced by fermentation process. A generalized illustration is provided in Fig. 2. The common step in all the three recovery processes is pretreatment of fermentation broth. Afterwards, the product has been recovered by different methods. The common pretreatment process includes neutralization of the fermentation broth with HCl or NaCl, three to fourfold dilution using distilled water, heat treatment for 30 min at 80 C, homogenization of the diluted broth, and centrifugation (10,000 g) for 30 min. After centrifugation, the pallets are obtained which are then subjected to washing process using distilled water followed by drying at 105 C. The obtained supernatant is then further used for the recovery of final product, that is, scleroglucan. The first method of scleroglucan recovery involves the cooling of supernatant to 5 C followed by addition of isopropanol or ethanol (96%) for precipitation. The
Fermentation broth
Homogenization
Neutralization
Supernatant Ethanol/isopropanol addition
Dilution
Dilution
Heat treatment Addition of CaCl2
Addition of CaCl2 Storage at 5ºC for 8 hrs. Separation of calcium oxalate Ethanol/isopropanol addition
Addition of KOH or NaOH to adjust pH value of 10-12
Separation of precipitate
Separation of precipitate
Drying
Drying
Separation of precipitate
Drying
Separation of calcium oxalate
Fig. 2 Methods for recovery of scleroglucan
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mixture is usually kept at this temperature for about 8 h to complete the precipitation of exopolysaccharide. After that, the scleroglucan is recovered through sieving and dissolved in distilled water. For better recovery, the crude mixture can be retreated with ethanol or isopropanol for reprecipitation. After that, the precipitated biopolymer can be freeze-dried or thermally dried at 55 C for about 8 h. Finally, the product is milled to obtain whitish scleroglucan powder which is then packed for further marketing (Farina et al. 2001). The second method used for the recovery of scleroglucan uses divalent cations such as calcium, iron, cobalt, magnesium, nickel, and manganese with organic solvent. Generally, calcium chloride is preferred for this purpose and can be used at concentration of 0.5–2.0%. Addition of calcium chloride to the fermentation broth results in the precipitation of calcium oxalate, which can be removed by filtration or centrifugation process. After that, 20–40% ethanol or isopropyl alcohol is added to the broth which results in further precipitation and the precipitates are again removed by filtration or centrifugation. The obtained scleroglucan is then purified through reprecipitation or rehydration processes. In the third method of scleroglucan recovery, 0.5–2% calcium chloride is added to the fermentation broth and then metal hydroxides are added to the mixture to maintain the pH of system under alkaline conditions. After completion of the reaction, calcium oxalate precipitates are removed and metal hydroxides (sodium or potassium hydroxide) are added to adjust the pH value at 10–12. The polysaccharide will be precipitated under alkaline conditions which will then be removed by filtration or centrifugation. The purity of the product can be further increased by repeating the treatment.
4.2
Production and Isolation of Schizophyllan
The fungus Schizophyllum commune ATCC 38548 is reported to produce schizophyllan. The produced homoglucan is appeared in the form of gum which is attached with the microbial cell wall or secreted in the fermentation broth. Additionally, shear stress, applied through agitation generally, decreases the pellet growth and enhances the secretion of biopolymer from the fungal cell wall. However, studies have reported that too high shear force results in the damage of fungal hyphae production of schizophyllan. The resultant cellular debris is subjected to downstream processing for separation of the product. The growth medium used for bioreactor cultivation of Schizophyllum commune mainly consists of deionized water, glucose, yeast extract, KH2PO4, and MgSO47H2O. The optimum temperature and pH values for cultivation of this fungus are 27 C and 5.3 (initial pH value). Generally, cultivation of fungal culture is carried out using bioreactors in 42 L capacity vessel with a working volume of 30 L. The medium is sterilized and then 5% inoculum is added to start the growth and fermentation activities (Rau 2008). A generalized process for production and isolation of schizophyllan is depicted in Fig. 3.
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Fig. 3 Production and isolation of schizophyllan
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Fermentation broth
Dilution
Homogenization Cell separation Cell-free schizophyllan solution Cross f low microfiltration
Concentration
5
Applications for Scleroglucan and Schizophyllan
Microbial exopolysaccharides (EPS) have found applications across a wide variety of domains, including the food industry, pharmaceuticals, biomedical solutions, and in the oil (petroleum) drilling operations. Being structurally versatile, these molecules exhibit diverse structural combinations, endowing them with unique properties. Conventional sources of polysaccharides included marine algae, higher plants, or animals, albeit with low resultant yields and high cost. However, microbial fermentation processes at commercial scale have emerged as a viable, industry significant, high yield, high purity, nontoxic, and biocompatible alternative (Survase et al. 2007) and consequently, have paved the way for greater utilization of these highly valuable biomolecules. Moreover, these microbial exopolysaccharides have a low environmental footprint and can be obtained from easily available and renewable natural resources (Survase et al. 2007a), as well as greater stability at elevated temperatures, and pH and salinity extremes (Brigand 2012; Jindal and Khattar 2018), thereby increasing their scope for utilization manifold. Apart from the inherent thermal stability characteristics, the research on solutions of scleroglucan (SG) has revealed a pseudoplastic behavior with a high yield value, rendering these solutions with high suspending power and excellent pouring properties. Consequently, this high yield value enables scleroglucan to be highly effective at keeping particles in suspension, in both static and dynamic conditions, and
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without any risk of sedimentation (Wüstenberg 2015). Scleroglucan (trade names include actigum, clearogel, polytetran, polytran FS, and sclerogum), with its excellent emulsifying capabilities, could be very suitable as a foam stabilizer. Moreover, its compatibility, without synergism, with various other thickening agents of hydrocolloid nature is also notable. Furthermore, scleroglucan has also shown compatibility with some of the most abundantly used surfactants, for example, sulfates, sulfonates, and quaternary ammonium salts. Also, scleroglucan has been recorded to be soluble even in mixtures consisting of almost 50% of polyols and glycols. With its exceptional rheological properties, as well as stability over a broad range of pH, salinity, and temperature conditions, scleroglucan is suitable for many applications (Wüstenberg 2015). Both scleroglucan and schizophyllan (SPG), over the years, have grown in significance owing to their superior functionality for various applications. For instance, scleroglucan found its initial utility in the oil industry, displaying greater stability (compared to xanthan that is currently in use) to shear stress, and over a broad range of temperature (20–90 C), salinity, and pH conditions, imparting enhanced viscosity characteristics, and therefore, improving the hydraulic pressure of the seawater or brine used for oil recovery procedures. Moreover, scleroglucan has also proven to be useful as an oil mud thickener and stabilizer, as well as a biopolymer with low sensitivity to field contaminants (Survase et al. 2007; Gao 2016; Liang et al. 2019). More recently, schizophyllan (trade name sizofiran, or sonifilan) has also been employed for enhanced oil recovery (EOR) or tertiary oil recovery processes in the oilfields, proved to be stable under the high temperature and high salinity conditions, and could substitute partially hydrolyzed polyacrylamide (HPAM) and xanthan gums for commercial EOR projects (Gao 2016; Liang et al. 2019). A recent application involved the use of scleroglucan as an emulsion stabilizer in bitumen formulations for road surfacing (El Asjadi et al. 2018). Refined scleroglucan has also been used in cosmetics manufacturing, in particular, hair control conditioning products and various skin care preparations such as creams, protective lotions. Similarly, schizophyllan has also been used in skin care products such as skin antiaging and healing preparations (cosmeceuticals), as well as an active ingredient in dermatological formulations aimed at reduction in skin irritation and recovery from sunburns (Gao 2016). Furthermore, scleroglucan has also found applications in the manufacturing of products such as adhesives, paints, watercolors, ceramic glazes, printing ink, various pesticides, and as a stabilizer in fire drencher foams. The utilization of these multipurpose and versatile biopolymers has also been documented for both food, biomedical, and pharmaceutical industries, and the proceeding paragraphs involve a discussion regarding their role in these industries in greater detail.
5.1
Applications in the Food Industry
Worldwide, the food industry uses several thousand tons of polysaccharides every year, and as food product innovation requirements continually demand a host of new,
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versatile, and potentially low-cost additives, microbial exopolysaccharides are becoming ubiquitous for various applications in the food industry. Polysaccharide gums such as modified starches, cellulose, curdlan, pectin, guar gum, pullulan, alginates, carrageenans, chitosan, gellan, and xanthan are a few examples being extensively used. In particular, xanthan has become an ingredient of great significance over the years and is being used as a thickening, gelling, and stabilizing agent in a variety of food products including jams, marmalades, soups, confectionery products, aqueous gels, frozen foods, yogurt, ice creams, and low calorie/nonfat products (Survase et al. 2007b; Goff and Guo 2019). However, the exploration for better alternatives continues with research on various polysaccharide-producing strains and novel microbial biopolymers.
5.1.1 As Stabilizers, Viscosifiers/Thickeners, and Gelling Agents In recent years, scleroglucan has been proposed as a possible substitute for xanthan, especially suitable for food manufacturing operations involving processing at elevated temperatures, owing to its greater thermal stability and process efficiency. Scleroglucan could, therefore, be ideally suited as a hydrocolloid, for stabilizing and viscosifying food products such as salad dressings, sauces, ice creams, and other desserts, as well as prevention of water loss (preservation of retrogradation) (Selvi et al. 2020). The stabilizing properties of scleroglucan were investigated in cooked starch pastes and the results indicated that scleroglucan can retain water in significant amounts, thus reducing syneresis during refrigeration. Moreover, there was evidence that the retention could be improved further by blending scleroglucan with corn starch before adding to the pastes (Selvi et al. 2020). Although, not inherently a surfactant, scleroglucan has the potential to stabilize oil-in-water (O/W) emulsions as well, by preventing coalescence (Giavasis 2013), to date, scleroglucan has not been approved either by the European Food Safety Authority (EFSA) or the US Food and Drug Administration (FDA), a major impediment in the way of its application on a commercial scale. Many Japanese patents, however, indicate growing acceptance in the form of application of scleroglucan for quality enhancements in a range of frozen/heat-treated and aerated food products, such as Japanese cakes, steamed edibles, rice crackers, and some bakery products (Brigand 2012; Jindal and Khattar 2018; Selvi et al. 2020), and there is increasing hope that scleroglucan could witness wider application in the food industry manufacturing processes worldwide. Since both scleroglucan and schizophyllan are structurally similar, as well as in terms of chemical properties, it is assumed that both can be used alternatively for applications in the food industry. The tendency of schizophyllan with regards to the formation of co-gels with gelatin has been studied, and it was observed that the concentration of schizophyllan directly influenced the elasticity or rigidness of the gels (higher elasticity was recorded at low gelatin/schizophyllan ratio). Schizophyllan, therefore, can potentially be used for food applications where gelatin is already being utilized as a thickener. Scleroglucan has shown good compatibility with various electrolytes. However, gel formation or flocculation of the solution may take place at higher electrolyte concentrations. Scleroglucan, as already mentioned, is also compatible
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with most of the commercially available thickeners, such as locust bean gum, alginates, xanthan, and carrageenans, as well as cellulose derivatives. Pure scleroglucan (90–98% EPS) solutions in the water at concentrations of 2 g/L, or lower, yielded highly viscous solutions with non-Newtonian, nonthixotropic, and pseudoplastic behavior (Survase et al. 2007b).
5.1.2 Preparation of Edible Films Owing to its chemical stability, biocompatibility, and biodegradability, scleroglucan could be used in the preparation of edible films and tablets for nutraceuticals (Giavasis 2013). Similarly, schizophyllan, in combination with lentinan, a polysaccharide obtained from the edible Japanese shiitake mushroom, Lentinus edodes, has been studied for possible applications in novel food products and nutraceuticals (Giavasis 2013). The use of schizophyllan for the formation of films with low oxygen permeability, aimed at food preservation, has also been investigated and involved the study of the physicochemical characteristics of films formed from schizophyllan and a chemically modified polymer derived from it. Owing to high water solubility and exceptionally low oxygen permeability, the utility of such films as oxygen barriers (easily removable by water), is a promising prospect (Sutivisedsak et al. 2013). 5.1.3 Production of Functional Foods A group of Japanese researchers, in a novel approach, added Schizophyllum commune (the source for schizophyllan) as a live starter culture for producing a functional cheese-like food. The fungal starter culture induced fermentation and coagulation of milk, owing to its lactate dehydrogenase and proteolytic and milkclotting enzymes. These proteolytic and clotting enzymes demonstrated higher activity of proteases, albeit a significantly lower clotting activity compared to rennin. The final product consisted of approximately 0.58% β-glucans, which had a significant antithrombotic function (Giavasis 2014). Schizophyllum commune has also been significant in terms of the discovery of a special family of small secreted, and moderately hydrophobic proteins, with a structurally characteristic spacing of eight cysteine residues. These proteins have been named hydrophobins and are capable of self-assembly into amphipathic membranes, resulting in the transformation of the attributes of contact surfaces. A highly promising application of hydrophobins involves their use as stabilizers for edible foams and emulsions. The emulsions based on these proteins resemble lipids (fats) in terms of their taste and mouthfeel and, therefore, have the potential for applications not only as natural highly efficient stabilizers but also as fat replacements from emulated products, presenting the opportunities for the creation of novel dietetic foods (Shamtsyan 2016). Scleroglucan, likewise, due to its nondigestibility, acts as a prebiotic (fiber) and has been linked with the formulation of low-calorie foods (Giavasis 2014).
5.2
Biomedical/Pharmaceutical Applications
It has been known for hundreds of years that the consumption of many species of fungi can impart favorable benefits to human health, with the practice widely
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prevalent in many of the Far East countries, such as China, Japan, Korea, and some parts of Russia, where they have been used either as dietary or medicinal supplements (Lemieszek and Rzeski 2012). More recently, though, the focus of the researchers has shifted towards the bioactive compounds present in these fungi and their potential impact on the functioning of the human body. This has led to these “mycopharmaceuticals,” or “fungal supplements” being introduced to the markets worldwide (Lemieszek and Rzeski 2012). Moreover, the significance of such fungi has warranted their inclusion towards many dietary regimens as “functional foods.” The bioactive compounds with the greatest potential in terms of biomedical applications are the β-glucan [(1!3), (1!6)] polysaccharides, including both scleroglucan and schizophyllan, among others.
5.2.1 As Immunostimulants in Anticancer Activities The anticancer potential of schizophyllan and other similar β-glucan polysaccharides, including scleroglucan, can be attributed to its origin, composition (in terms of types of sugar present) and chemical structure (tertiary), molecular weight, solubility in water, glycosidic linkages, branching rate and form, presence of ligands (such as proteins), and the methods used for isolation. Moreover, the activity and clinical attributes of schizophyllan can be significantly enhanced through chemical modifications, most importantly the Smith degradation (oxido-reducto-hydrolysis), activation by formolysis, and carboxymethylation. Polysaccharides that are water-soluble, have high molecular weights, consist of more β-(1!3) linkages in the main glucan chain, with β-(1!6) at the branch points, and where the value of the degree of branching (DB) with regards to the molecular weight lies between 0.20 and 0.33 have demonstrated the highest biological activity (Lemieszek and Rzeski 2012). The expression of the anticancer effects of β-glucan polysaccharides involves both direct (cell proliferation inhibition and/or induction of apoptosis) and indirect (immunostimulation) stimulation mechanisms. Indirect action manifests in the stimulation of host defense mechanisms, in particular, the activation of both T and B lymphocytes (tumor-infiltrating), macrophages, Langerhans cells (LCs), polymorphonuclear leukocytes (PMLs), lymphokine-activated killer (LAK) cells, and the natural killer (NK) cells [(also known as the large granular lymphocytes (LGLs)]. Moreover, these polysaccharides are also known to trigger the production of interferons (IFNs), interleukins (ILs), and other cytokines, often considered as the first line of defense in the host immune systems, as well as colony-stimulating factors (CSFs) by specialized receptor-mediated [Toll-like receptor (TLR), dectin-1, and complement receptor 3 (CR3)] induction of gene expression. A host of in vitro and in vivo studies also indicates a direct action of polysaccharides on cancer cells, whereby they have been found to either inhibit the proliferation of tumor cells or cause their death by apoptosis [characterized by the activity of caspase (casp)-3, and the ensuing cleavage of poly (ADP-ribose) polymerase (PARP)] (Lemieszek and Rzeski 2012). The mechanism for this direct action involves primarily the modulation of the nuclear factor kappa B (NF-κB) pathway. Disproportionate activation of NF-κB, a proinflammatory inducible transcription factor, has been observed in many types of
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cancer, and its activity has direct consequences in the form of promotion of tumor growth owing to increased transcription of genes, therefore, inducing proliferation of cancer cells, inhibition of apoptosis, as well as stimulation of angiogenesis and metastasis. The polysaccharides tend to inhibit the phosphorylation, ubiquitinylation, and degradation of the NF-κB inhibitor, IκBα, thereby preventing the activation of NF-κB, and ultimately, the expression of its subordinate genes (Lemieszek and Rzeski 2012; Giridharan and Srinivasan 2018). Another pathway for the direct action of polysaccharides involves polysaccharopeptide (PSP), a protein complex isolated from the common Turkey Tail fungus, Trametes versicolor, and has been shown to induce cell cycle arrest at restrictive points G1/S and G2/M in human myeloid leukemia cells U-937 and human breast cancer cells MDA-MB-231, thereby, also inhibiting the antiapoptotic proteins, resulting in suppression of cell division processes and a marked increase in apoptosis (Lemieszek and Rzeski 2012). Schizophyllan in conjunction with other anticancer drugs has proven effective for the treatment of cancers of the stomach, lungs, breast, cervix, etc., as well as the prevention of metastasis and side effects associated with chemotherapy treatments (Rakhra et al. 2020). However, the mode of action of schizophyllan specifically does not directly involve the cytotoxicity against cancer cells. Instead, the anticancer activity of schizophyllan manifests by the way of stimulating the secretion of the proinflammatory cytokine, TNF-α, by cells of the human immune system, such as monocytes, macrophages, and platelets (Nie et al. 2013). The bioactivity against various cancers in the case of schizophyllan can be attributed to its high molecular weight, triple-helix conformation, and according to some other studies, its random coil configuration. Moreover, the route of administration could be another significant factor to elucidate the high levels of bioactivity attributed to schizophyllan and other β-glucan polysaccharides (Rakhra et al. 2020). Scleroglucan, in its immunopharmacological form, Sinophilan, has also found applications for the clinical treatment of various cancers. The antitumor activity of scleroglucan is thought to be mediated by an increase in the number of macrophages in the presence of soluble glucan. Scleroglucan reportedly has a high affinity for human monocytic cells and has two main biological attributes, that is, the stimulation of phagocytic cells and the hemopoietic activity associated with monocytes, neutrophils, and platelets. The polysaccharide exhibited immunomodulating activities against cancerous tumor formation when it was administered either parenterally or orally. The oral administration characteristics of scleroglucan are unique among other glucans and cater favorably in terms of fewer side effects, as well as a significant reduction in pain. Moreover, the oral administration pathway presents the opportunities for incorporation of this biopolymer into foods designed for cancer patients (nutraceuticals), or the individuals at risk of developing the malignancies (Giavasis 2013).
5.2.2 As Immunomodulators in Antitumor Activities The antitumor action of schizophyllan is dependent on its capability to modulate immune responses (taking place only in the presence of T cells), and according to many reports, by the way of its immune-stimulating properties, it tends to enhance
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the ability of immune cells for identification and recognition of tumor cells as foreign entities, thereby increasing and consolidating the effectiveness of the host defense mechanisms. Schizophyllan induces the production of acute-phase proteins (APPs) and colony-stimulating factors, thereby triggering the proliferation of macrophages, peripheral blood mononuclear cells (PBMCs), as well as lymphocytes, and stimulation of the complement system (also termed as complement cascade). Also, this manifests in an increase in the production of the T Helper (Th) lymphocytes and macrophages, indicated by the strong activation of phagocytes, and this, in turn, contributes to increased production of reactive oxygen species (ROS), nitric oxide (NO), proinflammatory cytokines IL-6, IL-8, IL-12, and tumor necrosis factor (TNF-α), as well as increased expression of CD11b and CD69L markers on the surface of leukocytes (Lemieszek and Rzeski 2012). Schizophyllan was reported to demonstrate antitumor activity against both the solid and ascites forms of Sarcoma 180, as well as sarcoma 37 (the solid form only), Ehrlich-Lettre ascites sarcoma (EAC), Yoshida sarcoma, and Lewis lung carcinoma (LLC). The antitumor activity of schizophyllan was not detected in the thymectomized (thymus gland removed) individuals, indicating that the activity is T-cell mediated. Formyl methylated and aminoethylated derivatives of schizophyllan exhibited enhanced levels of antitumor activities and increased production for both the factors associated with tumor regression and the soluble cytotoxic factors when compared with nonderivatized schizophyllan (Nie et al. 2013). Pretreatment with schizophyllan did not exhibit a significant effect on the survival rates of sarcoma 180 xenografts. However, pre- and post-treatment with the polysaccharide revealed to have increased the chances of survival. The underlying antitumor mechanism involved in these studies can be linked to the improved cellular immunity, resulting from increased production of T lymphocytes, as well as that of the cytokines, interleukin-2, and interferon-γ, generated by the PMBCs, with the ensuing tumor inhibition estimated to be around 95% (Banerjee et al. 2015). Schizophyllan has been widely approved for clinical applications as a biological response modifier (BRM), as well as a nonspecific stimulator of the immune system in Japan, where multiple pharmaceutical companies are involved in the commercial manufacture of this polysaccharide as immunopotentiators for clinical treatment of patients undergoing various antitumor therapies. The use of schizophyllan, as an immunoadjuvant, in combination with conventional radiotherapy and chemotherapy (antineoplastic drugs) treatments [tegafur or mitomycin C, and 5-fluorouracil (5-FU)], in patients with recurrent and inoperable gastric cancer, has shown a significant improvement in median survival rates, without, however, any significant influence on the size of tumors (Rakhra et al. 2020). Schizophyllan appears to function in terms of controlling or eliminating the microscopic metastatic foci and/or small-sized macroscopic foci observed in stage III patients (Banerjee et al. 2015). Schizophyllan has also been shown to increase the life span for patients with cancers of the head and neck, aiding in the accelerated recovery of cells that are immunocompromised as a result of radiation, chemotherapy treatments, and surgical procedures. Schizophyllan enhanced the immune-associated function of the regional lymph nodes in these patients, as evidenced by the elevated production of IL-2,
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increased cytotoxic activity of effector cells (NK, LAK), as well as the increased numbers of Th lymphocytes (CD4+) in the tumor-uninvolved lymph nodes. However, a subsequent phase II, two-arm, randomized controlled trial (RCT) revealed that the clinical efficacy and tolerability of schizophyllan coupled with standard chemotherapy treatment, in a patient cohort with locally advanced head and neck squamous cell carcinoma, exhibited no significant difference in terms of overall response rate. Although, a comparatively greater number of complete responses (CR) were recorded in the case of Arm A (schizophyllan combined with chemotherapy), against Arm B (chemotherapy treatment regimen alone) (Banerjee et al. 2015). Schizophyllan is a potent BRM in terms of augmenting the immune function of regional lymph nodes in cervical cancer patients. When combined with radiotherapy treatments, schizophyllan significantly improved the survival time, as well as the time to recurrence (TTR) in stage II cervical cancer patients. However, the effect was not very well pronounced in the patients at Stage III. Another investigation reported that in comparison to the control group, a much higher complete response rate was recorded among patients for both stage II and III. Moreover, the lymphocyte counts that had suffered due to radiotherapy treatments showed rapid and significant improvement. An RCT involving 312 patients being treated with surgery, radiotherapy, chemotherapy (fluorouracil), and schizophyllan in various combinations, the patients who received schizophyllan survived longer. Another prospective RCT is recruiting participants (cervical cancer patients), aged between 20 and 75 years, in Seoul, the Republic of Korea, for a study aimed at evaluation of the impact of schizophyllan (sonifilan) on the quality of life (QOL) during radiation, and concurrent chemoradiotherapy, as well as the complications associated with the treatment involving schizophyllan (Banerjee et al. 2015). The study can be accessed at https:// clinicaltrials.gov/ (identifier: NCT01926821). In the case of chemically induced breast carcinomas, schizophyllan inhibited the incidence of tumors, also leading to reduced cell proliferation in tumor tissue. Schizophyllan and tamoxifen (a drug used for the prevention of breast cancer in women and treatment of breast cancer in both women and men) significantly reduced the incidence of mammary tumors induced by dimethylbenz(α)anthracene, in many cases up to 85% and 75%, respectively, as well as contributed to the suppression of the proliferating cell nuclear antigen (PCNA) labeling index relative to dimethylbenz (α)anthracene. After being exposed to the ultrasound procedures, the immunopotentiating activity of schizophyllan in the context of splenic lymphocytes and RAW264.7 macrophages as well as its antiproliferative action concerning human breast carcinoma T-47D cells increased noticeably (Wong et al. 2020). In ovarian cancer patients undergoing chemotherapy, schizophyllan has been found to be beneficial against myelosuppression by chemotherapy (Banerjee et al. 2015). Schizophyllan has also been attributed with possessing antiradiation properties, as it has demonstrated the ability to restore mitosis in bone marrow cells that were previously suppressed owing to treatment regimens involving antitumor drugs. Early administration of schizophyllan, either before the initiation of chemotherapy or irradiation, or concurrently for the duration of treatments, has been shown to improve their overall effectiveness (Lemieszek and Rzeski 2012). The radioprotective effects in
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bone marrow cell cultures can be attributed to the ability of schizophyllan to induce the expression of macrophage colony-stimulating factor (M-CSF) (Schepetkin and Quinn 2011). Also known as CSF-1 (encoded by the CSF1 gene), this hematopoietic growth factor plays a vital role in promoting the proliferation, differentiation, functional activation, and survival of the cells of the monocyte/macrophage family, reorganization of the cytoskeletal system, and ultimately, the cell spreading and migration of mature osteoclasts (Plotkin and Bivi 2014).
5.2.3 As Antiviral and Antimicrobial Agents Scleroglucan and schizophyllan have also demonstrated activity against various infectious maladies. In this regard, scleroglucan is significant for its antiviral effect, in particular against rubella (German measles) virus and the herpes simplex virus type 1 (HSV-1) (Survase et al. 2007). Scleroglucan was crucial not only in inhibiting the initial phases of rubella virus infection but in blocking HSV-1 infection as well, in the course of the very early phases of the viral replication (Freitas et al. 2015). The mode of action seems to involve the binding of scleroglucan to the host cell membrane. This binding of the polysaccharide to the glycoproteins of the host cell membrane may reduce or even prevent the viral attachment and the subsequent penetration into the cell. This should be noted, however, that the inhibitory action of scleroglucan has been found to occur only at the early stages of these infections. The key mechanism appears to be that the binding of scleroglucan manifests in disruption of the complex interplay between the virus and the host cell plasma. A further plausible explanation for the antiviral activity of scleroglucan can be that, following the entry of the virus into the cell, the polysaccharide is also internalized in the cell, consequently encapsulating the virus and thus inhibiting its activity. However, this course of action is not deemed possible for polysaccharides with high molecular masses, such as scleroglucan (Survase et al. 2007). The oral mode of administration of scleroglucan is significant in terms of its antimicrobial action against, for instance, fungal pathogens. The protective action following the ingestion involves the uptake of minute particles of the polysaccharide by the pinocytic microfold cells (or M cells) located in the gut-associated lymphoid tissue (GALT) of the Peyer’s patches in the small intestine. Scleroglucan directly binds to the intestinal epithelial cells and the GALT cells, in turn getting internalized by the two cohorts of cells. Once these cells have been activated, they can migrate to the lymph nodes and trigger the activation of other macrophages, NK cells, and T lymphocytes via the release of cytokines. An example of such a phenomenon has been observed in the case of low molecular weight scleroglucan hydrolysates ( 3)-α-L-Rha-(1- > 2)-α-L-Aco-(1->, while acidic portions were also revealed to be composed of repeating structures of O-hexuronosyl-rhamnose. However, a more systematic study recently appeared investigating also the influence of culture conditions on polysaccharides abundance and composition. After performing a meta-analysis in literature about culture conditions, the authors identified incident light as the most influencing parameter and proceeded to experimental quantification and analysis of the EPS/glycogen ratio (Phélippé et al. 2019). They reported about the accumulation and composition of polysaccharides in Arthrospira platensis. Although their meta-bibliographic analysis indicated a great variability, due to different operating conditions or extraction/analysis methods, this study allowed to define clear tendencies resulting that among the operating conditions, the light intensity can strongly modulate the accumulation of these molecules. They also found that uronic acids content decreased at high photon flux density, and the neutral monosaccharides components are modified, suggesting potential modulation of the biological activities modifying culture conditions. Extraction processes used for Arthrospira were studied for obtaining high-value chemicals such as phycocyanin, lipids/total fatty acids (TFA), and polysaccharides (Kurd and Samavati 2015; Chaiklahan et al. 2018). The study was contextualized in the economic analysis showing that producing phycocyanin alone was economically feasible, but producing coproducts (lipid/TFA and polysaccharides) was not. A
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microwave-assisted process has been introduced recently to study the efficiency of water extraction following the principles of green chemistry (de Sousa et al. 2018). During the cultivation process and study of growth (de Jesus et al. 2019), efforts for enhancement of production of extracellular polymeric substances (EPS) in Spirulina (Arthrospira sp.) were reported (Chentir et al. 2017) with the partial characterization of polysaccharidic moiety; however this study is limited to differential scanning calorimetry and infrared spectroscopy as general technique to assess the structures. A further study indicated that a spirulan like polysaccharide PUF2 from old culture medium had an effective anticoagulant activity and the culture medium of A. platensis may constitute a cheap and abundant source of this biomolecule that has been shown to be more effective than dermatan sulfate of porcine origin. However, its obtention by ultrafiltration may represent an extraction procedure of interest (Majdoub et al. 2009). Authors indicated generally that the sugar components in the structure of the sulfated polysaccharide were rhamnose (49.7%) and uronic acids (32% of total sugar) as glucuronic and galacturonic acids.
3.3
Chlorella
Microalgae not only have the potential to replace the use of antibiotics, but also can improve the growth of cultured animals owing to their high nutritional value. The effects of polysaccharides of the unicellular green eukaryotic microalgae Chlorella vulgaris when used as a feed or feed additive have been studied. One of the interesting aspects of this study is that in the adopted conditions of growth of C. vulgaris, the accumulation of polysaccharides from was up to 32.7%. An enhancement of the disease resistance of Trachemys scripta elegans due to functional role of these molecules was also reported (Gui et al. 2019). Two different fractions of EPS from Chlorella vulgaris were studied (Zhao et al. 2018) in the context of their roles in protecting cells against environmental stresses, in particular about cell damage caused by nano-ZnO and capability to adhere directly to the outside of cells. The amount of bound and soluble types of EPS were generally quantified but no structurally analyzed. The influence of extraction methodology on the bioactivity of polysaccharides is known. Very recently, extracts of polysaccharides were reported from Chlorella vulgaris (Yu et al. 2019) in the context of a comprehensive study exploring the antioxidant properties that these molecules can offer. The study is focused on the preparation, and limited to yields, monosaccharide composition and molecular weight distribution relating to different extraction methods (freeze-thawing, microwave-assisted-, ultrasonic wave-, alkali-, hot water-, and cellulase-based). The alkali extraction method had the highest yield of the crude polysaccharides. Anti-oxidant properties were also studied in vitro and in vivo. Ultrasonic wave extraction produces extracts with activities superior with respect to other methods. Significant production of extracellular polysaccharide (EPS) during the cultivation of Chlorella vulgaris has been reported (Barboríková et al. 2019). The report’s
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preliminary findings at the structural level indicated interesting complexity regarding interglycosidic linkages as determined by methylation analysis. In particular, the dominant galactose has been found involved in six different types of linkages in both pyranose and furanose forms while the second most abundant sugar, arabinose found in six types of different linkages in furanose form. Rhamnose with five different types of linkages was found along with glucose, and mannose residues in minor amounts. Significant effects in the in vivo animal testing were reported: bronchodilatory, anti-inflammatory, and antitussive. These exopolysaccharides thus appears to be interesting agents for the prevention of chronic inflammation in many respiratory diseases. Interestingly in a strain of Chlorella isolated from the coldest and driest arctic ecosystems, three polysaccharides fractions were also studied recently (Song et al. 2018). A crude yield of ca. 10% dry weight is reported after optimization of extraction using the response surface methodology. The most homogeneous fraction, based on infrared spectroscopy, high-performance liquid chromatography, and 1H and 13C nuclear magnetic resonance, was shown to possess sulfate group and α- and β-type linkages, and composed mainly of galactose (66%), with variable amounts of rhamnose (10%), arabinose (16.3%), and glucose (6.8%). NMR (nuclear magnetic resonance) details allowed the exclusion of furan rings as the resonance at δ107–109 ppm for their anomeric carbons resulted absent. Indeed, the presence of C-1 signals (δ93.46–103.87 ppm) indicated that the sugar residues were all in the pyranose form: H-1 signal at δ5.19 ppm and the C-1 signal at δ99.03 ppm were likely due to α-galactose residues. The H-1 signal at δ4.06 ppm and C-1 signal at δ101.43 ppm suggested that the polysaccharide contained β-arabinose residues and the H-1 signal at δ4.83 ppm and C-1 signal at δ103.87 ppm indicated a β-anomeric configuration for (1!4)-linked β-D-glucopyranosyl units. In a recent study, crude biomasses of six different representative microalgae were analyzed for their monosaccharide composition: Nannochloropsis salina, P. tricornutum, C. vulgaris and D. salina, P. purpureum and S. ovalternus were investigated. Trifluoroacetic acid (TFA) hydrolysis of algal biomasses and application of a high throughput method for the identification of complex carbohydrates were used. The latter is based on the selective derivatization by 1-phenyl-3-methyl5-pyrazolone, UHPLC separation, and MS analysis of the derivatives (Ortiz-Tena et al. 2016) allowing the identification of uncommon monosaccharides. Usually found neutral sugars, uronic acids, amino sugars, methylated and phosphorylated or sulfated monosaccharides were identified. This methodology is promising both in general utilization and in strain screening to maximize biomass development, allowing the detection of rare sugars. Polysaccharide-based nanomaterials can be used as drug carriers or tissueengineered scaffolds, in theranostic approach as the material constituting nanotools for cancer treatments, in wound dressings, as antimicrobial agents and biosensors, etc. The use of polysaccharides for the synthesis of metallic nanoparticles is a growing scientific topic and microalgal polysaccharides can be used as templates for the growth and stabilization of nanoparticles. Since these molecules are rich in reducing groups they can bind and reduce metal ions; chelation and bioaccumulation of heavy metals performed by microalgae are indeed mainly due to them. Chlorella
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pyrenoidosa was included in a study of production and investigation of exopolysaccharides in silver nanoparticles preparation (Gallón Navarro et al. 2019). With a novel approach, the authors described the green-based production of these nanoparticles and their activity as antibacterial agents. Additionally, for the topic of removal of ammonium and orthophosphate from wastewater by algae-based techniques, the role of cadmium ions was studied using Chlorella vulgaris polysaccharides showing the roles of these carbohydrate molecules in protecting the microalga in high cadmium environment (Chen et al. 2015). The polysaccharide fraction of Chlorella vulgaris was evaluated against herpes simplex virus type 1 although no direct virucidal activity against the virus was noticed. However, water and ethanol extracts were able to inhibit the in vitro virus replication in a significant manner (Santoyo et al. 2010).
3.4
Nannochloropsis
Water-soluble polysaccharides from Nannochloropsis oculata were structurally analyzed, and tested for their immunostimulatory activity (Pandeirada et al. 2019); complex structural features were discovered in the hot water extracted polysaccharides after defatting the biomass. The polysaccharidic material was fractionated by ethanol precipitation, size exclusion chromatography, and anion exchange chromatography, and the usual chemical technology were used for interglycosidic linkage detection. Carbohydrate microarrays technology was also used in this screening analysis [Liu et al. 2009). Solid matrices coupled with only a few amounts of sample were probed with proteins with well-known carbohydrate binding specificities to elucidate the carbohydrate structures; glucans and mannans of known structures were used as polysaccharide controls. The complete analysis of polysaccharides from Nannochloropsis oculata revealed the presence of mixed-linked (β1!3, β1!4)-glucans and (α1!3)-, (α1!4)-mannans, an important asset in considering this organism as a source of dietary fibers. Additionally, a complex structure of anionic sulfated heteropolysaccharide composed of GlcA, Rha, Xyl, Gal, Fuc, and a sulfated heterorhamnan was also found. These authors indicated also potential immunostimulatory activity of these molecules.
3.5
Tetraselmis
In the context of an evaluation of nutritional potential and toxicological effects of Tetraselmis sp. CTP4 biomass, produced in photobioreactors at an industrial level, the presence of starch-like polysaccharides was declared (Pereira et al. 2019). Interglycosidic linkage analysis of polysaccharides showed the presence of 1,4-linked Glc (57 mol%) and 1,4-linked Gal (22 mol%). A content of 4.4 mol% of 1,4-Glc is substituted at C6 (1,4,6–Glc) confirming the presence of starch-like polysaccharides containing a percentage of branching residues. Similarly, as inferred by the
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presence of 1,3,4–Gal (2 mol%), Tetraselmis polysaccharides seem to also be constituted by a galactan, with 1,4–Gal linkage in the backbone and substituted at C3. As for Tetraselmis suecica a study of enzymatic hydrolysis for polysaccharide characterization appeared in the literature (Kermanshahi-Pour et al. 2014); the authors used this technique as an analytical procedure for the characterization of carbohydrate composition. This analysis shows that this microalga consists of approximately 42 wt % glucose as starch and 5 wt% 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) on a dry weight basis of microalgae when grown under specific conditions (i.e., varying nitrate concentrations). Kdo, a crucial part of the structure of lipid A-based lipopolysaccharides, is very expensive than glucose per mole and a complex challenge molecule for chemical synthesis. However, a robust and cost-effective extraction process must be developed for this compound from Tetraselmis suecica. From the marine microalgae Tetraselmis sp. KCTC 12432BP and KCTC 12236BP, potential biodiesel producers, water soluble polysaccharides were identified as antioxidant materials for industrial applications (Dogra et al. 2017), their monosaccharide composition was identified by using HPAEC analysis and the authors partially concluded about galactosyl and glucosyl interglycosidic linkages without further details.
3.6
Isochrysis
Three polysaccharides were isolated from the marine microalgae Isochrysis galbana (Sun et al. 2014a) by ion-exchange chromatography. Previous work found that this species was rich in polysaccharides found at level up to 25% dry cell weight. Structural insights were obtained by infrared spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), and 1H nuclear magnetic resonance. The most effective fraction concerning antioxidant activity possessed a molecular weight of 15.934 kDa and was shown to be composed by variable amounts of glucose, galactose, and rhamnose. Anomeric hydrogen signals in 1H spectroscopy allowed the identification as β-forms for glucose and galactose and α-form for rhamnose. From the same organism, the group of Usov isolated at a very interesting yield (44 mg of total neutral glucan from 500 mg of lyophilized cells of Isochrysis galbana of a pure highly branched (1–3,1– 6)-β-D-glucan (4 mg) (Sadovskaya et al. 2014). The structure of this pure glucan was analyzed by methylation and Smith degradation, as well as ESI and MALDI TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and current proton and carbon 1-D and 2-D NMR spectroscopy. Interesting inhibition of the proliferation of U937 human leukemic monocyte lymphoma cells has been reported with a claimed potential of anti-tumor activity.
3.7
Thalassiosira
An investigation on extracellular carbohydrates of Thalassiosira pseudonana was reported in a collective study for three species including Cylindrotheca closterium
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and Skeletonema costatum. It was mainly aimed at providing insights into the potential role of these species in mucilage formation of the Adriatic Sea. However, this study hardly reports any detail about structures of substances under analysis (Urbani et al. 2005). The authors have expressed production rates by gas-chromatographic analysis of the dissolved extracellular fraction, indicating glucose as the most abundant monomer in exponentially growing cultures. Of the three species analyzed that the highest abundance of polysaccharides was found in T. pseudonana followed by C. closterium and S. costatum. However, the composition varied according to the phase of growth with increase in other sugars (galactose, mannose, xylose, rhamnose, and fucose content) evaluated as storage glucans and heteropolysaccharides. For Thalassiosira pseudonana the released polysaccharides and proteins, as major components of extracellular polymeric substances (EPS), were also measured to assess the potential effects of quantum dots in the aquatic environments by the interactions between quantum dots and the microalga (Zhang et al. 2013). The hypothesis of this work is that toxicity of quantum dots can be ameliorated by interaction with EPS from microalgae; the authors measured the extent of dissolution/aggregation of quantum dots relating it to environmental factors using T. pseudonana in different nutrient conditions. The production of carbohydrates increased with the addition of quantum dots but it did not show any relationship with particle’s stability; thus EPS proteins might be more involved in the detoxification. An interesting study related to the degradation of carbohydrates in Thalassiosira weissflogii during various stresses has been also published (Suroy et al. 2015); an increase in the carbon content was essentially reflected in the carbohydrate component of the TW cells. Three monosaccharides ribose, glucose, and galactose exhibited the highest degradation rate constants and the authors correlated this aspect to the differences in their initial macromolecular origin (e.g., storage vs structural carbohydrates).
3.8
Dunaliella
Five polysaccharide fractions were isolated from Duanaliella salina and studied by means of high-performance chromatography with precolumn derivatizations (Dai et al. 2010), a technique allowing that some minute quantities of polysaccharide samples can be analyzed with high resolution separation and simultaneous determination of many kinds of component sugars. Two fractions were acidic heteropolysaccharides mainly containing glucose and galactose, respectively, one possessing sulfate groups, two others were glucans, and the latter was a complex of polysaccharide linked with nucleic acids by covalent bonds. A linear (1–4)-α-D-glucan has been identified in the microalgae Dunaliella tertiolecta in a work (Goo et al. 2013) framed to evaluate the potential for the production of fermentable monomeric sugars. The structural assignment was based on enzymatic hydrolysis by α-amylase (endo-type) allowing the authors to conclude
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that EPS from D. tertiolecta are quite different from those obtained from other Dunaliella species, the structure was also confirmed by NMR analysis. In a study (Santoyo et al. 2012) about antiviral compounds sourced from microalgae used as carotenoid producers both Dunaliella salina and Haematococcus pluvialis were investigated. Although Dunaliella extracts were found with less potency, in both cases the antiviral activity was found to be related to polysaccharides since carbohydrate-rich fractions isolated from extracts showed higher activity.
3.9
Phaeodactylum
The cell wall of the diatom Phaeodactylum tricornutum is poor in silica and mainly composed of organic molecules, notably, a sulfated glucuronomannan identified in a pioneering work (Husemann 1968). Structural insights were recently gained (Costaouëc et al. 2017; Yang et al. 2019) on this polysaccharide using modern technology showing that the backbone is a linear poly-α-(1!3) mannan decorated with sulfate ester groups and β-D-glucuronic residues. This compound could play a role in shaping the architecture of the frustule. These authors, discussing monosaccharide composition of other diatoms, suggested that sulfated glucomannan structure is conserved, although with variations in the amount and modality of distribution of sulfate ester groups and glucuronic residues branched on the α-mannan backbone, depending on the diatom species and their morphotypes. A microalgal polysaccharide-enriched extract of this microalgae was used in the study of modulation of immune response in fish aquaculture (Carballo et al. 2019). The data reported by authors indicate that orally administrated insoluble yeast β-glucans in the sole aquaculture modulated the immune response and controlled the Vibrio abundance in the intestine of fishes; since microalgal polysaccharide caused a transient systemic anti-inflammatory response, they concluded that these polysaccharides are a promising source of prebiotics for the sole aquaculture industry.
3.10
Porphyridium
The red microalga Porphyridium sp. was shown to be a promising source of new sustainable thickening agents that shows a higher intrinsic viscosity compared to commercially used hydrocolloids. The EPS are composed of galactose, glucose, xylose, and glucuronic acid and their physicochemical properties were related with a different molecular structural organization of monosaccharides and sulfate groups constituting the polymer when compared to cell wall polysaccharides. Purification of carbohydrate fractions to obtain polysaccharide extracts with low protein and salt contents remain the main challenge for commercialization of extracellular polysaccharides of Porphyridium sp. (Bernaerts et al. 2018a). The process of purification has been indeed studied by membrane technology (Marcati et al. 2014) and also more recently (Balti et al. 2018) reporting about exopolysaccharide concentration
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and purification. In the first study the method used allowed recovering both B-phycoerythrin and soluble polysaccharides from Porphyridium biomass. The authors of the more recent study showed that exopolysaccharides purified by a ceramic membrane filtration with high yield of recovery, were concentrated more than six folds. For this microorganism too the antiviral activity has been recently reported (Radonić et al. 2018) as due to the presence of EPS from P. purpureum. The action is due to their anionic nature and the production of these compounds, which is possible in photo-bioreactors, is of great interest being the whole process easily scalable. The influence of sulfate on growth of microalgae was also monitored along with the composition and antibacterial and antiviral properties of the exopolysaccharide from Porphyridium cruentum thus showing that enrichment of the culture medium with sulfate improved sulfate content of exopolysaccharides and they presented a relevant activity against Vesicular stomatitis virus (De Jesus Raposo et al. 2014).
3.11
Botryococcus
In an investigation recently published, the interest toward the microalga Botryococcus braunii has increased despite slow growth rates of the organism; indeed some strains were grown relatively efficiently in continuous cultures in controlled photobioreactors and studies about milking process (extraction and recovery of polysaccharides (García-Cubero et al. 2018) were conducted obtaining good EPS productivity. A previous study (Jin et al. 2016) was focused on the ways to improve the return of hydrocarbons production with co-valorization of the biomass residues, according to a biorefinery approach to producing compounds of industrial interest such as polysaccharides. In another report (Atobe et al. 2015), it was shown that the water-soluble polymers that can be released from the extracellular matrix of Botryococcus braunii by thermal pretreatment, before hydrocarbon extraction, have to be reduced to less than 0.5% for optimal hydrocarbon recovery. The water-soluble polymers are high molecular weight polysaccharides, mainly comprised of galactose, arabinose, and uronic acid. The authors suggest that these polymers are desirable as industrial emulsifiers and thickeners. A more in-depth study showed also the presence of a protein to which polysaccharides are linked studied using mass spectroscopy and bioinformatics approach (Tatli et al. 2018).
3.12
Chaetoceros
Some reports on polysaccharide material from this diatom of importance in biofuels since long time (McGinnis et al. 1997) can be traced in more modern literature. In a study on issues induced by algal blooms in fresh and saline waters, aimed to minimizing adverse effects in membrane-based water treatment systems, Chaetoceros affinis was included among species studied. The algal organic matter
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was shown mainly composed by biopolymers (e.g., polysaccharides and proteins) containing fucose and sulfated functional groups (Villacorte et al. 2015) by lectinbinding affinity. Similar studies on organic matter generated by marine algal species were also present (Chowdhury et al. 2016; Dhakal et al. 2018). A modern study using lectinbinding demonstrated that conspicuous setae of the diatom Chaetoceros were preferred habitats for Polaribacter and these microorganisms are able to bind and degrade sulfated fucose-containing polysaccharides coating the Chaetoceros setae (Bennke et al. 2013), an interesting study for possible insight on structural elucidation of microalgae polysaccharide material.
3.13
Chlamydomonas
The genus of green alga Chlamydomonas is important in the study of biohydrogen production. A study of polysaccharides present in Chlamydomonas reinhardtii is dated back to 1975 showing the presence of a polysaccharide of molecular weight of 41,500 containing molar proportion of monosaccharides: arabinose, mannose, and galactose. The latter in the polymer was shown to consist of a mixture of the D and L enantiomers in a ratio of 84: 16. No more than monosaccharide composition was reported though (Shii and Barber 1975).
3.14
Pavlova
The scientific interest for the species Pavlova viridis and other Chrysophyta is to be found in fish feed preparation. The two articles found on Pavlova viridis focused on the study and potential of antioxidant activity of polysaccharides extracted in water and of their readily absorbable degradation fragments (Sun et al. 2014b) and on their potential about immunomodulation and antitumor activity (Sun et al. 2016). As for molecular structural details reported in these articles, the authors did not go in-depth more than sugar composition and sulfate content although they concluded interestingly that all the polysaccharide fragments evoked a significant dose-dependent scavenging ability and they could be considered as a potential antioxidant and that the polysaccharides of Pavlova viridis had potential antitumor activities by improving immune response; the bioactivities, however, depend on their molecular weights.
3.15
Scenedesmus
Efficient biomass harvesting is one of the bottlenecks to develop a cost-effective process in microalgal cultivation technology. Flocculation is one of the processes used in which freely suspended cells are aggregated together to form large particles, and cells can then be easily harvested by sedimentation. The study of flocculant agents is then important as a promising technology for low-cost biomass recovery.
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Scenedesmus obliquus, a green microalga that has been widely used in fish feed, is a self-flocculating microalga recently studied (Guo et al. 2013). The bioflocculant polymers were studied for composition and consist of glucose, mannose, galactose, rhamnose, and fructose with the molar ratio of 8:5:3:2:1 but no more precise details on structures were furnished although the author stated that this is the first report on the characterization of flocculation agent from self-flocculating microalga.
3.16
Synechococcus
Two interesting articles sorted from literature search for this species for production of two polymers, hyaluronic acid and heparosan using a photo-biorefinery approach. In the first (Zhang et al. 2019), the authors explored Synechococcus sp. PCC 7002, considered as safe hosts for biotechnological applications, for photosynthetic hyaluronic acid production system. For bacterial engineered solutions (Bacillus subtilis and Escherichia coli strains overexpressing different HA synthases) in fact, substantial amounts of carbon feedstocks, such as glucose or sucrose are necessary for fermentative processes on which these systems are based. The work emphasizes cyanobacteria as promising hosts to produce biomedically relevant sugar polymers solely by photosynthesis. In the second article, the authors used Synechococcus elongatus PCC 7942 for production of heparosan (Sarnaik et al. 2019); after cultivating the cells were under different environmental conditions they found yielding maximum heparosan production to 2.8 μg/L, which is useful for production of high-value chemicals from low-cost inputs.
3.17
Conclusion
Even before their structures were known, polysaccharides, including starch and cellulose from a historical perspective, have always been exploited before complete structural detail assessment. Only after the 1970s a serious resurgence of the interest for this class of biomolecules, considered as renewable resources, occurred and this new interest reflected and paralleled the advent of the new analytical techniques since the 1950s. Gas chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy together with permethylation procedure and selective enzymatic digestion induced major progress in the structural definition of polysaccharides. Currently, concern about the quality of polysaccharides preparations is still present; necessary standardization for commercial exploitation (Han 2018) and the poor repeatability of the methods used in sample preparation and lack of information about chemical characterization are still hampering research and product development. Even in a recent overview (Gray et al. 2019) of advanced solutions for the carbohydrate sequencing challenge, the authors stated that “our understanding of endogenous carbohydrate structure and function still lags behind that of other biomolecules, despite carbohydrates representing the most abundant biological class.” Whole extract utilization, at least for external or cosmetic applications can be partly
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possible but in the field of drug development a more demanding situation is real; solving unavailability of supply and acquiring detailed structural and composition information are necessary. The main conclusion of this report is based on the two aspects depicted above. Many review reports were generally found on natural products from microalgae with few ones specialized for polysaccharide material and widely indicated in the general parts above. Microalgae culturing seems to be a very active field in development due to successes of the genetic modification and this could have a positive impact avoiding issues for seasonality of macroalgae production. However, extraction procedures for polysaccharides, which can induce changes in the composition and chemical structures, have to be assessed and standardized and articles on these aspects are also present in literature about new systems of extraction. It is worth mentioning the microwave-assisted and the ultrasonic based processes to increase the efficiency of water extraction following the principles of green chemistry. Even if this report has been based on a literature search since 2013, articles reporting fine structural details of this biomaterial are hardly present. In fact very rare exceptions are the articles with plenty NMR details. If present they are obtained on whole material (Chlorella and Isochrysis polysaccharides) or after enzymatic degradation (Dunaliella). Structural analysis of polysaccharides of Nannochloropsis oculata by a very modern approach using proteins with well-known carbohydratebinding specificities to elucidate the carbohydrate structures is worth mentioning. A linear poly-α-(1!3) mannan decorated with sulfate ester groups has been reported for the diatom Phaeodactylum tricornutum. Biomedically relevant and structurally known sugar polymers can be obtained by photosynthesis in Synechococcus.
4
Conclusions
From this search in literature and previously published review reports, it seems that the situation for polysaccharide from microalgae is reflecting the historical one, with focusing first on collection of data on possible exploitation of potential in applications (rheological features, bioactivities, etc.) where the use of crude or partially purified and hardly standardized biomass is relatively possible in different fields. Hope is that, in future, with interdisciplinary efforts, new knowledge on structural details will increase with possible extension of applications in more demanding fields.
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Microalgae Polysaccharides with Potential Biomedical Application
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Michele Greque Morais, Gabriel Martins Rosa, Luiza Moraes, Ana Gabrielle Pires Alvarenga, Jacinta Lute´cia Vitorino da Silva, and Jorge Alberto Vieira Costa
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Polysaccharides and Extracellular Polymeric Substances Founded in Microalgae . . . . . . . . 3 The Cultivation Conditions that Increase Polysaccharides or Extracellular Polymeric Substances Production in Microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Extraction and Purification of Microalgae Polysaccharides or Extracellular Polymeric Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Uses of Polysaccharides or Extracellular Polymeric Substances from Microalgae in Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Antitumoral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Antiviral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Anti-Inflammatory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Other Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Final Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Microalgae and cyanobacteria contain several biomolecules of commercial interest, including pigments, fatty acids, proteins, lipids, and carbohydrates. Among these, polysaccharides (PS) and extracellular polymeric substances (EPS) have been highlighted for their high levels of biological activity. In this context, this chapter aimed to not only highlight the main types, growing conditions, and downstream processes that influence the PS and EPS yield of microalgae but also M. G. Morais (*) · A. G. P. Alvarenga Laboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil G. M. Rosa · L. Moraes · J. L. V. da Silva · J. A. V. Costa Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_20
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to report on the promising biomedical effects of their applications. The Increased production of PS or EPS by microalgae can be obtained mostly by providing stress conditions through nutritional limitation and/or adverse extrinsic growth conditions. In addition, the separation and purification stages can influence the acquisition of these biomolecules. Therefore, this chapter demonstrates that PS and EPS of microalgae, especially when a high concentration of sulfate is present, can act efficiently as bioactive molecules in the health field compared to specific reference drugs. Keywords
Biomass · EPS · PS · Sulfated polysaccharides
1
Introduction
Microalgae are photosynthetic microorganisms with potential for applications in many areas, including nutrition and health, due to their biochemical compositions, with high concentrations of proteins, lipids, carbohydrates, fatty acids, phenolic compounds, and carotenoids. As such, biomass, extracted bioactive compounds, or biomolecules recovered from the culture medium are widely used in the food, pharmaceutical, cosmetics, and nutraceutical industries (Costa et al. 2020). Polysaccharides (PS), a type of macromolecule produced by microalgae, play an important role in cells as energy and structural reserve components, as well as being produced and excreted into the extracellular environment (Delattre et al. 2016; Rossi and De Philippis 2016; Phélippé et al. 2019). This last group of biopolymers has a complex structure that consists mainly of different carbohydrate monomers linked in several structural arrangements. In addition, these biomolecules contain substituents other than sugars, such as sulfate and pyruvate groups (Delattre et al. 2016; Rossi and De Philippis 2016). Microalgal extracellular biopolymers, or exopolysaccharides, exist as extracellular polymeric substances (EPS), extracellular polysaccharides (ECPS), released polysaccharides (RPS), capsular polysaccharides (CPS), and sulfated polysaccharides (sPS) (Bhunia et al. 2018). Although the morphology of these molecules is distinct, they can be classified into two groups: (a) those that form capsules, establishing strong molecular bonds with the outer cell surface; (b) viscous biomolecules that are weakly attached to capsules or released into the surrounding environment cellular (Han et al. 2014). Under ideal growing conditions, these biomolecules are produced at different stages of microalgal growth. However, an increase in the production of PS or EPS can be promoted by stress, such as changes in nutritional factors (Bafana 2013; Tiwari et al. 2020), salinity (Chentir et al. 2017), or light intensity (Ge et al. 2014). The complexity of PS and EPS has made it is difficult to characterize the structures of and bonds between monomers and other non-sugar substituents present in their composition. However, their rheological and biological properties have led
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to increased interest in their study, with views to apply them in several industrial sectors, particularly in the biomedical area (Delattre et al. 2016; Phélippé et al. 2019). The following biological activities stand out among those listed in the literature: antioxidant (Chen et al. 2016; Belhaj et al. 2017), antitumor (Sheng et al. 2007; Zhang et al. 2019b), antiviral (Huleihel et al. 2001; Mader et al. 2016), anti-inflammatory (Motoyama et al. 2016), anticoagulant (Majdoub 2009), and antimicrobial (Belhaj et al. 2017). Therefore, this chapter addresses the main types of PS or EPS, the cultivation conditions that influence their production, and the techniques used for their extraction and purification, as well as evaluates the biological activities of these biomolecules, obtained from freshwater microalgae and cyanobacteria.
2
Polysaccharides and Extracellular Polymeric Substances Founded in Microalgae
Polysaccharides (PS) constitute the largest portion of carbohydrate macromolecules in microalgae and cyanobacteria. They are found in the cell wall, can be excreted by the cell, and serve as reserve components (Rossi and De Philippis 2016). Eukaryotic microalgae are capable of synthesizing starch molecules (composed of amylopectin and amylose) in plastids. They form their reserve compounds in the chloroplast, unlike prokaryotes, which form these compounds in the cytosol. Cyanobacteria are capable of synthesizing glycogen (α-polyglucan), with particle sizes 1:200 and fusions yielded 9 antibodies none of which were the desirable IgG1 isotype. IgG1 is the most desirable isotype because of its ability to promote phagocytosis, complement fixation, and bind the Fc receptor in macrophages, neutrophils, and lymphocytes. Then the first vaccine for C. neoformans was developed utilizing tetanus toxoid (TT) to boost the immune response to GXM when they were conjugated together (Devi et al. 1991). Immunizing mice with the GXM-TT conjugate, unlike utilizing whole C. neoformans infection, yielded an antibody response in all of the immunized mice (Casadevall et al. 1992). From these mice, which had antibody titers >1:1000, 33 mAb cell lines were isolated and characterized (Casadevall et al. 1992; Mukherjee et al. 1993). A number of these antibodies represent some of the most robust molecular tools utilized in cryptococcal research to this day. They have been utilized to develop ELISAs (Casadevall et al. 1992), in immunofluorescence to identify the different layers of the capsule (Maxson et al. 2007b), and one mAb 18B7 went through phase 1 clinical trials as a possible therapeutic for cryptococcosis (Larsen et al. 2005). Enzyme-Linked Immunosorbent Assays (ELISAs) have been used as diagnostics and for biochemical analysis since their development in the 1970s. ELISAs exploit the interaction between antibodies and their substrates by binding the substrate – often a protein – to a polystyrene plate and probing with polyclonal sera or mAb. When C. neoformans mAbs were being identified the interaction between purified secreted polysaccharides and polystyrene plates was reported to be weak. To get around this, the first cryptococcal polysaccharide-specific ELISAs were indirect or sandwich ELISAs using sets of anti-GXM mAbs (Casadevall et al. 1992). Here the plate would be coated with a secondary antibody of one isotype – often IgM – then the primary mAb, followed by the polysaccharide, then another primary mAb or a different isotype – often IgG – followed by a tagged secondary to that isotype conjugated with an indicator like horseradish peroxidase (HRP) or alkaline phosphatase (AP). In fact, the ELISA for the quantification of polysaccharides currently uses is this double sandwich with two mAbs identified in the 1992 Casadevall study – 2D10 (IgM) and 18B7 (IgG1). Since this time the interaction between polysaccharides and polystyrene plates has been further investigated and found to be stable. More recently direct ELISAs with purified exopolysaccharides or capsular polysaccharide have been used to assay the sera of mice in response to polysaccharideprotein conjugate vaccines (unpublished data). In addition to these direct and indirect ELISAs, competition ELISAs are used to determine if two antibodies bind to the same epitope of GXM. Additionally, this method can be used to determine the epitope(s) bound by a sera from an immunized animal. Beyond the quantification of polysaccharide and specificity of sera, these mAbs have been used to further characterize the C. neoformans polysaccharide capsule. Work by Maxson and colleagues utilized γ-IR in conjunction with immunofluorescence to identify different regions of the capsule and further our understanding of its architecture (Maxson et al. 2007b). Immunofluorescence allows imaging of an entire cell while fluorescently labeling just the epitope bound by an antibody. In this technique secondary antibodies conjugated with fluorophores, or directly conjugate primary antibodies efficiently label their epitope. When two antibodies with different
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epitopes, and different fluorophores are utilized, the regions occupied by the different epitopes can be elucidated. In their work Maxson and colleagues observed that two mAbs 12A1 and 18B7 occupy different locations within the capsule, 12A1 remaining at the outer edge while 18B7 occupied a region inside of 12A1 but still a distance from the cell wall (Maxson et al. 2007b). This suggests that there are at least 3 regions of the capsule – the outer edge which is labeled by 12A1, a middle region which is labeled by 18B7, and an inner region which is not bound by either antibody. In the future there are a number of further promising experiments using immunofluorescence to characterize architectural features of the capsule like examining other strains of C. neoformans, as all of these experiments were performed with H99 which is known to express a mixture of GXM motifs. In a bottom-up approach, these mAbs can be used to further define the polymers that make up the capsule of C. neoformans. Recently the Casadevall and Oscarson labs have collaborated to produce synthetic oligosaccharide arrays based upon the motifs of GXM (Guazzelli et al. 2020). These glycan arrays are printed with each synthesized oligosaccharide at a variety of concentrations, and in triplicate allowing for more precise analysis of antibody binding. While the GXM motifs were described some time ago, our understanding of how these 4–9 residue oligosaccharides fit together to form megadaltons-in-size polymers has been elusive. Utilizing these synthetic glycan arrays has shown that a decasaccharide containing two M2 motif repeats is bound by many of the anti-GXM mAbs that have been identified (Guazzelli et al. 2020). As this technology, and our abilities to manipulate it, improves it shows great promise in revealing larger and larger epitopes. In the future perhaps even showing how the motifs come together in GXM capsular polymers.
4
Future Application and Technologies for the Study of Cryptococcal Polysaccharides
Though we have known about microbial polysaccharides since the early 1900s, the molecular toolbox for their analysis has remained small. This is not terribly surprising when one considers the architecture of fungal polysaccharides in particular. Cryptococcal polysaccharides are complex with as many as seven repeating units or motifs and polymers containing a mixture of these motifs, their hydrated structure and the number of branch points only add to this complexity. In the capsule, GXM is relatively neutral, despite the charged carboxylic acid moieties, or perhaps because of calcium bridges between them holding polymers together (Nimrichter et al. 2007). This precludes the use of charge to aid in isolation. As noted earlier, though size fractions have been used for rough isolation, analysis shows that cryptococcal polysaccharides do not segregate according to the molecular weight cut offs (MWCO) of filters. While size exclusion chromatography (SEC) has been used for preliminary analysis, we must assume that the same would be true for SEC as for MWCO. So how can we characterize these polymers? Recent work by Crawford and colleagues utilized a hydroxylamine-armed fluorescent probe to label the reducing end of cryptococcal polysaccharides (Crawford et al.
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2019). This probe, referred to as the oxime probe, showed carbohydrate reducing ends exclusively at the cell wall-capsule interface of whole C. neoformans cells (Crawford et al. 2019). Additionally, the oxime probe labeled both EPS and CPS from C. neoformans suggesting that isolated polysaccharides contain reducing-ends as well (Crawford et al. 2019). The oxime probe, along with a recently developed reversible fluorenylmethyloxycarbonate 3-(methoxyamino)-propylamine linker (F-MAPA) (Wei et al. 2019), present the cryptococcal polysaccharide field with new molecular tools. Both the oxime probe and F-MAPA reversible linker are fluorescent and UV reactive, allowing for the isolation of single polymers from mixed samples using High Performances Liquid Chromatography (HPLC) or their visualization through other methods like gel electrophoresis. In the future the application of these molecular tools, and hopefully the development of others, have the potential to propel our understanding of cryptococcal polysaccharides forward. Early studies to develop a vaccine against C. neoformans using killed whole cells or crude polysaccharide preparations showed no protection, while purified polysaccharide showed an increase in survival, all immunized mice eventually succumbed to the infection (Gladebusch 1958). In the 1990s a new polysaccharide conjugate vaccine against cryptococcosis was developed with the GXM-TT conjugate mentioned above which was developed from the serotype A strain NIH 371 (Devi et al. 1991; Devi 1996). Two methods were used to develop conjugates, both of which yielded antibodies in BALB/c mice (Devi et al. 1991) and were shown to protect against C. neoformans infection (Devi 1996). While the authors note that the GXM-TT conjugate vaccine shows promise and that human immunization seems feasible, there are no further publications to show that human immunization was attempted. A synthetic heptasaccharide based on the M2 motif on GXM (see Fig. 1b) was conjugated to human serum albumin (HSA) to yield the conjugate vaccine M2-HSA (Oscarson et al. 2005). Unfortunately, this vaccine did not elicit protective antibodies or improve the survival of immunized mice challenged with C. neoformans (Nakouzi et al. 2009). However, the antibodies produced in response to M2-HSA were predominantly IgG and mAbs were isolated for further use in characterization of the capsule (Nakouzi et al. 2009). The authors note that antibody binding to GXM seems to be conformational and therefore may require larger synthetic oligosaccharides. One of the major hurdles to overcome in the development of a vaccine to C. neoformans, or any pathogen, is the fact that cryptococcal polysaccharides elicit both protective and non-protective antibodies. Studies to develop a cryptococcal vaccine have shown that any potential vaccine must push the balance toward the production of protective antibodies and limit the production of non-protective antibodies. In the future, characterization of the immune response to any potential polysaccharide for vaccine development – either synthetic or native purified – is essential prior to conjugation and further development as a vaccine. The current COVID-19 pandemic has revealed the utility of passive immunity for the treatment of disease once again. Currently, convalescent plasma, serum antibodies from an individual who has recovered from the disease, is one of the leading treatments for patients suffering from COVID-19 (Sheridan 2020). With the number of anti-GXM mAbs identified it is no wonder that antibodies have also been explored
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as therapeutics for cryptococcosis (Casadevall 1998). A phase 1 clinical trial for passive immunotherapy with the mAb 18B7 in patients suffering from cryptococcosis was the first such trial for a fungal disease in humans (Larsen et al. 2005). Since most patients suffering from cryptococcosis are immunocompromised, enhancing the immune response with a directed mAb is a logical method to investigate. The study, which examined an array of doses from 0.01 to 1 mg/kg in HIV-infected patients showed that mAb 18B7 was well tolerated (Larsen et al. 2005). Although the study suggested these results were promising and warranted further development of 18B7 as a therapeutic, it was not pursued further. While 18B7 was not pursued as a therapeutic, there are further anti-GXM mAbs and the continued development for further mAbs during the course of conjugate vaccine testing that may undergo this same analysis and be pursued further. It is clear that passive immunotherapy with mAbs has great potential for the treatment of cryptococcosis in the future.
5
Conclusion
Expounding the history of C. neoformans lays bare the dramatic advancements that have been made in the last century plus of work. Though, it also clarifies how vast our lack of understanding remains. It is our hope that advancements in glycosciences will soon allow for the identification of the remaining glycosyltransferases necessary for the synthesis of both GXM and GXMgal along with the other necessary enzymes for the elongation and size regulation of these polymers. With the new molecular tools, like the reducing end-linked probes (described above) allowing for HPLC analysis of polysaccharides, it is now possible to define how the identified GXM motifs fit together into the longer polymers observed. Further, new isolation techniques to preserve the GXM structure and prevent dehydration while maintaining sterility (unpublished data) will shortly allow for direct comparison between EPS and CPS. These comparisons will in turn allow for the characterization of similarities and differences between the two and resolve the variability in biophysical measurements (see Tables 1 and 2). Additionally, advances in both solution and solid-state NMR now allow us to examine how dehydration affects cryptococcal polysaccharide structure and determine if these effects are reversible. Further, utilization of the vast collection of anti-GXM mAbs to clarify their specific polysaccharide epitopes promises to advance our understanding of the polymers which elicit protective mAbs. Finally, the combination of reducing-end probes, HPLC purification, and NMR will allow for the mapping of 6-O-acetylation (6-OAc) adducts on cryptococcal polysaccharides. The 6-OAc mark is critical for antibody binding, though molecular modeling studies show very little alteration in structure, so the mapping of 6-OAc is of great interest. Together the utilization of these molecular techniques shows promise for the development of new antibody therapeutics as well as more targeted cryptococcal conjugate vaccines. With new tools to interrogate capsular architecture the need for a method of categorizing strains beyond serotype becomes clear. Defining the connection between GXM motifs to build capsular polymers necessitates codifying the
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difference between a serotype A strain expressing a single motif and one, like H99, with mixed motif expression. The use of chemotypes, previously developed from NMR analysis of a cadre of C. neoformans strains, seems the best course of action. The use of chemotype reference for C. neoformans strains will additionally allow for the better identification of antibody epitopes as well as the development of conjugate vaccines. While it is clear that the study of complex, branched polymers like those expressed by C. neoformans are exceedingly challenging, a review of the recent developments are quite inspiring. This bevy of recent advancement makes it clear that cryptococcal polysaccharides sit on the precipice of another great expansion in understanding and hopefully new therapeutic developments.
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Bioactivities of Bacterial Polysaccharides
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Karina Cruz-Aldaco, Mayela Govea-Salas, Rafael Gomes-Arau´jo, Miriam Desiree Da´vila-Medina, and Araceli Loredo-Trevin˜o
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Polysaccharide Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Polysaccharide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 EPS Activities and Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Antibacterial Activity of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Antiviral Activity of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Anticancer activity of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Other Bioactivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Cholesterol-Lowering Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Antioxidant Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Immunomodulator Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Production and Extraction of Polysaccharides of Microbial Origin . . . . . . . . . . . . . . . . . . . . . . . 8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Microorganisms produce a wide variety of molecules that they use to communicate with the environment around them. In the case of bacteria, this interaction is mainly through membrane proteins and polysaccharides, especially through extracellular polysaccharides and these molecules, additionally, provide K. Cruz-Aldaco Department of Food Research (DIPA), School of Chemistry, Universidad Autónoma de Querétaro, Querétaro, Mexico M. Govea-Salas · M. D. Dávila-Medina · A. Loredo-Treviño (*) School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico e-mail: [email protected] R. Gomes-Araújo School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, Mexico Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, Mexico © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_30
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protective functions for bacteria. Of importance to humans, it is that these polysaccharides have various beneficial actions such as antitumor, antiviral, antibacterial, antioxidant, and even immunostimulatory activities. This last activity is of utmost importance because through it, the optimal development of the human immune system is favored. In exchange, the host provides conditions for the microorganism to thrive. Likewise, these polysaccharides have properties that make them of interest not only for their specific biological properties, but they can also be used in various areas such as food, biomedical, and pharmacological areas to improve texture properties or as a means of drug delivery. Research about these properties as well as their production could be of advantage to humans since microbial culture, if well performed, can be a safe and cheap alternative to obtain these molecules. Keywords
Biological activities · Lactic acid bacteria · Immunostimulant · Antiviral · Antitumor
1
Introduction
Each human is an ecosystem that maintains 1013–1014 microorganisms with which we cohabit. One of the ways that microorganisms have for this is the action on our immune systems since we have coevolved with commensal bacteria and it seems that they have an important role in physiological development and health functions (Mazmanian and Kasper 2006). Commensal bacteria have been proposed to evolve to improve host health; this may be through the polysaccharides of the microorganisms. In this chapter, we will see the influence that bacterial polysaccharides have on various biological activities, as well as the influence of their structure on these activities. First, the structure will be reviewed, then the various biological activities, and finally, techniques for production, purification, and extraction of these polysaccharides will be explored.
2
Polysaccharide Structure
Carbohydrates are abundant on the bacterial surface and they are the means for the interaction between bacteria and host. They include lipopolysaccharides, lipid and teichoic acids, peptidoglycans, glycoproteins, as well as capsular polysaccharides. Variations in the composition of sugars, ring shapes, bond positions, isomers, and the different conformations will define the action of these epitopes. For example, purified polysaccharides induce specific IgM responses, with no detectable IgG response. Protein-conjugated polysaccharides activate T-cell responses and this is used in vaccine development (Nikaido 1988; Upreti et al. 2003).
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Microbial polysaccharides are divided into homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Examples of the former are cellulose, dextran, mutate, alternate, pullulan, levan, and curdlan. Of the latter are gellan and xanthan. In LAB, HoPS repeating units consist of a single type of sugar, such as D-glucose or D-fructose, and form glucans and fructans. Conversely, HePS are composed of glucose, galactose, rhamnose and in some cases N-acetyl-glucosamine and N-acetyl-D-galactosamine but they can also have phosphate or some other units in their structure. These heteropolysaccharides, their yield, composition, and structure, seem to be influenced by the type of culture and fermentation conditions (Caggianiello et al. 2016). Bacterial polysaccharides can be divided into capsule polysaccharides, lipopolysaccharide (LPS), and exopolysaccharide (EPS) depending on where they are located. The exopolysaccharide is the first molecule that has contact with the host organism and can be divided into HoPs or HePs; therefore, there could be many differences between them (Zhou et al. 2019). The size and composition of polysaccharides influence penetration capacity and molecular binding. For example, in studies it was found that a fraction of lipopolysaccharide rich in galactose has greater anti-inflammatory activity toward macrophages than others without as much of this carbohydrate. This is because monosaccharides appear to be associated with the affinity of receptors on immune cells. Furthermore, the sulfate groups, their position, and quantity are responsible for the activity. This is seen in trials where the antioxidant activity of chemically sulfated and “native” polysaccharides was compared and it was found that the latter had greater activity, as well as the addition of Se in immuno-stimulating activities resulted in increased stimulation in comparison with native structures (Chaisuwan et al. 2020). These differences in the structure of bacterial polysaccharides vary according to the environment in which the microorganism is cultured or where they thrive, that is, by the pH, temperature, water content, and concentration of mono and divalent cations (Brandenburg et al. 2003). This is just one example of the influence of structure on the biological activities that different polysaccharides can have, especially exopolysaccharides (EPS) (Zhou et al. 2019). Bacterial polysaccharides can also associate with other molecules and form glycoconjugates, glycolipids, and glycoproteins. For example, glycolipids have different functions such as acting as receptors in cell membranes, for cell aggregation, etc. These also modulate the immune response and can be toxins. Modulating the immune response includes things like mononuclear cell activation. For their part, glycoproteins contribute to the stability of the structures; in addition, in these the carbohydrate content varies much more (Brandenburg et al. 2003). An example of these carbohydrates is an immunomodulatory polysaccharide, expressed by commensal bacteria, which is involved in directing the proper development of the mammalian immune system. This is produced by Bacteroides fragilis and is a zwitterionic polysaccharide (ZPS) that activates CD4 + T cells and with it you can correct some immune defects (Mazmanian and Kasper 2006). Unlike virulent polysaccharides, this symbiotic bacteria ZPS is a member of a family of health-promoting molecules (Mcpherson and Harris 2004; Hooper et al. 2003). This
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polysaccharide has a positive and a negative charge in each unit that is repeated. This polysaccharide was initially thought to consist of A and B polysaccharide (LPS), but was found to have more diversity. Among the important features is that each LPSA and LPSB molecule is positively and negatively charged in each unit that is repeated. This is unusual in bacterial polysaccharides since most are negatively charged or neutral. This feature is suggested to be crucial for T cell activation (Mazmanian and Kasper 2006), since when the bacterium is endophaged by an antigen presenting cell, this polysaccharide is lysed into smaller sections and the negative “side” is binded to the presenter molecules and the positive “side” of the ZPS is the part that is presented to the CD4 + T cell. As for LPSA, it consists of several hundred repeating units of a tetrasaccharide. It is a right-handed helix with two repeating units per turn. The molecule is covered with positive and negative charges, which alternate along the sides of the helical backbone and are exposed on the outer surface. This favors the interaction with other molecules such as with the major histocompatibility complex (MHC) in the peptidebinding groove of MHC molecules as described briefly above. The sugars that conform to the PSA vary depending on the strains studied (Mazmanian and Kasper 2006).
2.1
Polysaccharide Synthesis
These EPS are produced in four ways. (1) the extracellular synthesis pathway; (2) the ATP-binding cassette (ABC) transporter-dependent pathway; (3) the synthasedependent pathway, and (4) the Wzx/Wzy-dependent pathway, which is the “universal” pathway for the synthesis of EPS in lactic acid bacteria. For the purpose of explanation, these microorganisms will be taken as an example. They use the extracellular synthesis pathway to synthesize homopolysaccharides. In summary, this is accomplished with the action of various enzymes such as fructansucrase and glucansucrase that will bind the specific monomers. Once the polysaccharide is synthesized, the chains are released into the extracellular environment (Zhou et al. 2019). For the synthesis of heteropolysaccharides, various enzymes are involved, such as glycosyltransferases, polysaccharide length regulation proteins, polymerization and export proteins, while homopolysaccharides are synthesized by a single enzyme (Zhou et al. 2019). EPS production in LABs has been correlated with clusters of eps or cps genes, located in Streptococcus thermophilus or in Lactobacillus plantarum on the bacterial chromosome or in plasmids in species such as Lactococcus lactis and Pediococcus damnosus. These clusters include genes that encode regulatory and enzyme factors involved in EPS biosynthesis, polymerization, and secretion. These enzymes include glycosyltransferases, which are responsible for the characteristics of the assembly of the repetitive units of EPS (Caggianiello et al. 2016). Regarding the Wzx/Wzy-dependent pathway, it is almost exclusive for lactic acid bacteria. It is more complicated than the HoPS route and involves more enzymes and sites to synthesize the heteropolysaccharides that will have a greater variability in terms of their structure. The next description is adapted from Mazmanian and Kasper
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Fig. 1 Exopolysaccharide synthesis via Wzx/Wzy. (Adapted from Mazmanian and Kasper 2006)
(2006) and Caggianiello et al. (2016). The route consists of five steps. (1) Transport and phosphorylation of mono and disaccharides (in the example of Fig. 1, glucose and galactose), which can be carried out either by the phosphotransferase system (PST) assisted, which can also do phosphorylation or permease assisted with which a specific kinase must act. In case there is lactose or phospho-lactose, there must be a galactosidase to obtain glucose and galactose/phospholactose. All this in order to obtain monosaccharides that will be used to form the extracellular heteropolysaccharide. (2) Formation of sugar nucleotides. Phosphoglucomutase and galactose-1-phosphate uridyltransferase mediate the transition from glucose-6-phosphate and from galactose-1-phosphate to monosaccharides with the phosphate group at carbon 1 and subsequently to UDP-glucose, dTDP-rhamnose, UDP-galactose with the action of other enzymes. Once these units are “ready,” the polymerization begins. (3) The synthesis of repetitive units. The individual units, attached to an undecaprenyl diphosphate anchor located on the inner surface of the membrane, are assembled by several glycosyltransferases in sequence. (4) Translocation of the repeating units from the intracellular to the extracellular surface. This is done by means of a flipase (WzX). (5) Polymerization of repeat units and release of the long chain. Repeated units are catalyzed by an outer membrane protein (Wzy) to form a long chain, and the length of the chain is regulated by a regulatory protein in Grampositive bacteria or by a protein complex formed by the polysaccharide co-polymerase and the outer membrane families in Gram-negative bacteria; eventually EPS is released into the extracellular space. This process is shown in Fig. 1. The Synthesis of sugars occurs in the cytoplasm; they are assembled in the lipid carrier molecule undecaprenyl phosphate through monosaccharides transfer from nucleotide sugars by specific glycosyltransferases; then a flipase (Wzx) moves the repeating units from the inside of the membrane to the outside, where they will be polymerized by Wzy (Caggianiello et al. 2016). It is important to note that the enzymes that act in this process are many more than those shown in the illustration. For example, the system for incorporating lactose is different from that of glucose or galactose, as well as there must be an enzyme that breaks down lactose into its
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monosaccharides. In the same way, there is an enzyme that is responsible for converting sugars into rhamnose (steps 1 and 2). In step 3 the sugars are polymerized and then in step 4, with the Wzx/Wzy complex, the “base” polysaccharide is transferred to the outside and, finally, there the polymerization is carried out to form the final EPS.
2.2
EPS Activities and Uses
EPS is used in various areas such as cell protection, adhesion, and biofilm formation, but it does not serve as a carbon source. Examples of monopolysaccharides are cellulose, curdlan, dextran, levan, and pullulan, while for heteropolysaccharides they are alginate, haloglycan, mauran, and succinoglycan (Chaisuwan et al. 2020). Polysaccharides are associated with various mechanisms such as prebiosis, probiosis, tolerance to stress associated with food processing, and technological properties in food as well as biological activities. In lactic acid bacteria (LAB), the exopolysaccharides are homo- or heteropolysaccharides, which are consistent with repeating units of sugars or derivatives thereof and can be branched. In some cases, EPS increases the viscosity of your environment (Caggianiello et al. 2016). EPS is thought to protect bacterial cells against extreme conditions such as abiotic or biotic stress, including temperature, light intensity, pH, or osmotic stress. Also, EPS can influence cell adhesion and recognition mechanisms (Caggianiello et al. 2016). It has been found that the structure of different exopolysaccharides affects their prebiotic properties and this same structure can confer probiotic and technological characteristics to the strains that produce them (Caggianiello et al. 2016). EPS contributes to viscosity and rheology, which can be used for industrial applications. Likewise, EPS has been shown to have physiological and biological functions such as antioxidant, antibacterial, immunoregulatory function, anti-tumor, antiviral, among others (Zhou et al. 2019). Within EPS there are hyperbranched polymers (HBP). These, obviously, have different characteristics in terms of rheology, low viscosity, high solubility, high density compared to unbranched polysaccharides, and a greater number of functional groups. For example, Leuconostoc citreum b-2 produces a highly branched EPS consisting of a backbone of α- (1–6) -D-Glcp (75%) with 19% α- (1–3) branched and only with some α- (1–2) branching. This HBP has a water solubility index of 80%, which is high, and a water absorption capacity of 450%; therefore it has a potential application in the food, cosmetic, and pharmaceutical industries (Chen et al. 2019). The modification of these HBPs has been explored, so that they have properties that can form rubber films and are flexible at room temperature to have applications as biomaterials and as biodegradable films. This has been achieved, for example, in Leuconostoc mesenteroides NRRL B-512 F where the HBP contained up to 20% α(1–3) -D-Glcp and long branches, which had non-Newtonian and non-thixotropic behaviors in solution (Chen et al. 2019). One of the most popular and most widely used HBPs is xantham gum, which is produced by various species of Xanthomonas, such as X. arboreal, X. axonopodis,
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X. campestris, X. fragaria, etc. This gum has good solubility in cold water and the aqueous solutions have highly pseudoplastic behavior and have synergistic action with galactomannans. It has been used in biomedical areas as a material for drug administration and tissue engineering (Chen et al. 2019).
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Antibacterial Activity of Bacterial Polysaccharides
Bacterial infections and their complications are extremely important worldwide, resulting from bacteria adaptations and multidrug resistance by abuse of antibiotics. The development of antibiotic substitutes is an urgent need to explore new sources of natural compounds with antibacterial activity. Food poisoning and nosocomial infections are the main way to bacterial infection by Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Escherichia coli, Candida spp., Salmonella spp., Pseudomonas spp., Clostridium difficile, Acinetobacter baumannii, Vancomycin-resistant Enterococcus, among others. Naturally, bacterial polysaccharides (BPS) are produced to provide various benefits to bacteria, such as protection against toxic or antimicrobial agents, predation by protozoa, attack by phages, and evasion of the immune system by other pathogenic bacteria (Zeidan et al. 2017; Lynch et al. 2018). Several studies have shown the antibacterial activity of lactic acid bacteria-derived EPS against pathogenic bacteria. Li et al. (2014a), after studying antibacterial activity, by agar diffusion, of exopolysaccharide (EPS) from 23 LAB found that the EPS of Bifidobacterium bifidum WBIN03 and Lactobacillus plantarum R315 at 300 μg/mL had a great activity against C. albicans Z1, Cronobacter sakazakii ATCC29544, E. coli O157: H7, L. monocytogenes CMCC54007, S. aureus CMCC26003, B. cereus ATCC14579, S. typhimurium ATCC13311, and Shigella sonnei ATCC25931. Jeong et al. (2017) showed bactericidal activity against L. monocytogenes and S. enteritidis using 1% (v/v) of EPS from Lactobacillus kefiranofaciens DN1 isolated from kefir. An EPS isolated from Lactobacillus sp. Ca6 at 10 mg/mL exhibited antibacterial activity against pathogenic bacteria such as S. enterica ATCC 43972 and Micrococcus luteus, in an agarwell diffusion assay (Trabelsi et al. 2017). In a recent report, four LAB were isolated from different Tunisian spontaneously fermented foods and beverages, bovine and turkey meat sausages, date palm sap and cow milk, and molecularly identified as Leuconostoc citreum, Leuconostoc mesenteroides, Pediococcus pentosaceus, and Leuconostoc pseudomesenteroides, respectively. EPS extracted from these bacteria showed high antibacterial activity at 1 mg/mL with an anti-biofilm activity against E. coli of 90%, 88% to Enterococcus faecalis, and 86.9% to S. aureus (Abid et al. 2018). Shahid et al. (2018) showed the antibacterial activity of EPS isolated from Lactobacillus reuteri SHA101 and Lactobacillus vaginalis SHA110 isolated from gut samples of healthy poultry (hen) against E. coli ATCC 25922, S. typhimurium CMCC (B) 50115, and S. petrasii subsp. pragensis KY196531 by agar diffusion with 5 mg/mL of EPS. Nehal et al. (2019) showed bactericidal activity with a new EPS from Lactococcus lactis F-mou against B. cereus ATCC 10702, E. coli ATCC 8739, Acinetobacter baumannii ATCC BAA-1710, Proteus mirabilis ATCC 7002,
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P. aeruginosa ATCC 49189, S. aureus ATCC 6538, Enterobacter cloacae ATCC13047, and L. monocytogenes ATCC 19115 with concentrations of EPS between 6.75 and 31.9 mg/mL, and fungicidal activity against C. albicans ATCC 10231 with 24 mg/mL. With the same emphasis, Aullybux et al. (2019) isolated bacteria from Mauritius seawater, identified as Bacillus, Halomonas, Psychrobacter, and Alcaligenes species and showed antibacterial activity with EPS extracted from these bacteria against E. cloacae, S. typhimurium, E. coli, S. aureus, S. saprophyticus, and P. mirabilis. Conclusively, LAB-derived EPS show an excellent and irrefutable antimicrobial activity against Gram-negative and Gram-positive bacteria pathogens. This activity could help probiotics in colonization of the surface of the gastrointestinal tract due the hydrophilic surface and reduction of auto-aggregation, inhibiting pathogenic bacteria growth by competitive inhibition in the host (Zhou et al. 2019). Probiotic bacteria produce self-inducing factors, generally acyl-homoserine lactone in Gramnegative and oligopeptide in Gram-positive bacteria, which sensing the density or size of surrounding pathogenic bacteria allows to regulate the biofilm formation that plays an important role in the range of infections, virulence, and cell communications (Chen et al. 2016; Alayande et al. 2018). Moreover, LAB-derived EPS are classified as biomolecules foreign to pathogenic bacteria, yet are not permeated or transported intracellularly, so a possible in vitro antibacterial activity mechanism is that they combine with the biofilm-related signal molecules or glycocalyx receptors in the surface of pathogenic bacteria, disrupting cell communication that interferes in the biofilm formation and eventually generating the antimicrobial effect (Spano et al. 2016). The mechanism of antimicrobial activity of EPS was elucidated in vitro; however, the functional mechanism in vivo is more complex and involves more processes from probiotic host. Nerveless, antibiotics are the main antimicrobial medicine and the EPS represent a resource with a high potential antimicrobial medicine against pathogens with increasing antibiotics resistance in biomedical area.
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Antiviral Activity of Bacterial Polysaccharides
There are many diseases caused by infection with various viruses; it occurs in humans and animals, in which various treatment alternatives have been sought to those already determined as antiviral drugs or medical therapies. However, in recent years much attention has been paid to the effects of bacterial polysaccharides (BPS) on different viral diseases and infections in vitro. The main reported mechanisms of antiviral activity caused by metabolites of lactic acid bacteria such as BPS are the inhibition of late stages of viral replication, causing reduced infectivity; inhibition of adsorption/penetration of the virus into cells due to the interaction of the bacterial-metabolite with the virus; and stimulating an immune system response (Yang et al. 2017a). Such is the case of the exopolysaccharides of Lactobacillus casei that it has been reported they can stimulate the synthesis and accumulation of interleukin 12, promoting the activity of
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natural killer cells and the synthesis of immunoglobulins in the spleen, allowing the host to have a better immune response against the Influenza A virus infection (Jung et al. 2017). Biliavska et al. (2019) evaluated the antiviral activity of the exopolysaccharides of lactic acid Bacteria (EPS) against Human Adenovirus Type 5 (HAdV-5), the cause of respiratory, intestinal, and urogenital tract infection diseases. EPS were obtained and purified from the Genera Pediococcus, Leuconostoc, and Lactobacillus (from pickled apple, sauerkraut, and pickled tomato juice). They infected Madin-Darby bovine kidney cells with HAdV-5 (infection titer 7.0 107 PFU/mL) and evaluated the viability and antiviral activity with the EPS (375, 750 and 1500 μg/mL). They found that cell viability reduces only up to 17% and show antiviral activity due to the degradation of the viral particles, blocking viral DNA replication, observing through the analysis of the cell cycle by flow cytometry causing decrease of the titer of viruses. Moreover, there are reports that EPS obtained of Lactobacillus plantarum LRCC5310 (isolated and identified from traditional Korean fermented vegetable food kimchi) showed antiviral activity against Human Rotavirus (HRV) Wa strain in vitro. MA104 cells were infected with HRV (0.01 MOI) and incubated with de EPS to continue the cell culture. The virus was quantified by qPCR and a decrease of viral RNA copy numbers reducing to 7.56 log compared with the untreated control was observed with 1.95 mg of EPS. Also, they observed that EPS generated a high percentage of adhesion, interfering with the binding of rotavirus to MA104 cells in vitro. Likewise, antiviral activity in vivo was evaluated in BALB/c mouse model by oral infected dose (10 μL of 2 104 FFU as infective dose 50%/mL and 1 mg/mice) of EPS by 5 days. Then they were euthanized and histopathological analyzes of the intestine were performed. They observed that EPS treatment in vivo decreased epithelial lesions of the intestine, decreased diarrhea symptoms while it reduced virus replication in the intestine (Kim et al. 2018). Similarly, other studies related to L. plantarum, indicating its antiviral effects against herpes simplex virus 1 and 2, and influenza virus due to a stimulation of the immune response by the induction of interleukins and immunoglobulins in the host (Yang et al. 2017b). In addition, EPS produced by microorganisms present in the microbiota of the human body have been shown to have antiviral activity, as in the case of Lactobacillus gasseri CMUL57 isolated from vaginal microbiota that showed antiviral activity against herpes simplex virus type 2 and Enterococcus faecium NCIMB 10415 antiviral effect against influenza virus (Jung et al. 2017). Likewise, the gut commensal Bacteroides fragilis NCTC9343 and its capsular polysaccharide A (PSA) has reported important results of antiviral activity against herpes virus simplex virus 1, strain 17+ syn causing encephalitis, with Vero cell culture in vitro and in vivo with 129 WT and Rag2 / mice. They found that virus replication decreased and viability was not affected, in vivo they observed a decrease in the inflammatory response, even only mice not treated with PSA died, a decrease in brain stem inflammation was also observed, and EPS promoted an immune response with interleukins (IL-10) to control inflammation (Ramakrishna et al. 2019).
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Anticancer activity of Bacterial Polysaccharides
Nowadays, cancer is a disease that affects the entire population and is considered one of the leading causes of death worldwide. Therefore, various therapies of natural origin have been sought to generate fewer side effects. There are several studies that indicate that the metabolites like bacterial polysaccharides (BPS) produced by lactic acid bacteria (LAB) have anticancer and antiproliferative activity by activating various cell signaling pathways, it modulates the host’s immune defense mechanism, promotes apoptosis, inhibits the onset of carcinogenesis and blocks its progression, and can also interfere with the downregulation of genes involved in tumor cell proliferation (Deepak et al. 2016; Saber et al. 2017). Furthermore, the PS being obtained from natural sources, have been shown to have few side effects and low cytotoxicity compared to the anticancer effect it generates, so it could be considered a good alternative for an antitumor agent. Another mechanism of action of BPS is the stimulation of components of the immune system such as macrophages, lymphocytes, NK cells and the induction of interleukins and decreases angiogenesis because it inhibits the growth of the vascular endothelium (Ismail and Nampoothiri 2013). In colon cancer cells, the effect of various EPS obtained from Lactobacillus spp. isolated from various dairy products was evaluated, observing a downregulation of the ErbB-2 and ErbB-3 genes, indicating an anticancer effect caused by the treatment of EPS in vitro (Faghfoori et al. 2017). Other authors isolated and purified three EPS fractions of Lactobacillus helveticus MB2–1 from traditional fermented milk (LHEPS-1, LHEPS-2, LHEPS3), elucidated their chemical structure, and incubated them with BGC-823 cells from human gastric cancer. They found an antiproliferative effect with all EPS but with LHEPS-2 they observed that it had a better growth inhibitory effect of up to 31.8% at a dose of 600 μm/mL, while LHEPS-1 of 27% and LHEPS- 3 25%, reporting that this polysaccharide has a different structure from the others (Li et al. 2014b). Moreover, a dose-dependent effect was observed in colon cancer Caco and HCT 15 cells with EPS obtained from Lactobacillus acidophilus. They determined the toxic dose of EPS that was 5 mg/mL and evaluated the effect of EPS on the messenger RNA (mRNA) of cells, where they found a downregulated expression of vascular endothelial growth factor and hypoxia-inducible factor (HIF-1α and HIF-2 α), also an increase in plasminogen activator inhibitor-1. This suggests that EPS induces an inhibitory effect on the expression of genes related to tumor angiogenesis (Deepak et al. 2016). In addition, in another colon cancer line (HT-29) the effect of EPS on the apoptotic pathway was evaluated, also to the composition of various EPS obtained from L. delbrueckii ssp. bulgaricus B3 isolated yogurt and L. plantarum GD2, L. rhamnosus E9, L. brevis LB63 isolated from healthy infant feces. These authors report a significant effect dependent on the variation of the molecular composition of EPS and time dependent. EPS induced apoptosis at 24 and 48 h, decreasing Bcl-2 and increasing expression of BAX, caspase 3 and 9, L. delbrueckii ssp. bulgaricus B3 being the best inducer of apoptosis of up to 42%. These analyses suggest that the high mannose and low glucose composition of EPS influences the apoptotic effect in
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tumor cells, because it is a signaling pathway that is commonly altered in cancer (Tukenmez et al. 2019). In general, the anticancer activity of BPS is reported to be related to its structure, chemical composition, such as molecular weight or sugar composition, as well as in a dose-dependent and time-dependent manner with BPS and the type of cancer. It also depends on the signaling or metabolic pathway that is intended to be modulated, such as apoptosis, angiogenesis, necrosis, immunotoxicity, or expression of oncogenes.
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Other Bioactivities
Polysaccharides are widespread in plants, animals, and microorganisms, and display chemical diversity, physiological properties, and biological activities. As it was stated, they could be divided into capsule polysaccharides, lipopolysaccharide, and EPS according to their locations in microorganisms. EPS, a biopolymer synthesized extracellularly or secreted into the extracellular of the microorganism during its growth for their survival in the increased temperature and pH conditions. The bacterial polysaccharides are of great interest for their immunostimulatory, immunomodulatory, antitumor, antiviral, anti-inflammatory, and antioxidant properties. EPS contribute to their inherent physiochemical and biological properties and render a multitude of functional activities that can be exploited for additives in health product or therapeutic medicine and adjunctive therapy in curing inflammation. It also has immunostimulatory, immunomodulatory, antitumor, antiviral, antiinflammatory, cholesterol-lowering and antioxidant properties (Kanmani et al. 2013; Abdhul et al. 2014; Chen and Huang 2018). Bacterial polysaccharides are always anchored to the cell surface by lipids. They are nontoxic natural biopolymers, and are reported as a kind of effective free radical scavenger and antioxidants, playing a critical role in protecting against oxidation damage in living organisms. On the other hand, many diseases, such as asthma, chronic obstructive pulmonary disease, inflammation, diabetes, myocardial infarction, and cardiovascular diseases, are reported to associate with oxidative stress (Wang et al. 2016). Numerous polysaccharides are able to activate cellular components involved in host defense mechanisms. Extracellular polysaccharides purified from Aureobasidium pullulans SM-2001 (Polycan) (EAP) have exhibited favorable antiinflammatory activities, potent immunomodulatory activities in immunosuppressed mice, showing powerful immunomodulatory, antioxidant, and anti-inflammatory mechanisms (Lim et al. 2018).
6.1
Cholesterol-Lowering Effect
The bacterial EPS is either covalently associated with the cell surface or loosely attached, or secreted into the surrounding environment. From studies, it was found that certain EPS-producing probiotic strains could bind free bile acids, hence
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increasing their discharge after assimilation. EPS has been reported to contribute toward autoaggregation and cell surface adhesion, thus enabling the colonization of probiotics in the host. Probiotic EPS has been reported to possess hypocholesterolemic bioactivities; this could bring about the synthesis of new bile acids from cholesterol by the liver and thus lower the level of circulating cholesterol (Zhang et al. 2016; Kanmani et al. 2018; Sasikumar et al. 2017). Cholesterol-lowering effect, related to EPS produced by Enterococcus faecium, displayed the cholesterol-lowering property compared with a negative control group. Since cardiovascular diseases are one of the major causes of global mortality, a decrease in serum cholesterol may decline the incidences of heart diseases. Incorporation of E. faecium K1 EPS in functional foods may help lowering the raised cholesterol level. Similarly, in an in vitro assay, 45% cholesterol level could be reduced by an EPS from Lactobacillus plantarum BR2 (Bhat and Bajaj 2018; Sasikumar et al. 2017; Zhou et al. 2019).
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Antioxidant Effect
Exogenous antioxidants are frequently added to food to aid preservation; however, many synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene, may exhibit cytotoxicity. For this reason, more attention has been paid to natural nontoxic antioxidants in an effort to protect the human body from free radicals and retard the progress of many chronic diseases (Pan and Mei 2010). Antioxidant activity has different reaction mechanisms including free radical scavenging, reductive capacity, binding of transition metal ion catalysts, inhibition of lipid chains oxidation, etc., and it could be due to the presence of the hydroxyl group and other functional groups of EPS, which converts the free radical into a more stable form (Kanmani et al. 2018). There is an increasing attention in exploiting the EPS-producing LAB, as the antioxidant activity (Abdhul et al. 2014) exhibited equal scavenging activity compared to the known standard, ascorbic acid. Kodali and Sen reported the EPS of probiotic strain B. coagulans Rk-02 to have strong super oxide and hydroxyl radical activity in vitro. One mechanism of action of EPS may be alleviation of cancers through the antioxidant (free radical scavenging) activity (Kodali and Sen 2008). Wang et al. (2016) reported that the EPS of Paenibacillus sp. TKU023 showed the strongest antioxidant activity. The antioxidant activity of various natural polysaccharides was examined and suggested that the chelating ions, including Fe2+ and Cu2+, play role in inhibition of hydroxyl radicals but not directly.
6.3
Immunomodulator Effect
One of the major pharmacological effect of EPSs lies in the modulation of immune system response, and its diverse composition dictates different immunomodulatory properties and their derivatives can enhance the body immunity. A large number of
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experiments show that polysaccharides can play an important role in regulating the immune system as an immunomodulator and accelerator, activate T lymphocytes, B lymphocytes, macrophages, and other immune cells to exert their immune activity. The regulatory effects of polysaccharides on macrophages include the number, morphology, phagocytic capacity, and cell secretion capacity (Chen and Huang 2018). Lactobacillus paraplantarum BGCG11, isolated from cheese, produces EPS composed of glucose, rhamnose, galactose, and mannose and exhibits an antiinflammatory effect on peripheral blood mononuclear cells (PBMC), unlike the non-EPS derivative strains, which induce the higher proinflammatory response of PBMC. Polysaccharides have been recognized as molecules with anti-inflammatory activity (Caggianiello et al. 2016; Dinic et al. 2018). Therefore, in the light of the rising application of bacterial EPSs, here we demonstrate the novel ability of EPS CG11 to suppress a pain sensation via regulation of inflammatory response. L. paraplantarum BGCG11 produces ropy, big-size polymer around 2 106 Da, mainly composed of glucose and rhamnose with the traces of galactose and mannose, but further information regarding stability of the polymer is missing (Dinic et al. 2018). An indirect benefit is prebiotic action. A “prebiotic” is a “selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, and thus, conferring health benefits on the host” (Caggianiello et al. 2016). A prebiotic effect has been found in EPS produced by LAB. For example, it was found that with α-D-glucan produced by L. plantarum, which had little digestibility in artificial gastric juice, it had a prebiotic effect in vitro, which was corroborated by the low growth of non-probiotic bacteria such as Enterobacteriaceae. Exopolysacharides of Weissella cibaria, Weissella confusa, L. plantarum, and Pediococcus pentosaceus had a high resistance to gastric and intestinal digestion as well as a selective increase in beneficial bacteria, in particular bifidobacteria (Caggianiello et al. 2016). LAB’s EPSs have an important role in biofilm formation and adhesion to surfaces, allowing colonization of different environments. Furthermore, EPS from probiotic bacteria adhere to the intestinal mucosa. These bacteria are associated with the digestion of nutrients, development and maintenance of the proper immune functions of the intestinal mucosa. Additionally, several strains provide essential vitamins such as folate, biotin, vitamin K, and produce short-chain fatty acids that are used as an energy source by cells in the colon (Caggianiello et al. 2016). Intestinal epithelial cells (IECs) or dendritic cells (DCs) can communicate with the intestinal microbiota through their pattern recognition receptors (PRRs), which can detect microorganisms-associated molecular patterns (MAMPs). The interaction between MAMPs and PRRs results in the induction of signaling cascades, which will determine a molecular response against the detected microorganisms, for example: cytokines, chemokines, and the immunomodulation of antimicrobial agents. The cell wall of Gram-positive bacteria has several structures that are essential in the interaction between probiotics and host receptors (Caggianiello et al. 2016). Some EPSs have immunomodulatory properties, with potential effect on human health. Some polysaccharides have a protective role for cells against the intestinal
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environment. For example, an L. lactis EPS film has been reported to have a protective effect against host phagocytosis by murine macrophages (Caggianiello et al. 2016). The ability of EPS to activate immune responses is different between LAB species and strains; these differences are related to the structure and size of the EPS produced. For example, acidic HePs are characterized by having phosphate in their composition and induce an immune response. Those with high molecular weight, on the other hand, appear to suppress the immune response. In Lactobacillus casei Shirota, high molecular weight EPS induced the production of various cytokines by macrophages (Caggianiello et al. 2016). Biofilms are resistant to the intestine environment and produce extracellular factors that have both immunomodulatory properties and the ability to inhibit the growth of pathogens (Caggianiello et al. 2016). All of these effects, that is, colonization of the gastrointestinal tract increases both nutrient absorption and immunomodulating activity. In experiments with bacteriafree mice, it has been found that the proportion of CD4 + T, CD8 + T and B cells is much lower than in mice colonized by bacteria. That is, the lack of colonization of the intestine by beneficial bacteria results in a defective development of the immune system (Mazmanian and Kasper 2006). Not only does the evidence show that certain bacteria are beneficial to humans, but it also appears that the host provides the right conditions for their growth. Several epidemiological data show that there has been an increase in allergies in recent decades, especially in developed countries. This seems to be influenced by an “excess” of hygiene, that is, by reducing exposure to microorganisms, especially in early stages of life, so that later in life, diseases and exaggerated responses to relatively innocuous antigens develop (Strachan 1989; Willis-Karp et al. 2001). A “balanced” gut microbiota has been found to contribute to an adequate state of health, whereas disturbances in either the type and/or amounts of non-beneficial microorganisms result in allergies, dermatitis, asthma, inflammatory bowel disease, etc. These changes are related to diet and modern lifestyle. It was found in an experiment that children from a developed country compared to a developing country had less colonization of Bacteroides species, which had a higher incidence of allergies (Alm et al. 2002). Because many of the beneficial effects of bacterial EPS come from LAB, which are present in fermented foods, the consumption of these foods may be related to these same beneficial effects.
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Production and Extraction of Polysaccharides of Microbial Origin
Bacteria produce exopolysaccharides for cell protection from phagocytosis by protozoans or white blood cells and to protect them from stressful environments. They are also produced for cell adherence and biofilm formation (Nwodo et al. 2012). Polysaccharide production is normally highest under aerobic conditions. Therefore, more polymer is usually excreted during growth on solid media than obtained from
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comparable amounts of cells grown in liquid media. The composition of most EPS found in bacterial slime or capsule appears to be independent of the carbon and energy source provided for growth and polymer synthesis (Cerning 1990). Exopolysaccharides produced by microorganisms represents an industrially untapped market; some microorganisms can produce and excrete over 40 gL 1 of EPS in simple but costly production conditions (Donot et al. 2012). Submerged fermentation (SmF) and Solid-State fermentation (SSF) are the methods used for the production of intracellular and extracellular polysaccharides. Both methods have been used to produce the maximum amount of microbial EPS and improve their bioactivities and other properties (Li et al. 2016). Fungi, bacteria, and archaea are the microorganisms responsible for synthesizing extracellular polysaccharides (Chaisuwan et al. 2020). The former is the cultivation of microorganisms in a liquid medium, while the latter involves the culture of microorganisms on solid supports in the absence of or with small amounts of free water. The selection of a method should consider the nature of microbes, the available equipment, and the production yield (Nwodo et al. 2012). The factors affecting the yield of EPS are inoculum age, inoculum size, compositions of the medium, and the physical parameters of growth conditions (Chaisuwan et al. 2020; Jenny Angel et al. 2018). The medium’s compositions, especially carbon and nitrogen sources, mainly involve microbial growth and production of EPS. Glucose is a common monosaccharide used as the sole carbon source for the production in many species, but some species use other types of sugar for fermentation that produce a higher yield of EPS (Chaisuwan et al. 2020). Some microorganisms prefer another carbon source as sucrose (glucose and fructose) that is suitable for Agrobacterium radiobacter, Bacillus spp., Leuconostoc mesenteroides, and Weissella confuse (Chaisuwan et al. 2020; Jenny Angel et al. 2018; Seesuriyachan et al. 2014). In case of xylose, maltose, sorbitol, lactose, and galactose are preferred by Streptomyces nasri, Brevibacillus thermoruber, Candida famata, Mycoleptodonoides aitchisonii, and Paenibacillus polymyxa, respectively (Chaisuwan et al. 2020; Choi et al. 2011; Gientka et al. 2016; Radchenkova et al. 2011; Raza et al. 2011). During fermentation is important to have an organic or inorganic such as yeast extract and/or (NH4)2HPO4. This is the case for development of Bacillus velezensis and Brevibacillus thermoruber (Radchenkova et al. 2011; Moghannem et al. 2018). Solid-state fermentation is a useful method for EPS production of microorganisms, especially lactic acid bacteria. The cultivation of some microbes on a solid medium increases EPS production. Seesuriyachan et al. (2014) reported that dextran from Weissella confusa cultivated by SSF was 86.4 g/l in 40.8 h, while by SmF it was 63.8 g/l in 24 h. Although SSF gave a higher amount of dextran, it took a long time more than SmF. Weissella confusa produced a higher amount of dextran on a modified MRS agar more than in the liquid medium at optimized conditions (Donot et al. 2012; Chaisuwan et al. 2020). Once the process of obtaining EPS has concluded, it is important to remove the biomass that has been generated. Usually, biomass is the easiest to remove impurities and it can be removed by decantation, filtration, centrifugation, etc. In the case of
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centrifugation, it is required to separate microbial cells, and ethanol precipitation is a widely used technique for precipitation of the EPS (Chaisuwan et al. 2020). These stages are where the product is concentrated and further purified. Here, the product is concentrated and renatured to move onto the final purification stages. These depend on the required final product purity. Pharmaceutical products require high purity while industrial products require lower purity. Chromatography and aqueous two-phase extraction are popular techniques (Aguilar et al. 2019). Isolation of EPS generally presents a few problems when it is secreted as an exocellular slime. The lack of physical attachment between polysaccharide and cell permits the use of differential centrifugation to be employed. The main problem in such preparations is the high viscosity of the slime solutions, which hinders the deposition of the cells. Different slime preparations vary in their intrinsic viscosity and there are no general rules for centrifugal speeds necessary to obtain an adequate separation. The addition of electrolytes (salts) may be useful in the precipitation by neutralizing charges on the polysaccharide. If the polysaccharide is in capsule form it must be detached from the cells. Again, it is difficult to generalize, as capsules are much more readily removed from some strains than from others. Gentle stirring or mixing in a homogenizer may suffice, but sometimes more drastic procedures may have to be employed. Depending on the culture medium used or methods employed, the isolation of a polymer containing various contaminations and some degradation may also occur (Cerning 1990). The importance of the quest for new chemical compounds from natural resources and bacteria both for food additives and for use in medicine is an active field (Mason et al. 2010). The application of green technologies as Ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, and hot-water extraction are an excellent alternative for obtaining and recovery chemical compounds as the polysaccharides (intracellular polysaccharides and exocellular polysaccharides) that could be used in food industry, pharmaceutical industry, and medicine. These methods have gained increasing attention due their environmentally friendly process, higher extraction efficiency, cost effectiveness, and structure preservation ability (Liu et al. 2015). Ultrasound-assisted extraction is a process that uses acoustic energy and solvents to extract target compounds from various plant matrices and biofilms. The generally accepted explanation for solvent extraction enhancement by using ultrasound is the propagation of ultrasound pressure waves and resulting cavitation phenomena, whereby the collapse of cavitation bubbles and highly localized temperature breaks cell walls and releases the contents of the cell into the extraction medium (Ebringerová and Hromádková 2010). Alboofetileh et al. 2019 studied the extraction of fucoidan from Nizamuddinia zanardini. They applied enzyme, ultrasound, enzyme-ultrasound methods, and evaluated their implications on yield, chemical and molecular structure, anticancer, and immunostimulatory properties. The results showed that the enzyme-ultrasonic isolated fucoidan showed the maximum extraction yield (7.87%) while that obtained by ultrasonic had the minimum value (3.6%). Fucoidans were composed of different levels of carbohydrates (52.78–58.65%), proteins (6.98–8.91%), sulfates (21.78–29.6%), and uronic acids (0.42–1.08%).
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On the other hand, microwave heating (2.45 GHz) occurs as the result of the dissipation of electromagnetic waves in the irradiated medium. The dissipated power in a medium depends on the dielectric properties and the local time average electric field strength. So, there is a fundamental difference between microwave and conventional extraction (Mason et al. 2010). De Cássia Da Fonseca et al. (2009) used a microwave oven to accelerate the levan acid hydrolysis from Zymomonas mobilis envisaging the production of oligofructans for antitumor uses. They concluded that the use of microwave oven in the levan acid hydrolysis is viable and leads to a considerable reduction in the hydrolysis time (5 min), and obtained oligofructans or fructooligosaccharides with a degree of polymerization varying from 4 to 14. But the traditional production of EPS always been an excellent alternative. An example of classic EPS production is hyaluronic acid. Streptococci strains for hyaluronan production generally use glucose as a carbon source, which is significantly higher than typical yields for complex polysaccharides in lactic acid bacteria (Duan et al. 2009). Extracting hyaluronan from microbial fermentation broth is a relatively simple process with high yields. An additional and important advantage of microbial hyaluronan production is that microbial cells can be physiologically and/or metabolically adapted to produce more hyaluronan of high molecular weight. Therefore, microbial hyaluronan production using either pathogenic streptococci or safe recombinant hosts, containing the necessary hyaluronan synthase, is nowadays more and more preferred (Boeriu et al. 2013). Due to the characteristics of hyaluronic acid (units containing N-acetyl-D-glucosamine and glucuronic acid), high molecular weight and unique viscoelastic and rheological properties, the principal applications are in biomedicine and cosmetic industry (Kogan et al. 2007). The EPS of LAB, as already mentioned, have the capacity of modifying the rheological properties, texture, and “mouthfeel” of food products, so with that they can find applications in the food industry as viscosifiers, stabilizers, emulsifiers, or gelling agents. Furthermore, LABs are “generally recognized as safe” (GRAS), due to their history in food production. One of the most used polysaccharides is xanthan gum, produced by Xanthomonas campestris, of which up to 50 g / L has been produced, which modifies the rheology of food. These substances, called “gums,” can be added to yogurth or bread, and they can be cheaper than the use of vegetable hydrocolloids (Caggianiello et al. 2016).
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Conclusion
In conclusion, bacterial polysaccharides, specifically EPS, are of great interest not only for their biological actions but also for their possible industrial applications. EPS, due to its biological actions, can prevent the prevention of diseases that are detected by oxidative stress or by the effect of high levels of cholesterol in the blood. Likewise, since EPS is the means by which the microorganism interacts with the environment, this molecule is capable of promoting, directing the development, or modulating the action of the host’s immune system. Obviously, there are EPS that are not beneficial, but there are many more that can be used to improve the quality of life
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of humans. This is closely related to the increase in recent decades of diseases such as allergies to foods or substances that are normally innocuous and this may be related to “exaggerated” hygiene, as well as the use of antimicrobial agents in every day products such as soap and antibiotics, so exposure to microorganisms and their respective EPS have been diminished resulting in these health conditions. As it was seen throughout the chapter, many of these microorganisms have coevolved with humans and one of its most important effects is the stimulation of the immune system, mainly through the intestinal mucosa; therefore contact with microorganisms through of their EPS is very important to achieve a good development of the immune system. Likewise, many of these polysaccharides not only serve for this stimulation, to prevent diseases such as cancer, or to take advantage of their antioxidant, antiviral, and even antibacterial properties, but they may also be important in various industries such as food industry to achieve the development of pleasant textures and flavors in food or the pharmaceutical industry as an agent for drug delivery. All in all, microbial polysaccharides are a group of very interesting molecules that have a lot of different applications.
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Production, Extraction, and Purification/Isolation of Exopolysaccharides . . . . . . . . . . . . . . . . 3 Physicochemical Properties of Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Xanthan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Chitin and Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Other Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Surface Modification and Functionalization of Microbial Polysaccharides . . . . . . . . . . . . . . . 4.1 Copolymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Cross-Linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Applications of Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Environmental Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Biological and Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Tissue Engineering Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Concluding Remarks and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Polysaccharides are sugar derivative polymers with high natural abundance existing in microorganisms, algae, plants, insects, and animals, where they play vital role in realizing cellular functions. On the contrary to synthetic polymers, E. Umut (*) Department of Medical Imaging Techniques, Dokuz Eylul University, School of Healthcare, Izmir, Turkey e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_31
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polysaccharides stem as a renewable source of value-added chemicals that can be synthesized from microorganisms (bacteria and fungi) by fermentation without harmful side products, although with relatively higher costs. Most of the microbial polysaccharides are hydrophilic, biocompatible, and biodegradable, which make them used in food, cosmetics, pharmaceutical, and biomedical applications. Moreover, their physicochemical properties can be improved either by in situ changing the bacterial culture conditions or post synthesis surface modification, that is, cross linking to fulfill the requirement of more applications. In this review, the structure-function relationship of the most commercially used polysaccharides is summarized and different surface modification strategies are discussed with the focus on microbial polysaccharides. Recent advances in the applications of microbial polysaccharides are given with examples from literature. Keywords
Microbial polysaccharides · Surface modification · Food applications · Pharmaceutical applications · Biomedical applications
1
Introduction
Polysaccharides are long chained biomacromolecules constituted of repeating monosaccharide (sugar) units either in neutral, that is, glucose, galactose, fructose, rhamnose, or acidic form such as glucuronic- and galacturonic acid. Their molecular weight may change in a broad range starting from a few kDa up to tens of thousands of kDa, in which the same (homopolysaccharides) or different types (heteropolysaccharides) of monosaccharides are linked to each other in varying sequences mostly forming linear shaped polymers for low molecular weights (cellulose, chitin) and branched or helical shaped ones (κ-carrageenan, xanthan gum) for high molecular weights. Polysaccharides are found in natural sources like plants (starch, cellulose, pectin), seaweeds (alginate, agar, carrageenan), animals (chitin, chitosan, gelatin), and microorganisms (dextran, xanthan gum, gellan gum, curdlan, pullulan) (Shanmugam and Abirami 2019). In the living organisms they can be located either inside (intracellular polysaccharides) or outside of the cells (extracellular polysaccharides) that are attached on the cell membrane in the form of capsules, granules, or slimes (Ahmad et al. 2015). Intracellular polysaccharides, that is, starch, glycogen store energy for the cell, tend to be branched or folded upon themselves and are generally insoluble in water, whereas extracellular polysaccharides (EPS) are responsible for maintaining the structural integrity of the cells (like cellulose in plants and chitin in insects) and have linear, rigid structures. Another important biological function of the EPS is that they act as targeting mediators for transferring the communication signals between different cells by conjugating to lipids and proteins hence forming glycolipids and glycoproteins. These EPS are generally in the form of water-soluble slimes that are released (secreted) from cells naturally or under stress into the external cell culture medium, hence it is easier to isolate such
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Fig. 1 Scanning electron microscopy (SEM) images EPS found on cyanobacterial soil crusts. (Reprinted from Mager and Thomas 2011 with permission of Elsevier Inc.)
EPS as compared to the ones attached on cell walls (Jindal and Khattar 2018). Figure 1 shows electron microscopy images of different forms of EPS found on cyanobacterial soil crusts (Mager and Thomas 2011).
2
Production, Extraction, and Purification/Isolation of Exopolysaccharides
The microbial polysaccharides exhibit unique physicochemical properties which make them commonly used in food industry, pharmaceutics, regenerative medicine, and cosmetics (Jindal and Khattar 2018; Smelcerovic et al. 2008; Ng et al. 2020). For this reason the production of pure and high quality EPS from microbial sources has crucial importance. EPS are synthesized via fermentation, that is, bacterial growth though enzymatic activity in the presence of nutrients, where fermentation parameters like pH, temperature, oxygen concentration as well as the strain (type) of the microorganisms determine the production amount and quality of the EPS. For the synthesis of each type of polysaccharide there is one or few specific type of bacteria (see Table 1) and regardless of the type of EPS that will be produced, in terms of the nitrogen-rich nutrient (biomass) availability as a general rule the carbon/nitrogen ratio should be kept maximum in batch culture medium (Oner 2013; Zhang et al. 2015; Jiang 2013; Sarilmiser 2015). The production of EPS is followed by the extraction, purification, and isolation steps that can include different processes depending on the type of produced EPS. The extraction of EPS in slime form or as capsules that are weakly associated to the
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Table 1 Different types of microbial polysaccharides and corresponding bacterial sources Polysaccharide Dextran Xanthan gum Cellulose Glucan Gellan gum Alginate Curdlan Levan
Microbial source Leuconostoc mesenteroides Xanthomonas campestris, and Xanthomonas pelargonii Gluconacetobacter xylinum Saccharomyces cerevisiae Sphingomonas elodea, Sphingomonas paucimobilis Pseudomonas aeruginosa, Pseudomonas putida A. faecalis Halomonas eurihalina, Zymomonas mobilis
Reference Siddiqui et al. (2014) Niknezhad et al. (2016) Ruka et al. (2012) Hunter et al. (2002) Zhang et al. (2015) Chang et al. (2007) Jiang (2013) Sarilmiser (2015)
cell membrane is done by centrifugation or preferably by ultracentrifugation also in order to filter the residuals and biomass remained in the culture. The centrifugation speed and time may change depending on the viscosity and molecular weight of EPS and sometimes performed with the aid of thermal treatment. On the other hand, for the case of capsular EPS that is tightly associated to microorganisms some chemical pretreatments such as alkaline, sodium chloride, EDTA, or alcohol precipitation or additional thermal steps, that is, heat bath in saline, phenol-water mixtures, are required before centrifugation. After the extraction, the EPS should be still purified and isolated from the crude extract including proteins and the secondary metabolites, which is achieved by using columns, membranes, or chromatography techniques, that is, size-exclusion chromatography, liquid chromatography, and anion-exchange chromatography (Harding 2005).
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Physicochemical Properties of Microbial Polysaccharides
The microbial EPS are rendered with a large variety of physicochemical properties in terms of charge neutrality (neutral, anionic, or cationic), hydrophilicity, water solubility, and viscosity. Since the structural units of an EPS are the monosaccharides, at first stage these properties naturally depend on the types of monomers, their absolute configuration (D or L), and anomeric configuration (α or β). The monosaccharides that most commonly involved in microbial EPS are D-glucose, D-galactose, D-mannose; L-rhamnose, L-fructose; and N-acetylhexosamines-N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Equally important factor is the glycosidic linkages (1 ! 2), (1 ! 3), (1 ! 4) and the sequence of monomers. For example, helix structured EPS including β(1 ! 4) linkages between monosaccharides are mostly water insoluble due to the strong interchain hydrogen bonding and exclusion of water, while EPS with β(1 ! 2) linkages have flexible coil structure and show substantial steric hindrance (Venugopal 2011). Another aspect affecting the physicochemical properties is the substitution groups, that is, sulfates and phosphates. Microbial EPS commonly include acyl substituents like ester-linked acetate or ketallinked pyruvate, where in terms of the charge neutrality of the polysaccharide
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molecule the first does not contribute to the overall charge on the contrary to the second. Other ester-linked substituents have been also identified in microbial EPS such as hydroxybutanyl, propionyl, and glyceryl residues (Ahmad et al. 2015). The ring type (size) constituting the EPS chain, that is, pyranose (six carbon – one oxygen) or furanose (five carbon – one oxygen) also was shown to be effective determining the rheological and mechanical properties. Marszalek et al. have shown that the elasticity of some microbial EPS (dextran, pullulan, amylose) results from the force-induced deformation of the pyranose rings eventually transforming from chair-like structures to boat-like structures (Marszalek et al. 1998).
3.1
Dextran
Dextan consists of linear α(1 ! 6) and branched α(1 ! 3) glycosidic linkages between D-glucose units, where the latter distinguishes it from dextrin which have branched α(1 ! 4) linkages (see Fig. 2) (Ng et al. 2020). It is the first commercially available microbial polysaccharide and produced by the fermentation of sucrose via the so-called dextransucrase enzyme secreted from Leuconostoc, Streptococcus, and Lactobacillus bacteria. Dextran synthesized by Leuconostoc mesenteroides mostly include α(1 ! 6) glycosidic linkages with rare α(1 ! 3) bonded side chains and are readily soluble in water, stable under mild acidic and basic conditions (Smelcerovic et al. 2008). On the contrary, dextrans including 50% branching through α(1 ! 3) linkages are water insoluble. It is a neutral biopolymer and available in a large range of molecular weights between 10 and 50,000 kDa (Kothari et al. 2014). Dextran forms highly viscous liquids at the same time easily degraded in human digestive tract, which makes it a promising material as viscosifier, emulsifier, gelling, and texturing agent for food industry. On the other hand, it is not degraded in blood and filtered by kidney allowing it to be used as blood plasma expander. Moreover, it has
Fig. 2 Chemical structure of dextran. (Reused from wikimedia.org under common license)
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been reported that water-soluble dextran derivatives including some extent of sulfonate groups, carboxymethyl, and benzylamide show antiviral properties (Neyts et al. 1995).
3.2
Xanthan
Xanthan gum is one of the first FDA (US Food and Drug Administration) approved microbial polysaccharide. It has a molecular weight around 2000 kDa and produced by the enzymatic activity of Xanthomonas campestris bacteria either from sucrose or glucose. Xanthan consists of D-glucose, D-mannose, and D-glucuronic acid in the molar ratio of 2:2:1, respectively, with lots of trisaccharide side chains (Ng et al. 2020). In particular, repeating β(1 ! 4) linked D-glucose builds the backbone and α(1 ! 3) linked side chains include D-glucuronic acid placed between two D-mannose units with β(1 ! 2) and β(1 ! 4) linkages. The presence of D-glucuronic acid attributes anionic character to branched xanthan polymer, which eventually causes the charged side chains to wrap around the backbone leading to helix structures. Depending on this ionic strength xanthan polymer can also get easily hydrated and dissolved in water. The solutions of xanthan gum show relatively high viscosity at low concentrations and for this reason they are used as thickeners and emulsion stabilizers in food products.
3.3
Gellan
Gellan is a linear, anionic, hetero-EPS produced from the bacteria Sphingomonas paucimobilis, also called as Pseudomonas elodea. It is built by β(1 ! 4) linked D-glucose, D-glucuronic acid, and α(1 ! 3) linked L-rhamnose sugar units and natively contains two acyl groups linked as side chains. It exists in two- or threefolded helix structure and can be soluble in water forming elastic, thermally reversible gels depending on the concentration. These gelation properties can be altered by deacylation of gellan, particularly O-acetylation on one of the two glucose residues, leading to a harder, brittle, and thermally irreversible form. Gellan gum is first approved in Japan and used in Japanese foods as gelation agent instead of agar (Jindal and Khattar 2018). Then, it became widespread and drawn attention as a modifiable biopolymer, whose gelation properties can be controlled by changing pH and temperature. In this respect in pharmaceutics, gellan was also shown to be a promising drug carrier for oral, nasal, and ophthalmic administration due to its in situ gelling capabilities (Coviello et al. 2007; Rajinikanth and Mishra 2008).
3.4
Cellulose
Cellulose is the most abundant biopolymer in nature as it exists in microorganisms, plants, and animals. It can be synthesized in large amounts from Acetobacter xylinum in
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Fig. 3 Chemical structure of cellulose. (Reused from wikimedia.org under common license)
biofilm form under static culture conditions. Independent of its source, molecular formulation of cellulose is same as it consists of β(1 ! 4) linked glucan chains (Fig. 3). However, only bacterial cellulose organizes as three-dimensional network of entangled nanofibers, and unlike the mostly crystalline form plant cellulose which shows very high mechanical strength, it exhibits great elasticity and hydrophilicity (Trovatti 2012). Thanks to its nanoporous surface, microbial cellulose can absorb water as hundreds times greater of its dry mass. This high amount of water content when combined with its flexibility makes cellulose very similar to extracellular matrix in human skin. Therefore, cellulose carries the unique properties to be used in regenerative medicine as it can both accommodate the drug molecules and serves as a physical barrier against any external infection (Czaja et al. 2006).
3.5
Chitin and Chitosan
Chitin is the second most abundant polysaccharide in nature after cellulose and its chemical structure can be described similar to cellulose with the difference that instead of a hydroxyl group on each D-glucose unit there exists an acetyl amine group, that is, the so-called N-acetyl-D-glucosamine. Chitin can be obtained from different microorganisms such as Allomyces, Penicillium, Aspergillus, and Fusarium. As in the case of most microbial EPS, chitin has been reported to show antiviral, antimicrobial, and antitumor activity as well as wound dressing property due to its nanofibrous structure similar to cellulose (Trovatti 2012). Chitosan, a much more popular biopolymer employed in many applications (cosmetics, pharmaceutics, food, biomedical, energy storage, etc.), is obtained by the deacetylation of chitin with NaOH. It is composed of randomly distributed anhydro-N-acetyl-D-glucosamine and anhydro-D-glucosamine residues with different degrees of deacetylation. This deacetylation pattern together with the molecular weight spanning from 10 kDA to more than 300 kDa determines the different physicochemical characteristics of chitosan. On the contrary to chitin, the native chitosan indeed has some disadvantageous such as poor water solubility, low surface area, and low porosity (Rajoka et al. 2020). However, it includes both hydroxyl and amino groups hence can be easily N-, O-, or N,O-modified to many derivatives each of them possessing a
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forthcoming property, that is, enhanced water solubility and pH or temperature resistance depending on the requirements of diverse applications. Chitosan derivatives like carboxymethyl chitosan (Shariatinia 2018), diethylaminoethyl chitosan (Viegas de Souza et al. 2018), trimethyl chitosan (Mourya and Inamdar 2009), and N-dodecylated chitosan (Liu et al. 2001) have been used in tissue engineering, gene delivery, and targeted and controlled drug delivery. Functionalized chitosan also was shown to exhibit enhanced antibacterial, antiviral, and antitumor properties.
3.6
Curdlan
Curdlan is a microbial EPS secreted by the bacteria Agrobacterium biobar and A. faecalis. Its building blocks are β(1 ! 3) bonded D-glucans, which are again polysaccharides constituted of D-glucose with α- or β-condensation linkages. In the linear chain these monosaccharides are partially esterified with succinic acid forming succinoglucan (Wustenberg 2014). Some intermolecular (1 ! 2) or (1 ! 6) linkages were also reported, which leads to branched and cyclic form of curdlan, respectively (McIntosh et al. 2005). Curdlan is a neutral EPS in triple helix structure with molecular weights changing between 50 kDa and 2000 kDa. At room temperature curdlan is insoluble in water, alcohols, and most organic solvents; whereas it can be dissolved in water at elevated temperatures. When heated up to 55 C aqueous solutions of curdlan form thermo-reversible gels dissociating to singe chain helix and further heating above 80 C and cooling results in thermo-irreversible gel form as it is used in most of the food applications. Due to its water and organic binding capabilities curdlan is proposed to be used in drug delivery applications as well. Carboxymethylether and sulfate modified curdlan reported to show enhanced water solubility and biological activity, that is, curdlan sulfates exhibit anticoagulant and anti-HIV properties (McIntosh et al. 2005).
3.7
Alginate
Alginate, also known as alginic acid, is a linear anionic copolymer constituting of β(1 ! 4) linked D-mannuronic acid (M) and α(1 ! 4) linked L-guluronic acid (G) residues both in homopolymeric (M or G) and heteropolymeric (MG) sequences. Alginate can be extracted from marine algae (seaweed) or produced by microorganisms Azotobacter vinelandii and Pseudomonas aeruginosa as biofilms. In terms of the homopolymeric chain lengths, M/G ratio and molecular weight of alginate synthesized from bacterial sources and algal sources generally differ from each other (Ahmad et al. 2015). Another aspect, which determines the viscosity and elasticity, is the acetylation degree i.e. alginates from algal origin do not include acetyl groups unlike the microbial alginates. Alginates from two different bacteria also show differences among each other. For example, the one produced from A. vinelandii contains G homopolymer component and interacts with (generally used) Ca+2 metal ions producing stronger gels, whereas alginate synthesized from P. aeruginosa produce flexible gels in the presence
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of Ca+2 (Urtuvia et al. 2017). Owing to their high water binding capacity and gelation properties alginates are good candidates as blood expanders, drug delivery vehicles, and scaffolds for tissues. Moreover, they are used as stabilizer, emulsifier, and thickeners in food products.
3.8
Other Examples
Some other microbial polysaccharides that are commercially produced and employed in biomedical, food, and pharmaceutical applications are Pullulan, Levan, and Scleroglucan. Pullulan composed of α(1 ! 6) bonded maltotriose units, which are three D-glucose linked with α(1 ! 4) condensation linkages and produced from fungi Aureobasidium pullulans and Cryphonectria parasitica. It dissolves in water forming slightly viscous solutions that is stable in wide range of pH, but does not form a gel even in the presence of ions. In food industry pullulan is used as a low-calorie food additive, binder, thickener, and coating agent. Scleroglucan is another fungal polysaccharide as it name comes from the fungi Sclerothinia and composed of β(1 ! 3) linked glucan backbone with β(1 ! 6) linked glucose as side chain for every three glucan residues (Jindal and Khattar 2018). It is neutral and soluble in water, and aqueous solutions of Scleroglucan are stable in a wide range of pH, temperature, and presence of ions. Scleroglucan is very commonly used in cosmetics and food products. Levan is produced by Bacillus subtilis, Zymomonas mobilis, Halomonas sp. and consists of many β(2 ! 6) linked fructan, a polymer of fructose. It can be both in linear and branched structure with relatively low molecular weight, where the latter is more stable and forms spherical shape. Levan shows cholesterol-lowering and prebiotic activity in food industry besides its use in some pharmaceutical and cosmetic applications (Oner et al. 2016). Although they cannot be exactly classified as microbial polysaccharides, due to their importance in industrial use two polysaccharides are worth to mention here: carrageenan and hyaluronan. Carrageenan cannot be produced by bacteria; however, it is extracted by processing of marine algae Kappaphycus alvarezii and Eucheuma denticulatum. It has a sulfated D-galactose based linear chain alternatively linked by β(1 ! 4) and α(1 ! 3) linkages (Ficko-Blean et al. 2017). Depending on the number and the location of the sulfate groups it takes different names such as (kappa) κ-, (iota) I-, and (lambda) λ-carrageenan including one, two, and three sulfate groups per disaccharide, respectively. Only λ-carrageenan is soluble in water at room temperature forming viscous solution and does not form a gel, whereas other two forms of carrageennan can be dissolved in water at higher temperatures and form thermo-reversible gels (see Fig. 4) (Blakemore 2016). Although there are continuing discussions about its safety for human health (David et al. 2018), due to its gelling and protein-binding capabilities carrageenan is commonly utilized in food industry and in cosmetic products. Another carbohydrate polymer, which is most commonly used in cosmetic and biomedical applications, is hyaluronan, which is also known as hyaluronic acid (HA). HA consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked with alternating β(1 ! 4) and α(1 ! 3) bonds among each
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Fig. 4 Thermo-reversible gelation mechanism of carrageenan. (Reprinted from Blakemore 2016 with permission of Elsevier Inc.)
other. Its molecular weight varies between 5 and 2000 kDa. HA is found in all living organisms as a main component of extracellular matrix and is particularly concentrated in synovial fluid, the vitreous fluid of the eye, and umbilical cords (Necas et al. 2008). HA is highly hydrophilic and besides its main role as hydrating the tissues and lubricating the joints and muscles, it realizes many other functions like cell proliferation and migration, tumor development, metastasis, and inflammation (Seton-Rogers 2012). HA is present in the skin and soft connective tissues in high concentrations, which together with abovementioned functions makes it a perfect material to be used as skin moisturizer, dermal fillers, and surgical wound repairers in cosmetics. Due to its viscoelasticity maintaining function HA is used in the local treatment for osteoarthritis via intra-articular injection. HA solutions also serve as a viscosity-enhancing component of eye drops and facilitated in dry eye care in ophtalmotology (Necas et al. 2008). Cross-linked HA hydrogels are utilized in sustained and controlled drug delivery as drug carrier agents.
4
Surface Modification and Functionalization of Microbial Polysaccharides
Although most of the microbial EPS natively carry unique physicochemical characteristics, which make them promising materials for many abovementioned applications, their properties can be further improved or extended in order to suit the
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requirements of more specific applications. The modification of microbial polysaccharides can be performed yet in the synthesis step by changing the fermentation conditions, that is, pH, temperature, oxygen, and nitrogen concentration in bacterial culture. The strain (family) of the source bacteria shown to be also effective on the structure and physicochemical characteristics of secreted EPS, hence this opens the way of synthesizing high quality EPS with desired properties by manipulating the bacterial genes encoding the enzymatic pathways, with a process so-called metabolic engineering (Schilling et al. 2020). This strategy successfully applied to produce functionalized xanthan, cellulose, and alginates so far and the biggest challenge seems to be to clarify the individual roles of genes in biosynthesis of EPS. All microbial polysaccharides include many hydroxyl groups, while some of them include carboxyl and amine groups, where the presence of such reactive groups allows their chemical modification and/or attachment of different moieties, that is, proteins, enzymes, drug molecules, etc. Esterification of EPS can be performed with compounds including carboxylic acid or in the presence of coupling agent N,N0 -dicyclohexylcarbodiimide at room temperature (Smelcerovic et al. 2008). Periodate oxidation, that is, another way of modification of polysaccharides with compounds lacking carboxylic acid function, is shown to be a proper way of conjugating proteins without any loss in their pharmacologic activity.
4.1
Copolymerization
The forthcoming properties of synthetic polymers and EPS can be combined either with the graft copolymerization of the synthetic polymers onto the backbone of EPS or synthesis of new amphiphilic block copolymers (Volokhova et al. 2020). Some examples are combinations of HEMA, PEG, PMAA, PLLA with alginate, dextran, chitosan, and pullulan. Synthesis of polysaccharide-based block copolymers can be done via direct coupling (linking a preformed polymer) or by chain extension approach including insertion of a polymer into the polysaccharide chain. The latter can be achieved by the polymerization of monomers with radical polymerization, ring opening polymerization, enzymatic polymerization, and atom transfer radical polymerization. On the other side, the graft copolymerization can be via microwave, UV, and gamma-ray irradiation, radical and atom transfer radical polymerization. Since polysaccharides are highly hydrophilic, both polysaccharide-based graft and block copolymers tend to form self-assembled structures in water as to maximize the water-polysaccharide interface area (see Fig. 5).
4.2
Cross-Linking
Polysaccharides easily undergo enzymatic degradation in physiological media and for this reason they are natively not suitable for some biomedical applications, that is, drug delivery. Besides the abovementioned preparation of graft and block copolymers, another way to protect the carbohydrate backbone from rapid degradation is
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Fig. 5 In water self-assembly of graft and block copolymer based on polysaccharides
cross-linking. The cross-linked polysaccharides both show mechanical stiffness at the same time preserve their hydrophilic and gelation properties leading to the formation of hydrogels, which are three-dimensional polymeric networks that can imbibe water much larger than their dry mass. Cross-linking of polysaccharides can be done following chemical or physical routes. Chemical cross-linking includes covalent bonding of polymers using of a cross-linking agent (cross-linker), hence forms hydrogels with higher mechanical stiffness compared to the ones prepared by physical cross-linking. However, these cross-linkers are generally toxic and should be avoided before using in vivo (Ng et al. 2020).
4.2.1 Physical Cross-Linking Physical interactions used in cross-linking are relatively weak and reversible, which is favorable for some applications where polysaccharide hydrogels should temporarily preserve its structural integrity but easily decompose under the influence of stimuli (pH, temperature change, etc.) as in the case of stimuli responsive controlled drug delivery. First approach to physically cross-link the polysaccharide chains is to facilitate –OH, COOH, –NH2 groups on their backbones that can form hydrogen bonds. Since protonation/deprotonation strongly depends on the pH, temperature, and the used solvent, such parameters should be selected as to promote the weak hydrogen bonding during cross-linking. For the case of anionic and cationic polysaccharides however, polyelectrolyte complexation between two oppositely charged polysaccharides or cross-linking of anionic polysaccharides in the presence of
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Fig. 6 Different physical approaches used for the cross-linking of polysaccharides. (Reproduced from Hu and Xu 2020 with permission of Royal Society of Chemistry)
counter metal ions, so-called as ionotropic gelation, is a better strategy to choose. This approach is particularly useful for sodium alginate, where the alpha-Lguluronate (G) blocks can electrostatically bind to divalent ions like Ca+2 and Cu+2 (Hu et al. 2018). Both divalent Ba+2 and trivalent Al+3 were chelated with gellan and due to its additional positive charge as compared to divalent Ba+2, trivalent Al+3 can bind one more carboxylate group on gellan hence forming stronger Al+3/gellan network (Patil et al. 2006; Reddy and Tammishetti 2002). Another physical and well-known method to form hydrogels is crystallization by repetitive freezing and thawing of aqueous polysaccharide solutions. Almost all polysaccharides can be gelated with this approach, where the microcrystals formed by subsequent freeze-thaw cycles function as joints for entangled polysaccharides. Crystallization can be performed with one type of polysaccharide or in the coexistence of another polymer such as polyvinyl alcohol (PVA) (Xiao and Gao 2008). An alternative way to form entangled hydrogels is introducing hydrophobic chemical moieties or micelles as joints onto the hydrophilic polysaccharide. In this approach the hydrophobic interactions lead to the organization of resulting amphiphilic polymers in a cross-linked structure. Figure 6 depicts the physical cross-linking methods applied to polysaccharides (Hu and Xu 2020).
4.2.2 Chemical Cross-Linking On the contrary to physical interactions chemical cross-linking includes strong covalent bonding of hydroxyl, carboxyl, amide, and amine groups on the backbone of one polysaccharide with a complementary group on another polysaccharide or on a molecular cross-linking agent. Depending on which of these reactive groups are present on the polysaccharide to be studied, different cross-linkers are used such as glutaraldehyde (GA), glycerin, epichlorohydrin, ethylene glycol, triethylene glycol, polyethylene glycol diglycidylether, diviniyl sulfone (DVS), and N,N0 -methylenebisacrylamide (MBA) (Sahiner 2013). Most of these cross-linkers are hazardous and
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toxic hence their residuals should be totally removed after the synthesis in order to be used in biomedical applications. On this respect, a safer additive-free method is polymerization triggered by high energy radiation. In this technique, aqueous solution of polysaccharides are irradiated either by microwaves, ultraviolet (UV), or gamma rays, which both decomposes the water molecules and breaks the C-H bonds on polysaccharide chain creating radicals, which then initiate cross-linkages with another polysaccharide (Dave and Gor 2018). By this method simultaneous sterilization of the resulting hydrogels is also achieved; however, the irradiation conditions, that is, irradiation time and radioactivity level of the gamma source should be considered in order to prohibit any degradation (scission) of the polysaccharide (see Fig. 7). Finally, an efficient though rarely employed alternative method is enzyme mediated cross-linking. As in the case of radiation initiated polymerization, no additional toxic cross-linkers are needed in this relatively novel method. However, for certain type of polysaccharides there are specific enzymes that catalyze the crosslinking, where this substrate specificity and high costs limit the application of the method. Recently, preparation of hydrogels based on ferrulic acid (FA) grafted neutral pullulan has been reported by the cross-linking using laccase enzyme obtained from Trametes versicolor, a common polypore mushroom (Hadrich et al. 2020). Cross-linking plays a crucial role in the mechanical and swelling properties of hydrogels, where the latter is closely related with the amount of water (or solvent) that the polysaccharide gel can absorb. Hydrogels are porous polymeric networks and when immersed in water, swelling (volume expansion) of a dry gel starts by the penetration of water into these interconnected pores. First, all hydrophilic groups of polysaccharide matrix on pore walls are bonded with water (first hydration shell). Subsequently more water molecules get partially bonded on the first layer building a second hydration layer, which is in continuous chemical exchange with bulk-like water that fills the rest of the pore volume. Finally, an equilibrium state is reached when all the pores are saturated by water and the osmotic pressure is balanced with elastic retraction. Such swelling dynamics are strongly dependent on the pore size distribution, which in turn is related with cross-linking density and the type of crosslinker. By increasing the concentration of the cross-linking agent during gel formation, more points on different polysaccharide backbones are connected to each other, building smaller meshes – the repeating units of gel network.
Fig. 7 Radiation-induced cross-linking among polysaccharides. (Reproduced from Gull et al. 2018 with permission of Elsevier Inc.)
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Fig. 8 Representative sketch showing the multiscale structure of a hydrogel for drug delivery application. (Reprinted from Li and Mooney 2016 with permission of Springer Nature Ltd)
Mesh size, which should not be confused with pore size, is another parameter determining the permeability against water or any other solute that is trying to diffuse through the gel matrix. This is especially important in drug release kinetics in sustained drug delivery application with hydrogels such that well-balanced drug encapsulation capacity and drug diffusivity depends on the mesh size (see Fig. 8) (Li and Mooney 2016).
4.3
Functionalization
Polysaccharide-based hydrogels often should be further modified and functionalized according to the special requirements of each different application. For example for wound dressing application in regenerative medicine, the hydrophilic nature of the polysaccharides is not favorable since it is repelled by the hydrophobic cell surface. Incorporation of many cell adhesion sites through the hydrogel network and further modifications to promote the cell proliferation is needed, besides a good mechanical strength and stretchability. On the contrary, for stimuli responsive drug delivery application hydrogel matrix should be degradable as to release its drug content under the influence of external stimuli (Gholamali 2019). This could be achieved by introducing cleavable bonds and groups on to the polysaccharide backbone, such that these bonds and groups break through hydrolysis by physiological enzymes or by the change of pH and temperature. For both procedures if transdermal application is planned, then the hydrogels should be injectable as well, that is, in the form of viscous liquid in vitro and gelation under the skin again by the effect of environmental conditions like pH and temperature (Liu et al. 2018).
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There are also numerous publications reporting the functionalization of polysaccharide-based hydrogels with a large variety of inorganic materials like metals, metal-oxides, magnetic nanoparticles, carbon nanofillers (graphene, graphene-oxide, carbon nanotube), and clays (Mergen et al. 2020). These materials are added into the gel matrix either by direct material blending during gelation or enzymatic incorporation, electrospinning, and electropolymerization. The main purpose of such studies is both to improve the intrinsic properties of hydrogels, that is, mechanical strength, elasticity, swelling capacity, and to design multifunctional composites, in which these improved properties are combined with individual forthcoming functions of the inorganic materials hosted in biocompatible gel matrix (Zheng et al. 2015).
5
Applications of Microbial Polysaccharides
5.1
Environmental Applications
One of the biggest sources of environmental pollution is the heavy metal ions like Cobalt (II), Nickel (II), Zinc (II), Aluminum (III), Chromium (VI), Mercury (II), and Lead (II), which are revealed during many industrial applications as side products and contaminates into the waste water, ground water, and soil. These heavy metal ions are threatening the living bodies’ health even in trace amounts, such that clinical reports showed development retardation, kidney damage, various cancers, and death cases for human patients who are continuously exposed to these ions. Among the recent technologies used for the clearance of heavy metal ions, ion-exchange, cation precipitation into inert form, and using of nanomaterials have certain limitations and include contaminants, which themselves indirectly cause pollution. On this respect, removal of heavy metal ions by microbial polysaccharide biosorbents stems as much cheaper and green chemistry approach. Many microorganisms (algae, fungi, bacteria) as part of their defense system are already able to remove the toxic metal ions by biosorption thanks to the EPS on their cell walls. These EPS have hydroxyl, carboxyl, amine, amide, sulfonate, and phosphonate groups that can bind the metal ions through different mechanisms like physical adsorption, ion exchange, complexation, and precipitation (Muthu et al. 2017). As mentioned above, the number of metal-binding sites and hence the affinity to metal ion uptake of EPS systems can be further improved by preparing nanocomposites based on polysaccharide hydrogels functionalized with magnetic nanoparticles, carbon nanotubes, titanium dioxide, zeolites, and clays benefiting the enhanced surface-to-volume ratio and porous structure. Among microbial polysaccharides chitin, chitosan, cellulose, and alginate are the most commonly studied ones for heavy metal ion removal due to the presence of multiple functional groups on their backbone (Na et al. 2020). For example Gokila et al. synthesized chitosan and alginate nanoparticles for Cr(IV) removal from wastewater and by investigating the adsorption kinetics they showed sorption capacity reaches maximum at pH ¼ 5 (Gokila et al. 2017). In another study, preparation of carboxymethyl chitosan (CMC)/sodium alginate (SA) hydrogel
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Fig. 9 CS-PGMA-PEI nanofiber membranes for Cr(VI), Cu(II), and Co(II) removal. (Reprinted from Yang et al. 2019 with permission from Elsevier Inc.)
nanocomposites that are functionalized by graphene oxide modified magnetite (Fe3O4) nanoparticles was described. It was shown that (CMC/SA/graphene oxide@Fe3O4) magnetic beads can successfully remove Cu(II), Cd(II), and Pd (II) ions and have 90% of the adsorption rates even after five cycles (Wu et al. 2019). A kind of different approach for the heavy metal removal was reported by Yang et al., where they prepared poly(glycidyl methacrylate) (PGMA) grafted chitosan electrospun nanofiber membranes, whose surface is further functionalized with polyethyleneimine (PEI) (Yang et al. 2019). They reported high adsorption rates and reusability of these CS-PGMA-PEI membranes for Co(II) and Cr (VI) removal (Fig. 9).
5.2
Biological and Pharmaceutical Applications
Native polysaccharides or its derivatives are known to exhibit many biological functions like antioxidant, antibacterial, antiviral, anticoagulant, antidiabetes, and antitumor activity. Unnecessarily high amount of reactive oxygen species, that is, superoxide anion, hydrogen peroxide, nitric oxide, and hydroxyl radicals, which are generated by cells during reduction of the molecular oxygen to water for energy production, cause oxidative stress, aging, and decrease in immune functions. Microbial EPS have the ability to remove these excess reactive oxygen species and in this
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manner said to carry antioxidant and antiaging activities (Yu et al. 2018). EPS were also shown to have hypoglycemic activity by increasing plasma insulin levels and decreasing plasma sugar levels, hence are used as a main component of several antidiabetic herbal medicines. Sulfated EPS, due to their anticoagulant activities, are good alternatives to heparin, the commonly used drug in medicine for preventing the blood coagulation, avoiding its severe side effects like thrombocytopenia and thrombosis syndrome. Regarding the antiviral activity, again mostly the sulfated EPS reported to inhibit the rapid replication of a wide spectrum of enveloped viruses including herpes simplex virus (HSV), human immunodeficiency virus (HIV) and influenza virus (Boission-Vidal et al. 1995). Nevertheless, among the different bioactivities of polysaccharides, maybe the most promising and investigated one is their antitumor/anticancer activity. It was demonstrated that EPS opposes to cancer in more than one way: i) inhibiting the growth of tumor cells and metastasis, ii) triggering the tumor cell apoptosis through different signaling pathways, iii) enhancing the immune functions by activating macrophages, T cells, and natural killer cells, and iv) potentiating the effect of chemotherapy drugs, where these functions were shown to be dependent on many factors like the molecular size, branching degree, type, and sequencing of monosaccharide units and number of sulfate groups. The long list of biological functions of EPS can be further extended, that is, involving hypocholesterolemic-, hypoglycemic-, hypolipidemic-effect, prebiotic activity (Liu et al. 2015). One of the most promising pharmaceutical application, in which the microbial polysaccharides are effectively used, is drug delivery. Drug delivery concerns with concentrating only the required quantity (dose) of drugs at target-specific locations with minimized side effects on healthy tissues both in spatial and temporal manner. The mechanical and compositional similarity of polysaccharide hydrogels to native extracellular matrix, when combined with advantages of nanoscale, that is, possibility of targeting to the close proximity of diseased tissues and enhanced surface-to-volume ratio promoting much higher amount of drug upload/release, make hydrogel micro/ nanoparticulate systems excellent candidates for drug delivery. Such polysaccharidebased micro/nanogels are prepared via emulsion cross-linking, reverse micelle, spraydrying, coacervation/precipitation, and emulsion droplet coalescence methods (Smelcerovic et al. 2008). The drug molecules that are incorporated into the gel matrix either by encapsulation or chemical binding can be released instantly by the influence of external (magnetic field, electric field, irradiation) or internal (pH, temperature, osmolality) stimuli via so-called stimuli responsive controlled drug delivery. As an example, pH-responsive xanthan gum and sodium alginate including hydrogels loaded with hydrophilic drug hydrocortisone sodium succinate (HSS) were site-specifically targeted to colon and used for ulcerative colitis therapy (You et al. 2015). However, in most cases, as similar in cancer therapy with chemotherapy agents, drug release should be realized in a time-controlled sustained process that the body can tolerate the released amount of drug in a certain time interval (sustained drug delivery). In contrast to other class of drug delivery vehicles, which mostly release its total drug content at an instant generally by the decomposition of their drug carrier matrix, polysaccharidebased hydrogels can do this job with a release rate (starting from a few days up to a few weeks) dependent on the diffusivity of active molecules or water through the gel
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network. In drug delivery, EPS-based hydrogels were applied in different particulate formulations such as micro/nanoparticles, liposomes, films, and membranes, and applied in almost all types of drug administration ways like oral, nasal, ocular, and transdermal routes (Li and Mooney 2016). Chitosan, alginate, and dextran are the most frequently studied EPS, where a wide spectrum of drugs including insulin, calcitonin, melatonin, paracetamol, ibuprofen, ampicilin, 5-Fluorouracil as well as proteins and genes were loaded on to the different forms of the hydrogels composed of these EPS (see Fig. 10) (Suner et al. 2020). Gellan due to its gelation tendency in tear fluid is utilized as drug carrier in ocular drug delivery. Despite their non-gelating characteristics in native form, derivatized and functionalized pullulan and hyaluronic acid (HA) are also used as drug delivery systems. Recently, Sahiner et al. synthesized HA-based hydrogel microparticles, which are loaded with several cancer drugs, corticosteroid, and antibiotics by encapsulation and conjugation methods. After performing blood compatibility tests and drug release studies in physiological conditions, they showed that prepared HA-microgels show sustainable and up to 300 hours long-term release for 5-fluorouracil, mitomycin C, doxorubicin, dexamethasone, and ciprofloxacin (Sahiner et al. 2020).
Fig. 10 Synthesis of drug-loaded biopolymer microparticles. (Reprinted from Suner et al. 2020 with permission from Springer Nature Ltd.)
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Tissue Engineering Applications
Actually in some extent being closely related with the field of abovementioned control drug delivery, tissue engineering and regenerative medicine define the replacement or repairing of damaged soft tissues often due to the injuries, burns, or infections. Traditional approaches for the treatment include surgical transplantation of healthy tissues obtained again from patient’s own body or from a donor, which is subject to considerable level of risks like tissue rejection and infections (Ng et al. 2020). For this reason, soft biopolymers that will inhibit the infection, trigger the proliferation of a small number of residual healthy cells, and eventually promote the formation of a new tissue on the wound are highly appealing. Microbial EPS have unique advantages due to their biocompatibility, biodegradability, antiinflammation, and antibacterial activities, and hence can be utilized in tissue engineering applications. There are already some commercial products like chitin-based Beschitin ® and oxidized cellulose-based Surgicel®, which are proved to accelerate postoperative wound healing and promote dermal regeneration in addition to anticoagulant activity (Smelcerovic et al. 2008). Owing to their additional porosity and mechanical stability, hydrogels prepared from EPS can be promising materials as tissue mimicking scaffolds. However, scaffolds should adhere on the hydrophobic surface of extracellular matrix containing integrin – a transmembrane protein responsible for cell-cell or cell-extracellular matrix adhesion; therefore the entirely hydrophilic EPS should be further derivatized and functionalized with integrin binding ligands, that is, collagen, laminin, fibronectin, and vitronectin. Such connections in microcellular environment are crucial for the signaling pathways can take place, which are necessary for cell viability, cell differentiation hence a good integration with surrounding tissues. Chitosan-collagen scaffolds were widely employed in cartilage, skin, and stem cell tissue engineering. Recently, injectable stem cell loaded alginate-laminin hydrogel microparticles showing in-situ gelation property were introduced for potential use as scaffolds in breast regeneration after lumpectomy (Yang et al. 2021). In the last decade parallel to the developments in biomedical technology, namely in three-dimensional (3D) printing of biopolymers, tissue engineering was pushed one step forward such that the first applications of skin replacements in this field are now extended to the fabrication of hydrogel-based scaffolds for bones, cartilage, blood vessels, and even organs. At his point, medical imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) provide highresolution detailed structures (3D maps) of the bones and tissues that will be printed. Most microbial EPS-based hydrogels suffer from the lack of mechanical strength required for 3D printing. Therefore particle-, fiber-, anisotropic filler-based reinforcements or another type of polymer/hydrogel (interpenetrating networks – IPN) are introduced in the host EPS-based hydrogels in order to tune the mechanical and rheological properties as to suit for printing purposes (Jang et al. 2018). Among different EPS chitosan, alginate, gelatin, hyaluronic acid based hydrogels, which are reinforced by the addition of nanocellulose, gellan gum, PEG, PLA, collagen, fibrinogen, hydroxyapatite, and laponite were frequently used as scaffold materials.
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Fig. 11 Three-dimensional printing of chitosan hydrogel-based scaffolds. (Reproduced from Zhou et al. 2020 with the permission of Royal Society of Chemistry)
These composite polymers, mostly in viscous solution form, are layer-by-layer deposited according to the 3D model using laser-based, nozzle-based, or inkjet printer-based 3D printing methods (see Fig. 11) (Zhou et al. 2020).
5.4
Food Applications
Food quality and safety is one of the most important aspects regarding the public health. Chemical ingredients used in the food products should meet the allowed limitations put by health authorities such as the FDA (US Food and Drug Administration). On this respect, naturally occurring polysaccharides that are inherently biocompatible and biodegradable have crucial importance in healthy and additive-free food production. Lots of attention are also focused on the taste, flavor, and mouthfeel of the food products, which are closely related with the texture and viscosity of these highly complex mixtures including proteins, lipids, fat acids, vitamins, minerals, sugar, and of course water. Another concern of the food industry is also to design food with unique textures to fulfill the requirements of some special groups of consumers, who have difficulty consuming normal food, that is, gluten-free, porcinefree, bovine-free, dietary products. Microbial polysaccharides are tasteless, odorless, and water soluble, hence can change the texture of the products by manipulating its rheological properties without interfering the original taste. Most of the foods exist in emulsion form, that is, composed of two or more nonmiscible components like oil-water, hence depending on the type and fraction of these components they can have very different viscosities from fluids (milk, fruit juice) to creamy (sauce, salad dressing, mayonnaise) and elastic materials (butter, cheese) (Yang et al. 2020). Here
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the low viscosity products suffer from time-dependent precipitation or separation of different phases and microbial EPS are used as emulsion stabilizers (emulsifiers), which ensure the preservation of the emulsion during shelf life. Dextran, alginate, and emulsan are the most commonly used emulsifiers that can significant increase the viscosity of added product even at very low concentrations (0.1%–1%). Gelation is another forthcoming characteristic of EPS in terms of applicability to food products. Thermo-reversible gelation is often required in the preparation of confectionary products, jellies, and jams. As gelling agent gelatin, which can be obtained from bovine and pork, is the most commonly used polysaccharide in the food market, but it is not preferred by vegetarian or Islamic consumers. For this reason gellan, xanthan, carrageenan, and agar are used as alternative gelling agents in food industry (Jindal and Khattar 2018). Recently, frozen foods are even more popular among consumers, so that it is practical and almost all types of products (meat, fish, vegetables, bakery, etc.) can be stored as frozen food. However, melting of the ice crystals present in frozen food matrix results in the loss of texture and softening. One way to overcome this quality retrogression is to reduce the amount of freezable water, namely the free water fraction in the food matrix. Due to their highly hydrophilic nature and water binding capacity, microbial EPS can do this job serving as water-binding agents and preserve the moisture in the food matrix during many freeze-thaw cycles. Dextran and curdlan were often employed as ice inhibitors in different kinds of frozen food products. The last but not least application of microbial EPS in food industry is the edible coatings on foods. This application is mostly based on the biofilm formation capability of some derivatized EPS and oriented to increase the shelf life of products. One typical example is the sausage casings made of cellulose or collagen, which are used in other protective coatings as well. Edible coatings are also used on some confectionary products both for protecting and decoration purposes. Finally, as a recent trend in food industry one should also mention the utilization of EPS-based hydrogel microparticles in fat replacement in dietary products, regulation of gastrointestinal functions, and satiety control. In this relatively new field, as similar to the case in drug delivery application different nutrients, reduced fat or prebiotics are encapsulated on microgels, which then release its content on target sites in digestive tract through swelling-deswelling (shrinking) mechanisms. The use of such colloidal microgel suspensions in food products was shown to increase the mouth-feel and satiety, while preserving the necessary sensitive and volatile ingredients in the product (Shewan and Stokes 2013).
6
Concluding Remarks and Future Perspectives
As discussed throughout the chapter, owing to their biocompatible, hydrophilic, and gellation properties together with suitability for further functionalization, microbial polysaccharides always have been popular in soft matter science. Especially in last two decades, developments in cutting-edge-technology characterization methods, such as small angle x-ray scattering (SAXS), x-ray crystallography, cryogenic
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electron microscopy (cryo-EM), and magic-angle spinning (MAS) NMR spectroscopy, allowed scientists to reveal the structure-function relationship in polysaccharides. Meanwhile, by the introduction of novel chemical approaches polysaccharidebased nanocomposites utilizing the size advantageous and enhanced surface-tovolume ratio in nanoscale was synthesized and replaced their bulk counterparts in biomedical, pharmaceutical, cosmetic, and food applications. Nevertheless, there are still remaining challenges in the commercialization of polysaccharides mostly related with the production costs. Commercial microbial polysaccharides are produced through fermentation by heterotrophic bacteria, which unfortunately require expensive organic substrates. Limited geographic availability of natural sources, nutrients, difficulties in cultivation, and downstream processing are other drawbacks in front of high purity large amount production of polysaccharides. At this point, cyanobacteria, which obtain energy via photosynthesis and able to grow also on inorganic media, are cheaper alternative sources of polysaccharides. Cyanobacterial polysaccharides have no noteworthy differences in monosaccharide composition found in EPS extracted from fungi, plants, and animals. Although their production amount is lesser than other bacteria and fungi, they show diverse functionalities which are similar to other microbial polysaccharides, and hence have the potential to substitute them in industrial applications (Bhatnagar and Bhatnagar 2019).
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Antioxidant and Antibacterial Activities of Polysaccharides
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Types of In Vitro Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Antioxidant Activity of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Mechanism and Factors Affecting the Antioxidant Activity of Polysaccharide . . . . . . . . . . . 5 Antibacterial Activity of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Mechanism and Factors Affecting the Antibacterial Activity of Polysaccharides . . . . . . . . . 7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Polysaccharide is a high molecular weight polymer, consisting of at least ten monosaccharides mutually joined by glycosidic linkages. Polysaccharides, present in almost all organisms, are important functional biological macromolecules because of their significant benefit to human health such as antioxidant, antidiabetic, immune potentiation, antitumor, anti-inflammatory, and hypoglycemic activities. More and more studies have shown evidence that polysaccharides have the capacity of scavenging free radicals and may be potential natural antioxidants. The antioxidant activity was found to be determined by multiple factors, including molecular weight, monosaccharide composition, sulfate position and its degree. To address this issue, this chapter summarizes the latest discoveries and advancements in the study of sources, chemical composition, structural characteristics, antimicrobial and antioxidant capacity of polysaccharides, and gives a detailed description of the possible mechanisms. S. C. Mohan (*) Division of Phytochemistry, Shanmuga Centre for Medicinal Plants Research, Thanjavur, Tamil Nadu, India A. Thirupathi Faculty of Sports Science, Ningbo University, Ningbo, China © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_32
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Keywords
Antioxidant activity · Polysaccharides · Chemical composition · Structural characteristics · Antibacterial activity · Mechanism · Factors
1
Introduction
Polysaccharides are polymeric carbohydrates, formed by repeating units joined together by glycosidic bonds. They have widely been investigated due to their chemical and biological activities (Rubavathi and Ramya 2016). Polysaccharides are also widely used as emulsifiers, gelling agents, thickeners, and fat replacers in functional food, cosmetics industries, and biological medicine, including drug delivery and tissue engineering (Bais et al. 2005). Polysaccharides present a large diversity of structures attributable to their variety in composition, substitutions, and glycosidic bonds. Polysaccharides widely distributed in plants, animals, fungi, yeasts, and algae have attracted considerable attention for their biological activities. Marine algae macromolecules include sulfated polysaccharides such as carrageenan and agar from red marine algae; alginate, fucan, and laminarin from brown marine algae; and cellulose and ulvan from green marine algae (Shannon and Ghannam 2016). Polysaccharides have proved to be potential sources of natural antibacterial, antioxidants, immunomodulatory, antitumor, hepato-cardioprotective, and neuroprotective compounds (Xie et al. 2016; Wei et al. 2015; Rjeibi et al. 2019). They have been increasingly applied because they are sourced naturally, and they impart less toxicity, biodegradability, and fewer side effects than synthetic ones. Antioxidant activity, a common bioactivity of natural-derived polysaccharides, was widely investigated for the prevention and treatment of diseases associated with oxidation damage (Wang et al. 2016). It is widely believed that the antioxidant activity of natural polysaccharides was influenced by raw materials, extraction procedures, and drying methods (Zhu et al. 2012; Zhao et al. 2015). The chemical mechanisms behind polysaccharide antioxidant activity have not systematically been elucidated to date. However, as literature suggest, the higher degree of sulfation in polysaccharides might be attributed to the observed antioxidant activity (Ananthi et al. 2010). Polyphenols bound to polysaccharides might also contribute to the antioxidant activity. Further investigation is needed to separate and further purify these polysaccharides and to identify their sequence, monosaccharide composition, and other structural properties. This chapter aims to review the antioxidative polysaccharides and summarize the possible mechanisms.
2
Types of In Vitro Antioxidant Activity
Many different in vitro models have been introduced to evaluate the antioxidant activities. The hydrogen atom transfer (HAT)-based methods usually measure the ability of quench free radical by hydrogen donation, that is, oxygen radical
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absorbance capacity (ORAC), total radical-trapping antioxidant parameter (TRAP), inhibition of induced low-density lipoprotein (LDL) oxidation, total oxyradical scavenging capacity assay, and so forth. On the other hand, single electron transfer (SET)-based methods detect the ability of transferring one electron to reduce any compound, including metals, carbonyls, and radicals, and result in a change in color when this compound is reduced, such as ferric ion reducing antioxidant power (FRAP) assay, and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging. Other assays, for example, superoxide radical scavenging, hydrogen peroxide scavenging, and singlet oxygen quenching, evaluate the scavenging ability for oxidants (Wang et al. 2016).
3
Antioxidant Activity of Polysaccharides
According to the extensive in vitro antioxidant studies, the polysaccharide is indeed an effective antioxidant. The underlying mechanism is, however, uncertain as the relationships between antioxidant activity and physicochemical properties or structural features are not comprehensively elucidated and confirmed. Besides, it is worth noting that conflicting results are observed in comparison with a number of literatures, since different sources, extraction methods, and even drying procedures can influence the evaluated polysaccharides. On the other hand, limited information is available on antioxidant activity of high-purity polysaccharides, and therefore other antioxidant substances, for instance, protein, peptide, and polyphenol, that may always be retained in polysaccharides in a form of either conjugation or mixture should be taken into account. Overall, the antioxidant potentials of polysaccharides are not determined by a single factor but a combination of several related factors. Numerous antioxidative polysaccharides have been discovered from plants, fungi, bacteria, and algae which are shown in Table 1.
4
Mechanism and Factors Affecting the Antioxidant Activity of Polysaccharide
Polysaccharides and polysaccharide-complex extracted from many natural sources. However, despite the great antioxidant potentials of polysaccharides exerted, their underlying mechanism is not systematically elucidated. Limited information is available on antioxidant activity of high-purity polysaccharides, and therefore other antioxidant substances, for instance, protein, peptide, and polyphenol, that may always be retained in polysaccharides in a form of either conjugation or mixture should be taken into account. As a result, the following sections summarize the current understanding of possible antioxidant mechanisms of polysaccharides. Structural Characteristics of Polysaccharide: It is widely believed that the bioactivity of polysaccharides is affected by their structure characteristics, such as chemical composition, molecular mass, types of glycosidic linkage, and conformation. Differences in origin materials, extraction procedures, and even drying
Source Brown Algae
Fucoidan
Fucoidan fraction
Fucoidan
Pigments free fucoidans Fucoidan
Fucoidan
Fucoidan
Fucoidan
Fucoidan
Fucoidan contains the monosaccharide composition such as fucose, galactose, mannose, and xylose Fucoidan
Crude sulfated polysaccharides (CSP)
Costaria costata
Dictyota ciliolata
Ecklonia cava
Laminaria japonica Lobophora variegate
Nemacystus decipients
Padinasanctae cruces
Sargassum cinereum
Sargassum fluitans
Sargassum polycystum
Turbinaria ornata
Turbinaria conoides
Polysaccharides Crude polysaccharides
Name Chnoospora minima
Table 1 Polysaccharides from various sources and their antioxidant activity
DPPH (61.22% at 1.0 mg/mL); reducing (67.56% at 1.0 mg/mL); TAC (65.30% at 1.0 mg/mL) DPPH (IC50 ¼ 534.45 μg/mL); ABTS (IC50 ¼ 323.8 μg/mL) ABTS (IC50 ¼ 88.71 1.01 μg/mL); DPPH (IC50 ¼ 440.07 4.43 μg/mL); superoxide (IC50 ¼ 352 4.58 μg/mL)
DPPH (14% at 2.0 mg/mL)
DPPH (51.99% at 80 μg/mL)
DPPH (IC50 ¼ 3.96 mg/mL); hydroxyl (IC50 ¼ 4.12 mg/mL) DPPH (22% at 2.0 mg/mL)
DPPH (IC50 ¼ 0.73 mg/mL); peroxyl (IC50 ¼ 0.48 mg/mL) DPPH (IC50 ¼ 4.64 mg/mL) DPPH (36.3% at 10 mg/mL)
DPPH (27% at 2.0 mg/mL)
Antioxidant activity DPPH (IC50 ¼ 3.22 g/mL); hydroxyl (IC50 ¼ 48.35 g/mL) Hydroxyl (63.3% at 10.0 mg/mL)
Delma et al. 2015 Guru et al. 2015
Chale-Dzul et al. 2017 Somasundaram et al. 2016 Chale-Dzul et al. 2017 Palanisamy et al. 2018
Zhao et al. 2018 Castro et al. 2015; Castro et al. 2016 Li et al. 2017
Ref. Fernando et al. 2017 Wang et al. 2014b Chale-Dzul et al. 2017 Kim et al. 2014
556 S. C. Mohan and A. Thirupathi
Green algae
Red algae
Crude polysaccharides
Crude polysaccharides
Crude polysaccharides
Crude polysaccharides
Crude polysaccharides
Sulfated polysaccharide
The four seaweed polysaccharides belong to β-type polysaccharides with pyranose groups, and have uronic acids Sulfated polysaccharides, main constituent of the SFP is iota-carrageenan Crude polysaccharides
Crude polysaccharides
Gracilaria foliifera
Ahnfeltiopsis pygmaea
Gracilaria corticata
Jania adhaerens
Gracilaria edulis
Gloiopeltis furcata
Sarcodia ceylonensis
Chaetomorpha antennina
Halimeda discoidea
Solieria filiformis
Crude polysaccharides
Gracilaria corticata var. ramalinoides
DPPH (88.93% at 4.0 mg/mL); ABTS (IC50 ¼ 2.01 mg/mL) DPPH (>2,000 μg/mL); alkyl (278.18 0.75 μg/mL); hydroxyl (102.68 16.00 μg/mL) DPPH (>2,000 μg/mL); alkyl (110.06 2.98 μg/mL); hydroxyl (1008.65 8.19 μg/mL)
DPPH (>2,000 μg/mL); alkyl (367.43 1.74 μg/mL); hydroxyl (654.13 9.14 μg/mL) DPPH (1,654 37.46 μg/mL); alkyl (382.55 1.23 μg/mL); hydroxyl (582.47 9.29 μg/mL) DPPH (>2,000 μg/mL); alkyl (377.24 6.10 μg/mL); hydroxyl (768.92 8.10 μg/mL) DPPH (603.38 40.3 μg/mL); alkyl (332.33 15.29 μg/mL); hydroxyl (287.63 13.68 μg/mL) DPPH (>2,000 μg/mL); alkyl (114.59 5.01 μg/mL); hydroxyl (281.70 4.96 μg/mL) DPPH (>2,000 μg/mL); alkyl (113.09 7.13 μg/mL); hydroxyl (602.95 12.26 μg/mL) Superoxide (64.37% at 90 μg/mL); DPPH (23.49% at 0.1 mg/mL) Hydroxyl (83.33% at 4 mg/mL); ABTS (IC50 ¼ 3.99 mg/mL)
Antioxidant and Antibacterial Activities of Polysaccharides (continued)
Fernando et al. 2017
Sousa et al. 2016 Fernando et al. 2017
He et al. 2016
Shao et al. 2013
Fernando et al. 2017
Fernando et al. 2017
Fernando et al. 2017
Fernando et al. 2017
Fernando et al. 2017
Fernando et al. 2017
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Fungi
Micro algae
Source
Aspergillus versicolor N(2) bC
Aspergillus terreus
Sarcinochrysis marina Geitler Alternaria sp. SP-32
A novel homogeneous extracellular polysaccharide An extracellular polysaccharide with a novel branched galactomannan Exopolysaccharide, the main chain consists of glucopyranose and mannopyranose units
β-type heteropolysaccharide with a pyran group Water-soluble polysaccharides which had β-pyranose and α-pyranose configurations Hetero-sulfated-anionic polysaccharides
Isochrysis galbana
Pavlova viridis
Hetero-sulfated-anionic polysaccharides
Sulfated polysaccharide
Ulva intestinalis
Graesiella sp.
Sulfated polysaccharide
Ulva fasciata
Chrysolaminarin, a glucan
Crude polysaccharides
Caulerpa racemosa var. racemosa f. Remota
Odontellaaurita K-1251
Polysaccharides Crude polysaccharides
Name Halimeda gracilis
Table 1 (continued)
Superoxide (EC50 ¼ 2.20 mg/mL); DPPH (EC50 ¼ 0.97 mg/mL)
DPPH (IC50 ¼ 0.77 mg/mL); hydroxyl (IC50 ¼ 0.70 mg/mL) DPPH (IC50 ¼ 0.91 mg/mL); hydroxyl (IC50 ¼ 0.91 mg/mL) DPPH (EC50 ¼ 3.4 mg/mL); hydroxyl (EC50 ¼ 4.2 mg/mL) Hydroxyl (EC50 ¼ 2.8 mg/mL)
DPPH (42.45% at 0.1 mg/mL); hydroxyl (83.54% at 10 mg/mL) Hydroxyl (IC50 ¼ 0.87 mg/mL); ferrous ion-chelating (IC50 ¼ 0.33 mg/mL) Superoxide (53.5% at 3.2 mg/mL)
Antioxidant activity DPPH (>2,000 μg/mL); alkyl (116.60 2.59 μg/mL); hydroxyl (1,006.90 6.40 μg/mL) DPPH (>2,000 μg/mL); alkyl (359.48 20.54 μg/mL); hydroxyl (200.08 8.17 μg/mL) Superoxide (81.45% at 90 μg/mL); DPPH (37.63% at 0.1 mg/mL) DPPH (the highest 82.23%)
Wang et al. 2013a Yan et al. 2016
Trabelsi et al. 2016 Chen et al. 2016
Trabelsi et al. 2016 Sun et al., 2014a Sun et al. 2014b
Shao et al., 2013 Peasura et al. 2016 Xia et al. 2014
Fernando et al. 2017
Ref. Fernando et al. 2017
558 S. C. Mohan and A. Thirupathi
Bacteria
Sulfated polysaccharide
Nitratireductor sp. PRIM-24
Labrenzia sp. PRIM-30
Sulfated heteropolysaccharide containing glucose, galactose, glucosamine, galactosamine, and glucuronic acid Sulfated polysaccharide
Acidic exopolysaccharide contained uronic acid (12.3%) and sulfate (22.8%) with constitution of glucose, galactose, and glucuronic acid Extracellular polysaccharide. Fructose and galactose were found as the major monosaccharides in the EPS Exopolysaccharide; galactose and glucose were found as main monosaccharides Exopolysaccharide; galactose and glucose were found as main monosaccharides
Extracellular polysaccharide, mainly composed of glucose and mannose with β-configurations Sulfated polysaccharides
Novel extracellular polysaccharide, the structure contains a backbone of (1 ! 6)linked β-D-galactofuranose residues with multiple side chains An extracellular polysaccharide
Haloterrigena turkmenica
Halolactibacillus miurensis
Enterobacter sp. PRIM-26
Bacillus thuringiensis
Bacillus amyloliquefaciens 3MS 2017
Alteromonas sp. PRIM-21
Streptomyces violaceus MM72 Aerococcus uriaeequi
Fusarium oxysporum
DPPH (IC50 ¼ 0.64 mg/mL); superoxide (IC50 ¼ 0.19 mg/mL) DPPH (IC50 ¼ 0.49 mg/mL); superoxide (not scavenging activity)
Antioxidant and Antibacterial Activities of Polysaccharides (continued)
Priyanka et al. 2015 Priyanka et al. 2015
Squillaci et al. 2015
Priyanka et al. 2015 Arun et al. 2017
DPPH (IC50 ¼ 0.44 mg/mL); superoxide (IC50 ¼ 0.33 mg/mL) DPPH (84% at 10 mg/mL); superoxide (89.15% at 0.5 mg/mL); hydroxyl (61% at 3.2 mg/mL) DPPH (IC50 ¼ 6.03 mg/mL)
Priyanka et al. 2015 El-Newary et al. 2017
DPPH (IC50 ¼ 0.61 mg/mL); superoxide (IC50 ¼ 0.65 mg/mL) DPPH (IC50 ¼ 0.21 μg/mL); H2O2 (IC50 ¼ 30.04 μg/mL); superoxide (IC50 ¼ 35.28 μg/mL)
Sathishkumar et al. 2018
Manivasagan et al. 2013 Wang et al. 2018
DPPH (IC50 ¼ 76.38 mg/mL); superoxide (IC50 ¼ 67.85 mg/mL) Hydroxyl (45.65% at 100 μg/mL); superoxide (67.31% at 250 μg/mL)
DPPH (79% at 1.0 mg/mL); superoxide (75.12% at 1.0 mg/mL)
Chen et al. 2015
Hydroxyl (EC50 ¼ 1.1 mg/mL); superoxide (EC50 ¼ 2.0 mg/mL); DPPH (EC50 ¼ 2.1 mg/mL)
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Leaves of Arctium lappa var. herkules,Aloe barbadensis, Althaea officinalis var. robusta, Plantago lanceolata var. libor, aerial parts and roots of Rudbeckia fulgida var. sullivantii, stems of Mahonia aquifolium, and peach-tree (Prunus persica) gum exudates Amaranthus hybridus L.
Plants
Chrysanthemum morifolium cv. Hangju
Name Polaribacter sp. SM1127
Source
Table 1 (continued)
Crude polysaccharides extracted by ultrasonic assisted extraction (CMP-U) and with heat reflex extraction (CMP-H)
Crude polysaccharides AHP-M-1 and AHP-M-2
Polysaccharides Exopolysaccharides and it mainly comprises N-acetyl glucosamine, mannose, and glucuronic acid residues bound by heterogeneous linkages Eleven polysaccharides have been isolated
Tang et al. 2020
The IC50 values of AHP-M-1 and AHP-M-2 were 1.194 mg/mL and 0.67 mg/mL, respectively DPPH (IC50 for CMP-U and CMP-H were 1.850 mg/mL and 3.587 mg/mL), BHA (0.047 mg/mL). Hydroxyl radical (IC50 for CMP-U and CMP-H were 2.612 mg/mL and 4.236 mg/mL, respectively). Ferrous chelating activity (IC50 for CMP-U and CMP-H were 2.523 mg/mL and 3.982 mg/ mL)
Hou et al. 2020
Kardošová and Machová (2006)
Ref. Sun et al. 2015
The polysaccharides were investigated for their ability to inhibit peroxidation of soyabean lecithin liposomes by OH radicals. The highest inhibition was found with glucuronoxylans of A. officinalis var. robusta and P. lanceolata var. libor, aerial parts. Their antioxidant activity accounted for 69% of the activity of the reference compound α-tocopherol.
Antioxidant activity DPPH (55.40% at 10 mg/mL); hydroxyl (52.1% at 10 mg/mL)
560 S. C. Mohan and A. Thirupathi
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technologies that influence the physiochemical properties, structure, or conformation of polysaccharides will lead to differences in antioxidant activity, speculating their possible relationships (Ma et al. 2013a; Chen et al. 2009; Shen et al. 2014; Gou et al. 2014). Specifically, a correlation between molecular weight and radical scavenging activity was well documented (Cheung et al. 2012). Additionally, it is suggested that the overall radical scavenging ability was associated with the number of hydroxyl or amino groups in polysaccharide molecules such as chitosan (Guo et al. 2005). Molecular weight of polysaccharides: The molecular weight affects the antioxidant ability of the polysaccharide. In addition, optimal antioxidant ability will vary depending on the type of polysaccharide. Liu et al. (2010a) reported that Ganoderma lucidum polysaccharide (GLPL1) with low molecular weight (5.2 kDa) has a higher ability to scavenge free radicals, superoxide radicals, and hydrogen peroxide than the component (GLPL2) with high molecular weight (15.4 kDa). At a concentration of 10 mg/mL, the scavenging rates of GLPL1 to hydroxyl radical and superoxide anion are close to 75% and 90%, respectively, whereas the scavenging ability of GLPL2 is only 50% and 60%, respectively. Ma et al. (2013) isolated four kinds of polysaccharides, namely, GLP1 (>10 kDa), GLP2 (8–10 kDa), GLP3 (2.5–8 kDa), and GLP4 ( GLP1 > GLP3 > GLP4, showing that the GLP2 polysaccharide with moderate molecular weight had the best reducing ability. Zha et al. (2009) obtained three polysaccharides from rice bran with a molecular weight ranging from 1.2 105 to 6.3 106 Da (PW1), 3.5 104 to 7.4 104 Da (PW2), and 5.3 103 to 2.3 104 Da (PW3), respectively. Results showed that PW3 exhibited the best potentials of reducing power, chelating metal ion, and scavenging abilities against DPPH and ABTS radical among three fractions, revealing that a relative low molecular weight fraction had high antioxidant abilities. Crude polysaccharides: In many reports, crude polysaccharide extracts exhibited notable antioxidant activity, but after further fractionation, the final purified polysaccharide showed moderate or low activity. It seemed that other antioxidant substances contained in the crude polysaccharide extract, such as pigments, flavones, peptide, protein, and polyphenol, might contribute to the antioxidant activity (Fan et al. 2014). Wang et al. (2013b) investigated the role of tea polyphenol (EGCG) in crude polysaccharide extracts from tea leaves (TPS) in the view of antioxidant ability. Results showed that the crude TPS exhibited strong antioxidant functions, whereas the further purified TPS fractions were hardly effective. But in the presence of EGCG, the reducing power and DPPH radical scavenging ability of TPS fractions were obviously enhanced. Meanwhile, the same results were also observed in dextran-EGCG system, indicating EGCG caused a synergistic increase in the antioxidant activity and tea polyphenol was the major antioxidant in the crude TPS. Mu et al. (2012) illustrated the existence of protein and pigment would influence the scavenging effect of both water-soluble and alkali-soluble crude polysaccharides from Inonotus obliquus. Wei et al. (2010) purified an acidic polysaccharide from Prunella vulgaris Linn., of which scavenging abilities against DPPH and hydroxyl radical were significantly lower than crude polysaccharide, possibly ascribed to
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other antioxidants, such as flavones and pigments contained in the crude polysaccharide extracts. Lin et al. (2009) compared the antioxidant properties of different polysaccharide fractions isolated from Lycium barbarum Linnaeus, including crude polysaccharide (CP), crude extract of polysaccharide (CE), deproteinated polysaccharide (DP), and deproteinated and dialyzed polysaccharide (DDP), as well as four purified fractions (one neutral and three acidic polysaccharides, named as LBPN, LBPa1, LBPa2, and LBPa3, respectively). In their study, it was suggested that the inhibition effect of superoxide and hydroxyl radical by hydroxyl groups in polysaccharides was minor due to lacking phenolic-type structure which was essential for scavenging free radicals. Many other factors, such as molecular weight, galacturonic acid, and other chemical components in polysaccharide fractions, were also supposed to play a role in their antioxidant activities. Crude and purified polysaccharide, obtained from G. atrum, was compared in terms of DPPH scavenging ability and self-oxidation of 1,2,3-phentriol. Although the high concentration of purified polysaccharide, PSG-1, showed noticeable antioxidant ability, it was much lower than the crude polysaccharide, probably attributing to other constituents contained in crude polysaccharides extracts, such as proteins, amino acids, peptides, cellulose, phytosterol, ascorbic acid, thiamine, nucleotide, nicotinic acid, organic acids, and microelements (Chen et al. 2008). Polysaccharide Chelating Metal: It is worth noting that one mechanism of antioxidant activity is to inhibit the generation of free radicals by chelating ions such as ferrous and copper instead of directly scavenging them. Transition metal ions could catalyze the generation of extremely reactive hydroxyl radicals from superoxide and hydrogen peroxide, known as Fenton reaction (Fe2+ + H2O2 ! Fe3+ + OH + OH.), especially ferrous ion, which is the most effective prooxidant in the food system (Yamauchi et al. 1998). Two polysaccharide fractions (GAPS-1 and SAPS-1) from A. barbadensis Miller were isolated and purified. The hydroxyl radical scavenging activity of GAPS-1 was significantly higher than SAPS-1. Meanwhile, GAPS-1 had a higher chelating ability against ferrous ion, indicating that the chelating effect might impart polysaccharides capable of antioxidant potentials (Chun-hui et al. 2007). A similar correlation was also revealed by Li et al. (2011) investigating an extracellular polysaccharide from N. commune. Generally, the structure of compounds containing more than one of the following functional groups, that is, -OH, -SH, -COOH, -PO3H2, -C¼O, -NR2, -S-, and -O-, is in favor of chelating ability (Gulcin 2006). Therefore, presence of uronic acid and sulfate groups appeared to be essential in demonstrating the chelating ability of polysaccharides. Chang et al. (2010) illustrated that the larger the content of galacturonic acid in polysaccharide, the higher the ability of chelating ferrous ion. Fan et al. (2014) fractionated four polysaccharides from the leaves of Ilex latifolia Thunb. by DEAE cellulose-52 chromatography (ILPS1, ILPS2, ILPS3, and ILPS4) with ILPS4 having the highest contents of sulfuric radical (3.7%) and uronic acid (23.2%). Results showed that IC50 of ferrous chelating activity for ILPS4 was 1965 8.1 μg/mL, while for other fractions the abilities were 4.7%, 11.3%, and 46.7%, respectively. This observation confirmed that the chelating effect might partly be due to the presence of functional groups such as carboxyl group and sulfuric radical in
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the polysaccharide structure. However, the ferrous ion chelating effect of a carboxymethylated polysaccharide (C-GLP) from G. lucidum was weak as compared to EDTA (Xu et al. 2009). The reason was probably attributed to its structural features unsuitability for chelating metal ion, as the chelating ability of ferrous was dependent upon hydroxyl numbers and the hydroxyl substitution in the ortho position (Wang et al. 2008). Types of substitution groups and degrees of substitution: Types of substitution groups and degrees of substitution (DS) appeared to have an effect on the physicochemical properties and conformation of native polysaccharides, such as molecular weight, polarity, solubility, and charge density. DS may also affect the activity through interruption of inter- and intramolecular hydrogen bonds. Chen et al. (2014) found that not only did the total sugar content of acetylated and carboxymethylated derivatives decrease significantly, but also its molecular weight was reduced in contrast to native G. atrum polysaccharide. Liu et al. (2010b) proved that sulfation effectively improved the water solubility and bile acid-binding capacities of a water-insoluble polysaccharide from G. lucidum (GLP). Furthermore, 13C NMR results showed that C-2, C-4, and C-6 position might be partially substituted, and C-4 was the most reactive. It was probably due to its special structure features and the influence of steric hindrance. A linear relationship between the degree of substitution and antioxidant potentials was not always observed, suggesting high DS was not necessary for antioxidant behavior. Xie et al. (2015) revealed that antioxidant activity of sulfated CP with a highest DS of 0.55 was not as effective as derivatives with middle DS (0.42 and 0.06). However, the influence of DS was still disputable, as a high DS could enhance the antioxidant activity evidenced in many reports. Yan et al. (2012) pointed out that the sulfation of exopolysaccharide, produced by Cordyceps sinensis fungus (Cs-HK1), occurred most frequently at hydroxyl groups of C-6 and caused a conformation change from random coils or aggregates to single helices in aqueous solution. The antioxidant activity of the sulfated derivatives for hydroxyl radical and ABTS radical scavenging effect was significantly enhanced with increasing DS and reducing molecular weight. Wang et al. (2014a) showed that C-6 substitution was predominately in phosphorylated derivatives of galactomannan (PGG) from guar gum according to 13C NMR analysis and PGG with high DS achieved a higher radical scavenging effect and stronger chelating ability than PGG with lower DS. Jung et al. found that DPPH radical scavenging ability of polysaccharide from Pleurotus eryngii was improved with increasing degree of sulfation (Jung et al. 2011). This finding was also consistent with the report that high degree of sulfated substitution (0.90) was more effective than that of low DS (0.43) in scavenging DPPH (Wang et al. 2013c). Another study also revealed a positive relationship between the degrees of acetylated substitution and scavenging effects against DPPH and superoxide radical, as well as reducing power (Song et al. 2013). One mechanism is that the introduction of these substitution groups into polysaccharide molecules leads to weaker dissociation energy of hydrogen bond. Therefore, the hydrogen donating ability of polysaccharide derivatives was increased. Another mechanism is speculated to activate the abstraction of the anomeric carbon.
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On the other hand, the chemical modification is sometimes accompanied with a decrease of molecular weight, hence improving the antioxidant potentials of polysaccharides. Among the derivatives, the sulfate polysaccharide is commonly reported as a stronger antioxidant, which is partly due to its ordered, extended structure. The sulfated polysaccharide usually traps free radicals in an electrostatic manner since the sulfate groups usually generate a highly acidic environment and the sulfur substitution may also weaken hydrogen bond interactions between polysaccharides (Wang et al. 2016). Sulfated polysaccharides: Sulfated polysaccharides demonstrated stronger antioxidant capacities than de-sulfated polysaccharides (Hu et al. 2010; Yang et al. 2011). Thus, the high degree of sulfation and low molecular weight showed the best antioxidant capacities (Wang et al. 2010). Yang et al. (2011) compared the antioxidant activity of sulfated polysaccharide from Corallina officinalis and its desulfated derivatives; the results showed that the native sulfated polysaccharides possessed more excellent radical scavenging activity and reducing power than the desulfated fractions. Generally, the biological activity of sulfated polysaccharides from marine algae is related to the molecular size, type of sugar, sulfate content, sulfate position, and type of linkage; also, molecular geometry is known to play a role in activity (Shanmugan and Mody 2000). Selenium enriched polysaccharides: Selenium (Se) is an essential trace element for nutrition of a capital importance in the human biology. The Se does not directly act as a ROS/RNS scavenger but is a cofactor of selenoprotein, for example, glutathione peroxidase, which exerts various antioxidant activities in vivo. Confirmed by FT-IR and NMR spectra, the selenylation modification by H2SeO3/HNO3 method predominantly happened at the C-6 position of polysaccharides and a distinct decrease of molecular weight was also induced due to the acid environment of selenized reaction. Additionally, it was proposed that the combination of Se in polysaccharides was possibly in the form of selenyl group (-SeH) or selenoacid ester (Xu and Huang 1994). Wei et al. (2015) synthesized a series of selenylated polysaccharide from Radix hedysari (Se-RHP), the content of Se ranging from 1.04 to 3.29 mg/g and the molecular weight decreasing from 62.7 kDa to 27.7 kDa, which showed better scavenging activity and reducing power in contrast to the native RHP. Se-containing derivatives from Artemisia sphaerocephala (Wang et al. 2012c) and Potentilla anserina L. (Zhao et al. 2013) have been acknowledged to improve the antioxidant activity compared to the native polysaccharides. The proposed mechanism might be involved in changes of conformation structure of polysaccharides and emerged the increasing amount of hydroxyl group, resulting in an influence on the antioxidant activity. Further analysis on polysaccharide obtained from Se-enriched materials confirmed the important role of Se in enhancing the antioxidant potentials of polysaccharides. As evidenced by the results of Yu et al. (2007), polysaccharide from Se-enriched green tea presented significant higher antioxidant capacity than that from regular green tea. In addition, all polysaccharides isolated from Se-enriched G. lucidum were more effective on attenuating the production of superoxide radicals (Zhao et al. 2008). Mao et al. (2014) revealed that although there was no significant
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Antioxidant and Antibacterial Activities of Polysaccharides
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difference of polysaccharide content and molecular weight of each Se-enriched G. frondosa polysaccharide (Se-GP) fraction and the corresponding GP, except for the Se content, Se-GPs were a more effective scavenger (against DPPH, ABTS, and hydroxyl radicals), especially for hydroxyl radical, reaching 71.32% at a concentration of 2 mg/mL. On the other hand, selenium-polysaccharide synthesized by adding selenium chloride oxide (SeCl2O) also exhibited a higher total antioxidant capacity, superoxide radical, and hydroxyl radical scavenging effect as reported by Guo et al. (2013). Phenolic compounds: Phenolic compounds, especially phenolic acids, play an important role in the overall radical scavenging ability of xylans and xylooligosaccharides from the wheat bran (Hromádková et al. 2008; Veenashri and Muralikrishna 2011). Hromádková et al. (2013) pointed out that both protein and phenolic compounds contributed to the radical scavenging effects of xylans, and the protein-free fraction displayed the highest hydroxyl radical scavenging ability indicating the distinct role of phenolic acids. Siu et al. (2014) study revealed that the antioxidant activities of all polysaccharide fractions from three mushrooms (L. edodes, G. frondosa, and T. versicolor) were significantly correlated with the total phenolic and protein content according to three in vitro assessments, including TEAC, FRAP, and ferrous ion chelating activity assay. However, no significant correlation was observed between the total sugar content and any of tested antioxidant assays. The results were similar to a study carried out by Wang et al. (2012a) that the neutral content was not apparently correlated with DPPH and FRAP antioxidant actions of polysaccharides from oolong tea. Furthermore, purified polysaccharide fractions, free of phenolics and proteins, hardly showed significant antioxidant activities. Indeed, polysaccharide-polyphenol residues have been demonstrated to have noticeable antioxidant functions in many reports. Li et al. (2007) found no statistical difference in scavenging linoleic acid radicals between the polysaccharides from Lycium barbarum fruits and the positive control (BHT). The coupled oxidation of β-carotene and linoleic acid developed free radicals, which oxidize unsaturated β-carotene molecules, leading to the discoloration of the system. In this model, the proposed mechanism in hindering β-carotene oxidation could be attributed to the polyphenolic-associated polysaccharide neutralizing the free radicals. In DPPH radical scavenging assay, the polysaccharide showed pronounced antioxidant ability as well, possibly attributing to polyphenolic-associated polysaccharide fraction formed between high molecular weight phenolics and polysaccharides. However, not all the conjugated moiety of polysaccharides was responsible for antioxidant power. After removing polyphenols, the tea polysaccharide conjugate from low grade green tea was found to possess strong antioxidant properties based on the results of free radical scavenging and lipid peroxidation inhibitory effect (Chen et al. 2005). Likewise, Wang et al. (2012b) evidenced that the DPPH radical scavenging effect of another tea polysaccharide fraction (TPS1) was beyond 90%, close to that of ascorbic acid, although both of the protein and polyphenol content were relatively low in TPS1, suggesting other factors such as carboxyl group other than polyphenol compounds that are of concern. In order to determine the molecular
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interactions between tea polyphenols and oat β-glucan, Wu et al. (2011) prepared complex and physical mixture of oat β-glucan and tea polyphenols, further using four in vitro antioxidant evaluations (DPPH radical, hydroxyl radical, superoxide radical, and reducing power) to compare the activity among tea polyphenols, βglucan, their complex, and physical mixture. Results showed that the complex had the strongest effect against superoxide radical, whereas the mixture had the strongest hydroxyl radical scavenging effect in the concentration of 0.5–2.5 mg/mL. With regard to reducing power assay, no synergistic effect was found between tea polyphenols and β-glucan, but it was observed in DPPH scavenging assay when β-glucan was combined with tea polyphenols at low concentration ( 7 deprotonation results later aggregation/breakdown in delivery of biomolecules/drugs). Mannose-TMC-chitosan loaded siRNA beats down TNF-α expression in gut allied macrophages by improved internalization by clatherin-free mannose (Joshi et al. 2019). Polycaprolactone conjugated hyaluronic acid bind chitosan imparts better delivery of anticancer drugs like paclitaxel and doxorubicin through oral route. Chitosan coated drugs found to shield and stabilize it in acidic pH, but at pH 7.4 wrap is released to aid drug loading (Dongre 2017). Assorted chitosan matrixes own biotechnology uses as shown in Fig. 12. Plenty amine groups of glucosamine unit of chitosan are accessible for protonation so as to attain positive charges and through electrostatic interaction gets complexed with negatively charged species like DNA. High-molecular-weight chitosan (>100 kDa) is soluble only in aqueous acids but low-molecular-weight chitosan ( 98 kDa > 48 kDa > 17 kDa. Chitosan with weight 15–70 kDa and deacetylation degree 80–90% showed high transfection efficiency (Dongre 2017, 2019). Rather degree of deacetylation is vital than molecular weight in functioning the cellular uptake and cytotoxicity of DNA– chitosan polyplexes (Dongre 2019; Joshi et al. 2019). Increasing charge density of deacetylated chitosan found to convey stability and utmost transfection efficiency to resultant polyplexes in vivo. The pH 6.5–7.5 is good for achieving higher transfection efficiency for cellular uptake of chitosan polyplexes due to unhindered endosomal release, but at pH > 7.5 DNA gets dissociated, so delaying
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Fig. 12 Assorted chitosan matrixes for biotechnology utility
cellular uptakes in 10–20% serum than serum-free medium (Dongre 2017). These formulated chitosan polyplexes appear less cytotoxic than Lipofectin™, Lipofectamine™, and PEI in vitro for 293 cells. Chitosan formulations show variable transfection efficiency inside diverse cells in-vitro, e.g., DNA–chitosan nanoparticles exhibit top transfection rate for HEK-293 than MG3 and mesenchymal stem-cells (Dongre 2017). Skeletal N-quaternization at terminal amine groups augment cationic property and improve transfection efficiency even as more cytotoxicity in such chitosan polyplexes. Further, cytotoxicity is reduced in such chitosan polyplexes through PEG/PLL grafting in its trimethyl skeleton than untreated chitosan (Dongre 2017, 2019; Joshi et al. 2019). Stearic acid and deoxycholic acid are hydrophobic species if conjugated into chitosan then it lessens aggregation and recovers cellular uptakes besides DNA release over underivatized chitosan-polyplexes. Cell-specific and transferrin choosy marked tumor cells are targeted through conjugated chitosan polyplexes against HEK293 cells than usual DNA–chitosan (Dongre 2017). Chitosan grafted mono/disaccharide formulation show hepatocytes state asialoglycoprotein receptors empathy for glycoproteins. Galactosyl/lactosyl chitosan yields via lactobionic acid coupling marks DNA delivery to hepatocyte/HepG2 cells in liver with great transfection efficiency. Galactosylated chitosan improve transfection efficiency in HepG2 due to increasing surface charge after complexation and interparticular aggregation besides avoiding enzymatic degradation by plasma protein (Dongre 2017, 2019).
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12.2.7 Chitosan in Biomedicals Biopolymer chitosan offered unique physicochemical properties such as biocompatibility, biodegradability, nontoxic and antimicrobial, so widely exploited matrix in pharmaceutics, biomedical innovation, and regenerative medicines. Such features of chitosan compel it as potent biomaterial for developing many systems like tissue engineering scaffolds, drug delivery/career, food-packing, and wound-healing besides chelating cholesterol, fats, proteins, and metals. Chitosan-based bone implants can offer unique features like mechanically robust, cheap, bio competent, bio-electronics, antibacterial, antifungal, hemostatic, osteo-conductive, and osteoinductive besides appear structural similarity to natural bone-stuff (Joshi et al. 2019). Certain chitosan formulated hydrogels and bandages are marketed in medical usage like wound healing, skin injury, and hemodialysis as shown in below Figure. Chitosan matrix/ scaffold are nontoxicity thus own admire utilities like as drug delivery/career and in dentistry to prevent tooth decay along with developing many dental care products (Dongre 2017). Chitosan is non-toxic and possess re-mineralizing power which aids hardening of tissues in tooth, so acts as desensitizer in toothpastes besides executes safe function in dentistry and oral health. Chitosan advances surgical healing of postextraction oral wounds and causes decline in bacterial bio-film usage in dental cements. Recently progressive scientific and biomedical fields have shaped novel biomaterials that are more capable and harmless. Frequently, processing of such biomaterials is tricky and expensive method, but many marine-based biomaterial including chitosan are incessantly explored to derive successful function in surgical, orthopedic, reconstructive medicines, plastic surgery, aesthetic, and dental (Jayakumar et al. 2010b). Often marine-derived biomaterials like alginate, chitosan and collagen are beneficial due to proven outputs. Chitosan is the most desirable marine-based biomaterial due to innate ability to shape up as films, sheets, particles, and gels thus, being exploited in diverse fields of medical including dentistry, tissue engineering, etc. In traditional dentistry and surgery chitosan formulations are used to stop tooth caries/ decay, involved in wound healings (Zohuriaan-Mehr 2005; Dongre 2018a). Innate antimicrobial activity is strongly influenced by formulating many materials based gels containing lactic acid, distilled water, and chlorhexidine through chitosan milieu to get desired functions performing at different pH. Such chitosan formulations are safe to use, with many prospective results in oral surgery and restorative dentistry with no side-effects (Dongre 2017; Böttcher et al. 2018). Chitosan-linked synthetic dental matrixes could improve its characteristics from a bacteriostatic to mycostatic purpose (Jayakumar et al. 2010a). Marine-derived chitin and chitosan are protagonists in gifted scientific and clinical interests being too, ascertained by their availability (Gortari and Hours 2013).
12.3
Carrageenan: C42H74O46S6
12.3.1 Structural Features Carrageenan is a renewable and sustainable biopolymer being extracted from edible red seaweeds class of marine origin (Dongre 2017). Carrageenan is a generic name of
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polysaccharides extracted through species of red seaweeds like Rhodophycea or Rhodophyta: Gigartina, Chondrus crispus, Eucheuma, and Hypnea. Carrageenan’s are identified in six Greek and IUPAC prefix nomenclature as Iota (i)-, Kappa (j)-, Lambda (k)-, Mu (l)-, Nu (m)-, and Theta (h) nomenclature as per chemical classification (Khotimchenko and Khotimchenko 2020). These carrageenan types found to vary in their degree of sulfation as Kappa-carrageenan has one sulfate group per disaccharide, iota-carrageenan has two, lambda-carrageenan has three, mu as four, nu has five, and theta has six sulphate functionalities per sugar unit. In fact carrageenan’s are hydrophilic linear sulphated galactan polymeric links owing alternating 3-linked b-D-galactopyranose (G-units) and 4-linked a-D-galactopyranose (D-units) or 4-linked 3, 6-anhydro-a-D-galactopyranose (DA-units). Assorted red algal found to yield varied carrageenan’s during their developmental span, e.g., Gigartina produces mainly kappa carrageenan’s during gametophytic phase while lambda carrageenan’s in sporophytic phase.
12.3.2 History, Chemistry Carrageenan or carrageenin means “little rock” a colloquial Irish carraigín family of linear sulphated polysaccharides as extracted from red edible seaweed (Dongre 2017, 2019). Gelatinous stuff was extracted from the Irish moss/seaweed called Chondrus crispus as food additives in the fifteenth century (Joshi et al. 2019). Carrageenan was used on industrial level in the 1930s, while was documented used in China around 600 B.C. and pioneered used ad Gigartina in Ireland around 400 A.D. Carrageenan own apt viscosity which is best for growing or gelling agent viable in many pharmaceuticals functions as thickening materials, cosmetics, polyelectrolyte complexes, prolonged and controlled drug delivery, wound dressing, and tissue engineering. It has limiting usages due to inherent hydrophilic property, but assorted derivatization like blending, reinforcement, and multi-layering can augment its restrictive properties beside expand prospective applications (Laurienzo 2010). 12.3.3 Property Carrageenan is bulky and highly flexible by virtue of curling helical arrangements and textures of constituting units with molecular weight 1507.4 g/mol. This constitutional orientation aids in imparting variable gelation at NTP conditions to be used as thickening and stabilizing agents in the food, dairy, and meat industries. All the six carrageenans owe high-molecular-weight consecutive sulfated and non-sulfated forms of galactose and 3,6 anhydrogalactose units joined via alternate α-1,3 links and β-1,4 links. The quantity and location of ester sulfated functionality exists in repeating galactoses primary differentiates and control their native properties. Huge ester sulphated groups lessen the solubility temperature of carrageenan which ultimately yields loose gelation and contribute to gel inhibition. All these carrageenans are soluble in hot water, only the lambda carrageenan is soluble in cold water along with sodium salts are cold water soluble. In food products, carrageenan owes the EU additive numbers E407 (E407a as processed eucheuma seaweed). Truly, carrageenans are considered a dietary fiber in many food products.
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Carrageenan is fragile to form blends with many polymeric skeleton like nanocellulose and advances its innate tensile strength by 50%. Carrageenan films layered through polylactic acid can reduce water vapor permeability up to high extent. Carrageenan polysaccharide is extracted from marine algae appears to be top renewable biomaterial substitute for conventional synthetic plastics/polymers. Carrageenan-derived matrixes are vast used as edible and flexible films beside coatings in various pharmaceutics and biomedical utility. Hydrophilic character of carrageenan films are improved to extend suitable applications by blending co-polymeric blocks like chitin/chitosan and reinforcements through plasticizers. Low molecular weight and highly sulfated carrageenan-based matrixes are the most active against tumors (Sashiwa and Aiba 2004).
12.3.4 Utility of Carrageenan Three main commercial classes of carrageenan, namely: kappa, iota, and lambda are often used in industries. Kappa form is mainly obtained from Kappaphycus alvarezii strong as rigid gels in presence of potassium ions, which can interact with proteins. Iota form subsists like soft gels with calcium ions as mainly obtained from Eucheuma denticulatum. While lambda carrageenan lacks gelation thus good to be used as thickening agent in assorted dairy products. Certain oligo-carrageenan blends apt G2/M phase and arrest apoptosis in cancer cells therapy besides abate immune suppression induced by antitumor drugs. Carrageenans formulations also act as adjuvant in vaccine based on dendrite cell actions. Carrageenans are known to offer anticancer and antitumor functions along with complete intracellular anti-proliferation. Novel agar/MMT hydrogels yield through cross-linking κ-carrageenan with triethyleneglycol-divinylether (TEGDE) polymer offer utmost inflammation and non-Fickian swelling capacity as per varying montmorillonite ratio. High montmorillonite reduces gelation growth, also display non-Fickian swelling capacity viable for biomedical applications (Joshi et al. 2019). Carrageenans are used in the food, dairy, and meat industry due to innate features like gelling, thickening, and stabilizing potential besides strap affinity to natural and synthetic proteins. Gelatinous extracts of the Chondrus crispus (Irish moss) seaweed have been used as food additives since approximately the fifteenth century (Dongre 2017). Carrageenan is a vegetarian and vegan alternative to gelatin in some applications or may be used to replace collagen-derived gelatine gummy polymer in many confectionery products. Carrageenans are broadly used in food processes as thickening, gelling, and protein-suspending agents along with excipient in pills/tablets (Joshi et al. 2019). Carrageenans offer prototype bio-activities like inflammation, immunity, herpes inhibition, and prohibition of HIV/AIDS (Joshi et al. 2019; Laurienzo 2010). Carrageenan advances texture of cottage cheese, puddings, and dairy desserts through controlled viscosity besides binds/stabilize patties, sausages, and low-fat hamburgers in meat-processing. Carrageenans are also preferred in cosmetics; printing, textile, wound dressings, and toothpaste stabilization by absorbing fluids. Carrageenans own superior properties like good compatibility, huge robustness, and constant visco-elasticity during compression so apt excipients for sustainedrelease formulations (Dongre 2017; Joshi et al. 2019). Carrageenans own broad
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bioactivity domain like antitumor, immuno-modulatory, anti-hyperlipidemic, anticoagulant, anti-inflammatory, and antiviral properties (Dongre 2017; Laurienzo 2010). But therapeutic applications of low molecular weight carrageenans are limited due to gastrointestinal toxicity which can be removed through assorted chemical modifications (Campo and Carvalho 2009).
13
Conclusion
Many marine organisms like crabs, lobsters, shrimps, molds, seaweeds, microbes, mollusks, arthropod, crustacean, fungi, yeasts, algae, and squid pen exoskeletons are resource for deriving such polysaccharides. Marine crustacean shells are rich in chitooligosaccharides owing 20–50% calcium carbonate, 20–40% proteins, and 15– 40% chitin ratios. An outline of three vital marine-based polysaccharides, namely, alginate, carrageenan, and chitin owing innate bio-activities in pharmaceutics and biomedicals are summarized including the aspects of preparation, formulation, and synthetic blending to be used for many purposes like water treatment, food, drug delivery, medicines, tissue engineering, wound healing, dentistry, cosmetics, and agriculture. Sea-based polysaccharides offer competent myriad applications due to native nontoxicity, biocompatibility, and biodegradability besides superior physicochemical/mechanical features. These polysaccharides offer distinctive field utilities in biotechnology, nanotechnology, medicine, cosmetics, food, diary, and agriculture.
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Bacterial Polysaccharides: Cosmetic Applications
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Main Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Biotechnological Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Cosmetic Applications of Bacterial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Skincare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Skin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Skin Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Cosmetic Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Definition and Categories of Cosmetics Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Cosmetic Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Main Cosmetic Vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Safety Requirements and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Bacterial polysaccharides are extensively used in cosmetic products, especially given their diverse properties and functionalities, assuming an important role in the R&D of new formulations and applications. In this chapter, the principal properties of the main polysaccharides present in this area are described and summarized. Advancements in skin biology knowledge, and the study of new formulations, support the research of active ingredients related to the maintenance of skin integrity and barrier function. Bacterial polysaccharides possess several
S. Baptista · F. Freitas (*) UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology, NOVA School of Science and Technology, Caparica, Portugal 73100 Lda., Lisboa, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_45
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properties (such as biocompatibility, biodegradability, film-forming, gelling, and thickening) that can provide protective effects on the skin, improving the efficacy of formulations while maintaining the skin in good conditions. Additionally, polysaccharides, which can be processed in emulsions, hydrogels, suspensions, and encapsulating structures, have been demonstrated to enhance the stability of formulations and promote sensorial properties. Data from several clinical studies revealed that the polysaccharide-based formulations promote skin health, with applications in several skin disorder treatments. Keywords
Polysaccharides · Cosmetic · Skin · Formulation · Emulsion · Hydrogel
1
Introduction
Despite being among the most important structures in Nature, polysaccharides were often overlooked in the past. However, due to the recent global demand for a sustainable economy, the previous paradigm has shifted (Shanmugam and Abirami 2019). These natural polymers have particular physical and chemical properties, and can be found in a wide range of life forms, from microorganisms to animals and plants, being essential in several biological functions (Yildiz and Karatas 2018; Shanmugam and Abirami 2019). In fact, polysaccharides are known to be decisive for cell adhesion, to serve as reservoirs in the cytoplasm, to be part of the cell membrane or cell wall, and to be a component of the defense, regulation, and information systems of the cell. Bacterial polysaccharides are extensively used in cosmetic products, especially given their diverse properties and functionalities (Freitas et al. 2014). Cosmetics can be defined as composite multiphase arrangements, with every single different component possessing a specific function on the final product (Freitas et al. 2014). Polysaccharides can be used as natural cosmetic components due to their unique physical and chemical properties, given the fact that they are biocompatible and biodegradable biopolymers (Freitas et al. 2014). Functional polysaccharides are claimed based on their functionalities in the cosmetics’ formulation technology, such as film formers, gelling agents, thickeners, suspending agents, conditioners, and emulsifiers, that mainly rely on the physicochemical properties of the biopolymer. On the other hand, cosmetic active polysaccharides promote water loss reduction, protecting the skin barrier function, and promoting a good sensorial (Kanlayavattanakul and Lourith 2014). In this context, polysaccharides are of great interest in cosmetics for the development of stable functional formulations. The continuous development of the cosmetic industry technology and the understanding of skin’s biology allowed the emergence of several novel active ingredients used in topical formulations. Still, cosmetic ingredients research is of utmost importance to further increase the stability, safety, and efficacy of cosmetic formulations. Natural ingredients are widely present in this industry because consumers have an
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interest in the benefits originated by these natural products. In this sense, polysaccharides present several interesting applications, due to their capability of creating gel structures in aqueous environments (through the hydration of the sugar building blocks), stabilizing emulsions and forming films, acting as binders, viscosity-increasing promotors, and rheology modifiers, as well as suspending agents (Freitas et al. 2014; Kanlayavattanakul and Lourith 2014). Polysaccharides interact, as active substances, with other ingredients of a cosmetic formulation, which grants them great benefits to be used, for example, as vehicles of dermatological formulations. This application is possible due to the non-reactive nature of polysaccharides when contacting the human skin. Moreover, these molecules can be metabolized and eliminated from the body using normal metabolic pathways (Ammala 2013; Freitas et al. 2014).
2
Bacterial Polysaccharides
In recent years, microbial polysaccharides have emerged as viable substitutes to animal and plant-based ingredients in many different applications, ranging from the food and agricultural industries to the pharmaceutical, medical, and cosmetic industries. This diversity in applications derives from polysaccharides’ fundamental and practical properties, granting them functions as stabilizers, emulsifiers, viscosifiers, and thickeners (Shanmugam and Abirami 2019). Therefore, the previously mentioned properties of bacterial polysaccharides contribute to their application in cosmetic formulations (Table 1), with xanthan and gellan gum being the most pertinent due to their use as cosmetic additives to control the viscosity, and as physicosensorial agents. Hyaluronic acid, bacterial cellulose, and levan are also relevant cosmetic ingredients, considering their application as bioactive components for skin regeneration/defense (Balkrishna et al. 2018; Freitas et al. 2014). Being high molecular weight polymers that may share components with other microbial cellular carbohydrates and having fundamental intricacy and variety, bacterial polysaccharides can be classified in several groups: capsular polysaccharides (CPS) and exopolysaccharides (EPS), which are observed in all types of bacteria; lipopolysaccharides (LPS) and cell-wall teichoic acids (WTA), which are reported, respectively, in gram-negative and gram-positive bacteria (Balkrishna et al. 2018; Shanmugam and Abirami 2019; Yildiz and Karatas 2018). CPS are extracellular polysaccharides that are important in bacteria pathogenicity due to their ability to encase microorganisms and promote adhesion and penetration of the host cell. It also acts as virulence promoters by screening the immune response, to avoid antibody reactions (an interesting feature in the advancement of vaccines) (Yildiz and Karatas 2018). EPS are high molecular weight (10–1000 kDa) extracellular polysaccharides secreted by many prokaryotes (both eubacteria and archaebacteria) and eukaryotes (phytoplankton, fungi, and algae), which present chemical structure and composition diversification, ranging from homopolymers to heteropolymers (Balkrishna et al. 2018; Roca et al. 2015). This diversity rendered EPS auspicious in terms of
Origin Xanthomonas spp
S. paucimobilis
Leuconostoc mesenteriodes, Streptococcus, Weissella, Pediococcus and Lactobacillus
Gluconacetobacter Xylinum, Agrobacterium, Alcaligenes, Rhizobium, Pseudomonas
Polysaccharide Xanthan gum
Gellan gum
Dextran
Bacterial cellulose glucopyranose
Glucose
Glucose Rhamnose Glucoronic acid
Monomers Glucose Mannose Glucuronic acid
~106
106– 108
5.0 x 105
Mw (2.0– 50) x 106
Table 1 Bacterial polysaccharides properties and their cosmetic application
Gelling capacity Hydrocolloid Water soluble Film form capacity Good stability (over wide temperature and pH ranges) Newtonian fluid behavior Insoluble in water and in most solvents High crystallinity High waterholding capacity High tensile strength moldability
Properties High viscosity Hydrocolloid
Thickener Emulsifier Viscosity controller Absorbent Skin regeneration Skin moisturizer Wound healing
Moisturizer Thickener
Cosmetic application Thickener Emulsifier Rheology modifier Stabilizer Stabilizer Gelling agent
Alves et al. (2016), Balkrishna et al. (2018), Freitas et al. (2014), Gallegos et al. (2016), Savary et al. (2016)
Yildiz and Karatas (2018)
Freitas et al. (2014)
Reference Savary et al. (2016)
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Acetobacter, Bacillus, Brenneria, Geobacillus, Halomonas, Lactobacillus
Streptococcus sp.
Levan
Hyaluronic acid
Acetylglucosamine, glucuronic acid
Fructose
2.0x106
3.0x106 High water solubility Low viscosity Stabilizer Film form capacity Water soluble Highly viscous hydrocolloid Viscoelastic High swelling capacity Humectant Moisturizer Softening agent Antiaging affect Immunostimulant Angiogenic
Cell-proliferating Skin moisturizing Skin irritation– alleviating effects Blending Alves et al. (2016), Freitas et al. (2014), Savary et al. (2016)
Alves et al. (2016), Yildiz and Karatas (2018)
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commercial applications, in several sectors such as paper manufacturing, pharmaceuticals, cosmetics, and food. For instance, since xanthan and gellan were allowed as food additives in Europe and the US, these two EPS gained great interest and are currently widely used (Roca et al. 2015). However, the global polymer market fraction related to bacterial EPS is still small, mainly due to production costs, in terms of substrates and bioreactors used for microbial growth, and purification procedures (Balkrishna et al. 2018). As shown in the previous paragraphs, bacterial polysaccharides present a great variety of functional properties, which is an important feature to broaden their research, development, and commercialization (Balkrishna et al. 2018).
2.1
Main Properties
Microbial polysaccharides present hydrophilic behavior, a feature that renders them major interest for commercial purposes, due to their capacity to attract and hold water molecules, as well as to change basic properties of aqueous systems (BeMiller 2019). Moreover, microbial polysaccharides present several relevant characteristics such as high viscosity at low concentrations (1), they also have gelling properties (2) that enables them to act as thickening and stabilizing agents (3) presenting compatibility with high salt concentrations (4) and stability against temperature changes and high shear pH (5); they have high water solubility and anti-freeze behavior (6), presenting polyelectrolyte and ion exchange potential (7), having a surface-active, dispersing and flocculating capacity (8); they also present versatile adhesive and film-forming properties (9), displaying capacity and selectivity for metal ions, proteins, lipids (10), with specific biodegradability (11) (BeMiller 2019). Microbial polysaccharides can adapt their molecular structure and rigidity by changing the intra and intermolecular interactions, which grants them unique rheological characteristics. In the cosmetics’ industry, polysaccharides’ capacity to present high viscosity at low concentrations is very important to allow a specific texture (of increased viscosity) to the formulation and to decrease elasticity-driven creaming of the droplets and other components of the emulsion. This ability derives from polysaccharides’ inflated molecular structure in aqueous solution, with a high effective volume fraction at low concentrations (BeMiller 2019). The main bacterial polysaccharides used in the cosmetic industry are xanthan gum, bacterial cellulose, levan, hyaluronic acid, gellan gum, and dextran because of their ability as film formers, emulsion stabilizers, binders, and viscosity increasing and skin conditioning agents (Balkrishna et al. 2018; Freitas et al. 2014; Bhavani and Nisha 2010).
2.2
Biotechnological Importance
The aforementioned features of polysaccharides (hydrophilicity, high molecular weight, and changeable molecular structure) allow them to have several
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applications, mainly related to their behavior in aqueous media, in high-value markets (Roca et al. 2015). The global hydrocolloids market, in which most polysaccharides are included, was estimated at USD 9 Billion in 2020 and to be at USD 11.7 Billion by 2027, and expected to grow at a compound annual growth rate (CAGR) of 3.7% between 2020 and 2027 (Research and Markets, official site). The cosmetics and Personal Care Products segment currently accounts for a 20.8% share of the global Hydrocolloids market (Research and Markets, official site). Even though this market is dominated by plant and algal polysaccharides, xanthan gum’s market value, for instance, is estimated to be USD 987.7 Million in 2020 and, expected to grow at a CAGR of 5.1% until the end of the year 2024 (Research and Markets, official site) Research and development are, therefore, important to further improve the biotechnological importance of bacterial polysaccharides. In the next paragraphs, it will be explained this importance in four pivotal areas: food, health, agriculture, and industrial sectors (Roca et al. 2015; Shanmugam and Abirami 2019).
2.2.1 Applications of Bacterial Polysaccharides in the Food Sector Bacterial polysaccharides have several roles in functional food, for example, as additives to probiotics (Alves et al. 2016). For instance, xanthan and gellan gums can be used for microencapsulation as stabilizers and protectors, to avoid nutraceuticals degradation in two different phases (food processing and intestinal transit) thus ensuring the release in specific sites to perform the desired health effect (Alves et al. 2016). Nowadays, specific foods demand a unique texture, viscosity, flavor, and watercontrol properties. For these purposes, bacterial polysaccharides are used to change food thickness, stability, and texture. For example, bacterial polysaccharides such as dextran and xanthan are used to change water-related rheology, thus altering the final product’s texture (Shanmugam and Abirami 2019). Moreover, they can be used to prevent/reduce the formation of ice crystals in frozen foods (Jindal and Khattar 2018) and also play a role in the color/flavor of prepared foodstuffs (Jindal and Khattar 2018). In terms of products, microbial polysaccharides can be found in instant foods, sauces, toppings, and dairy products (Jindal and Khattar 2018; Shanmugam and Abirami 2019). Some Food and Drug Administration (FDA) approved bacterial polysaccharides have an important role in food processing industries (Ramalingam et al. 2014). Xanthan gum is used in frozen foods (dextran also), beverages, and sauces (Ramaligam et al. Ramalingam et al. 2014; Shanmugam and Abirami 2019); gellan gum, xylinan, alginates, and curdlan polysaccharides are used as texturizers/gelling agents (Jindal and Khattar 2018; Ramalingam et al. 2014)); pullulan acts as pH stabilizer of food (Ramalingam et al. 2014). The health awareness of consumers and the multiple functions of hydrocolloids have contributed to the increased demand for these bacterial polysaccharides in the food and beverage industries (Shanmugam and Abirami 2019).
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2.2.2 Applications of Bacterial Polysaccharides in the Health Sector In the health sector, the use of bacterial polysaccharides is widespread in many applications that include drug delivery systems (Alves et al. 2016), dental impressions, absorbent dressings (Nwodo et al. 2012), anti-reflux therapies, and bioprinting (McCarthy et al. 2019). The importance of drug delivery systems has increased due to the request for pharmacologically active substances regulated distribution in specific areas of the human body. Accordingly, xanthan gum, gellan gum, hyaluronic acid, and levan have been studied as suitable for this type of formulation (Alves et al. 2016). In consequence, dextran and sulfated alginates have been reported to prevent blood disorders (Balkrishna et al. 2018) In terms of bioprinting, alginate, bacterial cellulose, hyaluronic acid, gellan, xanthan, and dextran have already been reported as appropriate for this type of application (McCarthy et al. 2019). There are several other medical applications for bacterial polysaccharides which include their use as an anticoagulant, anticancer, anti-angiogenesis, anti-inflammatory, antithrombotic, antidiabetic, antioxidant, antiviral, antiulcer, cholesterollowering, and prebiotic agents (Yildiz and Karatas 2018). One of the most important influences on the global market for xanthan gum is the increase of governments’ ventures in healthcare, and the key players include Danisco, Cargill, Pfizer Inc., Jungbunzlauer, Archer Daniels Midland, CP Kelco, and Fufeng Group Company Ltd. (Shanmugam and Abirami 2019). 2.2.3
Applications of Bacterial Polysaccharides in the Agricultural Sector In the agricultural sector, bacterial extracellular polysaccharides (EPS) are among the most important due to their utilization as soil nutrition for organic matter (Shanmugam and Abirami 2019) because they have a suitable suspending nature and affinity with salt, with promising features in toxic compounds transfer by decreasing its mobility to the soil (Balkrisha et al., Balkrishna et al. 2018). Moreover, EPS plays a role in controlling plant pathogens like fungus, and enhance pesticides, herbicides, fungicides, and insecticides (Balkrishna et al. 2018; Nwodo et al. 2012). They can also be used for environmental bacterial metabolism in metal detoxification of soils (Balkrisha et al., Balkrishna et al. 2018). Essentially, EPS improve plant growth by increasing soil fertility (Shanmugam and Abirami 2019). For example, the rheological properties of xanthan were demonstrated to increase two important pesticide properties: pesticide cling and pesticide permanence (Nwodo et al. 2012). 2.2.4 Applications of Bacterial Polysaccharides in the Industrial Sector In the industrial sector, bacterial polysaccharides applications include cosmetic, dairy, baking, textiles, and biofuel industries (Balkrishna et al. 2018). For example, microbial polysaccharides have been demonstrated to solve several problems encountered during yogurt manufacture (such as low viscosity, gel fracture, or whey separation) because they improve rheology, texture, stability, and mouthfeel of fermented milk products. Moreover, the use of EPS-producing microbial cultures during cheese manufacturing resulted in smoother, moister, and soften cheeses (Jindal and Khattar 2018). In the baking industries, in addition to their use as
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stabilizing agents that decrease the staling capacity of baked products, bacterial polysaccharides also play a role in water retention, moisture, texture, and bread volume by optimizing these important parameters of baked products. In the textile industry, bacterial polymers are used, for example, in the fabrication of waterresistant products such as raincoats and vehicle covers (Balkrishna et al. 2018). In the biofuels industry, bacterial polysaccharides are proposed to improve oil recovery fluids due to its high viscosity-enhancing ability at low concentrations (Balkrishna et al. 2018).
2.3
Cosmetic Applications of Bacterial Polysaccharides
According to their role in the product, polysaccharides applied in cosmetics can be classified as functional or active polysaccharides (Freitas et al. 2014).
2.3.1 Functional Polysaccharides Functional polysaccharides are usually classified based on their electrochemical charge in the product’s structure and are commonly integrated into cosmetic products to act as gelling agents, viscosity adjusters, thickeners, and emulsifiers (according to their polymerized network ability to hold water) (Kanlayavattanakul and Lourith 2014). Cationic Polysaccharides Polysaccharides with a cationic charge, which is not a widespread characteristic among natural polysaccharides, are often required by the cosmetic industry (Gruber 1999). For this reason, the cosmetic industry has focused mainly on synthetically derived polyglycans, which contain the advantage to tightly bind with anionic proteins of the human skin and hair. Therefore, polyglycans properties as filmforming agents are widespread with applications in hair damage controlling, hair fixing, and skin-conditioning products. The most commercialized cationic polyglycan is chitosan, which is a partially deacetylated (>50%) version of chitin to improve its solubility (Gruber 1999; Kanlayavattanakul and Lourith 2014). Anionic Polysaccharides Anionic polysaccharides are very interesting, in terms of cosmetic applications, and are comprised of a group of natural materials, of which the bacterial polysaccharides xanthan gum (Gruber 1999) and gellan gum (Zia et al. 2018) are the most representative. Regarding synthetically derived polysaccharides, carboxymethylcellulose and carboxymethyl-chitin are among the most commercially used for cosmetic uses (Gruber 1999). D-glucose, D-mannose, and D-glucuronic acid (molecular ratio of 3:3:2) building blocks linked through β-1,4 glycosidic linkages with a high number of trisaccharide side chains are the xanthan gum main constituents. With a molecular weight averaging 2000 kDa (Freitas et al. 2014; Jindal and Khattar 2018), xanthan gum is naturally synthesized, which is usually obtained via bacterial fermentation (Kanlayavattanakul
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and Lourith 2014). Its applications include the utilization as a thickener and dispersing agent and emulsion stabilizer (Lochhead 2017). Xanthan gum is a hydrocolloid with the ability to dissolve in water at room temperature (Jindal and Khattar 2018), acting as a non-gelling biopolymer and providing high viscosities at low concentrations (Lochhead 2017) because of the semirigid conformation conferred by its side chains containing mannose and glucuronic acid (Dubuisson et al. 2018). Xanthan can also polymerize and crystallize which gives it the ability to emulsify suspensions, a feature that is frequently adopted in cosmetic preparations to improve the stability against freeze-thaw challenges (Kanlayavattanakul and Lourith 2014). In emulsions, xanthan presents similar rheological properties: high viscosity at low shear rates, a strong non-thixotropic shear-thinning character, and a viscoelastic behavior (Dubuisson et al. 2018). In pharmaceutical cream formulations, as in barium sulfate preparations, the suspension stability provided by xanthan is of utmost importance. In toothpaste, this feature is also an advantage, improving ingredient suspension, due to high viscosity, and facilitating brushing onto and off the teeth. The uniform dispersal of pigments, long-term stability (in terms of pH, salinity, and temperature), and its ability to thicken, xanthan grants an extensive application as shampoo base (Yildiz and Karatas et al., Yildiz and Karatas 2018). Therefore, xanthan is generally used in cosmetic systems, being produced and commercialized by major companies like CP Kelco, Merck, Pfizer, Rhone Poulenc, Sanofi-Elf, and Jungbunzlauer (Alves et al. 2016; Freitas et al. 2014). Gellan gum is a heteropolysaccharide composed of a tetrasaccharide backbone with L-rhamnose, D-glucose, D-glucuronic acid, and D-glucose monomers and side chains of acetyl and glyceryl substituents (Alves et al. 2016). Among the main properties of gellan gum, its ability to withstand heat and acid stress in fabrication steps are most appreciated. Being a thermoresponsive, biocompatible, biodegradable, ductile, and non-toxic polysaccharide, it is used due to its gelling properties, its malleability, its texture, and its high efficiency in the final products (Zia et al. 2018). Gellan gum is mainly used in cosmetic formulations to increase viscosity and to stabilize emulsions, at low concentrations in dermal products (0.3–0.5%) and even lower in eye and hair products (0.0004%) (Freitas et al. 2014). Also, it forms gels at low concentrations (0.1%) which are available in the low acyl form (creating hard, brittle gels) and in the high acyl form (composing soft, elastic gels) (Freitas et al. 2014; Lochhead 2017). Gellan provides suspensions with low viscosity because it has significant yield stress for low viscosity ranges. So, gellan is another anionic polysaccharide used in the cosmetic industry given that it meets the requirements of the EC Cosmetics Regulation 1223/2009 (EC 2009) in which is stated that toxicological profile of all used ingredients and detailed knowledge of the product-specific exposure are required as fundamental for the safety assessment (Lochhead 2017). This polysaccharide is available with a trading name GelriteTM and KelcogelTM (Zia et al. 2018).
Nonionic Polysaccharides Nonionic polysaccharides, such as dextran, are not charged which allows them to interact with negatively or positively charged surfactants (Gruber 1999;
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Kanlayavattanakul and Lourith 2014), a feature that grants them use in the cosmetic industry as rheology modifiers and thickeners (Gruber 1999). Dextran is a bacterial homopolysaccharide constituted by chains of D – glucose and consisting of 97–50% α-(1–6) glycosidic bonds on the main chain with the remaining α-(1–2), α-(1–3) and α-(1–4) linkages that form branches, depending on the bacteria strains (Bhavani and Nisha 2010). It is soluble in water and other solvents, in addition to being biocompatible and biodegradable. Dextran and its derivatives have several applications in cosmetics as moisturizers and thickeners, specifically cationic dextran (CDC) because of its capacity to form complex salts with anionic or amphoteric surfactants, which can adsorb to hair/skin producing moisturizing effects, making it a useful conditioning agent. Dextran sulfate is another dextran derivative used in cosmetics as an anti-aging and anti-wrinkle product. Due to its moisture retention and increased lipase activity, it presents a smooth, fresh, and non-sticky feeling which results in supple skin with reduced weight (Bhavani and Nisha 2010). Amphoteric Polysaccharide Polyglycans that can have cationic and anionic charges on the same chain are called amphoteric polysaccharides. In a cosmetic formulation the pH determines if the amphoteric polysaccharide is cationic, anionic, or both (Gruber 1999). Amphoteric polysaccharides are mainly produced, via modification, from natural polysaccharides, being denominated as seminatural polysaccharides. Despite having limited use in the cosmetics industry, amphoteric polysaccharides can be found in personal care products formulations, acting as surfactants. For instance, Fucogel ®, which is developed by BioEurope and marketed by SOLABIA, is obtained through fermentation of a strain of K. pneumoniae (Roca et al. 2015). The result is a highly viscous and hydrophilic pol polysaccharide with a repeating unit of acetylated charged linear trisaccharide, fucose, galactose, and galacturonic acid. This product possesses several interesting characteristics, such as original physicosensorial qualities, moisturizing properties, and self-emulsifying properties (Guetta et al. 2003), which can explain its success in the cosmetic industry. Moreover, Fucogel ® was shown to act as a skin anti-aging agent, due to the stimulation of fibroblast proliferation and survival imposed by Fucogel ® oligosaccharides (Roca et al. 2015).
2.3.2 Bioactive Bacterial Polysaccharides Many bacterial polysaccharides are also frequently used in cosmetic applications as active ingredients (Freitas et al. 2014). Bacterial Cellulose Cellulose is a high molecular weight, anionic, and water-insoluble biopolymer extracted from natural sources, such as plants and bacteria (G. xylinus, G. hansenii, G. pasteurianus, Agrobacterium, Alcaligenes, Rhizobium, Pseudomonas, and acetobacter) (Kanlayavattanakul and Lourith 2014; Ullah et al. 2016; Balkrishna et al. 2018; Gallegos et al. 2016) which makes it the most abundant renewable polymer. Cellulose presents a homogenous and linear structure of
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D-glucopyranose sugar units connected through β linkages (Gallegos et al. 2016). Bacterial cellulose (BC) presents a porous network structure constituted by nanofibrous with high strength and low density, rendering it effective for membrane development for cosmetic products (Freitas et al. 2014). Cellulose ethers (mainly methyl and ethyl cellulose derivatives) are also observed in several cosmetic applications due to their physical and chemical properties (Kanlayavattanakul and Lourith 2014). The Hainan Guangyu Biotechnology Co. Ltd. is a major BC producer and promoter for BC applications in the cosmetic industry. Indeed, reports show the use of BC in cosmetic formulations produces stable oil-in-water emulsions which are non-irritating on the skin. Moreover, these emulsions can penetrate the skin and supply good hydration without the requirement of any surfactants (Gallegos et al. 2016; Ullah et al. 2016). BC also has other applications in components of artificial nails and fingernail polish. By presenting good water holding capacity and gas permeability, BC was also reported as an acceptable carrier in cosmetic active ingredients such as moisturizers, whitening ingredients, anti-wrinkling agents, growth factors, enzymes, or a combination thereof (Ullah et al. 2016). Additionally, BC can be used in personal cleansing formulations and contact lenses (Gallegos et al. 2016; Ullah et al. 2016. In fact, BC is a promising candidate for contact lenses production because of its transparency, light transmittance, and permeability to liquid and gases (Ullah et al. 2016).
Levan Levan is a homopolysaccharide composed of fructose units with β-2,6-glycoside bonds with β-2,1 side branches (Alves et al. 2016; Domżał-Kędzia et al. 2019; Siqueira et al. 2020) synthesized by several bacteria (B. subtilis, Z. mobilis, and E. herbicola) and fungi (Aspergillus sydowii and Aspergillus versicolor) having suitable properties for various cosmetic applications (Domżał-Kędzia et al. 2019). Among the interesting properties of this adhesive amphiphilic polymer, its solubility in oil, low viscosity, and the ability to generate films are valorized in hair-fixing products (Domżał-Kędzia et al. 2019). Moreover, levan can be used in discoloration products because it reduces tyrosinase activity, reducing melanin production. For increased stability to oxidation, levan products can be supplemented by ascorbic acid. It can also co-create a solid polymer matrix that dissipates at skin contact (Domżał-Kędzia et al. 2019). Additionally, levan has high compatibility with salts and surfactants, presents heat stability, water retention capacity, and is nontoxic (Siqueira et al. 2020). Levan can also be used as an encapsulation agent, due to its ability to create nanoparticles in water. The polymers’ biological activities (cell proliferation, skin repairing, and moisturizing) contributes to its use as part of three-dimensional artificial skin models, acting also as a protective agent against irritation. Furthermore, levan BPS diminishes skin water loss, keeping it moisturized (Balkrishna et al. 2018; Freitas et al. 2014). However, levan applications are still limited due to two major cutbacks which make its processing and preservation considerably difficult. Firstly, it has low stability in aqueous formulations (Alves et al. 2016) and secondly because, in acidic conditions or high temperatures,
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the polymer hydrolyzes into fructose and oligosaccharides (Balkrishna et al. 2018; Alves et al. 2016; Freitas et al. 2014). Hyaluronic Acid Hyaluronic acid (HA), also known as hyaluronan, is a linear glycosaminoglycan biopolymer with high molecular weight (up to 108 Da) formed by repeating disaccharides D-glucuronic acid and N-acetyl-D-glucosamine linked by a glucuronidic β-(1–3) bond (Huynh and Priefer 2020). It plays several roles in biological processes regulation like skin repairmen, cancer diagnosis, wound healing, tissue regeneration, anti-inflammatory, and immunomodulation (Bukhari et al. 2018; Huynh and Priefer 2020). There is nearly 15 g of HA in a 70 kg human being, which can be found in the synovial fluids, umbilical cords, vitreous humor of the eye, heart valves, skin, and skeletal tissues (Huynh and Priefer 2020). This biopolymer is biodegradable and biocompatible, which allows its use in the treatment of several animal and human diseases, preventing difficulties related to non-degradable treatments. Moreover, HA can be used as prognostic molecules and is a polysaccharide with a high natural abundance (found in human and animal bodies), which can hold/trap approximately 1000 times its weight of water (Bukhari et al. 2018; Huynh and Priefer 2020). Even though commercial HA was obtained, at first, from mammalian tissues, public health concerns ignited the research of alternative sources of this biopolymer, like microorganisms and marine organisms (Alves et al. 2016). HA activity is highly conditioned by its size but there are several specific applications of each molecular weight, with many utilizations in medicine and the cosmetic industry (Freitas et al. 2014). In fact, research and development of HA and its related products have been abundant, with reports showing its effectiveness as dermal fillers, anti-wrinkle agents, and tissue regeneration agents (Huynh and Priefer 2020). Hyaluronic acid cosmetic effects are mainly related to its performance as a soft tissue generator, a skin hydration improver, a collagen stimulator, and face rejuvenation inducer (Bukhari et al. 2018). The differences in percutaneous absorption of different molecular weight HA across the stratum corneum show that anti-wrinkle effect efficiency depends on the molecular weight of the used biopolymer (Bukhari et al. 2018). Pavicic et al., have conducted a clinical trial involving 76 females, with ages from 30 to 60 years old, who have periocular wrinkles. On these patients was applied, during 60 days (2 times a day), a 0.1% (w/w) cream formulation with different HA molecular weights (50, 130, 300, 800, 2000 kDa). The results showed tremendous enhancement in skin hydration levels and skin elasticity. Moreover, on the patients that used low HA molecular weight formulations, it was detected a reduction in periocular wrinkles (Pavicic et al. 2011). Anti-wrinkle efficacy of HA-based topical cream formulation has also been investigated by Poetschke et al. In this study, the authors studied, for 3 months, four topical cream formulations (Balea, Nivea, Lancome, Chanel) containing HA on 20 women with periorbital wrinkles. The results showed a significant improvement in skin elasticity and tightness by 13–30%, significant reduction in wrinkle depth by 10–20%, and improved hydration level in all treatment patients (Bukhari et al. 2018; Poetschke et al. 2016).
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Apart from the topical application advantages already explained, scientific research has reported the benefits of HA-based treatments, such as the intradermal injection of HA (as fillers, gels, and implants) in treating facial wrinkles and promoting skin rejuvenation. Moreover, the safety, tolerability, and patient satisfaction have contributed to the diffuse utilization of HA in cosmetics (Bukhari et al. 2018). In terms of HA gel injection, the effects of HA in the reconstruction of deficient interdental papilla, also known as the black triangle, which is the gingival portion area between two adjacent teeth, have also been studied. The results showed a reduction in 36.5% and 45% in the triangle surface after 3 months and 6 months, respectively. (Huynh and Priefer 2020). Regarding hyaluronic acid action as filler during the congenital and acquired lip asymmetries, the results showed that one of the disadvantages of this treatment is the injections required overtime to maintain the result. There are already several FDA-approved dermal HA fillers, which are mainly commercialized by Restylane® (Medicis, USA), Prevelle Silk ® (Mentor Corp., USA), Anika ® (Anika Therapeutics, Inc., MA), and Juvéderm™ (Allergan, USA) (Freitas et al. 2014). In conclusion, while dermal filler HA is indicated to treat skin aging-related issues, the topical formulation of HA is appropriate for skin treatment and to prevent skin roughness (Bukhari et al. 2018). An interesting research area to explore for hyaluronic acid could be the application of its properties in wound healing treatments/products (Huynh and Priefer 2020).
3
Skincare
3.1
Skin Structure
The skin is an integrated and dynamic organ, which accounts for 10 to 15% of our body mass, making it the largest organ of the human body. Besides serving as a barrier to the external environment, protecting our body from physical damage and pathogens, the skin is also important in maintaining the body’s homeostasis by preventing water loss and due to its functions in the immune-neuroendocrine system (Lai-Cheong and McGrath 2013; Monteiro-Riviere 2006). Free radicals (endogenous and exogenous) can interact with the skin, producing negative effects that may result in skin-based diseases such as psoriasis, eczema, urticaria, and skin cancer (Lai-Cheong and McGrath 2013). Therefore, the skin has a unique defense mechanism based on endogenous (derived from melanin) and exogenous (orally and topically administrated) antioxidants, in order to avoid these adverse effects of free radicals. This defense mechanism is intrinsically linked to the structure and function of skin layers elements (Kusumawati and Indrayanto 2013). Basically, the human skin is constituted by four layers (from top to bottom): the nonviable epidermis (the stratum corneum), the viable epidermis (the remaining layers of the epidermis), dermis, and hypodermis (Fig. 1).
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Fig. 1 Structure of the human skin
The epidermis is mainly composed of keratinocyte cells (95%), which are the producers of the protein keratin, while the remaining 5% are melanocytes, Langerhans cells, and Merkel cells (Lai-Cheong and McGrath 2013; Lawton 2019). Since this skin layer does not include blood vessels, the nutrient delivery and waste disposal are dependent on the second layer (dermis) through the basement membrane (Lawton 2019). Depending on keratinocyte differentiation, the epidermis is usually divided into four layers (Lai-Cheong and McGrath 2013; Lawton 2019): stratum corneum, stratum granulosum, stratum spinosum, and stratum germinativum/basale (Table 2) (Fig. 2) (Lai-Cheong and McGrath 2013; Lawton 2019; Kusumawati and Indrayanto 2013; Monteiro-Riviere 2006). The stratum lucidum is a fifth layer found in thick skin, such as the palms of the hands, the soles of the feet, and the digits) (Lawton 2019; Monteiro-Riviere 2006). Keratinocyte and melanocyte cells are the main components of the stratum basale, with the former being produced exclusively in this layer via division (Lawton 2019; Kusumawati and Indrayanto 2013). During the migration of keratinocytes to the upper layers (stratum spinosum and stratum granulosum) occurs a process called keratinization, which differentiates these cells to assemble a rigid internal structure of keratin, microfilaments, and microtubules (Lawton 2019). In a 40-day long process, keratinocytes proliferate and perform terminal differentiation, leading to the establishment of the stratum corneum (Lai-Cheong and McGrath 2013). In its turn, the stratum corneum features layers of dead cells that lost their nucleus (Lawton 2019; Monteiro-Riviere 2006), which suffer desquamation, a 28-day long process (Kusumawati and Indrayanto 2013; Lawton 2019). These dead cells, also known as corneocytes, are ingrained within a
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Table 2 Epidermis layers characteristics Epidermis layers Stratum corneum Stratum lucidum Stratum granulosum
Stratum spinosum Stratum basale
Description Comprised of several layers of completely keratinized flattened dead cells. Each stratum corneum cell is approximately 30 μm in diameter and 0.5 to 0.8 μm in thickness. Fully keratinized and homogeneous zone with dense cells without core localized between the stratum granulosum and stratum corneum. Can be found in the palms and soles, where the skin is extremely thick and lacks hair. Constituted for three to five layers of flattened cells and presents high lysosomal activity. This layer has lipids with a function for coating the cell membrane of the stratum corneum cells and are responsible for barrier to chemical absorption across the skin. These lipids include ceramides, fatty acids, cholesterol, and cholesterol sulphate. Consists of several layers of irregular polyhedral-shaped cells which are interconnected by desmosomes. Langerhans cells are mainly located in this layer. The basal cells layers of the epidermis are functionally heterogeneous. Can divide and proliferate or act as anchor cells that remain attached to the basement. It is composed by keratinocytes, which are either division phase or non-division phase. These cells contain keratin tonofibrils and melanocytes. Merkel cells are also found in the basal cell layer. These cells are a responsible for the in the skin such as touch feeling.
Fig. 2 Epidermis layers
hydrophobic extracellular matrix composed of lipid precursors and hydrolases, which results in the formation of fatty acids, cholesterol, and at least 10 ceramides (Lai-Cheong and McGrath 2013). Ceramide is essential for maintaining a
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moisturized skin due to its function as a water retention agent, playing also a role in the maintenance of skin defense by forming lipid barriers that prevent the penetration of allergens and irritants (Kusumawati and Indrayanto 2013; Lawton 2019). Therefore, ceramides contribute to high water content in the corneocytes, which causes them to swell and keep the stratum corneum flexible, preventing skin dryness and the appearance of fissures and cracks (Lawton 2019). The loss of the epidermis moisture retention ability causes dry skin, which can only be repaired using specific treatments to improve hydration and decrease water evaporation, such as topical application of moisturizers and the utilization of occlusive masks on the skin (Freitas et al. 2014). Summing up, the stratum corneum determines the extent and ratio of percutaneous absorption (Lawton 2019), and, given its highly impermeable lipophilic layer, acts as an important protector from the external environment, being determinant for a healthy skin appearance (Kusumawati and Indrayanto 2013). In the stratum basale, there can be found melanocytes, the synthesizers of melanin, the molecule responsible for the absorption of ultraviolet (UV) radiation (Kusumawati and Indrayanto 2013; Lawton 2019). By causing the peroxidation of the lipid matrix of the stratum corneum, which leads to loss of this structure barrier capacity, extended exposure to UV radiation can lead to mutations, causing premature skin aging or skin cancer (Freitas et al. 2014). Langerhans cells, which are bone marrow-derived, are dendritic cells with an antigen-presenting activity that can be found not only in the stratum spinosum but also in hair follicles, sebaceous glands, and apocrine glands (Lai-Cheong and McGrath 2013; Lawton 2019; Monteiro-Riviere 2006). On the other side, Merkel cells can only be found in the stratum basale and act as sensory information transmitting cells from the skin to the sensory nerves (Lawton 2019; Monteiro-Riviere 2006; Lai-Cheong and McGrath 2013). The next skin layer is the dermis, composed of fibroblasts, collagen, elastin, and hyaluronic acid (Kusumawati and Indrayanto 2013) and containing blood vessels, nerves, glands, and hair follicles (Ammala 2013; Monteiro-Riviere 2006). The major functions of this layer are the maintenance of skin characteristics and serving as a water reservoir for the skin (Kusumawati and Indrayanto 2013). As previously stated, the fibroblast is an important component of the dermis, being the major cell type observed in this layer, acting as a synthesizer of collagen, elastin, and viscous gel (Lai-Cheong and McGrath 2013; Lawton 2019). Collagen, responsible for the toughness and strength of the skin, constitutes 70% of the dermis layer, being continuously degraded and replaced. Elastin fibers are responsible for skin elasticity (Lai-Cheong and McGrath 2013; Lawton 2019). However, these two important molecules are influenced by increasing age and exposure to UV radiation, which usually results in dips and stretches of the skin (Lawton 2019). The hypodermis, which is the deepest skin layer, provides the main structural support for the skin, consisting of adipose tissue that serves as a thermal barrier to the skin. This skin layer contains collagen and extracellular matrix and is interlaced with blood vessels and nerves (Kusumawati and Indrayanto 2013; Lawton 2019).
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Skin Conditions
Skin disorders affect millions of people worldwide with physical and often traumatic psychological implications for quality of life (Long 2002). Therefore, many efforts have been made over the years to develop effective cosmetic and treatment solutions using bacterial polysaccharides.
3.2.1 Psoriasis Psoriasis is a chronic inflammatory, autoimmune disorder of the skin (Fig. 3) that affects approximately 120 million people worldwide. In this condition of unknown etiology, it is usually observed an increased proliferation of inflammatory cells within the dermis and epidermis and an expansion of the upper dermal capillaries (Long 2002; Song et al. 2019). Nowadays, there is a great focus, in the dermatology research field, to treat and prevent this condition (Song et al. 2019). The use of emollients as a topical therapy for psoriasis is widespread due to their properties for blocking transdermal water loss, which promotes skin softness and reduces skin scaling, making the patient feel more comfortable (Long 2002). Nevertheless, there are many other topical therapies available for psoriasis treatment, such as coal tar, topical steroids, calcipotriol, anthralin, and cyclosporine (Long 2002). In terms of bacterial polysaccharides utilization for the treatment/prevention of this condition, there are already several promising studies. For example, Song et al. (2019) investigated the use of bacterial exopolysaccharide as a moisturizer and drugloading material. For this purpose, the authors prepared an exopolysaccharides/ calcipotriol (EPS/CPT) emulsion (Fig. 4) using bacterial EPS as an emulsifier, sunflower oil as an oil phase, and CPT as the loaded drug. In vitro and in vivo animal experiments showed that the EPS/CPT emulsion could effectively treat psoriasis by increasing the accumulation of Calcipotriol in psoriatic skin lesions and reducing the levels of inflammatory cells and inflammatory factors. Additionally, the emulsion produced interesting results on reducing the side effects associated with Calcipotriol (Song et al. 2019). Xanthan gum has also been studied for the formulation of emulsions for the treatment of psoriasis (Musa et al. 2017). In that study, the active ingredient was cyclosporine, the oil phases were virgin coconut oil Fig. 3 Clinical manifestation of psoriasis: typical erythematous plaques with silvery scales. (Retrieved with permission from Boehncke and Schön 2015)
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Fig. 4 EPS emulsified sunflower oil at different concentrations. From left to right: 1.5%; 1.25%; 1%; 0.75%; 0.5%; 0% and control (Song et al. 2019)
and nutmeg oil. The nanoemulsion produced significant improvements in the stratum corneum, responsible for skin hydration (Musa et al. 2017). In another study, bacterial cellulose was combined with carboxymethylcellulose (CMC) to form a biocomposite for drug delivery systems (Fig. 5) (Lima Fontes et al. 2018). The authors loaded the BC/CMC biocomposite with methotrexate (MTX), a drug used to treat autoimmune diseases, to increase the effectiveness of the topical treatment of psoriasis (Lima Fontes et al. 2018). In a different study, hydrogels, composed of hyaluronic acid and polyvinyl alcohol, were loaded with methotrexate (Cheaburu Yilmaz et al. 2017). The resulting hydrogels revealed promising pH-responsive swelling and releasing abilities. In terms of biocompatibility, the toxicity studies performed in mice showed that the matrices are non-toxic and toxicologically safe when administered via intraperitoneal injection or topically. Therefore, these systems are a potential option for the development of novel topical formulations for psoriasis therapy (Cheaburu Yilmaz et al. 2017). Hyaluronic acid has also been reported in the development of solid microneedles (MNs) patch in an attempt to decrease the setbacks associated with MTX application in psoriasis therapy and also to increase the therapeutic effect (Du et al. 2019). That study illustrated the ability of HA to increase the solubility, biocompatibility, and biodegradability of MNs. Moreover, the presence of hyaluronic acid in the formulation conferred enough mechanic strength to the MNs to pierce the stratum corneum (Du et al. 2019).
3.2.2 Eczema Eczema or dermatitis is a chronic inflammatory skin disease characterized by frequent spontaneous flares and remissions (Fig. 6), which manifests dry skin and intense itching in patients (Long 2002). The treatment of this condition relies mainly upon moisturizers, such as hydrogels and humectant-enriched creams and lotions formulations of oil-in-water and water-in-oil emulsions. In fact, the incorporation of humectant ingredients like HA improves skin hydration (Draelos 2011). There are
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Fig. 5 BC/CMC biocomposite with methotrexate (MTX). (Adapted from Lima Fontes et al. 2018)
Fig. 6 Extensive atopic dermatitis (eczema). (Retrieved with permission from Mathe and Loffeld 2019)
several prescription devices already implemented to increase barrier function in atopic dermatitis patients. Those products are aimed to create an optimal healing environment, by using occlusive agents to decrease transepidermal water loss (Draelos 2011). For that purpose, research has been made and there are reports, for example, of HA-based foam technology aiming to boost moisture and distribute transepidermal water loss (Draelos 2011). In that study, 20 patients, who presented mild/moderate symmetrical atopic dermatitis in a body surface area higher than 10%, were treated with two products: in one side of the body the hyaluronic acid-based emollient foam was applied; on the other side, a reference ceramide-containing emulsion cream was applied. The patients and the researchers rated several symptoms and signs, such as
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erythema, scaling, lichenification, excoriation, itching, stinging, and burning, during the whole treatment. The results showed an overall improvement in eczema severity using HA foam, proving the effectiveness and soothing ability of the product (Draelos 2011). A different study assessed the utilization of a ceramide-HA emollient foam, in terms of short-term effectiveness, and compared the results with a pimecrolimus cream, during the treatment of atopic dermatitis. The authors reported that both products presented acceptable efficacy in patients with mild/moderate atopic eczema and that after a month of treatment, the ceramide-HA emollient foam continued to help the patients (children and adults) without side effects (Frankel et al. 2011). The HA-containing emollient can deposit ceramide proteins in the most important anatomic location, delivering the moisturizer through the stratum corneum and into the dermis (Hebert and Pacha 2012), an advantage that explains the increasing commercial availability of this safe and efficient type of product for the treatment of atopic dermatitis (Hebert and Pacha 2012). In another study, several subjects (aged between 18 and 75 years) were treated with low molecular weight HA gel, in an attempt to understand the efficiency and safety of this product for the treatment of facial seborrheic dermatitis (Schlesinger and Rowland Schlesinger and Rowland 2014). In that report, the researchers were interested in measuring scale, erythema, and pruritus, at baseline and weeks 2, 4 and 8. The results showed that HA sodium salt gel 0.2% presented improvements in the measured variables at week 4 with 76.9%, 64.3%, and 50% reductions in scale, erythema, and pruritus, respectively (Schlesinger and Rowland , Schlesinger and Rowland 2014). Resuming, HA-based medical creams are suitable for treating several dermatoses like atopic dermatitis and contact dermatitis and there are already various FDA-approved products on the market: Atopiclair ® (Sinclair Pharma), HylatopicPlus ® (Onset Dermatologics), and Bionect ® (Innocutis Holdings).
3.2.3 Acne Frequently present during adolescence, acne vulgaris is the most common skin condition, with 70% of the population developing recognized acne variants, which include infantile acne and occupational acne. This skin disorder is characterized by follicular dyskeratosis, increased sebum production, and inflammation induced by Cutibacterium acnes within the follicle (Fig. 7). Treatment for this skin condition includes the utilization of vitamin A-derived retinoids, to control the pathogenic pathways of acne, and topical applications of antibiotics such as erythromycin, clindamycin, and tetracycline. Azelaic acid is also effective in the treatment of acne (Long 2002). Acne scarring is an important aesthetic problem because it is widely prevalent, affects the face prominently, is poorly masked by cosmetics, and begins at an early age. Research for the prevention of this problem was performed, and a low-viscosity stabilized HA dermal filler was injected at microdoses into the mid-to-superficial dermis of twelve patients (7 men and 5 women) aged between 19 and 54 years, to improve the appearance of depressed acne scars. The treatment was well tolerated by the patients and the results showed an immediate visual improvement of all lesions (Halachmi et al. 2013).
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Fig. 7 Acne lesions. (Retrieved with permission from Vary 2015)
Bacterial cellulose is another bacterial polysaccharide used in acne treatment, being present, for example, in facial masks. Another example is the utilization of a bio-cellulose film (prepared from A. xylinum) incorporating several concentrations of P. granatum (pomegranate) peel extract for application as an anti-acne product. The authors of this study reported a satisfactory inhibition effect on S. aureus, S. epidermidis, and P. acne, when compared to antibiotics such as gentamicin and clindamycin, for BC with extracts of 5 or 10 mg/ml of pomegranate. A different study aimed to develop a system for the treatment and healing of skin prone to acne. By using a BC film loaded with natural propolis extract (with high water content), the authors expected to improve skin hydration and enhance the texture of the treated skin (Fig. 8). The product improved the skin’s mechanical properties, making it an efficient way to release active substances (Amorim et al. 2020).
3.2.4 Rosacea Rosacea, which is a chronic inflammatory skin disorder that mainly affects the facial skin, is usually characterized by skin dehydration, redness, erythema, and telangiectasia (Fig. 9). This skin condition derives from repeated environmental trauma (cold wind, ultraviolet radiation, and heat) which damages the upper dermal collagen and vasculature. Moreover, it can also be a result of prolonged use of topical steroids on the face. For the treatment of this skin disorder, topical applications of metronidazole and azelaic acid have been demonstrated to be effective, with persistent cases requiring the prescription of oral metronidazole, tetracyclines, or isotretinoin (Long 2002). Research using bacterial polysaccharides in rosacea treatment is widely available. For instance, researchers studied the efficacy and tolerability of HA containing sodium salt cream 0.2% during clinical trials on 15 patients (Chen et al. 2018). Several clinical symptoms, such as papules, erythema, burning or stinging, and dryness, were assessed and the results showed a great efficacy of low molecular weight HA cream in the reduction of all symptoms, coupled with good tolerability by patients (Chen et al. 2018).
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Fig. 8 BioMask prototype: BC film loaded with natural propolis extract (Amorim et al. 2020)
Fig. 9 Rosacea. (Retrieved with permission from Li et al. 2020)
3.2.5 Hyperpigmentation and Skin Aging Environmental pollution and overexposure to UV radiation can lead to skin oxidative stress caused by reactive oxygen species (ROS), which in turn may lead to skin disorders such as hyperpigmentation (Fig. 10, left image) and skin aging (Fig. 10, right image). Despite possessing a defense mechanism against ROS, the skin is usually affected by these external factors, characterized by freckles, dark spots, and wrinkle formation (Jesumani et al. 2019). Excessive presence of ROS prompts melanin production via the activity enhancement of the enzyme tyrosinase, leading
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Fig. 10 Hyperpigmentation (left) and facial wrinkles (right). (Retrieved with permission from Yamada and Prow 2020, and Leijs et al. 2018, respectively)
to hyperpigmentation. Moreover, high ROS concentration has an elastase-inducing effect, which degrades elastin, resulting in skin’s elasticity and strength loss, causing visible wrinkles (Jesumani et al. 2019). HA applications in anti-wrinkle products have already been mentioned in Sect. 2.3.2.3. HA is a moisturizing active ingredient, that can be used in anti-wrinkle cosmetic formulations, such as gels, emulsions, and serums, with the ability to recover a young skin typical physiological microenvironment (Fallacara et al. 2018). This is due to its strong water binding potential, which helos the maintenance of the skin elasticity, turgor, and moisture (Pavicic et al. 2011). For instance, Fillerina® (Labo Cosprophar Suisse), an HA-based cosmetic, is known to restore skin hydration and elasticity to exert an anti-wrinkle effect (Fallacara et al. 2018). The clinical efficacy of topical nano-HA formulation to reduce facial wrinkles and erythema intensity has also been the subject of research. The results showed major improvements in skin elasticity and firmness, and a higher hydration level with the consequent reduction in erythema intensity, wrinkles, and roughness of skin (Chen et al. 2018). Recently, HA utilization as a dermal filler has been widely applied. Dermal fillers (DFs) are class III medical devices that, injected into or under the skin, restore lost volumes and correct facial imperfections such as wrinkles (Fig. 11). The reversibility of the HA dermal filler effect can explain the success of this application: the correction of wrinkles is reversible, meaning that a medical error can be fixed using an injection of HYAL (Vitrase ®, ISTA Pharmaceuticals; Hylenex®, Halozyme Therapeutics). Depending on the used HA concentration, type and degree of crosslinking, and treatment area, HA DFs effects can last between 3 and 24 months
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Fig. 11 HA dermal filler process for facial imperfections such as wrinkles
(Fallacara et al. 2018). Sunscreens that contain HA also assist in the maintenance of youthful skin, because HA has free radical scavenging properties, which protects the skin against negative effects of UV radiation (Fallacara et al. 2018). As stated before, bacterial cellulose can be used in cosmetic masks. Considering its biodegradability, low toxicity, and ability to hydrate the skin, BC skin masks have been studied in the treatment and prevention of dry skin, in formulations with cosmetic substances such as sodium bicarbonate, ascorbic acid, and salicylic acid. These products have been shown to help the exfoliation and brightening of the skin, having an anti-wrinkle effect. Levan is also present in formulations because it improves skin elasticity, reduces wrinkles depth, and enhances the moisture of the skin (Kim et al. 2003). Dextran, conjugated with rosmarinic acid, was also reported in the development of a skin whitening agent, an innovative polymeric antioxidant. The studies (in vitro and in vivo) results showed that this product has a good skin whitening/lightening efficacy, being biocompatible. The cosmetic formulation has improved stability, with long-lasting effects, which means dextran is a suitable bioactive ingredient.
4
Cosmetic Products
Cosmetic and personal care products represent a massive market, providing an extensive range of properties and benefits to the consumer, with millions of consumers using cosmetics and their ingredients daily (Nohynek et al. 2010). In fact,
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cosmetic products global market was valued at USD 532.43 billion in 2017, and, through an expected annual growth rate of 7.14%, it can register USD 805.61 billion by 2023 (Bilal and Iqbal 2020).
4.1
Definition and Categories of Cosmetics Products
The European Union Council regulations define cosmetic products as “any substance or mixture intended to be placed in contact with the external parts of the human body (epidermis, hair system, nails, lips, and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition, or correcting body odors” (EC 2009, article 2.1). In general, a cosmetic product is applied for the direct treatment of the surface of the human body to maintain its good condition (1), to change the body appearance (2), to protect the body (3), or to correct the body odor (4) (Halla et al. 2018). These four functions must not affect the normal body functions or structure (Balkrishna et al. 2018; Ullah et al. 2016). Cosmeceuticals, also known as active cosmetics, which were created in the 1980s, are cosmetic products containing biologically active ingredients with medicinal or drug-like benefits, aimed to satisfy the customer’s health and beauty needs. In fact, cosmeceuticals can act as skin protectors, deodorants, whitening, tanning and anti-wrinkling (or antiaging) agents, and also possessing functions in nail and hair care products. In consonance with their field of applications and functions, cosmetic products can be divided into seven categories, as follows. Cosmetics for personal cleansing (1) (soaps, deodorants, shampoos); cosmetics for the skin, hair, and integument care (2) (toothpaste, products for external intimate care); cosmetics for embellishment (3) (perfumes, lip colors); protective cosmetics (4) (solar products, anti-wrinkle products); corrective cosmetics (5) (beauty masks, hair dyes); maintenance cosmetics (6) (shaving cream, moisturizing creams); and active cosmetics (7) (fluoridated toothpaste, antiseptics) (Halla et al. 2018). Bacterial polysaccharides are used in most of these cosmetic products, and Table 3 illustrates its level of utilization in several applications. Furthermore, there are several other bacterial polysaccharides with promising features for possible commercial applications. For example, Galactopol, an EPS composed of galactose, mannose, and rhamnose, has interesting properties (viscosity in an aqueous environment, emulsifier, film-forming) that could be used as alternatives to other cosmetic applications-related polysaccharides. Being anionically charged, Galactopol is usually synthesized by P. oleovorans and possesses a molecular weight of 1–5 106 Da. Another interesting EPS is FucoPol, a bacterial heteropolysaccharide constituted by fucose, glucose, galactose, and glucuronic acid. This BP has shown to form viscous solutions, with the ability to both form films and stabilize emulsions, which are interesting properties to use in the cosmetic filed. Moreover, it has already been shown its ability to encapsulate bioactive
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Table 3 Bacterial polysaccharides current applications in cosmetics Polysaccharide Xanthan gum
Gellan gum
Dextran
Bacterial cellulose
Levan Hyaluronic acid
Current applications in cosmetics Skin care, hair care, conditioners and shampoos, aftershave, shower gel and cream, body lotion, moisturizing products, sunscreen, toothpaste. Skin care, hair care, lotions and creams, makeup products, face masks and packs, toothpaste, suntans and sunscreens, toothpaste. Skin care, protective cosmetics, moisturizing products. Skin care, hair care, lipstick, eyeliner, moisturizing products, masks, hair dyes and colors, bath preparation, shampoos, toothpaste, antiperspirant. Skin care, hair care products, whitener products. Skin care, hair care, moisturizing and hydrating products, protective products, pre/after sun lotions, sunscreen.
References Kanlayavattanakul and Lourith (2014), Savary et al. (2016)
Bhavani and Nisha (2010), Kanlayavattanakul and Lourith (2014) Gallegos et al. (2016), Lima Fontes et al. 2018, Savary et al. (2016)
Fallacara et al. (2018), Pavicic et al. (2011), Savary et al. (2016)
compounds, which could be important for its application as an encapsulating matrix in cosmetics applications (Freitas et al. 2014; Lourenço et al. 2017). FucoPol (1.7 106–5.8 106 Da) is negatively charged due to the existence of glucuronic acid, succinyl, and pyruvyl in its constitution (Freitas et al. 2014).
4.2
Cosmetic Formulation
The abundant advances in studying and understanding the physicochemical properties formulation systems (and their ingredients) have contributed to the development of biologically stable products. The first generation of skincare products emphasized the relevance of free amino acids in skin hydration. In fact, water, oily substances, and humectants combinations can be efficient to keep the epidermis biological homeostasis, acting upon the physicochemical condition of the stratum corneum, since the barrier function of the skin is maintained by moisture, lipids, and natural moisturizing factors (Fig. 12) (Hosoi et al. 2017). Any cosmetic formulation includes in their formulation base substances, active agents, and additives (Table 4) with a different function on the final product. Base substances are natural skin components; active agents are substances with specific functions such as protection, preservation, and/or improvement of the skin’s natural condition; and additives are substances used to improve the product’s stability, providing protection against microorganisms, temperature shifts, oxygen and light related degradation, which improves the product’s shelf life (Freitas et al. 2014).
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Fig. 12 Moisture balance of the skin in the first generation of skin care. NMF - natural moisturizing factors. (Adapted from Hosoi et al. 2017)
Table 4 Substances used in cosmetic formulation Substances Base substances
Active agents
Additives
Ingredients Fatty acids Mineral oil products Triglycerides Wax esters Hyaluronic acid L-Fucose Valproic acid Vitamins (A, B3, C, E) Lactic acid Botanical extracts (Hesperidin, tea plant, ginseng, Resveratrol) Algal extract (B. braunii) Ubiquinone Water Antioxidants Dyes and pigments Emulsifiers Perfumes Preservatives (Parabens, organic acids) Thickening agents (Types: lipid, naturally derived, mineral and synthetic) UV filters
References Freitas et al. (2014)
Freitas et al. (2014), Lautenschläger (2004)
Freitas et al. (2014), Lautenschläger (2004)
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The active ingredients for cosmetics represent a large worldwide commercial market, projected to grow to US$1.6 Billion by the year 2025 (PRNewswire official website). These ingredients are declared using the International Nomenclature Cosmetic Ingredient (INCI) standards and are listed according to their weight ratio in decreasing order. However, aromatic principles (fragrances) are only declared as perfumes or essences. The modification of cosmetic products is a result of the overexposure to atmospheric oxygen, or due to the presence of microorganisms. Subsequently, antimicrobial and antioxidant preservatives can be added to cosmetic formulations to, respectively, inhibit microbial growth and suppress the formation of free radicals through oxidation (Halla et al. 2018). Generally, the used additives in cosmetic formulations surpass the INCI listed base substances or active agents. Except for UV filters and consistency control substances, these additives should be averted due to their prowess to cause allergies (Freitas et al. 2014). Given their ability to protect consumers from UV radiation, UV filters are important additives in cosmetic formulations that are controlled by international safety regulations, which are strictly evaluated before the marketing of these products. Other additives, like TiO2 and ZnO, were also analyzed due to consumers’ concerns and shown to be non-toxic and non-invasive, posing zero risk to human health (Nohynek et al. 2010). The active ingredients used can be naturally synthesized (peptides, ceramides, most vitamins), purified from natural sources (botanicals, herbal extracts), obtained by cell culture fermentation (enzymes and cofactors, polysaccharides and proteins), or extracted from animal sources. Still, the latter reduced greatly after the bovine spongiform encephalopathy related issues (Lintner et al. 2009). The specifications for any cosmetic ingredient are required to combine its chemical identification, in terms of structural formula, the raw material origins, the used extraction method; its physical arrangement, whether it is a powder, paste, gel or liquid; its molecular weight; its purity, with mandatory impurity characterization; its solubility in water and other relevant organic solvents; and additional specifications, like its organoleptic properties, flash point, melting/boiling point (Freitas et al. 2014).
4.3
Main Cosmetic Vehicles
In dermatology, the active substance is usually integrated with a carrier system called the vehicle, a term used to differentiate active from inactive ingredients/pharmaceuticals in the cosmetic and formulation areas (Bohnenblust-Woertz and Surber 2014; Buchmann 2001). They are tailormade for the cosmetic application that they are intended, with developers aiming to achieve stable and compatible vehicles. The vehicles’ patient/consumer acceptability, usage criteria, and bioavailability are also important aspects taken into consideration (Bohnenblust-Woertz and Surber 2014). The vehicle function is to carry and deliver the active ingredients to a specific localization in the skin or inside the human body. Despite not containing pharmacologically active substances for disease treatment, skincare cosmetic formulations
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rely heavily upon vehicles for cleansing, hydration, protection, and decoration purposes (Buchmann 2001; Freitas et al. 2014). Moreover, cosmetic formulations use bifunctional compounds that can act as vehicles and also have a desired effect on the skin. Therefore, there is a great variety of cosmetic vehicles available which, depending on the application, have different physical/chemical properties (Buchmann 2001). A good vehicle should be a homogenous, cosmetically acceptable ingredient with an easy application and removal; should be nontoxic, non-irritant, and nonallergenic, being chemically stable and bacteriostatic; and should be pharmacologically inert, releasing active agents in a controlled and targeted way (Bohnenblust-Woertz and Surber 2014). There are differences, in terms of drug absorption, between distinct cosmetic formulations for topical applications which are caused by the established interactions involving the drug, the vehicle, and the skin. In this regard, there can be three different interactions. The first is the vehicle-drug interactions determined by the drug’s thermodynamic activity on the vehicle; the second is the vehicle-skin interactions that can be enhanced using vehicle components that interact with the stratum corneum improving the drug solubility/diffusion; the third is the drug-skin interactions defined by the skin’s metabolism and binding of the drug in its receptors (Bohnenblust-Woertz and Surber 2014). In cosmetic formulations, vehicle-skin and vehicle-drug are the focused interactions, with many cosmetic ingredients being responsible for decreasing the skin barrier function and increasing the partitioning of active substances from the vehicle into the stratum corneum (Bohnenblust-Woertz and Surber 2014). Polysaccharides have been widely used as vehicle systems such as in emulsions, hydrogels, encapsulating structures, and suspensions (Freitas et al. 2014).
4.3.1 Emulsions Emulsions are the prevailing form of skincare products given their appealing feeling on the skin and ease of application when compared to waterless oils and lipids (Buchmann 2001; Epstein 2009). Lotions are flowing fluid emulsions, while creams are semisolid emulsions (Buchmann 2001; Freitas et al. 2014). An emulsion is constituted by two or more immiscible materials forming a system where one material is suspended/dispersed throughout another material. Since they are immiscible materials, the use of emulsifying agents is determinant because they act as solubilizers, spreading and dispersing the selected actives and other ingredients of the vehicle. Depending on the quantity and nature of the ingredients, an emulsion can feel oily or greasy. Emulsions can be classified into four categories. Macroemulsions (1) are simple emulsions that can be oil-in-water (O/W) or water-in-oil (W/O) like lotions and creams, with droplet size range between 0.1 and 100 μm; while nanoemulsions (2) are simple emulsions (O/W or W/O) with droplet size range between 20 and 200 nm. Thirdly, there are multiple emulsions (3) which are complex systems like oil-in-water-in-oil (O/W/O) or water-in-oil-in-water (W/O/W) with droplet sizes similar to macroemulsions; and microemulsions (4) which are water, oil, and surfactant/co-surfactant systems, constituting a single optically isotropic thermodynamically stable liquid solution, with droplet size range from 10 to 100 nm
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(Epstein 2009; Savary et al. 2016). Surface-active emulsifiers can be anionic, cationic, amphoteric, non-ionic, hydrophobic, lipophilic, ethoxylated, and non-ethyloxylated. All these characteristics will determine the type of emulsion (Epstein 2009). Additionally, there are several other components used in cosmetic emulsions like emollients (sensory properties increasers) such as silicon oil and isopropyl myristate; moisturizers and humectants like glycerol and urea; active substances such as UV sunscreens and vitamins; antimicrobial agents; perfumes and coloring agents; and viscosity-increasing agents (Freitas et al. 2014). Even though most polysaccharides are not surface-active, they increase aqueous phase viscosity and inhibit droplet movement, contributing to the stabilization of emulsions. Nevertheless, emulsifiers based on high molecular weight polysaccharides have already been demonstrated to effectively form and stabilize O/W nanoemulsions. As previously mentioned, xanthan gum is a widely used polysaccharide ingredient in cosmetic industries, acting as a thickener and stabilizer in many applications (Martins et al. 2020). In fact, xanthan gum is a beneficial hydrocolloid due to its solubility in hot/cold solutions, binding water promptly and productively (Martins et al. 2020). Xanthan gum improves the emulsification of W/O emulsions by suspending insoluble ingredients, due to its high viscosity, and can be combined with other substances to enhance texture, flow behavior, stability, and appearance of preparations (Martins et al. 2020). In fact, 1% (w/v) of xanthan gum is enough to largely increase a liquids’ viscosity, with many applications using as little as 0.1% (w/v) xanthan gum in their formulations (Sight et al. 2018). Gellan gum is also used to stabilize emulsions, protecting them against temperature fluctuations, improving quality in transit and shelf-time. In addition to xanthan gum and gellan gum, bacterial cellulose and hyaluronic acid have also been applied in emulsions (Freitas et al. 2014).
4.3.2 Suspensions Suspensions consist of active principles or functional excipients dispersed in a liquid or semisolid medium, acting as a vehicle (Buchmann 2001). If the particles are therapeutically active, the suspension is considered pharmaceutical. By using suspensions, a hydrophobic drug is effectively dispensed, avoiding cosolvents, and decreasing hydrolysis, oxidation, and microbial activity related degradation. For instance, suspensions are used for sunscreens, pigment-containing nail pearlescent lacquers, and also particle-containing shampoos or shower gels (Freitas et al. 2014; Savary et al. 2016). The main disadvantage of suspensions is the possibility of particle sedimentation if the particles possess a higher density than the medium (Freitas et al. 2014; Savary et al. 2016). By increasing the viscosity of the medium, sedimentation can be decreased or prevented. Xanthan gum has effectively been widely applied in suspensions for that end (Freitas et al. 2014). The benefits of using xanthan gum in suspensions are related to the bacterial polysaccharides’ rheological properties, the reduced viscosity at high shear rates promotes deagglomeration and mixing. In addition, gellan gum allows successful stabilization of hair products formulation
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(shampoo and conditioner) due to its thickening capacity and fluid gels development (Freitas et al. 2014).
4.3.3 Hydrogels Hydrogels are three-dimensional polymeric matrices with a hydrophilic nature, allowing the retainment of 10% of weight in water. Therefore, the combination of hydrogels with other chemicals may result in suitable cosmetic formulations (Mitura et al. 2020). A hydrogel may be classified depending on its origin (natural and/or synthetic), on its physical or mechanical properties, on the nature of its polymer side groups (ionic or non-ionic), the cross-linking type, and its response to chemical stimulation (Michelon et al. 2019). Natural hydrogels are constituted by either polysaccharide chains, like chitosan, cellulose, and hyaluronic acid, or by protein chains, like collagen. On the other hand, there are also synthetic hydrogels that are based on polymer like poly-ethylene glycol or poly-acrylamide. Hybrid hydrogels are comprised of natural and synthetic polymers combinations (Michelon et al. 2019). Regarding cosmetic formulations, hydrogels are used for skin, hair, and oral care applications; being based on several biopolymers like collagen, hyaluronic acid, alginate, chitosan, xanthan gum, pectin, starch, and cellulose (Mitura et al. 2020). Biopolymer based hydrogels are used for the development of new cosmetics like beauty masks, which promote skin hydration and restore skin elasticity, producing an anti-aging effect. Hyaluronic acid-based hydrogels have a wide variety of applications, acting as a wrinkle removal agent, treating nasolabial folds, treating skin augmentation, and promoting skin hydration and collagen stimulation (Mitura et al. 2020). Gellan gum characteristics (binding properties and gel structure) have been shown to have advantages in toothpaste formulations (Freitas et al. 2014). Levan was also demonstrated to have potential in dermal filling applications when combined, in a hydrogel, with pluronic and carboxymethylcellulose (CMC) (Choi et al. 2018). The results of this study showed that the novel levan-based hydrogel presented stability and strength enhancements, in vitro and in vivo. The product was established as a biocompatible material with the potential to stimulate dermal fibroblasts proliferation and to enhance gene expression for type I collagen production. For these reasons, the studied hydrogel was considered a valid, if not better, alternative to skin filler applications, when compared to the traditional hydrogel (based on hyaluronic acid) (Choi et al. 2018). 4.3.4 Encapsulating Structures Encapsulation is a common process in the cosmetic industry, aimed at the preservation of active ingredients during each stage of the product fabrication (formulation, storage, application) (Faieta et al. 2019). In fact, many personal care products require encapsulation, to increase the stability of its biologically active substances. For example, vitamins are not stable, being influenced by pH, light intensity, and oxidation, and the encapsulation technique ensures they are protected against degradation. Encapsulation also promotes the release of an active substance in a specific target (Ammala 2013; Faieta et al. 2019). The use of biodegradable polymeric carriers for the encapsulation increases efficacy and bioavailability of active
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ingredients, ensuring its degradation using normal metabolic pathways. The utilization of low moisture amorphous matrices is common given the high stability conferred to bioactive substances, as well as promoting the application of the encapsulated ingredients in formulated products (Faieta et al. 2019). The encapsulating materials available for cosmetic applications include polysaccharides, proteins, lipids, and natural/synthetic polymers. Moreover, second polymers can be comprised of inorganic materials (usually silicates, clays, and polyphosphates) (Casanova and Santos 2015). In skin delivery systems, the most interesting encapsulating materials are chitosan, aliphatic polyesters (e.g., poly lactic acid (PLA)), and copolymers of lactic and glycolic acids (e.g., PLGA – poly lactic-co-glycolic acid). They are natural and non-toxic materials, that do not react when contacting human tissues, which can be metabolized using normal metabolic pathways (Casanova and Santos 2015). The most used technique for encapsulation is spray drying, due to its simple, fast, and low-cost application. The first step of spray drying consists of dissolving, dispersing, or emulsifying the active compound and the encapsulating matrix in an aqueous solution. Afterward, the mixture is submitted to atomization and drying in contact with a hot gas flow (Lourenço et al. 2017). Other examples of encapsulation techniques are lyophilization, cocrystallization, fluidized-bed coating, and extrusion (Bodade and Bodade 2020). Nylon microparticles loaded with vitamins, sun filters, moisturizers, fragrances, and many other actives like caffeic acid ester, retinyl palmitate, D-panthenol, vitamin C, bioflavonoid, catechins, resveratrol, rosmarinic acid, vitamin P, linoleic acid, lycopene, benzoyl peroxide, tocopheryl acetate, dihydroxyacetone, salicylic acid, vitamin E, and dimethicone are among the examples of cosmetic substances encapsulated with a wide range of materials (Casanova and Santos 2015; Freitas et al. 2014). Using chitosan for encapsulation has proven to be possible, due to the increment on polymer matrix viscosity, which permitted the controlled release of the active ingredient. In terms of skin permeability, the encapsulation of retinoic acid with chitosan enabled a slower and controlled release rate of retinoic acid, when compared to free retinoic acid. Moreover, chitosan encapsulation may enhance the properties of encapsulated cosmetics while protecting against environmental agents, which could help to overcome retinoic acid and alpha-tocopherol limitations by improving their light and oxygen sensitivity, and reducing skin irritation. Hyaluronic acid, in its pure or modified form, is also able to encapsulate proteins, allowing their controlled release for several medical treatments. Hyaluronic acidbased nanoemulsions were tested as transdermal carriers, with acceptable stability. Moreover, these emulsions were tested for encapsulation with vitamin E, and the results showed a successful carrying of lipophilic additives (Kong et al. 2011). Additionally, skin penetration and histological experiments using vitamin E-hyaluronic acid nanoemulsions were made, with the results showing there was no dermis irritation and the delivery carrier of active lipophilic ingredient was effective (Kong et al. 2011). In another study, the encapsulation and stabilization of rosmarinic acid (RA) using poly(lactic-co-glycolic acid) (PLGA) was performed. The results showed that RA-loaded PLGA microparticles exhibit antioxidant and
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antibacterial activities, without releasing any harmful substance to human dermal fibroblasts, which suggests that electrosprayed RA-loaded PLGA microparticles could be used in cosmetic and pharmaceutical products. Dextran and gellan gum were also reported as materials used for microencapsulation (Bodade and Bodade 2020). FucoPol was also studied as a potential encapsulation matrix for a spray drying process. The technique produced spherical capsules with a smooth surface, which were loaded with gallic acid and oregano essential oil. The results acknowledged FucoPol’s potential to encapsulate bioactive compounds for application in the cosmetic industry (Lourenço et al. 2017).
4.4
Safety Requirements and Regulation
In general, cosmetic products have presented a good safety record throughout time but there are several examples of harmful experiences for costumers. For instance, until the early twentieth century, make-up dyes had toxic heavy metals, such as lead, mercury, and cadmium oxides in their constitution. In the 1930s, depilatory products caused severe intoxications, due to the thallium present in their formulation. Another example was the 1958/1959 epidemic of photo-allergic reactions in the UK caused by halogenated salicylanilide-containing cosmetics. During a decade (1950–1960), deodorants constituted by zirconium caused an outbreak of long-lasting allergic inflammatory skin reactions in consumers in Europe and the USA (Nohynek et al. 2010). A strict and rule-guided toxicological safety evaluation has been pivotal to prevent this type of occurrence, and for instance, in both the EU and US cosmetics safety evaluation has a particular legal position where they need to be proven safe under normal use conditions (Nohynek et al. 2010).
4.4.1 The Requirements of the EU Regulation The safety assessment of cosmetics requires a detailed toxicological profile of all used ingredients and manufacturers need to present a report regarding the effects of product-specific exposure (Hussain-Gambles 2020) in order to meet the requirements of the EC Cosmetics Regulation 1223/2009 (EC, 2009). Moreover, technical details of the ingredients and the final formulation placed on the consumer market need to be disclosed to receive EC approval (Basketter and Whitte 2016; HussainGambles 2020; Nohynek et al. 2010). Europe has been active in animal-rights related issues and companies are required to present toxicological data for hazard identification using ex vivo or in vitro methods, instead of the usual animal testing, which is strictly prohibited under the EC Cosmetic Regulation No. 1223/2009. Another particularity of the EC Regulation No. 1223/2009 is the use of the same legal text, with a binding nature in all member states, which simplifies regulations in the EU market, ensuring that all companies comply under the same rules (Basketter and Whitte 2016; Hussain-Gambles 2020). The Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) is responsible for the administration of Directive 76/768/EEC. SCCNFP belongs of the European Commission and is composed of knowledgeable scientists from the different Member States (Pauwels and Rogiers 2004). There are three stages
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for the safety evaluation of cosmetics conducted by SCCNFP. The first stage consists of identifying hazardous substances by analyzing a specific ingredient, and usually, a newly developed ingredient is required to be presented by individual firms and/or the European Toiletry and Perfumery Association (Colipa). The second stage is the risk assessment of the toxicology particularities of an ingredient under consideration, using available literature and studies, which allows safety evaluations for consumers exposed to the finished cosmetic product. The third stage is only used when additional toxicological tests are requested to reassess the safety profile of the ingredient under consideration (Pauwels and Rogiers 2004). The toxicological requirements for cosmetic ingredients according to Cosmetic Directive 76/768/ EEC are condensed in the SCCNFP Notes of Guidance (SCCNFP/0690/03, 20031) as follows: (1) Acute toxicity (if available); (2) Irritation and corrosivity; (3) Skin sensitization; (4) Dermal/percutaneous absorption; (5) Repeated dose toxicity; (6) Mutagenicity/genotoxicity; (7) Carcinogenicity; (8) Reproductive toxicity; (9) Toxicokinetics; (10) Photo-induced toxicity, and (11) Human data (Pauwels and Rogiers 2004; Nohynek et al. 2010). To perform the safety evaluation of the finished product (Table 5), all the available information (chemical, toxicological, and technical) are compiled in the TIF of the product, which is conjugated with the human exposure studies/information. The evaluated cosmetic risk is focused on irritation (and photo-irritation if relevant) and immunobiological reactions, such as contact allergy and eventually photo-allergic reactions. Potential systemic effects are also important in case of considerable skin penetration and/or oral intake of the product. The assessment must indicate, in an undersigned document, if a certain cosmetic product can be available on the EU market, posing no human health-related risks, considering a normal utilization (Pauwels and Rogiers 2004). Companies are also required to present a Cosmetic Product Safety Report (CPSR), which is divided into two main parts (Table 6). Part A compiles all the Table 5 Tools in safety evaluation for cosmetic ingredients and finished products (based on Pauwels and Rogiers 2004) Safety aspects Hazard
Exposure
Risk
Tools Animal studies Replacement studies Human data, epidemiological studies, clinical studies Product type Use pattern Application site Concentration Frequency Amount applied Chemical composition Stability Microbiological purity Target population Percutaneous absorption data Safety evaluation
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Table 6 Content of cosmetic product safety report to the European Directive 93/35/EEC (based on Basketter and White 2016) Part A- Cosmetic product safety information 1. Quantitative and qualitative composition of the cosmetic product 2. Physical/chemical characteristics and stability of the cosmetic product 3. Microbiological quality 4. Impurities, traces, information about the packaging material 5. Normal and reasonably foreseeable use 6. Exposure to the cosmetic product 7. Exposure to the substances 8. Toxicological profile of the substances 9. Undesirable effects and serious undesirable effects
Part B- Cosmetic product safety assessment 1. Assessment conclusion 2. Labelled warnings and instructions of use 3. Reasoning 4. Assessor’s credentials and approval of part B
information related to product composition, microbiological quality, physical/chemical characteristics, and stability; Part B is the safety assessment conducted by a qualified EU Safety Assessor (Turnbull 2018). There are several annexes in the EU Cosmetics Regulation, detailing the required content of the CPSR (Annex I), listing the prohibited substances to use in cosmetic products (Annex II), substances subjected to restrictions (Annex III), coloring agents (Annex IV), allowed preservatives (Annex V), and allowed UV filters (Annex VI) (Basketter and Whitte 2016).
4.4.2 The Requirements of the United States The US Food and Drug Administration (USFDA) is the government agency responsible for the safety of personal of cosmetics, designated in the U.S. Food, Drug and Cosmetic Act of 1938 (Nohynek et al. 2010). Specifically, cosmetics are regulated by a specialized branch of the USFDA called the Center for Food Safety and Applied Nutrition (CFSAN) (Turnbull 2018). Cosmetic drugs are considered to be over-the-counter (OTC) drugs, which are scoped under the definitions of both cosmetics and drugs. In fact, there are several products, listed in the EU as cosmetics, that are considered OTC drugs by the USFDA. Among these products, sunscreen products, fluoride-containing toothpastes, antiperspirant deodorants, antidandruff preparations, moisturizers, and makeup with sun-protection properties, are the most well-known cases. In these cases, if the manufacturer wants to sell the product in both the EU and US markets, they must meet both regulatory requirements (Nohynek et al. 2010; Turnbull 2018). For instance, an antidandruff shampoo is a cosmetic because it is focused on hair-cleaning, but it is also considered a drug due to the intention to control and help prevent the recurrence of dandruff symptoms, like flaking and itching (Turnbull 2018). Cosmetic Ingredient Review (CIR) and Good Clinical Practices (GCPs) The Cosmetic, Toiletry and Fragrance Association, now denominated the Personal Care Products Council (PCPC), determined the guidelines of the Cosmetic Ingredient Review (CIR) in 1976, a mechanism for voluntary self-regulation of the industry (Nohynek et al. 2010). The CIR has an independent expert panel (constituted by
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Tens of participants
Months Up to 500 participants
Confirm results Several years Up to thousands of participants
Phase IV
Weeks
Efficacy
Phase III
Humam safety
Phase II
Phase I
Toxicologists, Dermatologists, and Chemists from academic institutions) that review the available data on cosmetic ingredients and decides if they are considered safe (Boyer et al. 2017; Nohynek et al. 2010). The CIR panel also includes 3 nonvoting members appointed by the USFDA, Consumer Federation of America (CFA), and PCPC. These members represent, respectively, the government, consumer, and industry interests, acting as a link between the Expert Panel and the stakeholders (Boyer et al. 2017). The minimal data required to submit a cosmetic ingredient to CIR is the used concentration, the chemistry data, the skin irritation and sensitization, the dermal absorption data, and the genetic toxicity (Nohynek et al. 2010). The safety assessments of different ingredients used in the cosmetic industry are published in the International Journal of Toxicology (Nohynek et al. 2010). Regarding bacterial polysaccharides, CIR considered hyaluronic acid (Becker et al. 2009), bacterial cellulose (Final Report of the CIR) xanthan gum, gellan, levan, and dextran (Fiume et al. 2016) to be safe ingredients for cosmetic formulations. In fact, studies showed that hyaluronic acid did not provoke adverse reactions in topical applications and for the treatment of osteoarthritis and tissue augmentation with injected hyaluronic acid did not raise safety concerns regarding its use in cosmetics (Becker et al. 2009), being cytotoxic in high concentrations (Silva et al. 2017). Xanthan gum is also widely used in the cosmetic industry (3,470 reported uses), being present at up to 6% in leave-on formulations (Fiume et al. 2016). Moreover, xanthan gum and dextran are present pharmaceutical applications and have demonstrated to be of safe use. For example, Dextran (3,000 to 20,000 Da) cannot be absorbed through intact human skin (Fiume et al. 2016). Levan cytotoxicity has also been evaluated in human fibroblast and keratinocyte cell lines. The results showed that levan (100 mg/mL) was not cytotoxic to human fibroblasts, presenting a proliferative effect in keratinocytes, with proliferation over 30% at levan concentrations higher than 1 mg/mL (Fiume et al. 2016). Ocular irritation was also evaluated, with xanthan gum (1%) and gellan gum (0.8%) presenting no ocular irritancy in rabbits, while gellan gum (0.5%) did not have an irritation effect in humans (Fiume et al. 2016). After the ingredients and formulation safety assessment is complete and approved, USFDA requires a stage of human subject testing. These tests are executed under the control of Good Clinical Practices (GCPs) to demonstrate human tolerance and confirm the intended use of the product. An important note is that this stage of human testing is not intended to analyze the existence of hazards, but to confirm the safety of the cosmetic product (Fig. 13).
FDA Review Safety in typical pacientes Massive population
Fig. 13 Development of Good Clinical Practices steps (based on Foroughi-Heravani et al. 2020)
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Conclusions
A growing trend toward more sophisticated cosmetics and personal care products is observed due to the desire of formulators to obtain a competitive advantage to meet consumers’ expectations for product improvements and enhancements. The literature review revealed the positive effects of polysaccharide-based formulations on skin human, namely, the improvement of the skin barrier function and hydration. Moreover, polysaccharides rheological properties can increase formulation stability and enhance sensorial properties, which are important aspects of formulation technology. Accordingly, polysaccharides have been extensively used in cosmetic formulations. For example, hyaluronic acid is used as a bioactive ingredient, and in cosmetic vehicles; xanthan gum is used in cosmetic products due to its high viscosity-enhancing ability at low concentrations. Additionally, there are several promising bacterial polysaccharides, such as FucoPol and GalactoPol, which are still in the development studies to be used in cosmetic formulations. Finally, polysaccharide-based formulations are important to maintain physiological skin conditions and to prevent and treat skin disorders related to barrier function alterations.
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 The Specialty Bacterial Polysaccharides for Skin Care Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Chitin and Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Hyaluronic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Xanthan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Levan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 The Specialty Fungal Polysaccharides for Skin Care Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Lentinus edodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Ganoderma lucidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Agaricus subrufescens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Grifola frondosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Trametes versicolor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Pleurotus ostreatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Poria cocos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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M. Kanlayavattanakul (*) · N. Lourith School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, Thailand Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University, Chiang Rai, Thailand e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_46
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4 Challenges and Future Prospects of Microbial Polysaccharides for Cosmetics . . . . . . . . . . . 4.1 Biodegradable Material for Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Source and Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Polysaccharides are proved to be one of the important cosmetic ingredients especially for skin care products. Among which, those of renewable, eco-friendly, and accountable for circular bio-economy productions are highly in demands of the cosmetic consumers, which hit the interests of the researchers. Of which, biotechnological production route on the basis of microbial produced polysaccharides is the appointed choices. In this chapter, microbial polysaccharides applicable for cosmetics are summarized exclusively for those of skin care applications. The specialty bacterial and fungal polysaccharides, those of emerging mushroom, producing and commercializing for cosmetics are enclosed. In addition, challenges and future prospects of microbial polysaccharides in cosmetic industries in the sectors of packaging and delivery system are included in the context as well as the promising sources and productions. Keywords
Cosmetics · Skin care · Microbial polysaccharide · Cosmetic package · Active ingredient · Functional ingredient
1
Introduction
Classification of polysaccharides in cosmetics is on the basis of their actions in the products, i.e., functional and active polysaccharides. Functional polysaccharide is the compulsory ingredients to formulate the preparation with the desired preferences and textures. Thus, they act as gelling agent, viscosity adjuster, thickener and emulsifier, etc. Active polysaccharide is positively related with the claim efficacy of the skin care preparations, for instance, skin hydration, moisturizing, anti-wrinkle, or astringent agents. Furthermore, they can be categorized on the basis of their electrochemical charges in the structure that are anionic, cationic, nonionic and amphoteric polysaccharides. However, this categorization positively reflect onto those of functional polysaccharides rather than the active ones. In the view of the incorporated quantity of the polysaccharide in cosmetics, the high content of functional polysaccharide is required with the properties range from forming viscous solutions to exhibiting a pseudoplastic material nature, but low for active action (Kanlayavattanakul and Lourith 2015). Accordingly, the unit price for those of functional polysaccharides are lower than the active polysaccharides. Among which, naturally derived
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polysaccharides are highly in demand than the functional ones. Although polysaccharides suppling for cosmetic and personal care industries are majorly derived from plants, recent biotechnology formation of polysaccharides with modified structures for expected properties can be achieved by using of microorganisms (Freitas et al. 2011, 2014) with the safety requirements that meet the standard of cosmetics in particular safety. Polysaccharides derived from microorganisms, including bacteria, yeasts, fungi, and some algae, are emerging as an unexploited market. Polysaccharide biosynthesis and its accumulation generally take place after the growth phase of the microorganism. The polysaccharides produced by microorganisms can be classified into three main groups: (i) capsularpolysaccharides, which provide a carbon and energy sources for the cell; (ii) lipopolysaccharides that make up the cell wall; and (iii) exopolysaccharides (EPSs) that are exuded into the extracellular environment in the form of capsules or biofilm (Chen et al. 2010; Donot et al. 2012). In addition, microbial polysaccharides can be categorized into cellulose, xanthan, dextran, and alginate (Freitas et al. 2011, 2014). However, additional category of the microbial polysaccharides applicable in cosmetic industry are more versatile and feasibly suit with the dosage form and their expected claims as well. The two main industrially relevant microbial polysaccharides worldwide are cellulose and alginate with the revenue of over 22 billion USD annually and projected to reach 50 billion USD by 2025 (Anderson et al. 2018). Of which, microbial polysaccharides are regarded as the potential sources producing the specialty polysaccharides supplied for certain industries (Huertas and Matilla 2020) including skin care products. Amidst bacterial polysaccharides, exopolysaccharides are the main source of cosmetic industrial importance. Exopolysaccharides with different sugar moieties and the structural charge produced by a wide range of microorganisms used in skin care cosmetics are exemplified in Table 1. Polysaccharides used in cosmetic proposes can be in a modified form and composited with different materials in an order to tailor-made the expected properties. The most familiar cosmetic awareness on microbial polysaccharides are their active actions as the safe and efficient moisturizer and Table 1 Examples of microbial polysaccharides of different sources Polysaccharide Alginate Cellulose Chitin Chitosan Curdlan Gellan Hyaluronic acid Levan Pullulan Xanthan
Strain Azotobacter vinelandii, Pseudomonas aeruginosa Gluconacetobacter spp., Acetobacterium spp. Aspergillus fumigatus, Saccharomyces cerevisiae Bacillus pumilus, Streptomyces griseus Agrobacterium spp. Sphingomonas paucimobilis Streptococcus spp. Zymomonas mobilis, Halomonas smyrnensis, Bacillus subtilis Aureobasidium pullulans Xanthomonas campestris
Charge Anionic Nonionic Cationic Cationic Nonionic Anionic Anionic Nonionic Nonionic Anionic
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anti-wrinkle agent (Kanlayavattanakul and Lourith 2015, 2019; Lourith and Kanlayavattanakul 2016, 2019). Microbial polysaccharides are fond of more importance in cosmetic products accordingly. Those for skin care products are therefore exhaustively focused in this chapter. Of which, they are sometime known or called as specialty polysaccharides.
2
The Specialty Bacterial Polysaccharides for Skin Care Cosmetics
Bacterial polysaccharides have been exploited in a number of industrial uses. The particular application of a specific polysaccharide is a reflection of its unique physical properties. In cosmetic industry, different classes of polysaccharides are applied. Of which, cellulose, chitin and chitosan and hyaluronic acid are of importance in skin care products on the basis of their applications by functional and active actions (Lourith and Kanlayavattanakul 2018). The specialty bacterial polysaccharides, cellulose, chitin and chitosan, and hyaluronic acid will be therefore exclusively addressed in this section regarding on their widely used as skin hydrating agent besides their functional actions. In addition, xanthan gum, dextran, curdlan, gellan, levan, pullulan, and alginate are briefly addressed as per they are majorly implied as functional polysaccharides, although their skin hydrating capabilities are exhibited but in a moderate degree.
2.1
Cellulose
Cellulose, a homopolymer of (1-4)-β-linked glucose, is the most abundant biological polymer. Apart from being a major component of plant cell walls, cellulose is also synthesized by Acetobacterium, Rhizobium, Salmonella and Saricana spp. Bacterially derived cellulose is much purer than that extracted from plant tissue and as such it has been proposed for use in a number of medical applications including as an artificial skin, for topical drug delivery, and in wound dressings (Klemm et al. 2006; Rehm 2010; Shah et al. 2013), and of course for cosmetic applications. Cosmetic benefits of this polysaccharide is due to the nature of its hygroscopic material with a flow behavior as a shear thinning fluid. The natural anionic nature of cellulose enables its interfacial activity. Not only the native form of cellulose that is largely used in cosmetics, derivatization into ethers (Fig. 1) with a different of degree of substitution (DS) that tailor making the physicochemical properties is accounted as the major form used. In addition, physicochemical property adjustments can be also achieved in term of molar substation (MS) by propylene oxide. The resulting derivatives thereafter expand the applications for instance cellulose ether that prolonging its shelf-life and increase solubility in common vehicles used in skin care cosmetics. Derivatization of cellulose into ether forms change the anioinic nature of cellulose into nonionic
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Fig. 1 Preparation of cellulose ethers
polymers with the maximum DS of 3 (3 hydroxyl group derivatizable). Cellulose ethers are generally recognized as safe (GRAS) in similar to the native form. Therefore, they gain highly acceptance to be incorporated as functional polysaccharides in cosmetics as summarized in Table 2.
2.1.1 Methylcellulose Due to strong intermolecular hydrogen bonding between the hydroxyl groups on the cellulosic chains, unmodified cellulose is insoluble in water. However, methylation of cellulose introduces hydrophobic groups on the cellulosic chains producing methyl cellulose (MC), which can be easily dissolved in cold water. Furthermore,
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Table 2 Cosmetic functions of cellulose ethers Application Detergent
Celluloses CMC, HPMC
Cream, shampoo, lotion, ointment, gel
CMC, MC, HEC, HPMC
Function Anti-redeposition, wetting ability, suspender, emulsifier Thickener, binder, emulsifier, stabilizer, film former
MC can be modified into different form to fit with the desired properties for example hydroxypropyl methyl cellulose (HPMC) and so forth. Carboxymethyl cellulose, popularly known as CMC, is a carboxymethyl ether of cellulose. The ionic polysaccharide is water soluble. It can be produced and commercialized in free or salt forms. Of particular, commercial CMC are those with DS of 0.4–1.5 that make them pH media unaffected between 5 and 9. CMC is widely available in a number of grades for specific applications in the industry. For instance, extra pure CMC grade is used in food products, pharmaceuticals, and toothpastes, while semi-purified and technical grades are used for detergents, etc.
2.1.2 Hydroxyethyl Cellulose This cellulose ether is the most hydrophilic used cellulose ether with cloud point of more than 100 C. Hydroxyethyl cellulose (HEC) tolerates extremes of pH and salts. Therefore, it is accounted and extensively used as surfactant with a several form of derivatizations of HEC with a modified properties such as ethylhydroxyethyl cellulose (EHEC). 2.1.3 Hydroxypropyl Cellulose Hydroxypropyl cellulose (HPC) is the modified polysaccharide with cloud point arounds 45 C is more lipophilic than HEC. Therefore, it is easily dissolved in cosmetic solvents and giving a higher thickening effect. In addition, its thermoplastic nature (film forming during melts) with liquid crystallinity makes it insensitive to pH fluctuations. Once derivatized into ether forms, they turn to be nonionic polymer acting as thickening agents in the formulation with surface activity in addition to its task as film former. At a greater molecular weight, they become greater pseudoplasticity cellulose. Substitution with a higher methyl residue results in a harder gel. In contrary, if higher hydroxypropyl substitution took place (for example, HPMC), a softer gel will be offered. Therefore, MC & HPMC are DS unaffected viscosity cellulose ethers.
2.2
Chitin and Chitosan
Chitin is the second most abundant natural polysaccharide after cellulose. Chitin itself has low toxicity and is biodegradable polysaccharide with antibacterial properties, hydrophilicity, gel-forming properties, and affinity for proteins. Therefore, various applications according to its biological activities and wound healing effect are widely evidenced. Structural differences of chitin and its derivative, chitosan are shown in Fig. 2.
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OH
OH O
*
HO
O
OH
O * *
HO
B
A
O HO
NH2
C
O
*
OH
OH
*
NHAc
O
O * *
HO
HO O
NHAc
NH2 *
O
D
Fig. 2 Chemical structures of cellulose (a), chitin (b), chitosan (c) and partial deacetylated chitosan (d) repeating units
Chitin can be produced in the form of aminoglucan (poly-GlcNAc) by Saccharomyces cerevisiae (β-(1,6)-glucan), Aspergillus fumigatus (β-(1,3/1,4)-glucan), Candida albicans, Fusarium oxysporum, Microsporum fluvum, and Epidermophyton stockdaleae. On the means time, Aspergillus niger, Humicolo lutea, and Fusarium moniliforme are reported to be the producers of chitosan. Although two other strains that are commercially used to prepare chitosan are also reported, Bacillus pumilus chitosanse is more effective than Streptomyces griseus chitinase. The antimicrobial activities, wound healing, and antioxidant properties including asthma and atopic dermatitis curing effects enable high quality of chitin application in cosmetics. It is able to maintain skin moisture, treat acne, skin toning, improve suppleness of hair, and reduce electrostatic of hair as well as its efficient surfactant and humectant functions. High molecular weight polysaccharides are especially utilized for skin care cosmetics. Of which, the polysaccharides with a molecular weight of 104–106 Da are used as broad-spectrum ingredient in hair care cosmetics. Significant trans-epidermal water loss (TEWL) reduction of skin treated with high molecular weight chitosan was reported. This, consequently brings softer and more flexible skin accordingly in addition to its irritation reduction efficacy. Improvement of sun protection and product’s water resistance is attributed by the emulsion containing chitosan as well as adhering affinity of perfume containing chitosan onto skin that prolong wearing duration by a reduction of an evaporation rate. Moreover, its applications for deodorant and antiperspirant products are also available concerning to humectantcy of chitosan. The hydrophilicity of chitosan efficiently removes sebum and oils from hair, scalp, and skin (Lourith and Kanlayavattanakul 2018). Thus, it is appreciable to be used for greasy/oily skin treatment diminishing severity of acne and promote wear-ability of facial makeup applications.
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Hyaluronic Acid
Skin is composed of two distinct areas; epidermis and dermis. Epidermis consists of many layers of dead skin that are supported by the dermis. Dermis is a threedimensional network of collagen fibers and elastin fibers surrounded by gel-like material called the ground substance. Dermis accounts for most qualities considered on aesthetic property, i.e., the appearance of the skin. These include a moist, plump appearance and tautness of skin. The aging process is therefore considered skin that is less taut, less moist, less plump, and in some areas, sagging along lines that are flexed (also known as wrinkles) (Lourith and Kanlayavattanakul 2016, 2018, 2019). The extracellular matrix (ECM) is a gel-like material filling the extracellular space in skin tissues that holds cells together and provides a porous pathway for the diffusion of nutrients and oxygen to individual cells. This structure is composed by fibrous proteins (collagen, elastin, fibronectin, and laminin) and heteropolysaccharides, namely, glycosaminoglycans (GAGs). These heteropolysaccharides are unbranched polysaccharide chains, composed of repeating disaccharide units. GAGs are highly negatively charged, due to the presence of sulfate or carboxyl groups on most of their sugar residues. Based on the disaccharide composition, linkage type, and on the presence of sulfate groups, GAGs have been divided into four main groups: (i) hyaluronic acid (HA), (ii) chondroitin sulfate (CS) and dermatan sulfate (DS), (iii) heparan sulfate and heparin (HS), and (iv) keratan sulfate (DeAngelis 2012). They are called GAGs because one of the two sugar residues in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated. The second sugar is usually an uronic acid (glucuronic or iduronic), except for dermatan sulfate that contains galactose. GAGs are also known as mucopolysaccharides with biomedical benefits as summarized in Table 3. Hyaluronic acid (HA), also named hyaluronan, is the mostly used GAGs in skin care products. HA is a polyanion that can self-associate and can also bind to water molecules (when not bound to other molecules), giving it a stiff, viscous quality similar to gelatin. HA is one of the major elements in ECM of vertebrate tissues. It is available in almost all body fluids and tissues. It is also involved in several important biological functions, such as regulation of cell adhesion and cell motility, manipulation of cell differentiation and proliferation, and providing mechanical properties to tissues. HA is responsible for providing the viscoelasticity of some biological fluids and controlling tissue hydration and water transport. In addition, HA has been Table 3 Biomedical benefits of different GAGs GAG HA
CS DS HS
Biomedical benefits Surgical aid (ophthalmic; intra-abdominal anti-adhesive), viscoelastic supplementation (joint injection), moisturizer (eye drops, skin humectant), dermal filler, device coating, drug delivery, tissue engineering Surgical aid (ophthalmic, nutraceutical (joint health), tissue engineering) Anticoagulant Anticoagulant, device and stent coating, anti-proliferative, tissue engineering
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found during embryonic development in the umbilical cord, suggesting that materials composed of HA may persuade favorable conditions for tissue regeneration and growth. HA’s characteristics, including its consistency, biocompatibility, and hydrophilicity, have made it an excellent moisturizer in cosmetic dermatology and skin care products. In cosmetic applications, HA is simply divided into high molecular weight (MW) HA (HMWHA; 105–107 Da) and how molecular weight HA (LMWHA; 2 104–4.5 105 Da). HMWHA with anti-angiogenic activity is also exhibited fibrinogen binding enhancement assisting in clot formation in wound healing and therefore inhibit scar formation in addition to its anti-inflammatory activity. LMWHA is a good collagen type I & VIII synthesis enhancer and also expresses activities toward matrix metalloproteinase (MMP)-3, MMP-9, MMP-13, and hyaluronan synthase (HAS)-2 that are responsible for firmness of skin (Lourith and Kanlayavattanakul 2016, 2018, 2019). Biological functions of HA depend on chain length, molecular mass, and synthetic circumstances. Several microbes are differently used to generate hyaluronic acid, i.e., Agrobacterium sp., Bacillus sp., Escherichia coli, Lactococcos lactis, Bacillus subtilis, and Streptpcoccus sp. especially S. zooepidermicus (Mattila et al. 2018). HA has been approved as a dermal filler, and were known to be the second most popular non-surgical procedure for women and the third most popular procedure for men. Many dermal fillers have been developed, which the widely commercializing ones, Restylane ® and Juvéderm™ are made from Streptococci (Lourith and Kanlayavattanakul 2016, 2019).
2.4
Xanthan
Xanthan, a water soluble anionic polysaccharide, with the molecular weight of 2–50 106 Da has unique rheological properties demonstrating pseudo-plastic flow and viscoelasticity, retaining its physical properties over a broad temperature range (Freitas et al. 2011, 2014). Quantity of xanthan gum in cosmetics is positively affect onto sensory of the product especially integrity of shape, penetration force, wetness, ease of spreading, and gloss (Dubuisson et al. 2018). Xanthan gum is functionally used in cosmetics as stabilizer and thickening agent (Fiume et al. 2016). The main source of microbial production of xanthan gum for cosmetic products is Xanthomonas campestris, which can be further modified into xanthan gum cross polymer and xanthan gum hydroxypropyl trimonium chloride for variety of cosmetic function in regards with its good stability over a wide range of temperature, pH, and salt concentrations (Kanlayavattanakul and Lourith 2015).
2.5
Dextran
Dextran, a neutral polysaccharide, is low immunogenicity, has led to numerous clinical and pharmaceutical applications including cosmetics. Dextran is soluble in water, and
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a dextran solution behave as Newtonian fluids and has a viscosity that changes as a function of concentration, temperature, and average molecular mass. The most common forms of dextran to be used in skin care preparation are native dextran and dextran sulfate that are produced from Leuconostoc mesenteroides with the molecular size of 106–108 Da (Kanlayavattanakul and Lourith 2015) to function as skin conditioning and bulking/binding agent. In addition, modifications of dextran into sodium dextran, carboxymethyl dextran, sodium carboxymethyl dextran, and dextran hydroxypropyl trimonium for cosmetics proposes are conducted to vary their function as suspending agent, film former, binder, and conditioning agent, respectively (Fiume et al. 2016). In addition to Leuconostoc, Streptococcus, Weissella, Perdiococcus, and Lactobacillus are able to produce dextran (Kanlayavattanakul and Lourith 2015).
2.6
Curdlan
Curdlan is a neutral linear 1,3-β-D-glucan with the molecular weight range from 5 104 – 2 106 Da synthesized by several Gram negative bacteria, Alcaligenes faecalis including Agrobacterium spp., Rhizobium spp., Bacillus spp., and Cellulomonas spp. It is used for various industrial applications based on its uncharged structure. Although curdlan is insoluble in water, it could be dissolved in sodium hydroxide solutions and highly safe to be used not only for food products but also personal care preparations. It appeared to form triple helical structures under appropriate conditions. Thereafter, it serves as a gel forming agent in skin care products with a wide pH range application for either high or low setting gel (>80 or 55–60 C) depends on its molecular weight (Kanlayavattanakul and Lourith 2015). For its biological activity applicable in cosmetics, curlan is one of the antioxidant polysaccharides (Giese et al. 2015) capable to enhance superoxide dismutase or SOD activity (Lin et al. 2008). Moreover, its stimulating capacity was exhibited in the wounded treatment examined in an animal model (Berdal et al. 2007). The biological activities are relating with its glucan backbone. Of which, glucan is regarded as the active polysaccharide with collagen synthesis stimulating activity in addition to inductions of reepithelization and collagen deposition processes associating its potency against cellular aging (Kwon et al. 2009).
2.7
Gellan
Gellan has been proved to be one of the interesting recent microbial polysaccharide introductions as xanthan competitor. This anionic polysaccharide is produced commercially from a strain of Pseudomonas elodea with the molecular weight approximately 5 106 Da in addition to its procurable strain, Sphingomonas paucimobilis. It mainly functions as a stabilizer and thickener, which is stable over a wide range of pH. The gelling properties of this polysaccharide are only fully revealed after chemical deacylation to remove the O-acetyl and O-glyceryl substituents. The native
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polymer only formed soft, elastic gels but progressive removal of the acyl groups increased the brittleness of the gels produced in the presence of divalent cations. The extent of acetylation is thought to control the local crystallization of parts of the polysaccharide chains. Divalent ions linked the gellan molecules causing gelation. All possessed closely related chemical structures but none of the others formed gels although most yielded highly viscous solutions with considerable thermostability (Kanlayavattanakul and Lourith 2015).
2.8
Levan
Numerous Gram-positive bacteria, including Bacillus sp., and Gram-negative bacteria, including Z. mobilis, are able to produce levan. Fermenting saccharose with bacteria, such as Zymomonas mobilis or Bacillus subtilis or by enzymatic synthesis using saccharose as substrate produces the nonionic polysaccharide leven, with the molecular weight that less than 108 Da (Kanlayavattanakul and Lourith 2015). In addition to its film forming ability, levan is regarded as a thickening agent with a skin protecting property (Fiume et al. 2016). Antioxidant activity of levan was reported to be 81% of the standard vitamin C (Srikanth et al. 2015). Its safety was approved using human fibroblast cells at which its non-cytotoxicity dose was relatively high at 100μg/ml. Once it was tested on keratinocyte cell, cell proliferation effect was exhibited that was conformed to 3D-artifical skin test. Moreover, anti-inflammatory effect of levan was exhibited in 3D-artifical skin, at which IL-1α was suppressed. These information support the applications of the new promising polysaccharide, levan, in skin care cosmetics. Skin moisturizing efficacy of leven was evidenced in 10 female volunteers participating in a short terms efficacy test. Transepidermal water loss (TEWL) was suppressed by a single topically applied levan solution (1%) for 6 h monitoring by Vapometer ®. In addition, skin hydrating efficacy of levan (0.2%) assessed by Corneometer ® in the same group of the volunteers topically treated for 1 time with a monitoring time of 30 min. The efficacy was substantially with the benchmark hyaluronic acid at the same test concentration (Kim et al. 2005).
2.9
Alginate
Alginate or algin is microbial produced from two strains; Pseudomonas and Azotobacter in acid and salt forms, among which P. aeruginosa and A. vinelandii are the commercial available sources (Hay et al. 2013, 2014). Alginate, an anionic polysaccharide with 1.0–1.4 103 kDa, is used for applications such as thickening aqueous solutions, forming gel and water-soluble film, and stabilizer. Sodium alginate is a commonly applied form that is often used as a powder, either pure or mixed with other ingredients for topical products. Wound healing property of alginate was exhibited. The polysaccharide base stimulates reparative processes, prepares the wound for scarring, and displays protective and coating effects, shielding mucous
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membranes and damaged skin against irritation from unfavorable environments. Calcium alginate promotes the proliferation of fibroblasts and inhibits the proliferation of microvascular endothelial cells and keratinocytes. Profound wound healing effects have also been reported for a gelatin–alginate sponge impregnated with antiseptics and antibiotics (Balakrishnan et al. 2006). In addition, alginate possess antimicrobial activity with anti-elastase and anti-MMP-2, and anti-inflammatory as well which the activities pronounces for silver-containing alginate (Wiegand et al. 2009). This polysaccharide is therefore appointed for several skin care cosmetics for variety of claims.
3
The Specialty Fungal Polysaccharides for Skin Care Cosmetics
Fungal polysaccharides comprise a large group of biopolymers which are either part of the cell wall or may form intracellular inclusions and serve as energy reserve, or are excreted extracellularly providing a mechanism for cell protection or attachment to other surfaces (El Enshasy and Hatti-Kaul 2013). Of which, pullulan is the most widely known fungal polysaccharides supplied for cosmetics.
3.1
Pullulan
Pullulan is a linear homopolysaccharide composed of glucose. Aureobasidium pullulans is described in the literature as producing high pullulan concentrations with the molecular weight of 5 – 900 103 Da (Kanlayavattanakul and Lourith 2015). However, the commercial pullulan is available with the weight of 8 103 2 106 Da. The polysaccharide is highly soluble in water, and stable in a form of aqueous low viscosity solution over a wide pH range (Fiume et al. 2016) with the potency on oxidizing protection of the active cosmetic ingredients (Krochta and De MulderJohnson 1997). Besides an application of pullulan as the functional ingredients for cosmetics, its action as an active ingredient is continuously explored. Topical application of pullulan was exhibited to suppress UVB-induced skin damage in an animal model. Glutahione, depletion, MMP activation, and production of IL-1ß were attenuated as per the ability to decrease IL-10 and keratinocyte apoptosis. Thus, pullulan can be used as a protective agent against photoaging of skin (Kim et al. 2015). In regards with the fact of high cost pullulan, it is not commonly used as a single polysaccharide in skin care cosmetics. The most commonly used form is blending or compositing with another polysaccharides. In addition to pullulan, many of fungal polysaccharides derived from edible mushrooms, such as the Maitake or Shiitake or Oyster mushroom. Although many fungi are known for their health-promoting properties and have been widely consumed in Asia for centuries, those with pharmaceutical properties applicable for cosmetics are exemplified in Table 4.
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Table 4 Health beneficial mushroom
Scientific name Agaricus subrufescens Cordyceps sinensis Ganoderma lucidum Grifola fondosa Hericium erinaceus Inonotus obliquus Lentinula edodes Pleurotus ostreatus Poria cocos Trametes versicolor
835 Common name Almond/Himematusutake Caterpillar fungus Reishitake Maitake Lion’Mane Chaga Shiitake Oyster Tuckahoe Turkey tail
The biological activities of several fungal polysaccharides have been reported repeatedly in the last years and efforts have been made to elucidate their structure– function relationships. In the following section, the commercial available mushroom polysaccharides applicable for skin care cosmetics are summarized (Lourith and Kanlayavattanakul 2018).
3.2
Lentinus edodes
One of the most common and well-studied medicinal fungal polysaccharides is lentinan, a glucan elaborated by the edible mushroom Lentinus (or Lentinula) edodes, also known as Shiitake mushroom. It is composed of a main chain of β-(1,3)-D-glucose residues to which β-(1,6)-D-glucose side groups are attached (one branch to every third main chain unit), and an average molecular weight of about 5 105 Da. Lentinan is a high molecular weight glucan extracted from the cell wall of the fruiting body of L. edodes with mostly β(1!3)-glucose linkages in the regularly branched back-bone and β-(1!6)glucose side-chains.
3.3
Ganoderma lucidum
Ganoderma lucidum or Reishitake mushroom is another well-studied medicinal mushroom of the Basiodiomycetes family, which has been used in traditional East Asian medicine. It produces ganoderan, a typical β-(1,3) bioactive glucan branched at C-6 with β-(1,6) glucose units, with a varying molecular weight and degree of branching, especially when isolated from the water-soluble fraction of the fruit body, while the glucan isolated from filtrates of liquid-cultured mycelia which has a MW of 1.2–4.4 106 Da. Notably, as occurs with other mushroom biopolymers, the fruit bodies of G. lucidum also produce several more heteroglucans and proteoglucans with immunostimulating activity.
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Agaricus subrufescens
Another edible and medicinal mushroom, Agaricus subrufescens or Almond or Himematsutake mushroom, is the source of several polysaccharides contained in its fruit body. These include a β-(1,6);β-(1,3) glucan, an acidic β-(1,6);α-(1,3) glucan, and an acidic β-(1,6);α-(1,4) glucan. By contrast to most mushroom glucans, the above glucans have a main chain of β-(1,6) glycopyranose, instead of the more common β-(1,3) linked main chain. The fruit body also contains water-soluble proteoglucan of 3.8 105 Da with a α-(1,4) glucan main chain and β-(1,6) glucopyranoside branches at a ratio of 4:1, as well as two immunostimulating heteroglucans containing glucose, galactose, and mannose, one consisting of glucose and ribose and a xyloglucan.
3.5
Grifola frondosa
Other immunostimulating biopolymers from Basidiomycetes include grifolan, a gel-forming β-(1,3)-D-glucan with β-(1,6) glucosidic bonds at every third glucopyranosyl residue, found in Grifola frondosa or Maitake mushroom with a β(1,3)-D-glucan.
3.6
Trametes versicolor
The edible Turkey tail mushroom Coriolous versicolor or Trametes versicolor has been commercialized in Asia as an effective immunostimulative agent with the glucan structure similarly to polysaccharide from maitake.
3.7
Pleurotus ostreatus
The popular culinary Oyster mushroom, Pleurotus ostreatus, synthesizes bioactive β-glucans, such as pleuran, an insoluble β-(1,3/1,6)-D-glucan, which is another potential candidate for the development of nutraceuticals.
3.8
Poria cocos
Heteropolysaccharides isolated from P. cocos or Tuckahoe mushroom mainly contained glucose, galactose, and mannose, and exhibited anti-tumor activities both in vitro and in vivo. Nonetheless, commercial production of mushroom polysaccharides for cosmetic industry is infeasible in several aspects. Especially, high production or purification costs, low or erratic polysaccharide yields, and unstable chemical characteristics (i.e., composition, molecular weight, and degree of branching). Such problems are encountered mainly during the production of these biopolysaccharides from mushroom fruit bodies; however, they can be ameliorated to some extend by the use of
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fungal mycelium grown in submerged cultures under controlled process conditions (Lourith and Kanlayavattanakul 2018).
4
Challenges and Future Prospects of Microbial Polysaccharides for Cosmetics
The global demand on natural cosmetic ingredients derived from the renewable, eco-friendly, or sustainable sources of particular from the biotechnological production route is increasing year by year including for the sector of microbial polysaccharides that are biodegradable. This high demand is not only serve for the formulation of skin care cosmetics but including for cosmetic packaging and those of delivery system. Thus, challenges of microbial polysaccharides upon these issues are additionally addressed as well as the further uses and perspective production technology and source of microbial polysaccharides.
4.1
Biodegradable Material for Packaging
In addition to functional and active tasks of polysaccharides serve for skin care cosmetics, the polysaccharides are appointed to be used for cosmetic packaging proposes. Accordingly, general information on cosmetic packaging is briefly introduced. The beneficial applications of microbial polysaccharides for cosmetic packaging are highlighted (Lourith and Kanlayavattanakul 2018). Cosmetic packaging is the materials that enclose/surround the product since the time of manufacturing to till final usage. Cosmetic packaging functions in two aspects; protection and presentation. Protection function is environmental and physical/mechanical protections. While presentative functions of cosmetic packaging are identification of the product, providing information of the product with a convenience during handling, and advertising and marketing of product in the same time. The most important concerning issue that is needed for cosmetic packaging is the compatibility of the packaging and the containing cosmetics. To achieve compatibility there are according concerning issues as followings; 1. Adsorption onto the surface and absorption into the package When formulation components are removed by a package two different processes are involved: adsorption onto the surface and absorption into the package wall by diffusion. A component may also get desorbed from the outer surface of the package and pass into the atmosphere if it is volatile enough. Strong adsorption or absorption requires a strong chemical interaction between the component and the packaging material. In addition, a high level of absorption will occur when the packaging material is permeable to the component. When adsorption and absorption occur together, there is rarely a distinction made between the two and therefore the term “sorption” will be used to indicate both processes are taking place.
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2. Leaching Most leaching problems occur with plastics because of the presence of additives such as fillers, activators, and plasticizers. Leaching can cause discoloration, precipitation, change in pH, and contamination that can lead to increased toxicity or instability of the active ingredients. 3. Permeation The transmission of gases, vapors, or liquids through packaging materials can have an adverse effect on the shelf life of the cosmetic products. Permeation of water vapor and oxygen through the polymeric wall into the cosmetics can present a problem if the dosage form is sensitive to hydrolysis and oxidation. Temperature and humidity are important factors influencing the permeability of oxygen and water through the package. An increase in temperature reflects an increase in the permeability of the gas. Permeation can therefore alter properties of polymeric package and may also lead to degradation. Some solvent systems have been found to be responsible for considerable changes in the mechanical properties of the package. 4. Physical or chemical alteration The physical or chemical alteration of packaging materials by cosmetic products is called modification. Permeation, sorption, and leaching are physical alterations that can lead to degradation. Polysaccharide is biodegradable polymer or so called biopolymer can be found on its application for packaging including for cosmetic product. Biopolymer is simply divided into (i) biopolymers that are originated from living organisms and (ii) bioplastics or bio-based polymers that are manufactured from a natural or renewable sources and can be biodegraded. Microbial polysaccharide is accounted as biopolymer regarding on this two categories. Of which, the desired characters of cosmetic packaging can be summarized as followings; • • • •
Oxygen-scavenging Moisture-control Anti-microbial Natural & Eco-friendly
Nowadays, natural trend influences customer buying a product including skin care cosmetics. Use natural materials to imitate looks and add more trust and credibility to natural lovers. In addition, eco-friendly packing not only meets the emotional needs of consumers, but also reflects simple and eye-catching design, in particular the skin care cosmetic consumers of the niche market. The production biodegradable polysaccharides are expected to be increased to 2.44 million tons in 2020 (Gontard et al. 2018) to supply for the certain industries including cosmetics, of which the desired characters of skin care cosmetic package are illustrated as shown in Fig. 3.
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Fig. 3 Desired characters of cosmetic package
Fig. 4 Preparation of biodegradable or compostable polymer
Biodegradable material for cosmetic package is highly in demand. Biopolymeric materials with the desired characters for cosmetic package (Fig. 3) can be archived by several methods as shown in Fig. 4. Among which, cellulose is widely used as biodegradable cosmetic package nowadays. Cellulose esters, such as cellulose acetate (CA), cellulose acetate propionate (CAP), and cellulose acetate butyrate (CAB), that are characterized by their stiffness, moderate heat resistance, high moisture vapor transmission, grease resistance, clarity
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and appearance, and moderate impact resistance (Mekonnen et al. 2013; Rhim and Ng 2007). Benefits of the cellulose-based packaging materials is its wide heat sealing range (70–200 C), static-free, and can be varied in the levels of thickness (23 and 30μm). More important, it is claimed to be gas-selective and permeable (shelf life boost). In addition to an application based on a single polysaccharide. The mixture of polysaccharides in the forms of composites and blends with improvements of thermal and mechanical properties as well as biological properties in some cases are found more applicable to be used. Blending of pullulan with alginate by a ternary co-blended film technique was shown to improve water vapor permeability and oxygen barrier property of the films as per the moisture retention ability that positively achieve mechanical property (Pan et al. 2014). Pullulannanofibrillated cellulose composite had been developed and exhibited as the translucent film that is thermal stable and highly achieve on Young’s and tensile strength to 5500% and 8000%, respectively (Trovatti et al. 2012). In spite of the excellent property of pullulan-derived packaging, its unit price might infeasible the application.
4.2
Delivery System
A variety of natural polysaccharides are finding increasing applications in cosmetics and personal care markets (Ammala 2013). Encapsulation of active ingredients using biodegradable polymeric carriers can facilitate increased active efficacy and bioavailability. They can also be chemically functionalized to give enhanced properties over conventional carrier materials. Many of the conventional nano delivery system (e.g., liposomes, micelles, and polymer-based nanodevices) have reached the late stages of development, and some of them were approved. Various kinds of vehicles, i.e., liposomes, polymersomes, microemulsions, vesicles, and hydrogel have been developed in cosmetic industries, in which the duty of vehicles can be protection and delivery of actives. In particular, well-defined structure of synthetic polymers is attractive in percutaneous treatments due to the ease of synthesis and the wide diversity of material selection (Cho et al. 2014). However, the polymers used in topical delivery should meet several stringent requirements, e.g., low toxicity or skin irritation, biocompatibility or biodegradability, appropriate molecular weights and amphiphilicity (Lochhead 2010). Application of microbial polysaccharide in terms of a delivery system for skin care cosmetics is summarized as followings (Lourith and Kanlayavattanakul 2018). Cellulose is the polysaccharide used in delivery system especially in its microcrystalline form. In addition, chitosan has been reported on its ability to enhance permeation across the skin by altering the structure of keratin. It also increases the water content of the stratum corneum and cell membrane fluidity. Further, due to its positive charge under slightly acidic conditions, it can depolarize the negatively
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charged cell membrane and in doing so, it decreases the membrane potential and drives the active component or drug through the skin. Encapsulation of active ingredients using chitosan in the forms of nanoparticles, microspheres and hydrogels are possible. Which, a controlled release has been demonstrated by increasing the viscosity of the polymer matrix. Alginate is reported for delivery applications as they can form gels with divalent metal ions such as calcium. The fact that relatively mild conditions can be used to incorporate actives within alginates makes them excellent candidates for delivery of proteins that can minimize any denaturation. The spherical alginate capsules can contain water-soluble or dispersible active agents as well as liposoluble additives and can include biological compounds, colored pigments, sunscreens, and perfume. Hyaluronic acid is used as a delivery system to transfer keratinocyte cells from tissue culture to skin wounds, in particular burns, with high rates of healing. The stability of hyaluronic acid-based nanoemulsions as trans-dermal carriers was reported. Electrostatic, steric and hydrophobic effects were found to play a key role in the stability of the emulsions. Encapsulation was capable of lipophilic additives with desirable stratum corneum permeability, efficient partitioning capacity, and was able to be diffused deeper into the dermis. The small size of the emulsion droplets (50–200 nm) was also noted as significant as this enabled greater surface-tovolume ratio which has greater contact points between the emulsion and the skin (Ammla 2013). Gellan was evidenced as one of the appointed polysaccharide for delivery system applicable for skin care products. Nanohydrogel of gellan was proved to be the appointed delivery system for the cutaneous active agent. Of which, the system could enhance the retention of the active in the epidermis (Musazzi et al. 2018). Gellan-hyaluronic acid hydrogel was prepared and examined on its wound healing property in mice. It was shown to significantly increase epidermis thickness. The mechanism was by stimulating activities toward neoepidermis formation and neovascularization associating with its hydroscopic nature limiting inflammatory exudate promote cellular differentiation and angiogenesis and wound healing in terns (Cerqueira et al. 2014). Gellan had been developed into the form of gellantransfersomes lading with the skin active ingredient, baicalin. Skin restoration activity was shown with the significant inhibitory effect against inflammatory mediators, TNFα and IL-1ß in an animal model (Manconi et al. 2018). In addition to the incorporation of organic active with gellan, nanoparticle biofilm incorporated with gellan and titanium dioxide was developed and characterized including biological activity. The developed nanoscale particle exhibited anti-microbial activity, Staphylococcus aureus and Escherichia coli, the prohibited microbe in cosmetic products. Its cellular activity stimulating cell density and adherence. Moreover, it’s in vivo cell proliferation and migration promoting activities associated in wound healing process was evidenced in an animal model (Ismali et al. 2019). Moreover, levan can be prepared in the form of nanoparticle and loaded with vitamin E derivative to be used for skin care product (Nakapong et al. 2013).
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Calcium alginate nanocarrier was developed for hydrophobic active delivery, and the system was shown to be safe in keratinocyte with a rapidly enter the cell and dissociated. This delivery system was suggested as the potential carrier for cosmetic actives applicable with skin cell (Nguyen et al. 2016).
4.3
Source and Production
Microbial polysaccharides prepared from natural sources are of interests among the consumers and the researchers as well. Diversity of the natural sources surplus with an advancement on biotechnology feasibly serve the demands. In addition, ecological condition suitable with the growth environment of the specific microbial is one issue that can be set. In addition, new source of microbe producing polysaccharide applicable for cosmetic application is continuously revealed for instance Deinococcus radiodurans. Exopolysaccharide of 80–100 kDa was synthesized by D. radiodurans and it was exhibited to be constructed with xylose, galactose, fucose, glucose, arabinose, and fructose at the molar ratios of 10.6:6.1:4.2:3.8:2.6:1.0. The polysaccharide showed cellular antioxidant activity by suppression of intracellular radical formation induced by peroxyl radicals and ultraviolet exposure at the dose of 10μg. In addition, its protective effect against lethal doses of irradiation was evidenced in an animal model (Lin et al. 2020). However, production cost turns to be the disruptive issues for commercial development of microbial polysaccharides for certain industries. Of which, extraction and purification of the specific polysaccharides are obviously consume lots of cost during the production processes. Thus, biosynthetic control or metabolic engineering on the basis of gene and coding enzyme manipulations would be overwhelm this obstacles. Although the biosynthetic pathway of microorganisms have been continuously revealed, additional researches on the specific step in the pathway producing the desired polysaccharide are of challenge.
5
Conclusions
Microbial-derived polysaccharides are currently serving as the important cosmetic ingredients. They are applicable for different tasks not only for active and functional actions but include delivery systems and packaging as well. Their cosmetic benefits are enclosed therein. The biotechnological production of microbial polysaccharides is accountable as the natural and organic cosmetic ingredients that are highly appreciated among the consumers rather than those of the synthetic ones. The specialty microbial polysaccharides are therefore gaining the high interest in terms of the researchers’ point of view on challenging for the fabricated production of these sort of actives serving in health and personal care products industry, among which skin care products are the major section as enclosed. The cosmetic formulators will be widen in the choices on the formulation developments accordingly. In
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regard with the consumers’ demand and economically feasibility of microbial polysaccharides, safety is the mandatory issue that must be highly concerned.
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Nguyen HTP, Allard-Vannier E, Gaillard C, Eddaoudi I, Miloudi L, Soucé M, Courpa I, Munnier E. On the interactions of alginate-base core-shell nanocarriers with keratinocytes in vitro. Colloids Surf B Biointerfaces. 2016;142:272–80. Pan H, Jiang B, Chen J, Jin Z. Assessment of the physical, mechanical, and moisture-retention properties of pullulan-based ternary co-blended films. Carb Polym. 2014;112:94–101. Rehm BH. Bacterial polymers: biosynthesis, modifications and applications. Nature Rev Microbiol. 2010;8:578–92. Rhim J-W, Ng PKW. Natural polymer-based nanocomposite films for packaging application. Crit Rev Food Sci Nutr. 2007;47:411–33. Shah N, Ul-Islam M, Khattak WA, Park JK. Overview of bacterial cellulose composites: a multiprupose advanced material. Carb Polym. 2013;98:1585–98. Srikanth R, Siddartha G, Sundhar RCH, Harish BS, Janaki RM, Uppuluri KB. Antioxidant and antiinflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization. Carb Polym. 2015;123:8–16. Trovatti E, Fernandes SCM, Rubatat L, Perez DDS, Freire CSR, Silverstre AJD, Neto CP. Pullulannanofibrillated cellulose composite films with improved thermal and mechanical properties. Compos Sci Technol. 2012;72:1556–61. Wiegand C, Heinze T, Hipler UC. Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for photophysiological factors in chronic wounds of alginate and silvercontaining alginate. Wound Repair Regener. 2009;17:511–21.
Immunogenicity and Vaccines of Polysaccharides
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Benjamin Obukowho Emikpe and Michael Ayodele Odeniyi
Contents 1 Gums/Polymers from Phytogenic Origin Used for Vaccine Delivery . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Plant Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Mucosal Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Living Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Adjuvants of Phytogenic Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Gums and Mucilages: Salient Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Characterization of Gums and Mucilages from Excipient Analysis . . . . . . . . . . . . . . . . . 2.5 Gums as Delayed Transit and Continuous Release Systems . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Attempts at Use of Plant Polysaccharide for Vaccine Delivery . . . . . . . . . . . . . . . . . . . . . . 3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
There is an increased interest in the use of plant extracts as therapeutic agents exploring their ability to inhibit growth of pathogenic microorganisms; however, there has been a dearth of information on their application as adjuvants and vaccine delivery agents. Gums and mucilages from several plants have been evaluated for both bioadhesive and phytogenic properties. Gums were prepared from Abelmoschus esculentus, Irvingia gabonensis, and Boswellia carteri and were used as mucosal delivery agents for PPR vaccine in goats and sheep using the intranasal route of vaccine application. The application was comparable to the conventional subcutaneous route of PPR vaccination in terms of humoral immune response induction and duration of antibodies. Mixtures of gums were prepared B. O. Emikpe (*) Departments of Veterinary Pathology, University of Ibadan, Ibadan, Nigeria M. A. Odeniyi Department of Pharmaceutics and Industrial Pharmacy, University of Ibadan, Ibadan, Nigeria e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_47
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from Cedrela odorata, Khaya senegalensis, and Boswellia carteri as oral and ocular delivery agents for Newcastle Disease and Infectious Bursal Disease vaccines in broilers. The gums act as adjuvants to enhance immune response to the poultry diseases. Gums and mucilages of plant origin have great potential as both delivery agents and adjuvants in vaccines for the prevention and treatment of poultry and animal diseases. Keywords
Phytogenic gums · Vaccines · Poultry · Adjuvants
1
Gums/Polymers from Phytogenic Origin Used for Vaccine Delivery
1.1
Introduction
The use of plant extracts as therapeutic agents is enjoying increasing attention in the scientific research area. This interest has been geared toward the capacity for these plant extracts to inhibit the growth of pathogenic microorganisms and to be used as veterinary vaccine delivery agents. Reports by Emikpe et al. (2016) and Oyebanji et al. (2017a, b) have proven that gums from Khaya senegalensis and Cedrela odorata which are bioadhesives have shown the ability to interact with biologic tissues for veterinary applications such as vaccine delivery in Newcastle disease of poultry as well as in infectious disease vaccine of poultry (Adeniran et al. 2019, 2020a, b). Recent studies have also showed that gums prepared from Abelmoschus esculentus, Irvingia gabonensis, and Boswellia carteri are capable of serving as delivery agents for PPR vaccine in goats and sheep (Ezeasor et al. 2020a, b; Mumin et al. 2020) using the intranasal route of vaccine application. This application was comparable to the conventional subcutaneous route of PPR vaccination in terms of humoral immune response induction and duration of antibodies. The responses in the two animals, sheep and goats, are similar which gives a wider application of this plant polysaccharide as vaccine delivery agents in small ruminants. The probable explanation for the enhancement of immune response seen in studies done clearly revealed the adjuvant property of the gum.
1.2
Plant Gums
Gums, by definition, are substances made up of a mixture of chemical compounds that are able to conglomerate into gels that increase the viscosity of solutions as well as cause stability in foams and emulsions (Granzotto et al. 2017). Gums are commonly associated with water-soluble and modified polysaccharides and do not necessarily designate a class of chemical compounds. As plant gums are deemed to be natural, they have been reported to be obtained from different parts of plants including plant cell walls, seeds, trees or shrub exudates, and seaweed extracts (Granzotto et al. 2017).
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Gums obtained from plants have been identified to have several uses. Usage of gums from plants has been traced back to 2600 B.C. through the Fourth Dynasty of Egyptian time where they were used as binders and adhesives for mineral pigments on cartonnage and in the practices of mummification (Granzotto et al. 2017). Binding media and adhesives used in artworks and cultural artifacts have also been identified to be products made from plant gums. These benefits indicate strongly the significance of plant gums in the world. Currently, gums have been widely used, for example, as binders for watercolor and gouache paints. In reference to the distinguishing properties of plant gums, certain plant gums including that of gum Arabic (from Acacia senegal and seyal) and locust bean gum (LBG) (Ceratonia siliqua), Khaya senegalensis, Cedrela odorata, Abelmoschus esculentus, Irvingia gabonensis, and Boswellia carteri have been investigated for use in the food, cosmetics, textiles, and biomedical/pharmaceutical industries in recent studies (Ofoefule and Chukwu 2001; Stephen and Churms 2006; Kalu et al. 2007). Structurally, plant gum polysaccharides have been found to be complex in nature reflected in having high-molecular mass (up to several million Dalton), highly branched structures with a large variety of monosaccharides and glycosidic linkages, high polydispersity, and heterogeneity, while some have covalently attached protein moieties (Granzotto et al. 2017). In as much as polysaccharides are studied by different analytical techniques, limited information is provided in the literature about the makeup in their chemical structure. The structural analyses of plant gum polysaccharides focus on the monosaccharide composition and sequences, molecular weight and distribution, linkage positions between the glycosidic linkages and branches, ring size (furanosidic/pyranosidic rings), anomeric configuration (α/β), substitutions, and identification/localization of potentially attached proteins. With this knowledge on their structure, their use as mucoadhesive delivery agents for vaccine is limited in literature. Recent studies are on veterinary vaccines such as Newcastle disease (Emikpe et al. 2016; Oyebanji et al. 2017a, b), infectious disease vaccine (Adeniran et al. 2019, 2020a, b) of poultry, and PPR vaccine for control of morbillivirus of sheep and goats (Ezeasor et al. 2020a, b; Mumin et al. 2020).
2
Mucosal Adjuvants
Adjuvants are of major concern in recent scientific discourse. Most often, living antigens are used as adjuvants, but in recent times, emphasis is being laid on adjuvants that are phytogenic in nature. With this current shift and choice for phytogenic adjuvants for mucosal delivery, it is important to investigate their bioadhesive strength. There has been varied approaches and techniques to evaluate the mucoadhesive properties of gums either ex vivo or in vivo. Among these techniques are the Wilhelmy plate technique, electromagnetic force transducer for the tensile stress, while adhesion tests are based on measurement of the force required to separate two polymer-coated glass slides with a film of mucus sandwiched between, to know the shear stress as observed in the design of a flow chamber method (Calero et al. 2010). Other approaches are the rheological method (Prabhu et al. 2010) and the viscometric method (Thirawong et al. 2008) which evaluate in vivo behavior of mucoadhesive
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polymers and quantify mucin-polymer bioadhesive strength, respectively. In some recent studies, especially in limited resource environments, a tablet dissolution machine was adaptively used to evaluate the interaction between the gum polymer and mucosal tissue surfaces (Emikpe et al. 2016; Mumin et al. 2020). This often simulates the interplay of factors which plays out between the mucosal surface and the mucoadhesive polymer under an in vivo condition.
2.1
Living Antigens
Living antigens which have been used as adjuvants along mucosal surfaces, especially the gastrointestinal mucosal surface, had yielded positive results. This was evident by easy uptake of such microorganisms which seems to have a lectin-like interaction with specific carbohydrate receptors on M cells for easy uptake. Examples of such candidates are Salmonella typhi th21a, Vibro cholera, and Poliovirus strains. These have also been modified to express heterogeneous antigens for wider protection against a wide range of pathogens. Other adjuvant candidates for mucosal vaccines include liposomes, microparticles and nanoparticles, cytokines, ISCOMs, monophosphoryl lipid A, CpG, and detoxified ADP-ribosylating toxins (Hu et al. 2001).
2.2
Adjuvants of Phytogenic Origin
It is worth mentioning that the use of adjuvants in veterinary vaccines preparations seems to have broader candidates. However, the growing toxicity concerns of most adjuvant/delivery systems, the development of specialized vaccines, newer less invasive routes of applications, and the need for broader spectrum of responses, amid growing concerns of zoonotic infectious diseases have necessitated the review of available options for readily available, relatively stable adjuvants which are adaptable for less invasive routes, and broader response such as from phytogenic origin is inevitable. In this regard, plant products that have been generally explored are large carbohydrates, gums, and mucilages. Gamma inulins from Compositae family are identified as large carbohydrates which have proven to have strong stimulatory effects on cells of the immune and reticulo-endothelial system. Studies have shown they stimulate good cytotoxic and humoral responses without the observed associated adverse reaction seen with older adjuvants such as FCA (Petrovsky and Aguilar 2004). The combination of large phytogenic carbohydrates with some other traditional adjuvants such as Alum products often yields excellent and safe results with a more vigorous Th2 response. This result is easily attributable to Alum and is boosted by the addition of such phytogenic large carbohydrate. Candidate antigens tested in animal models include respiratory syncytia virus, diphtheria, tetanus toxoid, Hepatitis B surface antigens, Haemophilus influenza, and influenza hemagglutinin antigens. In another dimension, gums and mucilages which are natural polysaccharide products obtained from tree exudates have existed over 4,000 years. They are often produced by plants belonging to Leguminosae, Sterculiaceae, Bixaceae,
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Compositae, Combretaceae, and Gigarginaceae families, e.g., gum acacia, gum tragacanth, and gum karaya. Economically, they have been used as binding, stabilizing, thickening, emulsifying agents and matrix components in pharmaceutical and food industries. They have also gained uses in cosmetics, textiles, adhesives, lithography, paints, and paper industries (Bhosale et al. 2014).
2.3
Gums and Mucilages: Salient Features
Gums as they exist are formed when plants undergo pathological changes such as injuries as a result of unfavorable environmental conditions, such as drought which results into dissolution of cell walls. This process is termed as gummosis while mucilages are formed within the cell (intracellular formation) due to physiological metabolism of plants. Mucilages may serve as food reserves or water-holding structures of the plant (Bhosale et al. 2014). Certain gums (Acacia, Guar, and Tragacanth gums) have been observed to dissolve in water naturally while certain mucilages (Phoenix & Cassia Tora mucilages) form masses which are slimy in nature (Bhosale et al. 2014). In obtaining gums, incised trees barks or a tree trunk serves as the major source while mucilages can be harvested from other plant parts such as leaves (especially in the case of Aloe barbadensis mucilage). Despite the differences in their morphology and botanical source, gums and mucilages have been reported to have some similarities. Both products have been reported to be made up of hydrocolloids, translucent amorphous substances and polymers of monosaccharide units, or mixed monosaccharide constituents, such as starch instead of cellulose or hydrocellulose (Jania et al. 2004). Both products chemically yield mixtures of sugars and uronic acids upon hydrolysis. These hydrophilic molecules constituents could combine with water to form viscous solutions or gels depending on the nature of the containing compounds which ultimately reflects in the properties of different gums. Structurally, gums made up of linear polysaccharides are more viscous because they occupy more space than those containing highly branched compounds of the same molecular weight. The branched compounds form gels more easily and are more stable because of the impossibility of extensive interaction along the chains (Jania et al. 2004).
2.4
Characterization of Gums and Mucilages from Excipient Analysis
Gums and mucilages are often run through a panel of tests to detect primary structure, purity of the pure compound, and presence of any impurities, i.e., impurity profiling, physicochemical properties, and toxicity (Ansari 2006). Thereafter, a scientific dossier is developed for such gum or mucilage. Often for tablet/drug preparations, after incorporation into drug preparations, compatibility tests are performed using spectrophotometry, Fourier transfer Infra-Red (FTIR) spectroscopy, or Differential Scanning Calorimetry (Raymond et al. 2004).
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Purity: Purity tests for alkaloids, amino acids, steroids, terpenes, saponins, oils and fats, phenol, and tannins are often carried out to standardize the constituents of the gums. Toxicity: This is most often determined by following a fixed-dose method as recommended by OECD guidelines, No. 25. Determination of LD50 is often recommended by conducting a subacute toxicity study. An initial in vitro cytotoxicity test can also be performed while elimination of microbial load, or contamination is highly recommended.
2.4.1 Classification of Gums Gums have been classified based on varied criteria. They have been classified based on their ionic charge, origin, pH, and molecular shape or size. These varied classifications of gums have been described by Jania et al. (2004) as follows: • Based on the ionic charge, gums have been classified into anionic, cationic, and nonionic. – Anionic charged gums: tragacanth, Arabic, karaya, gellan, agar, pectin, algin, and carageenans – Cationic charged gums: chitosan – Nonionic charged gums: guar gum, locust bean gum, tamarind gum, arabinans, xanthan gum, amylase, and cellulose • Based on the origin, gums can be of the marine, animal, or plant origin: – Marine (sea weeds gum): alginates, agar, and carageenans – Animal origin: chitin and chitosan, chondroitin sulfate, and hyaluronic acid – Plant origin: Seed gums – locust bean, guar, starch, cellulose, and amylase Tree exudates – gum arabic, tragacanth, ghatti, and karaya Tubers – potato starch. iv) extracts-pectin. d) microbial origin (fungi and bacteria): glycan, pullulan, dextran, xanthan, and gellan • Based on the source, gums could be natural, modified, and synthetic in nature: – Natural, e.g., acacia, tragacanth, and xanthan – Modified or semi-synthetic, e.g., carboxymethylcellulose and microcrystalline cellulose – Synthetic, e.g., carboxypolymethylene and colloidal silicon dioxide • Based on the morphology or the shape of the gum, it can be linear or branched in nature: – Linear: amylase, pectin, and cellulose – Branched: Short branched-guar gum, locust bean gum Long branched-amylopectin, karaya gum, gum tragacanth, and gum Arabic • Based on pH: – Acidic, e.g., acacia, tragacanth, and albizia gums. – Neutral, e.g., asparagus gum and plantago seed gums. – Basic, no basic gum occurs in nature; natural gums are either acidic or neutral.
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Gums as Delayed Transit and Continuous Release Systems
Delayed transit and continuous release systems are drugs or delivery systems designed to prolong their residence time as well as release duration within the gastrointestinal tract (GIT). Often because of the hydro, chemical, and pH dynamics of the GIT (saliva, stomach, small intestinal juices, and large bowel system), the dosage form is fabricated to be able to withstand these dynamics in each region of the GIT and efficiently release the drug component to the targeted site. Systems included in this category are mucoadhesives and size-based systems. Most drugs or pharmaceutical preparations explored as delayed release are sensitive to stomach or intestinal juice, targeted at a specific site and, as such, need special formulation with excipients that could sequestrate agents such that gradual release at needed sites is achieved. Mainly, there are two types of delayed release systems, i.e., intestinal and colonic release systems. For orally administered drugs, addition of slow release excipients allows for optimal time of antigenic exposure and uptake by phagocytic cells and antigenpresenting cells which are situated along specialized mucosa structures along the gastrointestinal tract. However, for noninvasively administered vaccines, such as through the oral route, an excipient or vehicular base which could easily simulate what obtains with delayed drug release within the GIT is needed. This is against the backdrop that existing delivery systems for most common orally administered vaccines are water or Alum compounds which rapidly transit the GIT leaving behind little time for exposure and uptake. Hence, subsequent immune response is often nonvigorous, short lived, and nonprotective. Therefore, a repeated dose at close intervals is often needed for protection to be assured. Gums and mucilages from many plants have shown prospects as delayed or continuous release candidates for drugs. A few examples are given in Table 1.
2.6
Attempts at Use of Plant Polysaccharide for Vaccine Delivery
Vaccines are a prepared material that induces an immunologically mediated resistance to a disease but not necessarily an infection. Vaccines are generally composed of killed or attenuated organisms or subunits of organisms or DNA-encoding antigenic proteins of pathogens (Saroja et al. 2011). In order to induce an effective protective immunity, vaccines require boosting with agents called “adjuvants.” Adjuvants are believed to act by forming complexes with the agent to be delivered from whom immunogens are slowly released. Adjuvants potentiate the immunostimulatory property of the antigen while being nonimmunogenic, nontoxic, and biodegradable by themselves (Saroja et al. 2011). Conventional immunization which involves prime dose and booster doses sometimes fails while the administration of vaccines via gums allows for the incorporation of doses of antigens so that booster doses are no longer necessary as antigens are released slowly in a controlled manner. Again, the spatial and temporal presentation of antigens to the immune system could be controlled when administered via gums,
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Table 1 Examples of phytogenic gums and mucilages used in drug delivery systems Common name Acacia Bhara gum Chitosan Cordia gum Guar gum Gellan gum Karaya gum Locust bean gum Mucuna gum Okra gum Sodium alginate Xanthan gum
Botanical name Acacia Senegal Terminalia bellericaroxb –
Family Leguminosae Combretaceae
Pharmaceutical applications Osmotic drug delivery Microencapsulation
–
Colon-specific drug delivery, microspheres, and nanoparticles Oral sustained release matrix tablets
Cordia oblique willed Cyamompsis Tetraganolobus Pseudomonas elodea Sterculiaurens
Boraginaecae
Sterculiaceae
Colon-targeted drug delivery, microspheres Ophthalmic drug delivery, sustaining agent, beads, and hydrogels Mucoadhesive and buccoadhesive
Cerataniasiliqua
Leguminoseae
Controlled delivery
Mucunaflagillepes
Papillionaceae
Microspheres
Hibiscus esculentus Macrocytis pyrifera Xanthomonas lempestris
Malvaceae Lessoniaceae
Hydrophilic matrix for controlled release drug delivery Bioadhesive microspheres, nanoparticles
–
Pellets, controlled drug delivery system
Leguminoseae –
Source: (Reproduced from Bhosale et al. (2014) under the creative commons attribution – noncommercial (CC BY 4.0) license IJPPR)
thereby promoting their targeting of the immune cells. These benefits have encouraged the use of gums in the delivery of vaccines (Saroja et al. 2011). The use of mucoadhesive gums and biopolymers (Odeniyi et al. 2013, 2015) allows for the mucosal administration of vaccines, which offers protection against microorganisms which gain access to body via mucosal membranes. Patient compliance, ease of administration, reduction in possibility of needle-borne injections, and stimulation of both systemic and mucosal immunity are some of the advantages of this route of administration. This route has been exploited by Emikpe et al. (2016), thereby combining both the adjuvant and adhesive properties of the investigated gums; furthermore, some of the gum especially Boswellia carteri has been used to deliver Newcastle disease vaccine in microbeaded form (Fig. 1) for oral delivery in Indigenous and backyard poultry. Similarly, the Boswellia carteri-PPR vaccine combination administered through the intranasal route also exhibited a similar antibody titer with a subcutaneous route and hence could be an innocuous, safe, and immunogenic noninvasive alternative method than the invasive subcutaneous route commonly used for PPR vaccination (Mumin et al. 2020). Similar result was also obtained in another study using Irvingia gabonensis-PPR vaccine in goats (Ezeasor et al. 2020b).
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Fig. 1 Microbeads formulated from Boswellia carteri loaded with Newcastle disease ND vaccine
Another study evaluated the clinicopathological effects of the infectious bursal disease in broilers that were vaccinated orally and ocularly with a combination of infectious bursal disease vaccine (IBDV) and plant gums (Khaya senegalensis and Cedrela odorata) (Adeniran et al. 2020a, b). The prospects for phytogenic bioadhesives in vaccine delivery were explored by using mucilage from Cedrela odorata and Khaya senegalensis in established combination ratios of 1:1 (Emikpe et al. 2016). Their combined potential was harnessed for the optimal enhancement of the immune response to IBD virus and consequent protection against IBD virus (Adeniran et al. 2020a, b). Conclusively, this series of studies has clearly shown the importance of mucilage as adjuvant and immunomodulator; however, the mechanism for this needs further clarifications.
3
Conclusions
This chapter clearly showed that plant extracts and gum have potential as adjuvants and vaccine delivery agents. Though gums and mucilages from several plants have been evaluated for bioadhesive and phytogenic properties, especially those from Abelmoschus esculentus, Irvingia gabonensis, and Boswellia carteri, their evaluation for vaccine delivery should be the focus of research. The mucosal application of gum vaccine mixture, though comparable to the conventional subcutaneous route in some morbilliviral vaccines of small ruminant, should be explored for the eradication of PPR, a pneumo-enteritis viral infection in small ruminants. Mixtures of gum vaccine could be useful as oral and ocular delivery agents for Newcastle Disease and Infectious Bursal Disease vaccines in poultry especially Indigenous chicken in sub-Saharan Africa where poultry production is in subsistence form. This chapter revealed that gums and mucilages of plant origin, which are cheap and available in Africa, have great potential as both delivery agents and adjuvants of vaccines for the prevention and treatment of animal and poultry diseases in sub-Saharan Africa.
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Bacterial Polysaccharides Versatile Medical Uses
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Contents 1 New Developments of Well-Known, Commercial Bacterial Polysaccharides . . . . . . . . . . . . . 1.1 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Hyaluronic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Xanthan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Bacterial Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Levan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 New Discovered Bacterial Polysaccharides with Potential Medical Applications . . . . . . . . 3 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
During the last years, a lot of research results have been published concerning to new approaches to extend the use of microbial and particularly bacterial polysaccharides, in pharmaceutical and medical practice. Most of them were focused on new developments of already well-known, authorized on the health market, commercial polysaccharides (dextran, hyaluronic acid, xanthan, gellan, bacterial cellulose, levan), but regarding new derivatives and associations, having in view the progress in nanomedicine and preparative techniques. An overview of the state of the art in the main directions of research, starting from their present recognized applications, is presented, trying to notice challenges and perspectives. Efficient targeting and for controlled release drug carriers, especially of antitumor hydrophobic, low water soluble drugs, topical therapeutic agents, especially for wound healing, suitable scaffolds in tissue engineering, surgery, have been given major attention. Attempts of new producing strains and products were also mentioned. Among the challenges, the necessity of ensuring a compet-
M. Moscovici (*) · C. Balas National Institute for Chemical Pharmaceutical Research and Development, Bucharest, Romania © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_48
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itive ratio price/performance and full completion of the steps, especially in nonclinical (preclinical) development of an innovative/improved medicine or medical device, are highlighted. Keywords
Bacterial polysaccharides · Pharmaceutical · Medical · Applications · New developments
Introduction During recent years, increasing attention has been further given to microbial polysaccharides in health improvement. This chapter tries to highlight the progress of knowledge in the field regarding such bacterial polymers. The information is presented according to their main categories of applications based on valuable properties in the field of health. Bacterial polysaccharides have been used for decades in the medical field. Their biocompatible and biodegradable properties, as well as different rheology, other physicochemical properties associated to structure and molecular weight, have promoted them as valuable pharmaceutical ingredients, starting from blood plasma substitutes to thickeners, emulsifiers, stabilizers, as well as medical devices. For the already known and therapeutically approved bacterial polysaccharides, numerous studies presented modified derivatives acquiring new medical applications responding to current trends in therapy. To this field of research, the most important contributions are represented by their hydrogel forming property, as tunable matrices due to the hydrophilic structure and the numerous reactive hydroxyl groups. Meanwhile, new such biopolymers, isolated from different producing bacteria, with new properties, including useful biological activities, have been discovered.
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New Developments of Well-Known, Commercial Bacterial Polysaccharides
There are few bacterial polysaccharides which have up to date commercial applications in therapeutics or health improvement, but they have been the subject of intense research efforts with promising results during the last years. By processing a large number of reactive groups, a huge number of derivatives with potential therapeutic applications have been prepared and studied.
1.1
Dextran
This neutral glucose homo polymer containing α-(1!6) and α-(1!4) glucopyranosyl linkages could be considered the first remarkable example for a microbial polysaccharide used in medicine, as plasma volume expander for controlling wounds shock in
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Fig. 1 Chemical structure of dextran (www.dextran.com/ about-dextran/dextranchemistry/dextran-structure)
surgery since 1953, officially acknowledged by pharmaceutical authorities (United States Pharmacopeia [USP 43]; National Formulary [NF 38]; European Phamacopoeia [EP 9] 2017). Other pharmaceutical applications are in eye-drops, due to its lubricating action (to relieve dry/irritated eyes, e.g., Visine), or as carrier and stabilizer in vaccines (www.dextran.com). Almost linear, the degree of branching is approx. 5% in the native polymer, and the branches are mostly 1–2 glucose units long (Fig. 1). It is produced by Leuconostoc mesenteroides on sucrose-containing media. To medical applications of dextran seem to be dedicated the most publications of the late years. Some main considered ones are presented in Table 1.
1.1.1 Dextran Derivatives as Drug Carriers It is probably the most studied field of new applications of polysaccharides. As therapeutic direction, anticancer drug delivery systems are very well represented, having in view the importance and gravity, as well as the fact that free drugs against such diseases are hydrophobic, with low absorption, short resilience (rapid excretion), and exhibit severe adverse reactions. A better targeting and sustained release, permitting their lower dosage and superior effectiveness, is an ongoing objective of the research studies.
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Table 1 New developments of dextran medical applications Application Drug carrier
In systems for topical therapeutics
Formulation (drug/polymer system) Docetaxel/folate-dextran-polylactideco- glycolide DT/FA-Dex-PLGA Doxorubicin/carboxymethyl dextranblack hole quencer 3 Dox/CMD ex-BHQ3 PEG-ylated interferon-α-2a/Dextyramine PEG-IFN-α2a/Dex-Tyr Vaccine protein antigen+adjuvant (murabutide)/acetalated dextran Antigen+adjuvant/Ace-Dex Rivastigmine/dextran-polyacrylamide/ dextran-poly (vinylalcohol) (transdermal) RVS/Dex-PAA/Dex-PVA Camptothecin/2-methacryl ester hydroxyl-ethyl disulfide-dextran-poly [poly (ethyleneglycol)methyl ether methacrylate] (disulfide bond conjugated prodrug) CPT/Dex-POEGMA(DCO) Doxorubicin+campthotecin/dextran polyaldehyde-lipoic acid ester Dox+CPT/DexPA-LA Dexamethasone, indomethacin/dextrananiline trimer-hexamethylene diisocyanate DMT,IND/Dex-AT-HDI Curcumin/polyurethane-dextran Cur/PU-Dex Quaternary ammonium salts/dextransiloran-polyurethane QAS/Dex-SI-PU Quaternized chitosan-polyanilinedextran polyaldehyde QCh-PAN-DexPA Dextran-β-tricalciumphosphate Dex-βTCP Polylactic acid-dextran PLA-Dex Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-dextran PHBV-Dex
Potential therapy Breast cancer
Reference Alibolandi et al. (2016)
Head and neck Son et al. (2018) cancer Hepatitis C
Bae et al. (2015)
Vaccines
Chen et al. (2018)
Alzheimer
Patil et al. (2019)
Human Bai et al. (2018) cervical cancer Breast cancer
Breast cancer
Curcio et al. (2020)
Inflammations Qu et al. (2019)
Infected wounds Infected wounds
Sagitha et al. (2019) Gharibi et al. (2019)
Infected tissue Zhao et al. (2015) regeneration Bone regeneration Cardiac tissue engineering Bone repair
Ghaffari et al. (2020) Zhang et al. (2017) Zou et al. (2016)
(continued)
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Table 1 (continued) Application Gene and cell delivery
Other medical applications
Formulation (drug/polymer system) hSET1 antisense/thiolated carboxymethyl dextran-chitosan hSET1anti-TCMD-Ch miRNA(miR-145)-thiolated carboxymethyl dextran-aptamer AS1411/polyelectrolyte complexdeacetylated chitosan miR-145+AS1411/TCD-PEC-Ch Dextran-dimethyl/diethyl/ aminoacrylate ethyl methacrylatepEGFP-C1plasmid + (DNA)/siRNA (dextran based DNA, dextran based siRNA coacervates) p-EGFP-C1(DNA)/DexPDAA/DexPDEAEMA-siRNA Dex –DC, Dex-RC Arginylglycylaspartic acid (RGDpeptide)/ dextran RGD-Dex Chitosan-dextran-β-glycero-phosphate Ch-Dex-βGP Dextran antimicrobial Gentamicin/dextran (new nature isolated Leuconostoc mesenteroides strains) Dextran from a nature isolated Leuconostoc pseudomesenteroides strain Immunomodulatory Dextran from milk isolated Leuconostoc mesenteroides strains
Potential therapy Colon cancer
Reference Kiani et al. (2016)
Breast cancer
Tekie et al. (2018)
Ovarian cancer
Wang et al. (2020)
Epithelial tissue engineering Cardiac tissue repair Multidrugresistant urinary infections Infections with S. aureus and E. coli Inflammations
Riahi et al. (2017)
Ke et al. (2020) Salman et al. (2018)
Ye et al. (2019)
Zarour et al. (2017)
One of the main approaches is the encapsulation of the drug in amphiphilic polymer particles, obtained by grafting of hydrophobic segments onto the hydrophilic polymeric backbone, forming self-associated thermodynamically stable nanogel structures, with an inner hydrophobic core. An interesting example is represented by polymersomes (a synthetic alternative approach of natural liposomes), consisting of bi-layer vesicles of amphiphilic block-copolymers with an aqueous core. Such drug carriers were prepared by conjugation of dextran with poly(lactic-co-glycolic acid) (PLGA). A folate group was attached to the conjugated Dex-PLGA polymer as cell-receptor ligand. The poor water-soluble anticancer drug docetaxel was encapsulated in the bilayer. The antitumor effect was determined on human and murine breast cancer cells MCF-7 and AT1, respectively, overexpressing folate receptors. In vitro
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tests, confirmed by in vivo treatment of tumor-bearing mice, showed a higher cellular uptake and cytotoxicity of the loaded conjugate (FA-Dex-PLGA-DTX), compared to free drug formulation and nonfolate conjugate, corresponding to tumor-targeting capability, as well as a sustained drug-release reducing tumor growth and increasing survival rates of animals (Alibolandi et al. 2016). The low oxygen concentration under the oxygen demand (hypoxia) of tumor microenvironment, accompanied by a reduction potential, particularly by overexpression of nitro- and azoreductases, was exploited by a hypoxia-responsive carboxymethyldextran (CMD), chemically modified (amide conjugate) with BHQ3 (black hole quencer 3), containing an azo bond (-NH¼NH-) as hypoxia-sensitive moiety. The hydrophobic, poorly soluble anticancer doxorubicin was entrapped with a high loading capacity (71%) into the amphiphilic nanoparticles (DOX@CMD-BHQ3). The drug release was determined under hypoxic conditions (nitrogen, NADPH), noting a rapid release due to the bioreduction of azo bond, compared to normoxic (Fig. 2). The in vitro cytotoxicity and intracellular release were investigated in mouse head and neck carcinoma SCCT cells. Both were increased under hypoxic conditions. In vivo systemic administration into SCCT tumor-bearing mice showed high selective tumor accumulation, compared to the other tissues (Son et al. 2018). Protein incorporation in dextran derivatives was another direction of application for drug-delivery systems. The efficacy of protein drugs could be diminished by their proteinase-sensitivity leading to a biological short half-live and renal elimination. Covalent attachment to polyethylene glycol (PEGylation) is a way to avoid such inconveniences. An example is the PEGylated interferon-α2a (PEG-IFN- α2a), with a prolonged half-life than the free drug, which is used in hepatitis C therapy. However, frequent injections are necessary, increasing patient discomfort and the risk of adverse reactions. Considering the excellent biocompatibility and nonimmunogenicity and its ability to form aqueous two-phase system (ATPS) with PEG, a microstructured dextran derivative system was prepared aiming to a sustained release of PEG-IFNα2a. Thus, a dextran-tyramine conjugate crosslinked by catalytic oxidative coupling of tyramine moieties adding horseradish peroxidase and H2O2, incorporated PEG-IFNα2a as droplets in a continuous gel phase (Fig. 3). The drug release could occur by the gradually degradation of the gel, due to slow hydrolysis of carbamate (-O-CO-N-) bonds between dextran and tyramine. The dextran backbone could be further degraded by α-1-glucosidases present in various human organs and tissues. The system exhibited a drug sustained release for 3 months. The in vitro antiviral activity was determined on Huv-7 (human hepatoma) cells carrying hepatitis C virus (HCV) replicon. The retained activity after encapsulation depended on the microgel particles size, a 3 μm size proved to retain approx. 95% after 2 weeks and was chosen for in vivo experiments. In vivo tests were performed on humanized (human immune system) mice and showed a prolonged blood circulation of PEG-IFN-α2a after one administration of drug-loaded hydrogel (2 weeks), compared to free PEG-IFN-α2a, the plasma half-live increased from 1.5 to 15.6 days. After 8 weeks of treatment, the therapeutic effect of one administration of drug-loaded hydrogel was the same of weekly injections, proving the sustained release and corresponding efficacy (Bae et al. 2015).
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Fig. 2 Synthetic scheme of the doxorubicin-loaded hypoxia sensitive amphiphilic conjugate, tumor accumulation, and drug release (Elsevier permission, from Son et al. 2018)
Dextran-derivative microparticles with tunable degradation properties were investigated on possible controlled antigen and adjuvant release as vaccine ingredients, important for optimal immune response and safety. Acetalated dextran (Ace-Dex) with various degrees was prepared by reaction with 2-ethoxypropene.
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Fig. 3 Schematic preparation of microstructured hydrogels for sustained release of PEGylated proteins (Elsevier permission, from Bae et al. 2015)
Microparticles were obtained by electro-spraying. The loading substances (murabutide), a vaccine adjuvant, and ovalbumin, as model protein antigen, were encapsulated in the hydrogel during the same electrospray process. The degradation behavior of the polymer was studied at a physiological environment pH (7.4) and at pH 6.5, resembling the acidic conditions within lysosomes of phagocytic cells, the vaccine target (pH 6.5). The polymer showed an acidic sensitivity, depending on the proportion of cyclic acetals, more stable, and on the electrospray procedure, favoring polymer stability. Protein release was faster for lower cyclic acetals, so the polymer could be a platform of immunization process control. The intracellular internalization and transport of antigen were revealed in murine bone marrow-derived dendritic cells (BMDCs). After an in vivo study of 42 h on mice, different kinetics responses were noted for encapsulated adjuvant and antigen, but permitting, by polymer cyclic acetal degree, a control of adjuvant and antigen release, enhancing the safety and effectiveness of both vaccine subunits (Chen et al. 2018). An electro-responsive transdermal delivery system was prepared consisting of a hydrogel reservoir containing polyacrylamide-grafted dextran linked with glutaraldehyde, with encapsulated rivastigmine, a cholinesterase inhibitor in Alzheimer disease treatment, and rate controlling membranes composed of cross-linked dextran-poly (vinyl) alcohol films. The drug permeation release, which was determined in vitro on rats’ skin excised samples, reached only 66% in the absence of electric current, decreasing with the crosslinking density of the hydrogel (higher content of glutaraldehyde), but increased with direct current application of 2–8 mA at every 30 min
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intervals, to 97.5%. The electric responsiveness of the hydrogel is attributed to carbonyl groups resulted from partial hydrolysis of the amide ones (Patil et al. 2019). The advantage of transdermal delivery is the direct systemic transfer of the drug, avoiding the higher doses requested by oral administration, with their associated side effects. Regarding the above presented transdermal system, its distinctive feature could be considered the drug delivery on demand, not only for rivastigmine. Concerning this drug, a commercial therapeutic transdermal medicine exists (Exelon 9.5 mg/24h transdermal patch of Novartis), formulated with poly (alkyl) methacrylates and polyester (www.medicines.org.uk/eme/product/7764). The effect of combining two anticancer low water-soluble and bioavailable drugs (doxorubicin and camptothecin) by an oxidized dextran-lipoic acid prodrug formation and encapsulation in the self-assembled vesicles of the prodrug, respectively, was investigated. Dextran was oxidized to polyaldehyde and its nonoxidized hydroxyl groups were esterified by carboxylic group of lipoic acid, a natural antioxidant produced by the human body, which contains a disulfide bond. Then, doxorubicin was covalently linked to the dextran polyaldehyde group by a Schiff-base formation (imine bond CH¼N- between the amine group of the drug and the aldehyde group of the polymer). The second anticancer drug (camptothecin) was entrapped in the prodrug obtained vesicles. The system was two stimuli release responsive: pH 5.0, specific to the cancer cell environment (favoring the hydrolysis of doxorubicin imino-bond) and redox, the presence of added glutathione enhancing the hydrolysis and reducing the disulfide bonds of lipoic acid residues. Thus, the destabilization of the vesicle structure allowed the selective intracellular release of the drugs. Comparative results on breast cancer MCF-7 cells and healthy MCF-10A cells showed a better internalization of the loaded drugs than free drugs in the cancer cells and enhanced toxicity on cancer cells, compared to the combination of free drugs. The selectivity was also higher, proved by increased healthy cells viability (Curcio et al. 2020).
1.1.2 Dextran Derivatives in Systems for Topical Therapeutics An electro-responsive delivery system was prepared from dextran cross-linked with electroactive aniline trimer (obtained by condensation of two aniline molecules and one of p-phenilenediamine, resulting N¼ intermolecular bonds) and hexamethylene diisocyanate, forming polyuretane groups (NH-CO-O-) with dextran hydroxyl and free aminogroups of aniline trimer (Dex-AT/HDI). The hydrogel exhibited good conductivity and corresponding electro-responsive properties. Different such hydrogels (with aniline trimer content of 0–11%, corresponding to pore size 5–15 μm) were loaded with anti-inflammatory dexamethasone and indomethacin. In vitro drug release at pH 7.4 was as expected, increasing with pore size and reaching for dexamethasone 77% after 180 min. Electric-driver release was determined applying different voltages (3,1,0 V) to the 5μm hydrogel, leading to significant improvement of cumulative dexamethasone release, reaching 90% after 120 min. In vitro cell compatibility on L 929 fibroblast cells showed a good cell viability, even cell proliferation for 10 μm hydrogel, confirmed by a desirable biocompatibility
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during in vivo (rats) tests. Thus, such hydrogels were considered as smart drug carriers for localized sustained release (Qu et al. 2019). pH responsive curcumin-loaded polyurethane-dextran nanofibrous membranes were considered as potential wound dressing materials. Membrane composites of polyurethane/dextran with different concentration of dextran (5–25%) aiming to increase hydrophilicity, sorption capacity, hemostatic potential, mechanical reinforcement, and biodegradability were prepared by electrospinning. Encapsulating curcumin, for a synergistic antibacterial activity, such composites showed the best results of blood clotting, wettability, biocompatibility (max. viability of 3T3 mouse fibroblasts), and biodegradability for 25% dextran. The drug releasing performance was noticed at pH 5.8, compared with pH 1.2 and pH 7.4 (explained by poor solubility at acidic and neutral pH, but a complete protonation of dextran hydroxyl groups and interaction with β-diketone of curcumin). The in vitro antibacterial activity against S. aureus was exhibited by more than 20% dextran containing membranes (Sagitha et al. 2019). The hemolytic activity of antimicrobial quaternary ammonium salts was reduced by grafting dextran on a siloran-quaternary ammonium functionalized polyurethane dressing membrane, permitting to obtain a nonhemolytic dressing membrane with a preserved very good antimicrobial activity against meticillin-resistant S. aureus, Ps. aeruginosa, and Candida albicans (Gharibi et al. 2019). Dextran polymers were also applied as tissue scaffolds, contributing to build new functional 3D tissues, despite the high water solubility of the polymer. Electroactive antibacterial injectable hydrogels were prepared from chitosan grafted with polyaniline, crosslinked with oxidized dextran (Schiff-base). The presence of polyaniline in the copolymer, as electroconductive component, favored the proliferation of C2C12 mouse skeletal muscle cells and enhanced the antibacterial activity, assayed against E. coli and S. aureus. In vivo subcutaneous injection (rat) of the hydrogel encapsulating bacteria showed a gel formation and an antibacterial activity confirming the in vitro study results (Zhao et al. 2015). A composite of dextran and crystalline β-tricalcium phosphate (β-TCP) nanoparticles was prepared by incorporation of TCP in the hydrogel during crosslinking with epichlorhydrin. The highest porosity was obtained with 5% wt TCP. In vitro biomineralization by formation of hydroxyapatite in simulated body fluid (SBF) and cellular proliferation, assayed with 3T3 fibroblasts, were observed, thus considering a promising scaffold in bone regeneration (Ghaffari et al. 2020). A similar dextran functionalization to methacrylate derivative was used to UV photo-crosslinking in situ of poly (lactic acid) (PLA) grafted with dextran through maleic anhydride, obtaining nanofibers by co-axial electrospinning. Variable dextran compositions of PLA-dextran nanofibers scaffold produced different cell attachment, proliferation, and differentiation of mouse V1 embryonic stem cells to cardiac muscle ones (Zhang et al. 2017). An interesting application was covalently surface grafting dextran onto polyhydroxyalkanoate: [poly(3-hydroxybutyrate-co-3-hydroxyvalerate)] (PHBV) electrospun fibers scaffold methacrylic acid functionalized. This hydrophilic modification significantly enhanced the proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) (Zou et al. 2016).
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1.1.3 Dextran Derivatives in Gene and Cell Delivery Gene therapy, introducing new genes into the cells, represents a new hopeful way in therapy, especially in cancer, when their introduction into a tumor cell or its surrounding tissue aims to cause cell death or its growth rate reduction. Nanoparticles are considered possible efficient carriers. Polyelectrolyte complexes of carboxymethyl dextran, thiolated carboxymethyl dextran, by conjugation with cysteine (amide formation) and chitosan nanoparticles, were prepared; loading the antisense of hSET1 enzyme, overexpressed in malignant cells, could determine their regression, without affecting normal ones. After a stability study in serum, simulated gastric and intestinal fluids, a mucoadhesive study, the specific biological activity was assayed by cell toxicity on SW480 colon cancer cells, cellular uptake, and expression of hSET1 (gene silencing). The best results were obtained with high degree thiolated carboxymethyl dextran (Kiani et al. 2016). A similar role to thiolated carboxymethyl dextran and chitosan polyelectrolyte complex (TCD/Ch) nanoparticles was given to carry miR-145, a tumor suppressive miRNA, whose presence is abnormally reduced in some cancers. That gene silencing agent was accompanied by antinucleolin aptamer AS1411, a guanine-rich oligodeoxy nucleotide aptamer, potential inducer of apoptosis, which binds to nucleolin, overexpressed on the surface of certain cancer cells, a nucleolar protein involved in antiapoptosis. The components of the polysaccharide complex were synthesized as above (conjugation of carboxymethyldextran with L-cysteine and depolymerization of fully de-acethylated chitosan). MiR-145 was firstly conjugated with TCD and the aptamer was finally added to the polyelectrolytic complex. The system was redox-responsive, the disulfide bonds being susceptible to be broken by reductive action of the high cytoplasmatic concentration of glutathione. Contrary to naked miR-145, the system proved to be gene protective against RNase. In vitro studies on human breast cancer MCF-7 cells showed that miR-145 concentration significantly increased in the treated cells (RT-PCR), the cell proliferation was reduced, and the apoptosis was considerably increased. This double targeted gene delivery system was considered helpful in breast cancer therapy (Tekie et al. 2018). A dextran-based system aimed to solve the problem of cationic polymers toxicity, an obstacle in their efficient use to transport negative charged genes. Thus, a dextran graft copolymer was obtained introducing cationic monomers (dimethyl/diethyl/ amino acrylate/methacrylate) in a dextran solution, a polymerization initiator, and a disulfide bond containing monomer (dialyl disulfide) as crosslinker. Dextran-based coacervates were prepared adding pEGFP-C1 plasmid (DNA) and a small interfering RNA (siRNA), respectively. The coacervates were formed by electrostatic interaction between amino cationic groups and the anionic genes. In vitro experiments were performed on human embryonic kidney cells 293T and ovarian cancer cells OVCAR-3. After endocytosis, the gene release was due to disulfide bond cleavage by glutathione reduction, as above. The efficient transfection was proved by protein expression (green fluorescent protein, GFP) of pEGFP-C1 into 293T cells and detection of fluorescent labeled si-RNA in the cancer cells (Wang et al. 2020). Cell delivery could be considered a first step of tissue engineering. A method to graft the RGD peptide (arginylglycylaspartic acid) to dextran through a divinyl
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sulfone linker to obtain a cell delivery system to achieve a porous, three dimensional matrix, was developed. A dextran conjugate with divinyl sulfone was firstly obtained, by covalent binding of one vinyl group to dextran C2 glucopyranosyl unit hydroxyl (-SO2-CH2-CH2-O-). The free vinyl sulfone reacted with a free amino group of RGD peptide by a Michael type addition (-NH-CH2-SO2-). The peptide dextran derivative was crosslinked with sodium trimetaphosphate. RGD, known as cell adhesion agent, supported the attachment, proliferation, and migration into the hydrogel scaffold of the human ombilical vein endothelial cells (HUVEC). Optimal conditions, especially regarding RGD content, were determined (Riahi et al. 2017). A thermosensitive hydrogel, capable of controlled gelation at 37oC, and therefore considered injectable, was prepared from chitosan, dextran, and β-glycero-phosphate. The biodegradability tests at pH 7.4 showed a 70–80% degradation after 28 days, more pronounced in enzymatic conditions (lysozyme). With an excellent biocompatibility (3T3 and HUVEC cells), the hydrogel exhibited a linear cumulative cell delivery during 7 days (HUVEC), effective proliferation cell viability after injection by dual syringe applicator, and genetic differentiation (umbilical cord mesenchymal stem cells UCMSC). The hydrogel was considered a promising vehicle for injectable cell therapy of cardiac repair after myocardial infarction (Ke et al. 2020).
1.1.4
Dextran in Other Medical Applications
Dextran antimicrobial Dextran-type biopolymers produced by nature isolated Leuconostoc mesenteriodes strains exhibited antibacterial activities as their own property: one of them, single or blended with gentamycin, was active against a biofilm of multidrug-resistant microorganisms on urinary catheters (Salman et al. 2018) and another, produced by a Leuconostoc pseudomesenteroides isolated from mango juice, presented against S. aureus and E. coli MIC of 3 mg/ml and 2 mg/ml, respectively (Ye et al. 2019). Immunomodulatory Dextrans produced by Lc. mesenteroides strains isolated from human milk, Lc. mesenteriodes and Lactobacillus sakei isolated from meat products, exhibited immunomodulatory activity changing the ratio TNF-α/IL-10 in human macrophages costimulated with E. coli lipopolysaccharides, corresponding to an anti-inflammatory effect (Zarour et al. 2017).
1.2
Hyaluronic Acid
It was discovered in 1934 and its chemical structure was established in the 1950s as anionic linear glucosaminoglycan composed of disaccharide units of (1!4)-β-Dglucuronic acid and (1!3)-β-N-acetyl-D-glucosamine. Physiologically present in almost all mammalian tissues, including human body, hyaluronic acid entered easily between the officially approved pharmaceutical ingredients: as sodium salt (hyaluronan), it has a monograph in the European Pharmacopoeia (EP9, 2017) (Fig. 4).
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Fig. 4 Chemical structure of hyaluronic acid
Its first and remained medical application was considered a vitreous substitution/replacement during eye surgery in the late 1950s. Originally extracted from animal tissues, especially rooster combs, it is now produced by recombinant bacteria. The bacterial biopolymer was firstly approved only for topical applications: chronic, difficult wound healing, for example, Hyiodine, Sorelex (Contipro), and cosmetics. Lately, medical devices with bacterial hyaluronan were approved for eye surgery and intra-articular injections in osteoarthrities, for example, BioLon (Altacor-BTG), approved EU in 1995 (EC), USA-FDA 1998, EUFLEXXA (Ferring Pharmaceuticals, Savient Pharmaceuticals BTG), EC2004, FDA 2011, DUROLANE (Bioventus), EC 2001, FDA 2017. A sustained release injectable formulation of somatropin (recombinant human growth hormone), SP-hGH-Declage (L G Life Sciences), using sodium hyaluronate microparticles, has been approved by Korean FDA since 2008 and is now in extended clinical trials as LGO3002 (Eutropin Plus inj.) (Hwang et al. 2018). The first formulation approved by FDA for treatment of vesicoureteral reflux was a stabilized dextran/hyaluronic acid composite (NASHA/Dx) and gained European approval for the treatment of stress urinary incontinence (Huerta-Ángeles et al. 2018; Elzayat and Corcos 2008). Another cited commercial hydrogel wound dressing containing hyaluronic acid promoting autolytic debridement was Restore Hydrogel produced by Hollister Inc. (Aswathy et al. 2020). New published results are presented in Table 2.
1.2.1 Hyaluronic Acid in Wound Healing Hydrogels are considered the most promising material for wound dressing, providing a moist environment, the removal of exudates, infection prevention, and suitable environment for tissue regeneration. The healing process includes hemostasis, reducing inflammation, cell proliferation, and tissue remodeling (Aswathy et al. 2020). The microbial hydrogels should compete with synthetic ones.
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Table 2 New developments of hyaluronic acid medical applications Application Wound healing
Drug and gene therapy
Tissue engineering
Formulation Hyaluronan alginate
Potential therapy Wound closure
Gentamycin, vancomycin/ hyaluronan-D,L-lactide HA-PLA (Defensive Antibacterial Coating (DAC)), antiseptic Antiseptic+β-lactam antibiotic/hyaluronic acidcellulose in ionic liquid GUMBOS/HA-CEL SN38 Active metabolite of irinotecan(CPT-11)/hyaluronic acid bioconjugate prodrug ONCOFID-S miRNA/hyaluronan/ cholanic acid/Zndipicolylamine miRNA/hyaluronic acid/ chitosan miRNA/hyaluronic acid/ protamine sulfate miRNA/hyaluronic acid/ dipalmitoyl phosphatidylcholine-PLGApluronic F127 Hyaluronic acid methacrylated-hyaluronic acid nanoparticles (pastelike) in vivo photo crosslinked Cytomodulin-2-in vivo crosslinked hyaluronic acid Cx-HA-CM (injectable) Thiolated adamantanehyaluronic acidmethacrylated β-cyclodextrin-hyaluronic acid Dual crosslinked (second in vivo: Michael addition) (injectable)
Coating for implantable biomaterials (orthopedics, traumatology, dentistry, maxillo-facial surgery)
Reference Catanzano et al. (2015) Gaetano et al. (2018)
Patches for (infected) wound Lopez et al. (2020) care
Ovarian cancer
Montagner et al. (2015)
Gene silencing in colon, breast, prostate cancer
Fernandez-Piñeiro et al. (2017)
Bone implants
Beck et al. (2015)
Cartilage-like tissues
Park et al. (2019)
Soft tissue reconstruction (nucleus pulposus, myocardial after infarct)
Rodell et al. (2015)
The presence of hyaluronan through cross-linking with alginate promoted wound closure in vivo (rats) (Catanzano et al. 2015). A hyaluronic-based antibacterial hydrogel coating for implantable biomaterials in orthopedics, traumatology, dentistry, maxillofacial surgery was developed through a
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EU-FP7 project. It consisted of hyaluronan grafted with hydrophobic poly-D, L-lactide (HLA-g-PLA). The hydrogel formed a uniform, resistant coating, entrapping antibiotics, as antiadhesive antimicrobial film on the prosthetic materials. Antibiotic (gentamicin/vancomycin)-loaded hydrogel reduced by 75–80% biofilm formation with S. epidermidis and S. aureus. Drug release started after the implant, providing a concentration higher than MIC, ensuring the effectiveness. The product was administered as prefilled syringe of sterile powder, filled at surgery with sterile water and antibiotic. After preclinical studies (rabbits), with 72–99% bacterial load reduction, without side effects, the product, developed by the Italian company Novagenit, with the commercial name DAC (Defensive Antibacterial Coating) was clinically studied on two groups of patients (380 and 256) with good results (Gaetano et al. 2018). The wound healing process is considered containing three main events, every one involving hyaluronic acid (HA): a matrix rich in HA laid down in a cell-depleted space; stimulated mesenchymal cell migration from adjacent tissues which infiltrate the HA matrix; cells within the HA matrix secrete hyaluronidase which degrades the HA, sulfated glucosaminoglycans, and collagen which replace the HA and the tissue is remodeled (Gupta et al. 2019). Composites of hyaluronic acid and cellulose were prepared dissolving the biopolymers in an ionic liquid (green technology, full solvent recovery), aiming at exploiting favorable biomedical properties of both biopolymers in patches for wound care (Fig. 5). Antibacterial ionic synergic combinations of antiseptic, betalactam antibiotic, composed of chlorhexidine-oxacillin/cephalothin, known as GUMBOS, active against multidrug-resistant bacteria, were loaded. In vitro burst drug release, S. aureus inhibition zones, and high swelling capacities (to efficiently absorbing exudates) were presented as promising for wound healing (Lopez et al. 2020).
Fig. 5 Scheme of preparing hyaluronic acid-cellulose composites in an ionic liquid (green technology) as patches for loading antibacterial agents in wound care (ACS permission for Lopez et al. 2020; further permission related to the material excerpted should be directed to the ACS).
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1.2.2 Hyaluronic Acid in Drug and Gene Therapy A bioconjugate of SN-38, the active metabolite of anticancer pro-drug irinotecan (CPT-11) and hyaluronan, named ONCOFID-S, was obtained and studied as anticancer agent in vitro and in vivo by local-intraperitoneal administration in ovarian cancer. A selective internalization in tumor cells was proved in vitro through the binding to the CD44 receptor of three ovarian tumor cell lines: 1-GROV-1, OVCAR3, and SKOV-3. The in vivo experiments on tumor bearing mice with the irinotecan conjugate showed a strong antitumor activity vs. controls and even an increased therapeutic effect and survival, compared to irinotecan, for 1-GROV-1 bearing animals (Montagner et al. 2015). Four systems containing hyaluronic acid were cited as miRNA nanocarriers for targeted delivery to gene silencing in cancer therapy which passed in vivo studies: hyaluronan/cholanic acid/Zn dipicolylamine, hyaluronic acid/chitosan, hyaluronic acid/protamine sulfate, hyaluronic acid/dipalmitoyl phosphatidylcholine-PLGApluronic F127 for different cancer types: colon, breast, and prostate. Administered intravenously, they ensured a selective delivery, permitting to reduce doses and unwanted effects (Fernandez-Piñeiro et al. 2017). 1.2.3 Hyaluronic Derivatives in Tissue Engineering The capacity of hyaluronic acid-based colloidal gels to fully recover after compression was exploited aiming at applications including cartilage regeneration. Cytomodulin-2 (CM), a chondrogenic differentiation peptide factor, was chemically immobilized in hyaluronic acid (HA), crosslinked (Cx) by click reaction with tetrazine and transcyclooctene, separately and mixed using a double barrel syringe: Cx-HA+CM!Cx-HA-CM (Fig. 6). This injectable formulation was tested in vitro and in vivo (mice) for enhancing chondrogenic differentiation of human periodontal ligament stem cells (hPLSC). The chemical attachment of CM resulted in long time persistence (28 days after injection), favoring a significant expression and induction of the formation of cartilage-like tissues. This injectable system was considered suitable for repair of damaged articular cartilage (Park et al. 2019). A supramolecular injectable assembly of two guest-host, HA derivatives (HA-adamantane and HA-β-cyclodextrin), was developed at the aim to obtain firstly an ex vivo crosslinking with shear thinning delivery as liquid with high retention at the target site, followed by a secondary covalent crosslinking in situ, time-controlled, clinically required, to enforce and stabilize the hydrogel network. The covalent bonds were created by Michael addition reaction with slow kinetics between thiol and methacrylate groups previously provided at the HA before modifications. In vivo experiments on rats with myocardial infarct showed stabilization, progressive remodeling, and trend toward increased contractile function. The results were thought potentially useful for soft tissue reconstruction, for example, nucleus pulposus replacement, treatment of myocardial infarct (Rodell et al. 2015). Comprehensive reviews regarding a very wide range of possible chemical modifications of HA and potential product applications including tissue engineering were published (Khunmanee et al. 2017; Highley et al. 2016).
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Fig. 6 Scheme showing injection of a formulation of human periodontal ligament stem cells (hPLSC)-loaded hyaluronic acid-tetrazine-cytomodulin (HA-Tet-CM) and hPLSC-loaded hyaluronic acid-transcyclooctene-cytomodulin (HA-TCO-CM) for chondrogenic differentiation in an in vivo formed crosslinked-hyaluronic acid-cytomodulin (Cx-HA-CM) (permission confirmed by Copyright Clearance Center, from Park et al. 2019)
1.3
Xanthan
Discovered in 1950, it is an anionic branched heteropolysaccharide with a backbone of (1!4)β-D-glucose disaccharide units with alternate branches consisting of three other hexose units: glucuronic acid between two mannose units, the whole monomer being a pentasaccharide. The terminal mannose can have a pyruvate group attached to C5–C6 and the mannose linked to the backbone glucose may have an acetyl group to C6. The ratio pyruvate/acetate depends on fermentation conditions (Fig. 7). Xanthan is produced in aerobic fermentation of Xanthomonas campestris. With high molecular weights (more than 1000 kDa) and viscosities, it was firstly used for enhanced oil recovery and later on in food industry, as a thickener and suspension stabilizer. Approved as food additive in the USA (1969), by FAO in 1974, in Europe in 1982, it was subsequently included in the USA and European Pharmacopeias (USP 43-NF38, EP9, 2017), as pharmaceutical ingredient.
1.3.1 Xanthan as Drug Carrier Polyelectrolyte complexes of chitosan and xanthan were used for coating rifampicinloaded liposomes (chitosomes). The microparticle vesicles aimed to lung delivery by
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Fig. 7 Chemical structure of xanthan
nebulization. A chitosan-xanthan ratio of 1:0.5 was found the best to improve the drug total mass output and deposition (Manca et al. 2012). To improve its low mechanical stability and excessive swelling, which limited xanthan application in drug delivery and tissue engineering, the polysaccharide was blended with silk fibroin to prepare a robust and with controlled porosity drug delivery scaffold. The chloramphenicol antibiotic was loaded as model drug and in vitro drug release (phosphate buffer pH 7.4, 37oC) kinetics was studied and optimized by response surface methodology (RSM) and artificial neural network modeling (ANN), using cumulative percentage release of antibiotic. The predictive model with blend combination, porosity, and swelling as variables was verified. The ANN was found as better predicting than RSM (Shera et al. 2018).
1.3.2 Xanthan in Tissue Engineering and Wound Healing Magnetically responsive nanoparticles of xanthan-chitosan polyelectrolyte complex hydrogels for tissue engineering were prepared, incorporating iron oxide magnetic nanoparticles. Deacetylated chitosan was added to a xanthan solution containing D-(+)-glucuronic acid δ-lactone as acidifying agent with dispersed Fe3O4 nanoparticles, and an electrostatic complex was formed followed by hydrogen-bonding
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interactions. Cell adhesion and proliferation of NIH3T3 fibroblasts were favored under magnetic field acting on the incorporated magnetic nanoparticles. Such tunable magnetic hydrogels were considered suitable for skin, cartilage, muscle, and connective tissue engineering (Rao et al. 2018). Xanthan/konjac glucomannan blend hydrogels were obtained. Konjac glucomannan is a polysaccharide extracted from the tubers of Amorphophallus konjac plant. Xanthan presents in aqueous solutions at low temperature an ordered double helical strand structure which at about 40–50oC forms a three dimensional network gel-like. This property and a mechanical strength effect on konjac glucomannan were exploited in the blend, obtained by autoclaving the polymeric solutions at 121oC and decreasing the temperature to 37oC, when the sol-gel transition forms a gel-like morphology, permitting a direct application at the wound site, acquiring its shape and size. The biocompatibility, cell adhesion and infiltration, as well as cell migration in a wound healing scratch assay were proved on cultures of human dermal fibroblast (NHDF) cells (Alves et al. 2020).
1.4
Gellan
Discovered in 1978, gellan is a linear anionic heteropolysaccharide, with a tetrasaccharide repeating unit of 1,3-β-D-glucose, 1,4-β-D-glucuronic acid, 1,4-β-Dglucose, and 1,4-α-L-rhamnose. The native form contains glyceryl and acetyl groups (Fig. 8). Gellan is produced by fermentation of Sphingomonas paucimobilis, also referred to as Sphingomonas elodea. It is produced and marketed under different trade names: GelriteTM, KelcogellTM, Phytagel, Gelzan, Gelrite, and Applied gel. Acetyl groups can be easily removed by alkaline hydrolysis and deacylated gellans are mostly used in tissue engineering and pharmaceuticals. The average
Fig. 8 Chemical structure of gellan
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molecular weight is about 500 kDa. Dissolved in water, heated and mixed with cations (mono or divalent), gellan forms a gel by decreasing temperature. The gels are thermoreversible. The acetylated form yields soft and elastic gels, the fully deacetylated, hard, and brittle gels. With a large area of applications in food industry, it has been approved by FDA as a stabilizer and thickener in food since 1990 and in Europe as food additive (E418) since 2012. A low acyl form is used in solid dosage pharmaceutical formulations of immediate or sustained release (Osmalek et al. 2014). In ophthalmic preparations, low acetyl gellan is exploited for gel forming behavior in situ, in the presence of cations (tear fluids) ensuring a prolonged contact time and sustained release, for example, antiglaucoma timolol maleate gel forming solution, marketed as Timoptic-XE (www.rxlist.com/timoptic-xe-drug) or Blocadren Depot (www.ndrugs.com). Such applications determined the inclusion of low and high acyl forms of gellan in US Pharmacopeia (USP 43-NF38, 2020).
1.4.1 Gellan Developments as Drug Carriers Dozens of delivery systems containing gellan and derivatives have been developed based on its gelling stimuli responsive properties, mainly temperature and ionic strength (Palumbo et al. 2020). A drug delivery pH-responsive nanosystem was obtained consisting of sericin/ rice bran albumin embedded gellan. Gellan was conjugated with sericin protein by CO-NH amide groups. The anticancer doxorubicin was loaded by encapsulation. The drug release was faster and greater at acidic pH (4.0), possibly due to the polymer ester bonds hydrolysis. The sustained release favored a decrease of the survival rate of the tumor MCF-7 cells, compared to single drug. A strong tumor cell internalization of the nanoparticles was proved (Arjama et al. 2018). A thiol derivative of gellan was also prepared via amide bond formation by conjugation with 2-(2-aminoethyldisulfanyl)nicotinic acid, obtaining different degrees of thiolation. Keeping biocompatible, vaginal films casted from the obtained biopolymer exhibited a several fold increased dynamic viscosity in porcine vaginal mucus and 3-fold improved adhesion on mucosal surface compared to gellan films. It ensured a sustained release of metronidazol, thus proving to be a promising novel excipient for coating vaginal films (Jalil et al. 2019). 1.4.2 Gellan in Wound Healing and Tissue Engineering Gellan-methacrylate was combined with up to 1% laponite XLG, a synthetic layered silicate clay, water insoluble, but swellable, forming colloidal dispersions. The mixture appeared as a nanocomposite hydrogel, which was UV photocrosslinked. It was considered a novel possible wound dressing material, having also in view the drug sustained release of a model antimicrobial, ofloxacin (Pacelli et al. 2016). Gellan was added to gelatin, forming a blank hydrogel (heating and cooling) which incorporated tannic acid as antibacterial. Thus, the tannic acid-loaded hydrogel proved to be active in vitro against E. coli, S. aureus, and methicillin multidrugresistant S. aureus. Tested in vivo on mice, that electrostatic complexation hydrogel showed a good injectability and synergistic gel formation and promoted cell adhesion and migration, leading to complete healing by skin regeneration after 12 days,
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without scars, compared to slower and unsatisfactory results of a control, treated with single tannic acid (Zheng et al. 2018). A comprehensive and well-ordered review on medical applications of gellanbased systems was published by Palumbo et al. (2020), highlighting the exploited important properties of that biopolymer, as gelling ability, thermal and ion sensitivity mucoadhesion. Cell therapy by tissue engineering and scaffolds formation for regenerative medicine seems to be the major perspective application of gellans. As collagen-derived protein, hydrazide-modified gelatin and oxidized to aldehyde gellan were covalently crosslinked to a 3D matrix mimicking the extracellular one. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC) were successfully cultivated in that matrix and they retained their normal beating behavior (contraction-relaxation), the gelatin-gellan hydrogel proving to be suitable for 3D cardiac tissue modeling and study of such diseases (Koivisto et al. 2019). A low-acyl gellan-hydroxyapatite spongy-like composite hydrogel obtained by Manda et al. (2018) was aimed to mimic the extracellular matrix of bone formation. CaCl2 was used as crosslinker. When human adipose-derived stem cells hASC were seeded into the polymeric 3D structure, cell adhesions and spreading, as well as the precipitation of apatite similar to bone-apatite in modified simulated body fluid were noticed. Santhanam et al. (2019) prepared injectable fibrillary hydrogels composed of deacylated gellan and semiflexible polyelectrolyte copolymer of poly[methacrylamideco-methacrylic acid], both thiolated and crosslinked by disulfide bonds. Two products proved good and superior properties as vitreous biomimetic-biocompatible replacers on rabbit experiment, compared to chemically accepted silicone oil. They were stable at least for 30 days, maintaining optical transparency, physiological intraocular pressure, intact retinal layers. Vilela et al. (2018) obtained a low acyl methacrylated gellan and used it as chondrogenic polymer for cartilage repair. In vitro chondrogenesis was studied on hydrogel encapsulated human nasal cartilage cells (NC) and human adipose stem cells (hASC). The in vivo study was performed on a rabbit model, by chondral lesion induction and repair with autologous rabbit ASC encapsulated in the hydrogel. After 8 weeks, a full thickness regeneration of size lesions and good integration with native cartilage were noticed. The biopolymer-derivative was considered a support for cartilage repairing surgery.
1.5
Bacterial Cellulose
Firstly observed by ancient Chinese producing a fermented beverage from acetic bacteria and firstly described by J. M. Brown in 1886 (Azeredo et al. 2019; Abeer et al. 2014), bacterial cellulose (BC) presents the same chemical structure as plant cellulose, as linear homopolysaccharide composed of β-1,4-glucose units (Fig. 9). It is produced by fermentation of numerous bacterial species, but Gluconacetobacter or Komagataeibacter xylinus is the most studied and considered for
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Fig. 9 Chemical structure of bacterial cellulose
commercial production, and static batch cultivation is preferred for the surface pellicle product morphology, even limited by the too large requested surface. Apart from the advantage of the possibility to control the product characteristics (shape, network) and yield by bioprocess parameters, BC shows high purity, high degree of polymerization (2000–10000 glucose units), high crystallinity (80–95%), high fiber stability (similar to steel), very high water holding capacity (more than 99%), forming stable hydrogels (Moscovici et al. 2017), but due to process limitations, its applications are currently limited to high value, of justified performance products (Freitas et al. 2014). Approved by FDA and the European Food Safety Authority (EFSA) as a dietary fiber in foods, it was also approved as component of other products for various clinical indications and as primary and unique component of some wound dressings. Numerous such products were cited as existing on the market, including vessel implants, artificial skin, tendon repair (Abeer et al. 2014; Ludwicka et al. 2016), as well as US and EU patents regarding similar products. Despite the existence of commercial products and challenges in efficient production, the bacterial cellulose medical applications are continuously developed, as shown by the increased number of publications (Gorgieva 2020).
1.5.1
Bacterial Cellulose Developments in Wound Healing with Antimicrobials Delivery and Tissue Engineering A dressing saturated with gentamycin antibiotic was obtained and tested for bone film infections. After in vitro experiments on a microbial biofilm on a simulated bone environment and evaluation of cytotoxicity of the dressing product in osteoblast (U2-OS) cultures, ex vivo experiments were performed on rat femur and jaw. Efficient inhibition was reached against S. aureus and Ps. aeruginosa biofilms and
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an antibiotic prolonged release, compared to gentamycin-loaded collagen sponge (Junka et al. 2019). Silver nanoparticles were deposited in the BC matrix by Ag+ ions reduction of a silver nitrate solution impregnated in the BC membrane, using UV light irradiation. A strong inhibition of E. coli was reached at a concentration of 10-2 M Ag/BC sample (Pal et al. 2017). A “mussel” mimetic transdermal patch was prepared on a moiety of dopamine (cathecol containing) modified by amidation-carboxymethyl BC/reduced graphene oxide (by CM-BC hydroxyl groups) composite, doped with Ag+ nanoparticles. In vitro tests regarded cytotoxicity on 3T3 fibroblast cells and wound healing of scratched cell layers of 3T3 and A549 human lung epithelial cell lines and antimicrobial activity against S. aureus, Lysinibacillus fusiformis, E. coli, and Ps. aeruginosa. The composite enhanced proliferation of 3T3 cells, especially by the presence of Ag nanoparticles compared to a control; efficient wound healing for both cell types was noticed, and the antibacterial activity was significant, even compared to a ciprofloxacin containing control (Khamrai et al. 2019). Roman et al. (2019) published an evaluation of BC-based tissue engineering scaffolds, starting from the specific criteria requested to the tissue scaffolds for implantation: biocompatible (nontoxic, nonimmunogenic), biodegradable, biomimetic, and ideally, bioactive, stimulating cell differentiation and tissue regeneration. Thus, BC was considered accordingly. With the advantage of a relative physical and chemical stability, permitting heat, chemical, and radiation sterilization, with a prolonged structural integrity of implants, its resistance to in vivo biodegradation in the absence of cellulolytic enzymes in mammalian tissues was considered the main limitation of this biomaterial. To overcome it, some approaches were cited, starting from incorporation of commercial enzymes for a gradual biodegradation, to metabolic engineering and irradiation. Thus, Yadav et al. (2015) obtained a genetically modified Gluconacetobacter xylinus which produced modified bacterial cellulose lysozyme susceptible as chondrogenic scaffold. A genetically modified BC producer Komagataeibacter hansenii culture produced BC membranes with increased pore size and relaxed fiber structure, superior to membranes produced by a wild-type strain, as scaffold of chondrogenic model ATDC5 cells in vitro (Jacek et al. 2018).
1.5.2 Bacterial Cellulose in Drug Delivery Transdermal drug delivery of antimicrobials from BC membranes has been firstly applied in wound dressings, including commercial ones, but it has been extended to other drugs to penetrate the skin, due to the significant advantage of direct transport with controlled release to the bloodstream through skin penetration and to the target site, avoiding the gastrointestinal tract, where the drug substance may undergo degradation or cause adverse side effects. A transdermal potential system from BC was investigated by a study on crocin, an antioxidant carotenoid extracted from safron (Crocus sativa L.) with several medicinal properties, including antitumorigenic and selective antitumor activity. BC
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membranes were loaded by drug adsorption and the diffusion experiments through mouse epidermal skin showed a slow release profile (Abba et al. 2019). A multifunctional theranostic platform was designed consisting of magnetic hydrogel nanoparticles containing immobilized folic acid, encapsulated anticancer doxorubicin, and hematoporphyrin monomethyl ether which loaded bacterial cellulose membranes. So, folic acid was a target molecule, enhancing the accumulation of nanocomposite, the magnetic nanoparticles absorb a 633 nm laser radiation and are photosensitizer for the oxidation damage of tumor cells by the singlet oxygen generated from the hematoporphyrin derivative under light exposure. Thus, the tumor cells could suffer a dual attack, drug and singlet oxygen O (photodynamic treatment). The in vivo study of the composite immobilized on a medical adhesive tape on the skin of MCF-7 breast cancer cells tumor bearing mice exposed to laser irradiation and magnetic field showed a synergistic effect of approx. 80% inhibition after 14 days (Zhang et al. 2019).
1.6
Levan
With production insight since 1930, it is a neutral, water-soluble homopolysaccharide composed of fructose units linked in a backbone of β-2,6, with β-2,1 side branches, produced by a few plants (Agropyron cristatum, Dactylis glomerata, Poa secunda) and numerous microorganisms: fungi (e.g., Aspergillus sydowii, A. versicolor) and a wide range of bacteria, including species of Erwinia, Streptococcus, Pseudomonas, Zymomonas, Lactobacillus, Bacillus, Aerobacter, with different MW (Fig. 10). Levan
Fig. 10 Chemical structure of levan
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is usually synthesized from sucrose by the enzyme levansucrase (EC 2.4.1.10) (Freitas et al. 2014; Srikanth et al. 2015). It has been approved as food additive in the USA, Europe, Japan. As different from other microbial polysaccharides, levan exhibits biological activities by itself.
1.6.1
Biological Activities of Levan for Health
Antioxidant Products with antioxidant activity are very important, combating oxidative stresses, as a cause of numerous serious diseases. Those of natural origin are preferred. Hydrogen donating ability for free radical scavenging and heavy metal ions chelation by their functional groups are considered the main features of exopolysaccharides as antioxidants (Andrew and Jayaraman 2020). A high MW (20 Mda) levan produced by a B. subtilis strain showed a very strong antioxidant activity in vitro, much superior to butylated hydroxyanisole (BHA) (Bouallegue et al. 2020). Levan produced by B. licheniformis as cholesterol hydrophobized derivative incorporated fullerene C60 nanotubes, forming a noncovalent supramolecular hybrid exhibiting a high dose dependent antioxidant activity (Kop et al. 2020). A levan produced by Leuconostoc mesenteroides showed a strong antioxidant activity, but also an immunomodulatory role, inducing a dose-dependent antiinflammatory cytokine IL-4 production in HT-29 (human colon adenocarcinoma) cells (Taylan et al. 2019). Antitumor A dose-related antiproliferative effect of levan produced by a halophilic bacterium Halomonas smyrnensis on human MCF-7 breast cancer cells was noticed and associated with cell apoptosis and oxidative stress, which was considered dependent on cell type and redox status (Queiroz et al. 2017). Similar results were obtained with the oxidized aldehyde derivative of the same levan on other human cancer cell lines: A549 (lung), Hep2/C3A (liver), AGS (gastric) (Sarilmiser and Oner 2014). Prebiotic Mild acid hydrolysis of levan produced by a strain of Erwinia sp. led to hydrolysate (MW approx. 3kDa) which stimulated Bifidobacterium and Eubacterium rectale-Clostridium coccoides growth in a gut model system stronger than inulin (Liu et al. 2020). Two levan producing Bacillus subtilis strains isolated from honey were evaluated as probiotic-prebiotic systems (synbiotics) in vivo in a mouse model. Positive results were noticed regarding gut microbiota, immune system improvement, and protection from Salmonella typhimurium liver infection; levans probiotics showed antioxidant activity (Hamdi et al. 2017).
1.6.2 Levan in Tissue Engineering Blends of poly (ε-caprolactone), gelatin, and hydrolysate of Halomonas produced levan were prepared in suitable compositions for 3D-bioprinting. The presence and
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increased concentration of levan hydrolysate in the scaffolds increased the proliferation of human osteoblast cells (Duymaz et al. 2019). Free-standing layer-by-layer adhesive films prepared from chitosan, alginate, with levan sulfate addition, non- or crosslinked with genipin, were obtained and tested on mouse C2C12 myoblast cell line culture. The presence of sulfated levan and crosslinking conferred a higher mechanical resistance to the membranes and enhanced cell compatibility and proliferation, for a possible application in cardiac tissue engineering (Gomes et al. 2018).
1.6.3 Levan as Drug Carrier A carboxymethyl derivative of Halomonas levan hydrolysate was incorporated as crosslinker in poly-N-isopropyl acrylamide. 5-Aminosalycylic acid, an antiinflammatory drug for bowel diseases (ulcerative colitis, Crohn’s disease), was encapsulated in the obtained hydrogels, which were tested for biocompatibility on mouse fibroblast L929 cells. The increased levan concentration favored the biocompatibility and a controlled release (8 h) of the drug at 37oC and pH 7.4 (Osman et al. 2017). Another levan produced by a strain of Acinetobacter nectaris and its carboxymethyl derivative were studied as anticancer 5-fluorouracil carriers. The drug was loaded by encapsulation. Drug loading reached a maximum (approx. 60%) at basic pH (12) and the drug release was higher for levan at pH 7 (more than 70%) than for carboxymethyl derivative, which retained the drug by an amide formed with a NH group of the drug (Tabernero et al. 2017).
2
New Discovered Bacterial Polysaccharides with Potential Medical Applications
Some of the new polysaccharides showed biological activities of interest regarding widespread serious diseases. An exopolysaccharide (EPS) produced by a Bacillus subtilis strain isolated from mangrove marine sediment, with a composition of mannuronic, glucuronic acids, glucose, galactose, mannose (1.6:1.5: 1.0:2.3:1.4), showed antioxidant activity and improved hyperglycemia, dyslipidemia (molecular markers), and cardiovascular disease risk (histopathological) in streptozocin-induced diabetic rats (Ghoneim et al. 2016). A strain of Bacillus mycoides isolated from a gas station was found as producer of an exopolysaccharide, composed of galactose, mannose, glucose, and glucuronic acid, which exhibited selective antitumor activity against cancer cells HepG2 and Caco-2 with IC50 of 138 μg.ml-1 and 151 mg.ml-1, respectively (Farag et al. 2020). 60 Lactobacilli were isolated from 55 herbal plants and 42 dairy samples, among them 21 producing significant concentrations of EPS. The produced EPS exhibited antioxidant, different anticollagenase, antielastase activities. No significant toxicity on normal primary human fibroblasts was observed, but some cell proliferation. Positive results were noticed in a scratch test for wound healing. The EPS from L. casei showed positive effect regarding down-regulation and inhibition of matrix-
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metal proteinases gene-expression, skin-aging related. D-glucuronic acid was found in some of the EPS compositions and was suggested as a key molecule in the skin antiaging process. The EPS products were considered promising for skin antiaging agents to tissue engineering scaffolds (Shirzad et al. 2018). An exopolysaccharide produced by a new isolated Bacillus strain from mangrove marine sediment composed of galacturonic and glucuronic acid (1:1) was studied for 90 days in aluminum chloride-induced Alzheimer disease rats. Reduction of oxidative stress parameters (malondialdehyde, hydrogen peroxide, nitric oxide), stimulation of antioxidant ones (catalase, superoxide dismutase), and especially, inhibition of cholinesterase, a key activity in the treatment of neurodegenerative diseases, were considered potent anti-Alzheimer effects. A subchronic study revealed the EPS as nontoxic (Asker et al. 2015).
3
Conclusions and Perspectives
Bacterial polysaccharides already play an established role in medicine and health improvement. During the last years, numerous smart medical developments of known bacterial polysaccharides have been published, as well as bioprospecting for new strains producing valuable polymers of this class continued. From the medical-pharmaceutical authorization point of view, their current developing applications could be divided into two major classes: drugs and medical devices. In the drug class, a very large area belongs to new drug carriers, especially as ingredients of smart targeted nanopharmaceuticals. Here, there are still limitations/barriers to overcome and corresponding challenges. So far, no polysaccharide nanoparticle formulations have obtained regulatory approval, only drug nanocrystals, liposome and lipid nanoparticles, PEG-conjugated nanodrugs, other polymers: protein-based nanopharmaceuticals (e.g., albumin), synthetic polymers (Farjadian et al. 2018) have been approved. As a general remark, physiological polymers or with a very known past are preferred. Apart from financial, ethical issues, common to all new pharmaceuticals, we consider the regulatory issues the most important. In this view, larger in vivo studies, in different complexity targets, confirming in vitro results, should prove the efficacy and safety. As other innovative medicines, the nanomedicines should present a complete Common Technical Document according the guidelines of the International Council for Harmonization (ICH), with the very important safety chapter (M4-5) containing pharmacology, pharmacokinetics, and toxicology preclinical reports. Generally, nanoparticle formulated drugs, by their advantages (reducing doses and side effects, possibly cheaper, due to their more precise delivery to the target) and a higher accumulation in the targeted cells and tissues, could represent a major direction in the future therapy, including personalized medicine. Concerning to bacterial polysaccharides, reliable, reproducible products, with stable properties, in vivo confirmed and quantitatively detectable in physiological fluids and tissues, are necessary conditions for their authorized entry in therapy.
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A different image, with already existing approved bacterial polysaccharide content, even as nanomaterials, presents the medical devices. Though mostly as wound dressings, new developments of bacterial polysaccharides have a good chance to enter the market, easier as physiological polymers, like hyaluronic acid. Some requirements are common with medicines, regarding the chemical purity determined according to the Pharmacopeias and other physicochemical properties depending on destination (viscosity, molecular weight and its distribution, mechanical properties). Generally, and particular for Europe and USA, there are three medical devices categories, depending on the type of device (surface or implant), duration of tissue contact, and mainly on type of tissue in contact with the device (intact skin, bone, damaged tissue, blood, etc.). Cytotoxicity, sensitization, and irritation assessments are recommended for all medical devices (Huerta-Ángeles et al. 2018). Open wound dressings and implants in contact with damage tissues are generally considered in second or third category and safety conditions are more severe. Preclinical reports should demonstrate that the product is nonpyrogenic, mutagenic, toxigenic, hemolytic, and immunogenic (norm ISO 10933). It is important to select adequate cell lines for biocompatibility/cytotoxicity test. If the device is in contact with blood, quantitative detection and systemic in vivo toxicity are necessary. As intensive research is going on and a lot of results are relatively new, certainly novel medical applications of bacterial polysaccharides, efficient and safe, will reach the market and satisfy people’s needs.
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Alcina M. M. B. Morais, Ana Alves, Decha Kumla, and Rui M. S. C. Morais
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Structural Characteristics of Sulphated Polysaccharides Produced by Algae . . . . . . . . . . . . . . 2.1 Macroalgae/Seaweed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Microalgae and Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Other Sulphated Polysaccharides Produced by Animals, Plants, or Other Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Bioactivity of Sulphated Polysaccharides from Algae. Relation with Chemical Features of Their Structures and Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Polysaccharides from Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Bioactivities of Sulphated Polysaccharides from Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Case Study: Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Potential Medical/Biomedical Applications of Sulphated Polysaccharides from Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Anticancer Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Immune Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Antipathogenic and Anti-Inflammatory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
In recent years, sulphated polysaccharides (SPs) have emerged as an increasingly important class of biopolymers with potential innovative applications in the biomedical and pharmaceutical fields due to their diverse physicochemical A. M. M. B. Morais · A. Alves · D. Kumla · R. M. S. C. Morais (*) CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal e-mail: [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_49
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properties and biological activities. The focus of the present chapter is to describe these biological activities and pharmacological properties of SPs and to highlight their potential application in the pharmaceutical and biomedical areas, as well. These compounds obtained from algae (macroalgae and microalgae) exhibit many beneficial biological activities, such as anticoagulant and/or antithrombotic, immunomodulator, antitumor and cancer preventive, hypoglycemic, antibiotics and anti-inflammatory, and antioxidant. In addition, the interest in using algal polysaccharides as biomedical vehicles for drug delivery has increased steadily. The isolation, structure elucidation, and bioactivity testing of algal SPs always bring perspectives for the discovery of new drugs and new applications, as well as an economic development, both by introducing a high diversity of new products and, eventually, a product with high added value. Keywords
Sulphated polysaccharides · Biological activities · Macroalge · Microalgae · Biomedical applications
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Introduction
Sulphated polysaccharides (SPs) comprise a complex group of macromolecules, consisting of single or different types of monosaccharides with one or more sulphate group attached in different positions, with a wide range of important biological properties (Cunha et al. 2009). Many organisms are very different sources of polysaccharides with several interesting functional properties, in which sulphate substitution patterns can provide specific biological functions. In the last years, SPs from algae have emerged as an important class of natural biopolymers with applications of great interest. The truth is that the marine environment offers a tremendous biodiversity, and original polysaccharides have been discovered showing a high chemical diversity possessing variations in the carbohydrate backbone, location of the sulphate group(s), and degree of sulphation that is specific of the algae species, as mentioned above. Polysaccharides, in general, and sulphated exopolysaccharides, in particular, are produced by many species of algae (macroalgae and microalgae) and with a broad range of applications: bone joints and used in healthy foods, as well as other applications in various industrial areas (Muhamad et al. 2019). A study on the biological properties of polysaccharides from marine eukaryotes and marine prokaryotes revealed that polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides. However, SPs are one of the most interesting biopolymers found in nature with the diverse range of potentials for use in medical, pharmaceutical, and biotechnological applications, such as antioxidant, anticoagulant, anti-inflammatory, antiviral, antitumor, and immunostimulatory agents. From the wide range of these compounds, the SPs of red algae have been extensively studied (Lee et al. 2012).
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This review focuses on SPs from algae and their biological activities with potential for pharmaceutical and biomedical applications.
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Structural Characteristics of Sulphated Polysaccharides Produced by Algae
Sulphated polysaccharides are compounds widespread from different sources in nature, having been isolated from animals, plants, and microorganisms. From previous researches it is possible to extract SPs from freshwater algae and seaweed, because their cellular walls are rich in SPs, such as carrageenan in red algae, fucoidans in brown algae, and ulvans in green algae. SPs have been reported to display main biological activities, such as anticoagulant, antiviral, antioxidant, antitumor, antiprotozoal, anti-inflammatory, anticomplementary, immunomodulatory, antibacterial, and antilipemic properties (Lee et al. 2012). SPs isolated from algae also include sulphated galactan produced by red algae (Gelidium crinale), sulphated fucans produced by brown algae (Ascophyllum nodosum), and sulphated glucans and sulphated arabinogalactans produced by green algae (Codium latum and Codium divaricatum). Fucoidan and laminarin are SPs extracted from brown algae and showed for their antitumor antimicrobial, immunostimulatory, and anti-inflammatory activities (Dobrinčić et al. 2020).
2.1
Macroalgae/Seaweed
Marine macroalgae, or seaweeds, are plant-like organisms that grow attached to hard surfaces, such as dead coral or rock, or other hard substrates in coastal areas. Macroalgae are classified belonging to three major groups: green algae (phylum Chlorophyta, classes Bryopsidophyceae, Chlorophyceae, Dasycladophyceae, Prasinophyceae, and Ulvophyceae), brown algae (phylum Ochrophyta, class Phaeophyceae), and red algae (phylum Rhodophyta). Macroalgae represent an important role in the natural product field, because they have been reported as an important source of biomedical compounds, such as antimicrobial, antiviral, anticoagulant, anticancer, antifouling, and antioxidant. Recently, SPs have been developed and rosen interest as medical products, because SPs show many beneficial biological activities, such as anticoagulant, antiviral, antioxidative, antitumor, immunomodulating, antihyperlipidemic, and antihepatotoxic (Jesus et al. 2015).
2.1.1 Freshwater Algae (Macroalgae) Freshwater algae as other sources of sulphated polysaccharides allow to explore interesting and important biological activities with potential health benefits. At present, SPs have only been reported from marine algae belonging to the genera Ulva (38%), Enteromorpha (14%), Monostroma (14%), and other (34%) (Wang et al. 2014). However, SPs have also been reported from freshwater algae (Table 1).
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Table 1 Sources of sulphated polysaccharides from freshwater algae (macroalgae)
Algae group Green algae
Species Cladophora glomerata Cladophora surera Ulva flexuosa
Adapted from Ciancia et al. (2020)
2.1.2 Marine Algae (Macroalgae) Marine macroalgae are potentially novel sources of sustainable biologically active secondary metabolites, which may exhibit various biotechnological applications. Among marine organisms, marine algae have been identified as simple unicellular or multicellular organisms, including seaweeds (macroalgae). Sulphated polysaccharides from marine macroalgae are commercially beneficial compounds with a wide range of pharmaceutical and biomedical applications. The SPs found mainly in marine seaweeds and their structures depend on the seaweed classes, Chlorophyta (green algae), Phaeophyta (brown algae), and Rhodophyta (red algae) (Ngo and Kim 2013). Well-known sulfated polysaccharides in seaweeds include galactans from red algae, ulvans from green algae, and fucans and fucoidans from brown algae. Therefore, SPs from different marine algae have been isolated and are summarized in Table 2.
2.2
Microalgae and Cyanobacteria
Microalgae and cyanobacteria are specifically attractive as natural sources of bioactive metabolites for drug discovery. Most bioactive compounds produced by microalgae include carotenoids, glycolipids, polysaccharides, and proteins. Furthermore, microalgae are valuable sources of structurally diverse bioactive compounds, including alkaloids, polyketides, cyclic peptides, polysaccharides, phlorotannins, diterpenoids, sterols, quinones, and lipids found in macroalgae as well (Talero et al. 2015). Sulphated polysaccharides are found from many microalgae strains. Thus, microalgae display an important source of polysaccharides, the main producers, such as diatoms, chlorophytes, prasinophytes, haptophytes, rhodophytes, and dinoflagellates (Jesus et al. 2015).
2.2.1 Freshwater Microalgae Currently, sulphated polysaccharides were found mostly in marine seaweeds and in the animal kingdom. Many researchers reported SPs from marine seaweeds and have studied their pharmacological activities, and some of these sulphate polysaccharides have been developed as new drugs for antitumor, antivirus, anticoagulant, and antihyperlipidemia treatments (Sigamani et al. 2016). Thus, some of freshwater algae are producers of SPs (Table 3).
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Table 2 Sources of sulphated polysaccharides from different marine algae (macroalgae) Algae group Red algae
Brown algae
Species Acanthophora spicifera Achanthophora muscoides Agardhiella ramosissima Ahnfeltiopsis flabelliformis Chondrus crispus Corallina officinalis Gloiopeltis furcate Gracilaria caudata Gracilaria cornea Gracilaria corticate Gracilaria cervicornis Gracilaria debilis Gracilaria rubra Gracilariopsis lemaneiformis Grateloupia filicina Grateloupia indica Grateloupia longifolia Grateloupia livida Hypnea musciformis Jania rubens Laurencia obtusa Laurencia papillosa Lithothamnion muelleri Mastocarpus stellatus Nemalion helminthoides Porphyra haitanensis Porphyra yezoensis Pterocladia capillacea Schizymenia dubyi Solieria filiformis Adenocystis utricularis Ascophyllum nodosum Cystoseria barbata Cystoseira compressa Dictyopteris divaricata Dictyota cervicornis Dicty-ota mertensii Ecklonia cava Ecklonia kurome Fucus evanescens Fucus vesiculosus Hizikia fusiforme Kjellmaniella crassifolia (continued)
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Table 2 (continued) Algae group
Green algae
Species Laminaria japonica Lessonia vadosa Lobophora variegata Padina tetrastromatica Sargassum binderi Sargassum cristaefolium Sargassum duplicatum Sargassum fulvellum Sargassum fusiforme Sargassum hemiphyllum Sargassum honeri Sargassum pallidum Sargassum vulgare Spatoglossum schroederi Turbinaria conoides Turbinaria turbinata Undaria pinnatifida Capsosiphon fulvescens Caulerpa racemosa Codium cylindricum Codium divaricatum Codium fragile Codium pugniformis Enteromorpha intestinalis Enteromorpha prolifera Monostroma angicava Monostroma latissimum Monostroma oxysperma Ulva armoricana Ulva conglobata Ulva fasciata Ulva lactula Ulva rigida Undaria pinnatifida
Adapted from Silva et al. (2012), Ghazali et al. (2017), Manlusoc et al. (2019)
2.2.2 Marine Microalgae Most of the marine microalgae have been of great interest because they were shown to produce a huge variety of bioactive compounds with biotechnological potential, particularly in the biomedical, pharmaceutical, nutraceutical, and cosmetic areas. Sulphated polysaccharides derived from microalgae have shown to possess a variety of bioactivities with potential applications in the pharmaceutical field, but also in the
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Table 3 Sources of sulphated polysaccharides from freshwater algae (Microalgae)
Algae group Green algae
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Species Chlamydomonas debaryana Chlorella stigmatophora Chlorella vulgaris Chlorella variabilis Chlorella sp. Haematococcus pluvialis Tetraselmis sp.
Adapted from Sigamani et al. (2016) Table 4 Sources of sulphated polysaccharides from different marine algae (Microalgae)
Algae group Red algae
Green algae
Diatom
Species Ahnfeltia tobuchiensis Cochlodinium polykrikoides Gyrodinium impudicum strain KG03 Gyrodinium impudicum Porphyridium cruentum Porphyridium purpureum Porphyridium sp. Rhodella reticulata Isochrysis galbana Navicula directa Navicula sp. Tetraselmis suecica Phaeodactylum tricornutum
Adapted from Talero et al. (2015)
food and cosmetic industries (Muhamad et al. 2019). SPs found in marine microalgae are, for example, red and green algae (Table 4).
2.2.3 Cyanobacteria (Blue-Green Algae) Cyanobacteria or blue-green algae (Table 5) are an ancient and diverse group of photosynthetic microorganisms, and live in different environments, being known to produce extracellular and intracellular secondary metabolites. Many of secondary metabolites from cyanobacteria have been reported to present biological activities, for example, antibacterial, antifungal, algaecide, immunosuppressive, and antiviral (Jesus et al. 2015). Cyanobacteria also constitute an important source of SPs. Thus, some of cyanobacteria are producers of SPs (Table 5).
2.3
Other Sulphated Polysaccharides Produced by Animals, Plants, or Other Microorganisms
Sulphated polysaccharides have been reported mostly from marine algae sources, such as microalgae and macroalgae. Actually, SPs may be isolated not only from
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Table 5 Sources of sulphated polysaccharides from cyanobacteria (blue-green algae) Algae group Blue-green algae
Species Anabaena sp. Aphanocapsa sp. Aphanothece halophytica Arthrospira platensis (formely Spirulina platensis) Cyanothece sp. Gloeothece sp. Nostoc sp. Nostoc flagelliforme Phormidium sp. Synechocystis sp.
Adapted from Raposo et al. (2013)
marine algae, but also from other organisms such as bacteria, fungi, plants, and animals, for example, sulphated polysaccharide from fungi, such as Antrodia cinnamomea, Agaricus blazei, Hypsizigus marmoreus, and Poria cocos (Cheng et al. 2012). In addition, four SPs have been identified from plants, including two species of mangrove, Avicennia schaueriana and Rhizophora mangle, and three species of marine angiosperms, Ruppia maritima, Halophila decipiens, and Halodule wrightii (Aquino et al. 2011). Later on, SPs have been reported in three freshwater plants, Eichhornia crassipes, Hydrocotyle bonariensis, and Nymphaea ampla; moreover, SPs have also been isolated from Gossipium hirsutum L (cotton) and Helianthus tuberosus (sunroot), Eleocharis dulcis (water chestnut), Psyllium ovata (blond plantain), and Moringa oleifera (drumstick) (Mukherjee et al. 2019). Animals also are natural sources of SPs, such as three species of sea urchins, Echinometra lucunter, Arbacia lixula, and Lytechinus variegatus, Meretrix petechialis (marine clam), Haliotis discus hannai Ino (abalone shell), and Solen marginatus (grooved razor shell) (Souissi et al. 2019). Unfortunately, there is scarce information available in the literature about the structural elucidation of the SPs isolated from different organisms in order to allow highlighting the differences.
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Bioactivity of Sulphated Polysaccharides from Algae. Relation with Chemical Features of Their Structures and Mechanisms of Action
3.1
Polysaccharides from Marine Algae
Polysaccharides are natural polymers, which consist of a single or several types of monosaccharides (Cunha et al. 2009). Sulphated polysaccharides comprise a complex group of macromolecules, whose specific structures are strongly related to a wide range of important biological properties. Thus, these compounds have gained
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prominence due to their great applicability, mainly in the areas of foods, biomedical, pharmaceutical, and cosmetics (Lee et al. 2012; Muhamad et al. 2019). Despite the various sources of polysaccharides, those with industrial applications are essentially extracted from plants (including algae), animals, and fungi, or are obtained via microbiological fermentation (Cunha et al. 2009). SPs from algae are rich sources to obtain various types of polysaccharides and are known as one of the most promising substances for drug administration. This is due to the various low-cost sources, their relative abundance, biocompatibility, biodegradation, unique chemical composition, low toxicity, and low immunogenicity. Thus, SPs obtained from algae have emerged as an important class of natural biopolymers with potential pharmacological applications (Lee et al. 2012). In general, aquatic and marine algae have been of great interest as excellent sources of nutrients. However, polysaccharides are the main components of algae, although they can vary significantly in seaweed (4–76% w/w) and microalgae (8–64% w/w), according to the species and time of harvest. Therefore, much attention was paid to the isolation and characterization of seaweed polysaccharides due to their number of health benefits (Muhamad et al. 2019). Algae polysaccharides are important sources of raw materials used in food, pharmaceutical, and nutraceuticals industries. In the food industry, the search is for a gelling agent. In the pharmaceutical area, the following biological activities are relevant: antiviral, antithrombotic, anticoagulants, antioxidants, antitumor and antithrombotic agents, and other activities. As algae are routinely harvested in large quantities to be used as food, they become a cheap resource worldwide. Historically, as algae have been consumed for centuries without any identifiable toxicity, they have also gained interest at other industrial levels (Patil et al. 2018). Thus, there has been a focus on red and brown algae for the extraction of polysaccharides of interest (Cunha et al. 2009). However, it is important to note that the polysaccharides from seaweed exhibit structural variations depending on the species, geographic origin, time of harvest, extraction method, processing conditions, and environmental conditions. Structural variations can be in the form of position and degree of sulphation and/or composition of monosaccharides and percentage of uronic acid (Muhamad et al. 2019). In general, there is a high degree of heterogeneity in size, sulphation, and branching in many polysaccharides used in natural sources. Thus, marine-based polysaccharides have a wide variety of structures that are still underexplored and should, therefore, be considered an extraordinary source of chemical diversity for many applications.
3.1.1 Green Algae Versus Red Algae The sulphated polysaccharides of green algae are by far the least studied in terms of biological activities. These compounds present more heterogeneity, which makes it difficult to understand the relationship of the polysaccharide with its biological activity (Costa 2008). In addition, green algae remain a poorly explored biomass, particularly in areas where other polysaccharides of algal origin have already proved their value, such as the impressive commercial success of carrageenan (Silva et al. 2012). Although the number of works carried out with these polysaccharides is still minimalist, these
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compounds have shown pharmacological importance since they present interesting biological activities, as can be seen in Table 6. The SPs of green algae are rich in galactose, mannose, xylose, arabinose, and/or uronic acids, but there are homopolysaccharides, such as arabinans and galactans. In recent years, the number of studies on SPs of green algae has increased, and several of these studies have reported their molecular structures, which may facilitate the elucidation of the mechanisms of action of these compounds, as well as the activity/structure relationship. For example, ulvan, a sulphated polysaccharide extracted from the green algae Ulva lactuta, had low antioxidant power; however, when modified by the addition of benzoyl and acetyl groups, it started to show excellent antioxidant activity, mainly for the superoxide radicals, which was even higher than that observed for vitamin C (Lahaye and Robic 2007).
Table 6 Algae sulphate polysaccharides and their biological activity Types of polysaccharides HWE Arabinogalactans
Ulvan
Ulvan S-rhamnan Heterorahmnans Carrageenans
Agarans
Hybrid galactans Fucans
Species Caulerpa recemosa Codium fragile Codium vermilara Codium isthmocladum Codium cillindricum Codium latum Ulva pertusa Ulva lactuta Ulva rígida Ulva reticulata Monostroma sp Gayralia oxysperma Maristella gelidium Hypnea musciformis Gymnogongrus griffthsiae Botryocladia occidentais Gracilaria birdiae Gracilaria domingensis Gracilaria cornea Porphyra spiralis Bostrychia montagnei Acanthophora spicifera Cryptonemia crenulata Dyctyota menstrualis Dyctyota mertensii Padina gymnospora Spatoglossum schröederi Sargassum stenophyllum Sargassum wightii
Adapted from Costa (2008), Cunha et al. (2009)
Biological activities Antiviral Anticoagulant Anticoagulant Anticoagulant Antiangiogenic Antiviral Antihyperlipidemic Antitumor, antiviral Immunomodulatory Antihepatotoxic Anticoagulant, antiviral Antiviral: Herpes Antiviral: Dengue and herpes Antiviral: Dengue and herpes Antiviral: Dengue and herpes Antiviral: Dengue and herpes
Antiviral: Herpes Antiviral: Herpes Antiviral: Dengue and herpes
Anticoagulant, antithrombotic Antiviral: Herpes, antitumor Antimicrobial
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On the other hand, in red algae (Rhodophyta) the most characteristic SPs in their composition are the sulphated galactans, which are subdivided into carrageenans and agarans. Carrageenans are found, essentially, in the species Soliera, Eucheuma, Maristiella, and Callophucis, while the agarans in the species Gracilaria, Gelidium, and Pterocladias (Cunha et al. 2009). There are several reports in the literature on pharmacological/biological activities attributed to red algae, one of the most studied is the antiviral activity. All of these studies demonstrate that polysaccharides extracted from Rhodophyta (red algae), essentially galactans, exhibit antiviral activity against a broad spectrum of viruses, including human pathogens, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV), and cytomegalovirus (CMV), in which the modes of action of these compounds are generally attributed to blocking some early stages of the virus replication cycle (Matsuhiro et al. 2005). However, other types of polysaccharides can also be found in these algae, as we can see in Table 6. Phaeophyceas, a class of brown algae, have in their constitution a single group of SPs: fucans. These polymers are characterized by the presence of sulphated L-fucose in their structure and can be found in the form of homo- or heteropolysaccharides (Costa 2008). Fucans are assigned a wide range of pharmacological activities. In addition to those previously reported, many other biological activities are shown in the following table (Table 7).
3.2
Bioactivities of Sulphated Polysaccharides from Algae
Polysaccharide bioactivities are closely correlated with their chemical properties, such as molecular sizes, molecular weight, rheological properties, types and proportions of constituent monosaccharides, and characteristics of glycosidic bonds. Algae-derived polysaccharides with clearly elucidated compositions/structures identified cellular activities and desirable physical properties have shown the potential that can create new opportunities to exploit them to the fullest to serve as therapeutic tools for biomedical applications, such as immunoregulatory agents or pharmaceutical delivery vehicles. Thus, a basic understanding of the physicochemical properties and biological activities of algal polysaccharides is essential for successful application and will help to access their multifunctional applications (Lee et al. 2012). The therapeutic potential of natural bioactive compounds, such as polysaccharides, combined with natural biodiversity will allow the development of a new generation of therapies. In particular, algal polysaccharides have real potential for the natural discovery of therapeutic applications, of which sulphated fucans and sulphated galactans are the most studied (Muhamad et al. 2019).
3.2.1 Antiviral, Antibacterial, and Antifungal Activities Sulphated polysaccharides, in particular carrageenan, fucoidan, and ulvan, have shown to be of great interest, due to their ability to act as antimicrobial agents against human pathogens (Pierre et al. 2011; Jun et al. 2018). Antiviral activity is probably the most studied activity exhibited by SPs from marine microalgae. Its
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Table 7 Pharmacological activities associated with fucans obtained from algae Biological activities Angiogenic Anticomplement
Anti-immigration Antiadhesive
Anticoagulant
Antioxidant Antiproliferative Antithrombotic Antitumor
Anti-ulcer Antiviral Antimetastatic Cell-cell binding block via selectin Sperm-epithelium binding block in the oviduct Fibrinolytic
Algae Fucus vesiculosus Ascophyllum nodosum Fucus evanescens Laminaria cichorioide Laminaria japonica Ascophyllum nodosum Fucus vesiculosus Ascophyllum nodosum Laminaria brasiliensis Sargassum schröederi Sargassum stenophyllum Ascophyllum nodosum Dictyota menstrualis Eisenia bicyclis Ecklonia kurome Padina gmynospora Fucus vesiculosus Laminaria japónica Ascophyllum nodosum Ascophyllum nodosum Ascophyllum nodosum Eisenia bicyclis Graffenrieda caudata Sargassum thumbergii Cladosiphon okamuranus Cystoseira indica Sargassum horneri Fucus vesiculosus Fucus vesiculosus Fucus vesiculosus Ecklonia kurome Fucus vesiculosus
Adapted from Costa (2008)
inhibitory effects on viral replication have been reported for more than six decades. Polysaccharides and SPs have shown viral activity against a wide variety of viruses involved, including human papilloma virus (HPV), myocarditis encephaloma virus, hepatitis virus (types A and B), dengue virus and yellow fever, and the human immunodeficiency virus (HIV) (type 1 and 2) (Wijesinghe and Jeon 2011; Muhamad et al. 2019). The action of inhibiting the sulphated polysaccharide involves blocking the virus from binding to the surface of the host cell, inhibiting virus-induced syncytium formation or even inhibiting the replication of the virus involved, such as HIV, human cytomegalovirus (HCMV), and the respiratory syncytial virus (RSV), inhibiting, therefore, virus adsorption or virus entry into host cells (Wijesinghe and Jeon 2011). Some SPs are effective only if applied simultaneously to the virus or
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immediately after infection. In general, and as mentioned in the previous paragraph, in relation to HIV, SPs act preventing the replication of the virus, even at low temperatures, whereas antiviral activity increases with the level of sulphation and the molecular weight (MW). Anti-HIV activity has also been reported for several algae, such as Schizymenia pacifica, Eucheuma cottoni, and Nothogenia fastigiate. A more detailed study demonstrated that the antiviral activity of sulphated galactans from Chondrus ocellatus was related to MW, those of smaller size presenting higher activity. However, further studies are needed to prove this theory (Zhou et al. 2004). An article published by Matsuhiro et al. reported antiviral activity of a sulphated galactan isolated from Spirulina binderi. This compound was highly selective against herpes simplex virus types 1 and 2, with selectivity indexes (cytotoxicity/antiviral activity) higher than 1000 for all tested virus strains (Matsuhiro et al. 2005). In addition, no cytotoxic effects were observed when cell viability was assessed. The authors thought these results regarding the high selectivity of the compound were directly related to its unusual sulphation pattern. Bouhlal et al. evaluated the antiviral activity of SPs isolated from two species of red algae: Sphaerococcus coronopifolius and Boergeseniella thuyoides. The results were positive regarding the inhibition of the replication of the HSV-1 and HIV-1 viruses in vitro at concentrations that have no effect on cell viability. The HSV-1 adsorption stage in the host cell appears to be the specific target of the polysaccharide action. While for HIV-1, these results suggest a direct inhibitory effect on HIV-1 replication, controlling the appearance of new generations of viruses and having a potential virucidal effect (Bouhlal et al. 2011). Also, Gomaa and Elshoubaky developed a study on the antiviral activity of algal polysaccharides. This study showed that the natural product carrageenan, a polysaccharide from red algae Acanthophora specifira and brown algae Hydroclathrus clathratus, could inhibit the herpes simplex virus (HSV-1) and the rift valley fever virus (RVFV), and presented low cytotoxicity (Gomaa and Elshoubaky 2016). Certain SPs from marine algae are also known for their antimicrobial activities against human bacterial pathogens, in addition to their physiological benefits. Thus, the antimicrobial and antibiofilm activities of SPs from different marine algae have been evaluated in several studies (Jun et al. 2018). For example, Pierre et al. tested, in vitro, the antimicrobial activity of green marine algae Chaetomorpha aerial. The polysaccharide isolated from this alga, composed of 18% arabinose, 24% glucose, and 58% galactose, exhibited selective antibacterial activity against three gram-positive bacteria, including Staphylococcus aureus (ATCC 25923) (Pierre et al. 2011). In another research, Berri et al. reported the study of an aqueous extract of marine sulphated polysaccharide, prepared from the green macroalgae Ulva armoricana, regarding the antibacterial activity against 42 bacterial strains and isolates found in animals. The results show that the growth of gram-positive and gram-negative bacteria was affected, the most susceptible pathogens being Pasteurella multocida, Mannheimia haemolytica, Erysipelothrix rhusiopathiae, Staphylococcus aureus, and Streptococcus suis, with the minimum inhibitory concentration ranging from 0.16 to 6.25 mg mL 1 (Berri et al. 2016). Also, Jun et al. reported that among several sulphated polysaccharide obtained from seaweed, the fucoidan isolated from Fucus vesiculosus showed remarkable antimicrobial activity against dental plaque bacteria,
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as well as some foodborne pathogenic bacteria. The minimum inhibitory concentrations were from 125 to 1000 μg mL 1. Regarding the antibiofilm activity, the fucoidan at concentrations above 250 μg mL 1 completely suppressed the biofilm formations and planktonic cell growths of Streptococcus mutans and S. sobrinus. However, no eliminative effect on the completed biofilm was observed (Jun et al. 2018). Regarding the antifungal activity, studies on SPs are scarce and their activity against human pathogens is poorly documented.
3.2.2
Antiproliferative, Tumor Suppressor, Apoptotic, and Cytotoxicity Activities There has been an increasing demand for natural therapies with bioactive compounds against cancer, since the side effects of synthetic chemicals and other types of treatment against tumor are sometimes very aggressive for the patient (Raposo et al. 2013). There are some works in the literature that have shown SPs from marine algae to present antitumor activities. These sulphated compounds can directly affect tumor cells by acting through the inhibition of metastasis and/or the proliferation of tumor cells by binding to growth factors and cell adhesion molecules. On the other hand, other sulphated polysaccharide can induce apoptosis and differentiation of tumor cells, although the mechanism is uncertain. Thus, the antitumor mechanism of sulphated polysaccharide can, in part, be explained by the direct effect on tumor biology (Manlusoc et al. 2019). In addition to all the activities mentioned so far in relation to sulphated fucoidans, these compounds may also exhibit antiproliferative activity. For fucans, the blocking of the cell cycle preventing the proliferation of tumor cells and the activation of different apoptosis pathways, especially via caspases seem to be the main antitumor mechanisms. However, other studies show fucans acting through different mechanisms, in particular by potentiating NK cells and increasing the production of IFN (interferon gamma) in T cells or preventing cell adhesion to the extracellular matrix (Maruyama et al. 2006). Another study, carried out by Zhou et al., highlights the antitumor activity of galactans, but, this time, isolated from the seaweed Chondrus ocellatus. The tests were performed in vivo on the S-180 sarcoma and H-22 hepatoma strains. The results demonstrate a positive correlation between the tumor inhibition and an increased activity of NK (Natural Killer) cells and lymphocyte proliferation (Zhou et al. 2004). 3.2.3 Anticoagulant and Antithrombotic Activities Polysaccharides derived from algae may act as an anticoagulant, as they can inhibit thrombin, activate antithrombin III, or prolong the duration of intrinsic (via activation of contact of all components present in the blood) and extrinsic (via the tissue factor TF) pathways. However, some research attested that the molecules were involved in the prothrombin pathway, resulting in effect only in the intrinsic pathway. On the other hand, SPs play a more important role, as they contain sulphate, in the processes of coagulation and/or platelet aggregation. Anticoagulant activity depends on the type of polysaccharides, for example, sulphated galactans depend
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on the nature of the sugar residue, the sulphating position and the sulphating content (Ngo and Kim 2013). Red algae polysaccharides are by far the most studied for anticoagulant potential, since 1941, when this potential was discovered in extracts of algae of the genus Laminaria (Costa 2008). SPs from algae, as well as those obtained from invertebrate animals, have been investigated as anticoagulant and antithrombotic agents, due to the structural similarity with heparin (Cunha et al. 2009). Although heparin is a potent anticoagulant widely used in the clinic area, it has some limitations, which include pharmacokinetic varieties, residual hemorrhagic effect, thrombocytopenia, and inhibition of platelet function, among others. These limitations have led to the development of new anticoagulant agents, and among the alternative sources we have the SPs of marine algae, such as fucans (Lee et al. 2012). Studies carried out on seaweed present their SPs as one of the main groups of compounds with anticoagulant/antithrombotic potential. Thus, these compounds are seen as promising drugs for the replacement of heparin, mainly due to the high structural diversity, which provides the possibility of presenting a different mechanism of action than heparin, but also because they reduce the possibility of contamination by viral particles. These polysaccharides are prone to form soluble complexes with plasma proteins, such as fibrinogen, and their mechanism of action occurs mainly through thrombin inhibition. For example, SPs isolated from the Rhodophyta B. occidentalis and G. crinale showed different biological effects, which are depending of their structural variations. Such structural variations produced anti- and procoagulant actions, as well as anti- and prothrombotic (Fonseca et al. 2008). Due to their high sulphate content, SPs, in particular carrageenan, also show interest in coagulation processes. Patil et al. presented the hypothesis that highly sulphated groups of carrageenan could act as heparin sulphate and be a potent anticoagulant. However, they failed to find a conclusive link between carrageenan and coagulation. Carrageenan was suggested to have an antithrombin effect and disrupted platelet aggregation (Patil et al. 2018). Fucoidans exhibit anticoagulant activity that is largely dependent on the seaweed from which they are extracted. For most fucoidans, increases in aPTT and thromboplastin time (TT) were observed on a scale comparable or superior to heparin; many fucans can also act indirectly in altering the coagulation process, such as promoting the release of TFPI (tissue factor pathway inhibitor) or heparan sulphate, a sulphated glycosaminoglycan produced by endothelial cells that has antithrombotic action. Therefore, these compounds have the potential to be a natural alternative to heparin Also, rhamnan sulphate, an equally important but less studied compound than other marine polysaccharides, has shown some anticoagulant properties. A low MW form has shown to have higher anticoagulant activity than heparin at high concentrations, as determined by the aPTT test (Wang et al. 2012; Manlusoc et al. 2019). Although green algae polysaccharides are the least explored, there are reports in the literature on their anticoagulant activity. The first report was by Deacon-Smith, who worked with 45 species of Codium and found that the polysaccharides of these
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algae, mostly galactans, showed anticoagulant activity (Costa 2008). In general, these algae synthesize homo- and sulphated heteropolymers rich in galactose, arabinose, mannose, or glucose. However, the presence of these monosaccharides was detected in several ketone fractions, which indicates that the alga does not synthesize a single type of polysaccharide formed by galactose or arabinose, but two families of sulphated galactans and of sulphated arabinans.
3.2.4 Antilipidemic Activities Sulphated polysaccharides from seaweeds are potent inhibitors of human pancreatic cholesterol esterase, an enzyme that promotes its absorption at the intestinal level; this inhibitory effect is enhanced by higher MW and degree of sulphation. Qi and Sun conducted an in vivo study on a sulphated polysaccharide derivative obtained from the species Ulva pertusa (Chlorophyta) in order to assess its antioxidant activity (it will be described in the Sect. 3.2.6) (Qi and Sun 2015). The study carried out on the liver of hyperlipidemic rats allowed to conclude that this SP can be used as an antihyperlipidemia agent. 3.2.5 Anti-Inflammatory and Immunomodulatory Activities Inflammation is part of the complex biological response of the immune system to harmful stimuli, such as pathogens, damaged cells, and toxic compounds. Algal polysaccharides can reduce the pro-inflammatory state or other harmful conditions, such as allergic reactions to this activity during the intrinsic immune response. The immunomodulatory properties of algal polysaccharides can make the immune system to function better and can be strengthened in the right way to respond to certain infections. These properties can be demonstrated in several ways depending on the polysaccharide, its source, and type as well as the inflammation site (Muhamad et al. 2019). Fucoidans are a complex heterogeneous group of SPs that have strong antiinflammatory effects. Fucoidan has been established as an inhibitor of L-selectin, which mediates leukocyte rolling and neutrophil adhesion to inflamed endothelial cells, and it was used as a blocking agent that could reduce inflammation. Recently, Fernando et al. carried out a study on fucoidans from two species of brown algae, Chnoospora minimal (CMF) and Sargassum polycystum (SPF). Both species showed good dose-dependent anti-inflammatory effects, reducing the levels of the nitric oxide (NO) inflammation mediator. In addition, the corresponding increase in the viability of the cells tested meant that these fucoidans had protective effects against inflammation induced by lipopolysaccharides (LPS). The reduction in NO levels was related to the reduced production or activity of iNOS. In addition, fucoidan samples regulated the dose of PGE2, thus revealing its anti-inflammatory effects as inhibitors of prostaglandin production, which was related to the reduction of COX-2 levels. The ability to downregulate pro-inflammatory cytokines TNF-α, IL1β, and IL6 was another interesting feature of these compounds. These observations reflected that both CMF and SPF had a wide range of anti-inflammatory effects (Fernando et al. 2018).
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Many animal models submitted to anti-inflammatory treatments are centered on the ability of carrageenan to induce local inflammation, playing the role of a proinflammatory agent. This is normally associated with the number of sulphate groups. However, unlike other modes of inflammation, histamine and 5-hydroxytryptamine do not participate in inflammation induced by carrageenan. Purified polysaccharides extracted from an edible green alga, Caulerpa lentillifera, were evaluated in vitro for their anti-inflammatory activity. One of the tested compounds had a better anti-inflammatory effect, which might probably be related to the presence of sulphate groups; another study carried out on polysaccharides obtained from this algae species identified a new type of xylogalactomannans, which differ in MW, composition of monosaccharides, and in the content of uronic acids and sulphate groups leading to various activities, including immunomodulatory activity. The in vitro evaluation of immunostimulatory activity revealed that all fractions significantly stimulated macrophages, improving phagocytosis, NO production, and acid phosphatase activity in macrophages (Sun et al. 2020). In addition, Pérez-Recalde et al. tested the SPs extracted from the red algae Nemalion helminthoide to determine, in vitro and in vivo, their immunomodulatory activities. Two fractions of xylomanan induced in vitro proliferation of macrophages from the murine cell line RAW 264.7 and significantly stimulated the production of NO and cytokines (IL-6 and TNF-α) in the same cells. On the other hand, the fraction of mannan did not exhibit this effect. In tests carried out in vivo, xylomanans were also shown to be immunomodulatory agents of interest (Pérez-Recalde et al. 2014).
3.2.6 Antioxidant Activities and Sequestration of Free Radicals Recently, studies on polysaccharides of marine origin have shown that these polymers play an important role in the sequestration of free radicals responsible for a wide range of disorders (Fig. 1), including cardiovascular diseases, ischemia, and Alzheimer’s disease, in addition to being directly involved in inflammation, aging, and cancer formation. The main mechanism of antioxidant action of algal sulphated polysaccharide consists in the elimination or in the inhibition of appearance of free radicals (free radicals of superoxide, hydroxyl, 1,1-diphenyl-2-picryl-hydrazil (DPPH)). In addition, they have total antioxidant capacity and a strong capacity as reducing agents and ferrous chelators (Wang et al. 2014). Ulvans are water-soluble sulfated heteropolysaccharides obtained from the cell walls of green marine macroalgae of genus Ulva; this natural polymer can be chemically modified, an example of which is the addition of functional groups to promote its antioxidant abilities: acetylated and benzoylated ulvan exhibited stronger antioxidant effects compared to natural ulvan. High sulphate content and ulvan MW led to an increased antioxidant activity. The sulphate content appears to be especially important in the antioxidant effect of ulvan. Also, Qi and Sun concluded that, due to the antioxidant activity, a polysaccharide derivative with a high sulphated content obtained from the species U. pertusa (Chlorophyta) was effective in protecting the liver tissue of hyperlipidemic rats (Qi and Sun 2015).
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Fig. 1 Dysfunctions associated with free radicals. (Adapted from Costa 2008)
Antiaging Activity The collagen and elastin fibers produced by fibroblasts are two of the main components of fibrous connective tissues that maintain the smooth and healthy appearance of human skin. The degradation of these fibers is mainly mediated by matrix metalloproteases (MMPs), which include the enzymes collagenase, serine protease, and elastase (Fig. 2). Thus, assessing the inhibition of these enzymes is a general approach for assessing the anti-wrinkle potential of a substance. Fucoidan, one of the most known SPs, is an effective inhibitor of collagenase and elastase. For example, Wijesinghe and Jeon reported that the incorporation of fucoidans in cosmetic preparations showed improvements of the skin or in the cosmetic action, preventing and relieving skin aging conditions, such as freckles, wrinkles, and diseases (Wijesinghe and Jeon 2011). Fucoidan can effectively deregulate matrix metalloproteases (MMPs) involved in the degradation of connective tissues by activating MMP inhibitors. In addition, fucoidans exert mixed reversible
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Fig. 2 Schematic example of one extrinsic factor, the UV radiation, that promotes aging. It penetrates into epidermal tissues, causing exfoliation of the dermal matrix. Degradation is caused by the activation of cytokines and enzymes collagenase and elastase. These enzymes further accelerate the generation of ROS, which in turn generate DNA damage. The combined effect of UVA and UVB leads to ROS generation, which directly activate MMPs, following multiple cascade pathways transcribing pro-inflammatory and/or proapoptotic genes. (Adapted from Thomas and Kim 2013)
(competitive and noncompetitive) inhibitory actions on tyrosinosis, which is a critical enzyme mediator in melanin biosynthesis. The antimelanogenic activity will be discussed in Sect. 3.2.7. Wang et al. evaluated the antiaging activity by comparing the absorption and moisture retention properties of polysaccharide extracts from five different species of seaweed. With this study they concluded that the polysaccharides extracted from
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brown seaweed (more precisely the fucoidan obtained from Saccharina japonica) exhibited the best capacity for absorption and moisture retention, while the green ones showed worse results. This ability of polysaccharides was influenced by their sulphated content, MW (chain length), and type of algae from which they are extracted (Wang et al. 2014). Also, Fernando et al. concluded that SPs from brown algae had antioxidant and anti-inflammatory properties and were capable of inhibiting collagenase and elastase, showing potential as anti-wrinkle cosmeceutical ingredients (Fernando et al. 2018).
3.2.7 Other Biological Activities Other pharmacologic/biological activities attributed to the presence of sulphated polysaccharides in marine algae have been documented, especially in vivo tested. Wang et al. described fucoidan tyrosinase inhibiting activity as being of a reversible mixed type, and in silico studies have revealed that fucoidan pentameric and hexameric units have the best binding interactions with the localized copper ion tyrosinase active site. Thus, once the activity of the tyrosinase enzyme is inhibited, there is a greater control of the early reactions of the melanin synthesis pathway, preventing the appearance of spots and/or the darkening of the skin (Wang et al. 2012). Fernando et al. also developed some studies, which showed that fucoidans isolated from brown algae, Chnoospora minima and Sargassum polycystum, exert a direct inhibitory effect on tyrosinase, as well as the inhibition of tyrosinase during the synthesis of melanin in keratinocytes (Fernando et al. 2018). Thus, these compounds are promising to be incorporated as skin-lightening agents in cosmeceuticals due to their biocompatible properties. Among the different sources of polysaccharides, algal polysaccharides, such as fucoidans and, especially, their low MW derivatives, may play an important role in the future development of cell therapy and regenerative medicine. Thus, the desirable physical properties of polysaccharides derived from algae can create new opportunities that can serve as therapeutic tools for biomedical applications, such as immunoregulatory agents or medication delivery vehicles (Muhamad et al. 2019).
3.3
Case Study: Atherosclerosis
It was the year 1970 that boosted the interest in polysaccharides derived from seaweed as lipid-lowering drugs and inhibitors of the formation of atherosclerotic plaques. Currently, there is a great clinical need for therapies that can reduce the incidence and progression of atherosclerotic vascular disease beyond what is already possible with current treatments. Polysaccharides are, thus, seen as attractive therapies for atherosclerosis due to their low cost, availability, and low toxicity. SPs have potential against risk factors for atherosclerosis, including lipid reduction, coagulation, oxidative stress, inflammation, and modulation of the microbiome. In particular, fucoidan is a polysaccharide of great interest in this area. A potential mechanism of action for fucoidan activity in atherosclerosis is modified lipid uptake and metabolism, where levels of total cholesterol, triglycerides, and low-density
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lipoprotein (LDL) were significantly reduced, while levels of high-density lipoprotein (HDL) suffered an increase. On the other hand, it has also been shown that fucoidan altered signaling through ROS. As ROS can modify LDL and LOX-1 is regulated under pro-inflammatory conditions, its decreased expression under treatment with fucoidan suggested that this marine polysaccharide reduced oxidative stress in atherosclerosis. Although the reduction of oxidative stress and inflammatory markers in the aorta might be one of the side effects of hepatic lipid metabolism, ROS was known to cause an interruption in the liver homeostasis. However, there was no consensus on the mechanism of action. Nevertheless, due to the additional effect of fucoidan on dyslipidemia, the liver was identified as a possible target for research focused on hepatic lipid metabolism. This suggested that fucoidan modified the development of atherosclerotic plaque by increasing lipid metabolism and decreasing lipid synthesis and absorption. With respect to carrageenan, it is considered to be a complex candidate for the treatment of atherosclerosis, including against the formation of atherosclerotic plaques (Patil et al. 2018).
4
Potential Medical/Biomedical Applications of Sulphated Polysaccharides from Marine Algae
The materials found in the marine environment are of great interest, since its chemical and biological diversity is unique. The biomedical field is constantly looking for new biomaterials, with innovative properties. Thus, natural polymers appear as the materials of choice for this purpose due to their biocompatibility and biodegradability. In particular, SPs have received increasing attention for their applicability in healthrelated fields (Silva et al. 2012). SPs may be isolated from plants, animals, and microbial organisms. Marine algae (red, brown, and green) are rich sources of SPs and they may display many health beneficial nutraceutical effects, such as anticancer, antioxidant, antitumor, anti-allergic, immunity enhancement, anticoagulant, antiviral, antihyperlipidemic, and antihepatotoxic activities. Therefore, SPs are a multipurpose group of materials widely explored to develop biomaterials for fulfilling various biomedical applications, such as drug delivery, tissue engineering scaffolds, wound healing materials, and anticancer agents (Ngo and Kim 2013). Thus, in marine algae, the presence of sulphate radicals and their positions in the chemical structure have been prerequisites for these macromolecules to exhibit important biological activities of interest in biomedicine.
4.1
Tissue Engineering
As a response to trauma or tissue disease, the human body responds in order to remodel the injured tissue. However, when these efforts are not achieved the result starts in a dysfunctionality and may culminate in tissue failure. To solve this problem, science has focused on developing new alternative therapeutic solutions to overcome the drawbacks of current clinical practices. Thus, tissue engineering
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emerges as a multidisciplinary approach that uses the principles of engineering and life sciences to create a new tissue that will be implanted at the patient’s injury site. Thus, it is necessary that certain properties, especially biocompatibility, both in the implanted form and after degradation, are present throughout the process and that it occurs in a controlled way. Regenerative medicine has brought new hope to the treatment of numerous diseases and conditions. In this context, several techniques and a wide range of materials have been proposed, including natural and synthetic polymers, or a combination of both. It should be noted that the degree of sulphation, MW, and structural composition may influence cellular behavior, with changes being observed in different physiological but also pathological processes. These are important aspects to be considered in the process of tissue regeneration using polymers as alternative therapy material. Due to the enormous potential of sulphate groups, scientific communities have been looking for alternative ways to obtain them. These polysaccharides can be obtained from marine organisms, in particular, macroalgae (Jesus et al. 2015). As an underexploited resource, marine environments are an extraordinary source of biomaterials and, in particular, SPs. In addition to their biological activity and potential pharmaceutical use, SPs from green algae can also be used for biomedical applications, in areas as demanding as regenerative medicine. In this sense, several researches have already been carried out related to the processing and development of biomaterials as well as work inherent to the modification of polysaccharides, mainly using ulvan as starting material (Silva et al. 2012). Fucoidan is one of the most studied SPs in terms of tissue engineering. This compound, generally extracted from brown seaweed, in combination with different polymers, such as chitosan or polycaprolactone (PCL), and processed in hydrogels, scaffolding films and nanofibers, has an enormous potential for cell support systems. An example of this is a study by Sezer et al. who propose the use of a fucoidanchitosan hydrogel as a fast healing agent. The use of chitosan is, thus, proposed due to its hydrogel-forming properties and advantageous use in applications as dressings, adding to the excellent anticoagulant activity of fucoidan, among other properties (Sezer et al. 2008). Another study addresses a rapid prototyping methodology resulting in a mixture of fucoidan with polyesters (PCL). This procedure resulted in a porous structure suitable for bone tissue regeneration, where low MW fucoidan promoted cell proliferation in addition to osteoconductive properties, including alkaline phosphatase activity, type I collagen expression, and deposition minerals (Lee et al. 2012). However, not all polysaccharides are suitable for tissue engineering, mainly due to their gelatinous consistency and insufficient mechanical properties. Thus, and as mentioned in this topic, SPs are generally combined with other natural or synthetic polymers, or reinforced with inorganic substances.
4.2
Drug Delivery
Sulphated polysaccharides for drug delivery substances are usually not administered as they are in their pure state, but rather as part of a formulation where they are
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frequently combined with other agents (excipients). Often, SPs used for excipients act as simple inert supports of the active molecule(s). Currently, the specific application of some polysaccharides and/or SPs are in pharmaceutical formulations; for example, they are used in the manufacture of solid monolithic matrix systems, implants, films, beads, microparticles, nanoparticles, inhalable, injectable systems, and hydrogel formulations. Fucoidan is a SP with application in pharmaceutical, as fucoidan-based drug carriers, such as nanoparticles, microparticles, and hydrogels, while chitosan sulphates have been used to design microcapsules, as carrier systems for drug delivery. Nevertheless, Sezer and Akbuğa reported fucoidan from brown seaweed Fucus vesiculosus for the development of a new microspheres delivery system based on cross-linking of fucoidan with chitosan named fucosphere, and evaluated it as a drug carrier with extent of drug release being dependent on the concentrations of the polymers and protein (Sezer and Akbuğa 2006). In addition, Tziveleka et al. discovered that ulvan, a sulphated polysaccharide isolated from the green algae Ulva rigida, showed antibacterial activity with gram-positive bacterial strain Staphylococcus aureus, while this compound has potential for the development of drug delivery nanoplatforms (Tziveleka et al. 2018).
4.3
Anticancer Agents
Currently, there are more than 200 different types of cancers, such as lung cancer, breast cancer, liver cancer, cervical carcinoma, and pancreatic cancer. Chemotherapy and radiotherapy are cancer treatments, but they can cause many adverse secondary reactions in the patients. Thus, sulphated polysaccharides can be an important and nontoxic natural source for potential use in cancer treatments. Some SPs have already been reported to demonstrate anticancer and cancer-preventive properties. Fucoidan isolated from the seaweed Cladosiphon novae-caledoniae was capable of enhancing the anticancer effect of a chemotherapeutic drug against MDA-MB-231 and MCF-7 breast cancer cells (Zhang et al. 2013). Another fucoidan isolated from the brown seaweed Fucus vesiculosus displayed cytotoxic effects on MCF-7 and MDA-MB-231 human breast cancer cell lines in in vitro tests. Moreover, sulphated polysaccharide isolated from brown seaweeds, Saccharina japonica and Undaria pinnatifida, showed high antitumor activity and inhibited the proliferation and colony formation of human breast cancer T-47D and melanoma SK-MEL-28 cell lines (Vishchuk et al. 2011). In addition, sulphated polysaccharide with a MW of 197 kDa isolated from the filamentous algae Tribonema sp. showed significant anticancer activity on HepG2 cells with a 66.8% inhibition at a concentration of 250 μg/mL (Chen et al. 2019).
4.4
Immune Function
Nowadays, immunomodulatory studies on sulphated polysaccharides from marine algae have been performed, mainly from Undaria pinnatifida, Caulerpa lentillifera,
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and Hypnea spinella. On the other hand, SPs from microalgae, Phaeodactylum tricornutum, Chlorella stigmatophora, Pavlova viridis, Thraustochytriidae, and Spirulina, have been studied regarding the human immune system (Muhamad et al. 2019). Fucoidan from the brown alga Undaria piaantifida has a variety of immunomodulatory effects on lactic acid bacteria (LAB), by stimulating cytokine production from antigen-presenting cells (APCs). The results indicated that fucoidan could enhance a variety of beneficial effects of LAB on immune functions (Kawashima et al. 2012). Karnjanapratum et al. showed that sulphated polysaccharides isolated from Capsosiphon fulvescens strongly stimulated macrophage cells, RAW264.7 cell line, producing considerable amounts of nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines, which suggested that they could be strong immunostimulators (Karnjanapratum et al. 2012). In other in vitro and in vivo tests, sulphated xylomannans, a water-soluble sulphated polysaccharide from the red seaweed Nemalion helminthoides, exhibited strong immunomodulators (PérezRecalde et al. 2014). In addition, a sulphated polysaccharide named UP2-1 isolated from Ulothrix flacca exhibited significant in vitro immunomodulating activity and another sulphated polysaccharide isolated from microalgae Tribonema sp. exhibited significant immune-modulatory activity on cytokines, including upregulating interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor α (TNF-α), by enhancing macrophage cell (Chen et al. 2019).
4.5
Wound Healing
Natural polysaccharides can stimulate the extracellular matrix synthesis and facilitate the wound healing. In general, wound healing concerns different phases, for example, hemostasis, inflammation, proliferation, angiogenesis, and scar formation. Sulphated polysaccharides are emerging as high-value drugs and biomaterials for use in wound management and tissue engineering. Some SPs have shown therapeutic potential for using in wound healing. Sezer et al. reported a chitosan film with fucoidan from Fucus vesiculosus for the treatment of dermal burns on rabbits, the results showing that chitosan-fucoidan films might be a potential treatment system for dermal burns (Sezer et al. 2008). In another study, fucoidan polysaccharides were isolated from two species of seaweeds, Padina tetrastromatica and Padina boergesenii, which exhibited significant wound healing effects (Kordjazi et al. 2017).
4.6
Antipathogenic and Anti-Inflammatory
In a study of Chen et al., fucoidan isolated from the brown seaweed Ulva pinnatifida inhibited the invasion of erythrocytes by Plasmodium falciparum (Chen et al. 2009). However, fucoidan extracted from the brown seaweed Fucus vesiculosus displayed anti-inflammatory effects, through the inhibition of production of inflammatory mediators, nitric oxide (NO), and prostaglandin E2 (PGE2), in LPS-stimulated BV2 microglia, by suppressing nuclear factor kappa B (NF-κB), p38 mitogen-
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activated protein kinase (MAPK), and Akt pathways. In addition, fucoidanssulphated polysaccharide from brown algae, Chnoospora minima and Sargassum polycystum, showed anti-inflammatory effects on LPS-stimulated RAW 264.7 cell macrophages (Fernando et al. 2018). Sanjeewa et al. reported the anti-inflammatory effect of sulphated polysaccharide purified from Sargassum horneri, using LPS-stimulated RAW 264.7 cells. This sulphated polysaccharide inhibited the LPS-induced NO production and PGE2 production, by inhibiting the MAPK and NF-kB signaling pathways (Sanjeewa et al. 2017).
5
Clinical Trials
A phase II of clinical trial involved oral administration of seaweed extracts (Fucus vesiculosus 10%, w/w; Macrocystis pyrifera 10%, w/w; Laminaria japonica, 5% w/w; zinc, vitamin B6 and manganese). After 12 weeks, the results revealed a decrease in the dose-dependent osteoarthritis in five female and seven male participants, with no adverse health effects (Myers et al. 2010). Carrageenan a sulphated polysaccharide was incorporated into four food items and distributed to 20 human volunteers to determine its effects on cholesterol, blood, and lipid levels. The results showed that carrageenan could significantly reduce serum cholesterol and triglyceride levels, presenting anticoagulant properties and also a regulatory role, mainly in growth factors (Panlasigui et al. 2003). In addition to the benefits of regular algae consumption, many studies have indicated the health benefits of algae supplementation along with a regular diet. Hence these organisms continue to be increasingly explored in the areas of nutrition, pharmacology, cosmetics, and many other.
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Conclusions
Unlike many artificial chemical inhibitors in certain pharmaceutical and cosmetic products, sulphated polysaccharides are biocompatible polymers with a wide range of desirable functionalities. SPs from seaweed and microalgae exhibit many beneficial biological activities such as anticoagulant and/or antithrombotic, immunomodulator, antitumor and cancer preventive, hypoglycemic, antibiotics and antiinflammatory, and antioxidant properties. In addition, the interest in using algal polysaccharides as biomedical vehicles for drug delivery has increased steadily. Thus, the structural analyses of polysaccharides from algae, as well as specific biological tests on them, are useful tools to investigate molecular mechanisms of possible biological activities, in addition to their obvious practical implications. Therefore, the isolation of a new algal sulphated polysaccharide always brings perspectives for the discovery of new drugs, as well as an increased economic development, by introducing a higher diversity of products and, eventually, products with high added value.
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Acknowledgments This work was supported by National Funds from FCT – Fundação para a Ciência e a Tecnologia through Project UID/Multi/50016/2019 and project EXTRATOTECACOMPETE2020 POCI-01-0247-FEDER-033784.
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Rita Rebelo, Mariana Caldas, Miguel A. D. Neves, Subhas C. Kundu, Rui L. Reis, and Vitor Correlo
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Applications of Biophotonics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Photoacoustic Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Surface-Enhanced Raman Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Optical Coherence Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Photodynamic Therapy (PDT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Photothermal Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Natural Polymers in Biophotonics – Current Uses and Applications . . . . . . . . . . . . . . . . . . . . . . 3.1 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Silk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Rita Rebelo and Mariana Caldas contributed equally with all other contributors. R. Rebelo · S. C. Kundu · R. L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal M. Caldas · M. A. D. Neves · V. Correlo (*) Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, I3B’s Research Institute on Biomaterials Biodegradables and Biomimetics, Universidade do Minho, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associated Laboratory, Braga/Guimarães, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_50
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Abstract
The constant development of optical techniques for medical diagnostics and therapies has led to a growing need to develop and optimize biophotonic applications. Through biophotonics, it is possible to precisely control light propagation, scattering, and emission, enabling a different range of applications, from light-activated therapies to sensing and drug delivery technologies. Particularly, the use of biocompatible materials with desired mechanical, optical, biological, and chemical properties is essential to enable clinically relevant biophotonic devices. Thus, natural polymers such as chitosan, cellulose, keratin, silk, and agarose are promising building blocks for the development of multifunctional biophotonic structures due to their biocompatibility, mechanical characteristics, renewability, and surface chemistry. In this chapter is presented an overview of recent advances in medical biophotonics applications, using natural polymers, from imaging to sensing and phototherapy uses. Herein, the light phenomena in biophotonics, the most used natural polymer properties and state-of-the-art biophotonics applications will be explored. Keywords
Natural polymers · Optical applications · Biocompatibility · Photonic structures
1
Introduction
Photonics is the science of photon (light) generation, detection, and processing. The term originated somewhere in the 1960s and 1970s with the integration of the first practical semiconductor light emitters with optical fibers. The emergence of low intensity loss optical fibers led to the replacement of copper wires for most long distance and transcontinental telecommunications with fiberoptic wires, which now serve as the backbone of high-speed data transmission networks (Quimby 2006). Biophotonics is the science of production and use of photons or light to study optical procedures in biological systems. It results from the integration of four major technologies: lasers, photonics, biotechnology, and nanotechnology and it is applied in several fields. Biomedical applications include light interactions in medicine and biology for the purposes of health care. More specifically, biophotonics can be used for imaging and detecting cells and tissues, drug delivery systems, sensing devices, and injection of fluorescent markers, to track cells or molecules. Additionally, biophotonics can also be used for light scattering applications, in a micro- or macroscale, such as for microscopy or tomography, respectively.
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The development of this technology allows for monitoring the progression of and early detection of diseases, light-guided and light-activated therapies that allow a better understanding of the pathobiology (Shan et al. 2018). These techniques are able to precisely control light propagation, scattering, and emission via hierarchical structures and chemistry, allowing biophotonic applications for transparency, camouflaging, protection, mimicking, and signaling (Xiong et al. 2020). The features that affect the most the potential of biophotonic structures are the optical properties, mechanical properties, chemical structures, and the biological functionalities. However, the most fundamental requirement for optical materials suitable for biophotonic is its capability to achieve high-efficiency light delivery with diminished loss. In the current materials, silica is the most used of the optical material platform due to its considerable optical properties that include a high transparency over a large spectral range from visible to near-infrared (NIR), which makes it suitable for different possible applications (Tong et al. 2003). Nevertheless, its fragile properties make it a risk to use in biological tissues; it is also nonbiodegradable and, as the traditional silica, has low biocompatibility. Therefore, there is a need to find better materials that are biodegradable and biocompatible, and natural occurring polymers can be a great substitute as next-generation materials in optical sensing (Shan et al. 2017). For biophotonics applications, the materials used are essential to produce optical structures, such as waveguides, i.e., a physical structure that guides electromagnetic waves in the optical spectrum, and lenses, which are optical components that are designed to either focus or diverge light that will transmit, detect, and transform light. Thus, an optical material can be defined as the material that can control or alter the electromagnetic radiation in the ultraviolet, visible, and infrared spectral region (Shan et al. 2018). The most important property of this material is its degree of transparency and its refractive index, so when a material has high transparency, it will have low reflection, absorption, and scattering, which are the desired features. Polymeric biomaterials are typically nonhomogeneous due to a somewhat disordered network of polymer chains and macromolecules and usually are not considered for optical devices applications (Shanmugam and Sahadevan 2018). Nevertheless, despite the relatively high optical loss, polymeric optical biomaterials can still meet the distance of interest between the network spaces in most biomedical applications. Therefore, optically transparent polymeric biomaterials are emerging as key materials, as they often have advantages over inorganic silica like soft mechanical properties, biocompatibility, biodegradability, as well as desired chemical and biological functionalities. Furthermore, polymeric optical biomaterials can be easily manufactured into complex structures with high optical efficiency. The advantages mentioned above have motivated researchers to pay more attention to the development of advanced natural polymer structures for biophotonic applications.
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Applications of Biophotonics
One of the branches of biophotonics is optical bioimaging, which refers to cellular imaging techniques that acquire images of biological matter using fluorescent probes, labels, or nanomaterials. These can be small fluorescent organic molecules, fluorescent proteins, and nanoparticles; the last of which has been gaining major focus over the last decade (Yang et al. 2019a). When it comes to fluorescent probes, they must have emission in a suitable range of detection (400–800 nm), a specific excitation wavelength, a high quantum yield, molar absorption coefficient, and photostability. However, a fluorophore can lose its ability to fluoresce when excited several times or for long durations, a phenomenon known as photobleaching. The loss of fluorescence is caused by the absorbed excitation energy, rather than being emitted as fluorescence, is absorbed by the molecule causing the cleavage of covalent bonds or nonspecific covalent reactions with surrounding molecules. This phenomenon is a major problem in the use of these molecules for high-intensity excitation and long-lifetime applications, such as imaging or sensing (Ma et al. 2017). Some nature-inspired optical systems based on bacteria have been produced to overcome photobleaching. However, these systems present several disadvantages such as: inherent variability, limited sources, and restrictive engineering potential. Such disadvantages led to the use of other natural or synthetic polymers and inorganic materials for the development of biophotonic systems (Shan et al. 2018; Prasad 2004). Therefore, nanoparticles and nanomaterials have arisen as a promising alternative. The majority of these fluorescent nanoparticles and nanomaterials are inorganic; however many have been constructed of inorganic natural (bio)polymer hybrids and natural polymers. When compared to conventional small molecule fluorophores, these materials present good emission, contrast, and photostability, along with their nanometric size, allow for efficient and fast cellular internalization, similar to that of small molecule fluorophores (Owens et al. 2015). Thus, optical bioimaging is a noninvasive, high-sensitivity, and high-resolution tool that is able to perform effective and low-cost diagnostics of different wide-ranging diseases and imaging in wide-ranging cells and tissues. The techniques range from the traditional luminescence to more recent, state-of-the-art techniques, such as photoacoustic imaging and surface-enhanced Raman spectroscopy (SERS); where biophotonic techniques are used as a mean to diagnose several diseases using light-responsive materials (Son et al. 2019), natural biopolymer biphotonic materials have shown to provide improved biocompatibility over inorganic and hybrid materials, hence are preferred for certain applications, which has led to increased development (Ma et al. 2017). Luminescence is based on the light (energy) emission of certain materials by either chemical or physical processes and includes photoluminescence (fluorescence or phosphorescence), chemiluminescence, and electroluminescence. Regarding these, the fluorescence imaging is the most representative on biophotonics because
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Fig. 1 In vivo optical properties of injected fluorophores along with wavelength
it uses light from microscope imaging to be used in intraoperative image-guided surgery. The major problems regarding this technique are the autofluorescence of the endogenous biomolecules, which is the light emitted from the background tissues that leads to an increase of excitation light, and the depth of the tissue penetration. One way to counterbalance this effect is to use NIR, which wavelength of the electromagnetic radiation spectrum ranges 650–900 nm (Owens et al. 2015). NIR fluorescence can be advantageous for biophotonics because of the low absorption of endogenous hemoglobin and water that can disperse the light, the scattering effect, and also minimize the autofluorescence, resulting in a better tissue penetration of NIR light (Fig. 1). It is important to understand that fluorophores are at the ground state level on energy (S0) in a singlet state and, when they absorb light, they move to an excited state (Sn) (Fig. 2). When they return to the S0 level of energy is when fluorescence is emitted; therefore, the controlling of energy and retention in the excited state is very important for imaging purposes (Son et al. 2019).
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Internal conversion
S2
Absorbance
Increasing energy
Fig. 2 A Simplified Jablonski diagram. (Owens et al. 2016). (Figure reproduced with permission from American Chemical Society)
Fluorescence Intersystem crossing Phosphorescence
S1
hv
T1 hv hv
S0
2.1
Photoacoustic Imaging
A hybrid technique based on the generation of acoustic signals by the absorption of optical energy, photoacoustic imaging (PAI), has emerged to overcome the limitations of current optical imaging, such as light diffusion and photon penetration depth. This process consists in the absorption of energy by endogenous chromophores within a tissue (or in some cases an injected tracer); these chromophores convert the absorbed light energy into heat energy that is thermoelastically converted into acoustic waves able to be transduced by an ultrasound probe into a signal (Xia et al. 2014). This technique greatly outperformed the aforementioned optical imaging methods on account of the increased sensitivity, resolution, and the superior depth achieved by the nonionizing laser light source. PAI is a noninvasive technique and can be performed without using any contrast agents because of the endogenous molecules, such as hemoglobin, melanin, lipids, and water, which will absorb electromagnetic energy and generate acoustic waves. Each one of these chromophores has their own characteristic absorption spectra, so multiple wavelengths allow their relative quantification and analyze physiological changes. Nevertheless, the use of exogenous probes (contrast agents), such as fluorophores, IR active molecules (i.e., indocyanine green), polymers, carbon, or gold nanoparticles, will enhance the photoacoustic (PA) signal if the endogenous contrast is not sufficient due to the similarity of absorption spectra of one or more chromophore or in the case of monitoring circulation (Attia et al. 2019; Steinberg et al. 2019; Chaudhary et al. 2019).
2.2
Surface-Enhanced Raman Spectroscopy
Surface-enhanced Raman spectroscopy (SERS) is a noninvasive analytical tool that combines molecular fingerprint information provided by Raman scattering
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with the signal-enhancing power of the metallic nanostructures of gold or silver. The Raman effect consists of inelastic light scattering by chemical species with a change in the wavelength of light that occurs when a light beam is deflected by molecules. Through SERS it is possible to obtain qualitative and quantitative chemical information of biomolecules in situ without the need of any external labeling and reach single-molecule detection levels (De Marchi et al. 2019; Fan et al. 2020). In vitro applications of SERS include primarily detection of infectious pathogens, including virus and bacteria via their building blocks, such as DNA and proteins, using hybridization or immunoassay concepts. Another application is cancer diagnostics by analyzing proteins, nucleic acids, circulating tumor cells, immunophenotyping of cancer cells, as well as the detection of several tumor biomarkers produced by cancer cells. SERS is advantageous for its rapid screening, capability of facile multiplexing, and for high sensitivity. Regarding in vivo applications, clinical SERS can be used for guided intraoperative imaging for tumor resection and endoscope-based imaging (Prochazka 2016).
2.3
Optical Coherence Tomography
Optical coherence tomography (OCT) is a noninvasive diagnostic technique that provides cross-sectional images of biological structures based on the optical properties of a different tissue. It is an interferometric technique that uses NIR light waves that reflect off the internal microstructure in a way that, in principle, is analogous to an ultrasonic pulse echo. It is possible to obtain real-time images with excellent axial resolution. OCT offers tomographic imaging of internal tissues with high-resolution 2D or 3D cross-sectional images. In particular, OCT has been highly useful in ophthalmology to analyze optic nerves, the retinal nerve layer and for measuring the stiffness of the cornea (Katkar et al. 2018). When small nanorods, nanostructured objects with a rod shape, are located in the matrix, their Brownian movements are changed by the intermittent collision with neighboring macromolecules. The polarized light scattering produced can reveal the nanotopology of the tissue by OCT. For example, gold nanorods can provide anisotropic optical scattering, which makes it easier to track in native tissue using polarization-sensitive OCT, and in a tissue where there is increased collagen and cell density, the polarization is decreased (Son et al. 2019). Another possible application is biosensing, where analytical devices enable the sensing of molecular interactions and convert them into a detectable electrical signal (Solaimuthu et al. 2020). Therefore, optical biosensors use optical responses, like intensity, wavelength, or polarization variations, as a result of chemical or biological changes that are correlated with the physiological disorder. Using these devices, it is possible to obtain real-time multiplexed information. To date, many biosensors have been developed to detect biomarkers of innumerous cancer types such as breast cancer, lung cancer, or melanoma and other diseases including Alzheimer’s disease (Jayanthi et al. 2017; Yang et al. 2019b; Eftekhari et al. 2019; Carneiro et al. 2020).
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Photodynamic Therapy (PDT)
An area of biophotonics that has received a great deal of attention and development is light-activated therapy. Photodynamic therapy (PDT) is a modern noninvasive therapy, utilized for the treatment of several diseases including a variety of cancers (Beack et al. 2015; Kim et al. 2020; Sun et al. 2019). The technique relies on photosensitive compounds, photosensitizers, which accumulate in the pathological tissues. The photosensitizers absorb the light at a specific wavelength and initiate the activation process, which leads to the destruction of surrounding cells. It is a selective therapy as the photo-cytotoxic reaction only occurs in the pathological tissues where the agent has been administered. Under laser irradiation, the photosensitizer is excited into the excited state leading to the production of singlet oxygen species and free radicals in the surrounding tissue causing damage to local cells; for example, if the photosensitizer accumulated in a tumor, excitation would lead to tumor cell death in the local area around the photosensitizer (Zhang and Li 2018). Photosensitizers accumulate in higher concentrations in cancer cells due to their tendency to combine preferentially with low density lipoproteins (LDL), which supply cholesterol to create cellular membranes. In cancer cells, the division and mitotic process is accelerated where an increased uptake of LDL takes place and in that way the LDL can act as a transporter of the photosensitizer to the tumor. If cancer cells escape the photo-cytotoxic effect directly, they can be destroyed by the indirect influence of PDT on tumor vessels, as a result of reactive oxygen species damage of blood vessel endothelial cells. Furthermore, this blood vessel damage leads to an inflammatory state causing platelet activation and aggregation resulting in the formation of platelet thrombi, which causes local hypoxia resulting in cell death (Kwiatkowski et al. 2018). PDT is proven effective alone or in combination with chemotherapy for the treatment of many cancers, including skin and breast cancer and leukemia (Beack et al. 2015; Kim et al. 2020; Sun et al. 2019).
2.5
Photothermal Therapy
Photothermal therapy (PTT) is another form of biophotonic light-responsive targeted therapy, where the photosensitizer applies heat for tumor ablation. PTT has emerged as a minimally invasive approach to kill cancer cells without damaging the healthy tissue around the pathogenic cells. This strategy is possible as the absorbance of energy by the photosensitizer causes it to release a large amount of vibrational energy, which turns into heat energy (Batul et al. 2017; Cheng et al. 2014). Literature reports indicate that, to cause the destruction of cancer cells, the local temperature should be maintained for a duration of 15–60 min at approximately 43 C, or for 5 min at over 50 C (Habash et al. 2006). Owing to the superior tissue penetration and high-resolution adjustability in both duration and three-dimensional space, NIR has been extensively utilized for PTT, as precise control can be achieved. The possibility of inducing heat by NIR light irradiation at the specific tumor site improves the targeting and efficacy of the therapy. Due to the low heat tolerance
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of tumor cells, the high temperature is able to destroy them while avoiding damage to normal healthy cells (Wei et al. 2019). Light-responsive agents, such as nanoparticles, are useful for photo-triggered drug release upon irradiation, which commonly utilized photo-degradable materials, or a photo-induced decrease in hydrophobicity. These strategies allow the stable storage of drugs in these agents while in blood circulation and controlled in situ drug release only in the specific tissue at the desired time. A large number of light-responsive materials that have been developed respond to UV light. However, UV light has a limited tissue penetration depth due to light scattering and absorption by water or other biological substances. Furthermore, UV light is harmful for all cells, both pathogenic and health, which greatly limits applications. Alternatively, NIR can penetrate deep into tissues without damage and, therefore, has been gaining an increased interest for on-demand therapeutic and drug delivery purposes (Cho et al. 2015). To achieve the full potential of biophotonic applications, the characteristics of the material used are of crucial importance, influencing the overall performance of the system (Shan et al. 2018).
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Natural Polymers in Biophotonics – Current Uses and Applications
Besides the optical properties needed for biophotonics applications, the biological, chemical, and mechanical properties are also crucial. Thus, optically transparent polymers are emerging as key materials for biophotonics, since they present advantages over traditional optical materials (e.g., silica), including soft mechanical properties, biodegradability, biocompatibility, as well as desired chemical and biological functionalities. Natural polymers are produced by animals, plants, and microorganisms and present excellent biocompatibility and biodegradability. Additionally, many of those polymers also present good optical properties and are constructed into several well-organized photonic structures (Xiong et al. 2020). These structures offer good mechanical properties, are lightweight, renewable, and of low-cost to produce at large scale. Moreover, many natural polymers present strong adhesion and good responsive properties, can be self-healing, and have tunable properties. Furthermore, natural polymers can suppress the major disadvantage of synthetic polymers: nondegradability and lack of biocompatibility (Xiong et al. 2020; Shan et al. 2018). The natural polymers most used in biophotonics, as well as their main properties and applications, are presented in Table 1.
3.1
Chitosan
Chitosan, a natural biopolymer, is a cationic polysaccharide produced by deacetylation of chitin, a polysaccharide found in the exoskeleton of almost all vertebrates, obtained from an alkalizing process at high temperatures (Fig. 3) (Younes and Rinaudo 2015).
Natural source Insect cuticles/ wings, mollusk shells
Fruits, flowers
Avian feathers
Silkworm
Natural polymers Chitosan
Cellulose
Keratin
Silk
Photonic crystals
Multilayered structure, photonic crystals
Chiral nematic structures, gratings
Photonic structure Chiral nematic structures, multilayered structure, disordered networks
Crystals
Multilayer interference
Chiral photonic crystals, diffraction gratings
Optical mechanisms Chiral photonic crystals, multilayer interference, light scattering, diffraction gratings
Properties Antibacterial effect Biocompatibility Biodegradability Nontoxicity High humidity absorption Optical clarity Mechanical stability High transmittance of visible light Favorable permeability for water and ions High tensile and compressive strength Nontoxic Biodegradability Durability High toughness and modulus High content of amino acids Good mechanical strength and
Table 1 Summary of polymeric biomaterials, their properties and applications for biophotonics
Eco-dye and multifunctionalization of fabric
Photonic and plasmonic
Photonic and plasmonic Raman spectroscopy Sensing
Applications Sensing
Diao et al. (2013), Burke et al. (2016), Arsenault et al. (2007), Colusso et al.
Zhu et al. (2019)
Mu and Gray (2014), Espinha et al. (2018), Orelma et al. (2020) and Peng et al. (2011)
Reference Chen et al. (2012), Voznesenskiy et al. (2013), Mironenko et al. (2017) and Razali et al. (2020)
930 R. Rebelo et al.
Agarose
Seaweeds
Photonic crystals
Crystals
toughness Flexibility Biocompatibility High transparency High gel strength Agarose gels are thermo reversible Nontoxic Drug delivery Sensing Imaging
Sensing Photothermal therapy Imaging
Jain et al. (2012), Fujiwara et al. (2020) and Gao et al. (2014)
(2017), Li et al. (2017), He et al. (2019) and Xu et al. (2018)
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Fig. 3 Schematic representation of chitin and chitosan chemical structures. (Figure reproduced from Younes and Rinaudo (2015))
Chemically, chitin is a cationic amino polysaccharide polymer randomly composed of β-1,4 linked N-(acetyl-D-glucosamine). As chitosan is produced after partial deacetylation of chitin, it presents in its structure D-glucosamine as the major constituent (80%) and N-(acetyl-D-glucosamine) as the minor one (20%). Thus, chitin and chitosan are structurally very similar, with the exception of the less acetyl groups present in the later form (Younes and Rinaudo 2015; Kumar and Kumar 2017; Rebelo et al. 2017). Widely known for its antimicrobial properties, chitosan is also biodegradable and highly biocompatible. Furthermore, chitosan is polycationic; it can easily interact with polyanionic polymers, is insoluble in water but soluble in weakly acidic aqueous solutions, it is non-immunogenic and mucoadhesive. Due to such versatile properties, chitosan has been broadly used in different forms (membranes, gels, particles, films, or scaffolds) in several applications: from biomedical to industrial areas (Kumar and Kumar 2017; Rebelo et al. 2017). Chitosan has been used to coat an optical fiber tip (Fig. 4a) to detect Pb2+ at 0–70 ppm levels, in water. Chitosan has excellent adsorbent properties for Pb2+ due to metal chelating moieties in its structure (as amine and hydroxyl groups). As a result, chitosan refractive index (RI) is modulated by the amount of Pb2+ present in the analyte and, consequently, modulates the Fresnel reflectivity at the fiber tip region. The use of the chitosan coating greatly enhanced the sensitivity for the detection of Pb2+; seven-fold higher than the uncoated sensor (Fig. 4b) (Jain et al. 2012). Moreover, chitosan was also applied to form the waveguide as a sensing element of a relative humidity sensor. Different forms of chitosan (salt and neutral) were evaluated, and both led to the production of integrated-optical sensors based on waveguide films with high sensitivity and short response time (He et al. 2019).
3.2
Cellulose
Cellulose is one of the most abundant natural polymers on earth; generally produced by plants, but it can also be synthetized by some bacteria. This natural polymer is a
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Fig. 4 (a) SEM images of sensor (i) before chitosan coating and (ii) after chitosan coating; (b) Sensing spectrum of (i) uncoated sensor and (ii) chitosan-coated sensor (Razali et al. 2020); (c) (i) Photographs of the hydroxypropyl cellulose (HPC) photonic films (square lattice of imprinted cylindrical holes), for lattice parameters of 400, 500, and 600 nm. Square lateral size is 1 cm, (ii) images of the HPC photonic films acquired with an optical microscope, (iii) SEM micrographs of the HPC photonic films (top view). Scale bar, 5 μm, (iv) Specular reflectance characterization of the samples (solid curves), along with theoretical modeling calculations (dashed curves) (Espinha et al. 2018); (d) A cycle test of the color change between humidity levels of 10% and 90%. The change can take place in 5 s, with recovery in 1 s upon pumping wet or dry air (Li et al. 2017); and (e) (i) agarose-based structured optical fiber: (ii) cross-section view of the end-face and (iii) output speckle field of the core-guided modes. The fiber has 60 mm length, diameters of 0.64 mm, 2.5 mm, and 0.5 mm for core, cladding, and holes, respectively, and bridges of ~0.08 mm width (Fujiwara et al. 2020). (Figures reproduced with permission from AIP Publishing, Springer Nature and Royal society of chemistry)
homopolymer of glucose, in which the monomers are joined by β-1,4 linkages. Due to its long and linear molecular structure, cellulose does not dissolve readily in water. Cellulose chains are arranged in microfibrils, which are organized into fibrils. This arrangement not only provides stability for the plant structures but also infers the high strength and superior mechanical properties of cellulose, when compared with other natural polymers (Torok and Dransfield 2017; Pandey et al. 2019; Nishiyama 2009). The hydrogen atoms of cellulose are all in the axial position, whereas all the hydroxyl groups are equatorial. These equatorial hydroxyl groups can hydrogenbond with their nearest neighbors, allowing cellulose to readily crystallize. Additionally, cellulose can be functionalized with many modifiers through these hydroxyl groups in order to form cellulose derivatives with useful properties, like cellulose esters of cellulose butyrate, cellulose acetate, or cellulose triacetate (Pandey et al. 2019; Andreana and Stolow 2014).
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Moreover, cellulose features a high transmittance of visible light and favorable permeability for water and ions, which can be useful in biophotonic applications, like optical fibers and sensors (Shan et al. 2018; Andreana and Stolow 2014). Regenerated cellulose and cellulose acetate have been used as optical sensing fibers. These fibers were prepared by coating regenerated cellulose filaments with cellulose acetate. While cellulose acetate passed the whole light wavelength range, regenerated cellulose absorbed UV light, passing the visible light wavelengths. The prepared optical fiber guided light in the range of 500–1400 nm and when fiber was placed in water, a clear attenuation in the light intensity was observed (Younes and Rinaudo 2015). In a different strategy, hydroxypropyl cellulose was used to produce photonic and plasmonic structures (Fig. 4c), using soft lithography. This led to a structural color change in this material, which was obtained by exploiting its chiral nematic phase. Patterned cellulose membranes possessed tunable colors and may be used to boost the photoluminescence of a host organic dye. Additionally, metal coating these cellulose photonic structures resulted in plasmonic crystals that presented excellent optical properties and can act as SERS substrates (Gao et al. 2014).
3.3
Keratin
Keratin represents a family of structural proteins that are abundant in nature, specifically in the outer layer of skin, hair, nails, horns, and feathers of animals. Keratin is rich in half cysteine and it possesses the ability to self-assemble into bundles of fibers. Within these fiber bundles, individual strands are further crosslinked through disulfide bonds, mediated by the cysteine side chains. This high sulfur content introduces several inter-/intra-molecular disulfide bridges, allowing a complex hierarchical structure of keratinous materials with polypeptide chains and filament-matrix, forming lamellar/sandwich structures (D’Alba et al. 2011; Xiong et al. 2020). Keratin can be classified as: α-pattern, β-pattern, feather-pattern, or amorphous pattern, determined by X-ray diffraction. α-pattern keratin can be found in mammals, for example, in wool, hair, and nails, while β-pattern keratin are present in feathers (Xiong et al. 2020; Wang et al. 2016). Thus, keratin forms durable insoluble structures that are of particularly high toughness (5.6–22.8 kJ/m2 to 10–80 kJ/m2) and modulus (10 MPa to 2.5 GPa), which are among the strongest nonmineralized tissues found in nature (Xiong et al. 2020; Wang et al. 2016; Singh et al. 2017). The capability to pattern natural polymers at different scales is extremely important in several biomedical applications, including biophotonics. Zhu et al. used wool keratin for production of precise protein microarchitectures. Through straightforward biochemical processes, modified wool keratin proteins become intrinsically photoreactive without major changes in protein function or structure. The resultant photoreactive keratin can be photo-crosslinked using a photomask and UV-light. The un-crosslinked water-soluble area can be removed, leaving periodic microstructures for creating brilliant structural colors (Rebelo et al. 2017).
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Silk
Silks are natural protein fibers produced by insect larvae of silkworms, moths, butterflies, and spiders. The raw silk thread, produced by silkworms, is composed of a fibroid core, silk fibroin, and a glue-like coating consisting of sericin proteins. Their hydrophobic β-sheet structures grant high mechanical strength and toughness while providing comparable in vitro and in vivo biocompatibility with other commonly used biomaterials, like collagen. This behavior can be attributed to the β-sheet configuration of silk fibroin, in which van der Waals forces and strong hydrogen bonds generate a structure, which is thermodynamically stable (Shan et al. 2018; Rebelo et al. 2017). The high flexibility of silk allows the production of several silk forms: films, gels, or scaffolds. Moreover, molecular designability and moderate aqueous processing permits the incorporation of growth factors, cells, or peptides into the silk structures, which is extremely useful in biomedical applications. Regarding biophotonics, silk films with 20 and 100 μm of thickness were found to be great candidates for optical devices, due to their low surface roughness (95%) across the visible spectrum (Shan et al. 2018; Khalid et al. 2020). For instance, Li et al. used silk fibroin to develop sensing applications. Silk fibroin films were produced by spin coating and demonstrated bright color and high sensitivity to humidity. The optical properties of silk fibroin can be easily tuned by the coating rates. Moreover, due to the high hydrophilicity of silk fibroin, the film presented fast responses to humidity, in just few seconds (Fig. 4d). At humidity above 80%, the red shift of the reflectance peak in the visible spectrum was even larger than 130 nm, corresponding to a significant color change as distinct as yellow to violet. Thus, the silk fibroin film is superior to several other multilayered or photonic crystal-based humidity sensors (Li et al. 2017). In a different application, a silk fibroin nanofiber hydrogel system complexed with upconversion nanoparticles and nano-graphene oxide was developed for upconversion luminescence imaging and photothermal therapy. The injectable system showed excellent upconversion luminescence imaging properties and a photothermal therapy effect under NIR laser irradiation (He et al. 2019).
3.5
Agarose
Agarose is a nontoxic marine-based polysaccharide, derived from agar and extracted from red seaweeds, that has been widely used as a biocompatible material for cell encapsulation, DNA electrophoresis, and tissue regeneration. Agarose is composed by an alternating copolymer of β-1,3-linked d-galactose and α-1,4-linked 3,6-anhydro-α-lgalactose residues. It is a thermally gelling polymer: at temperatures below 35 C the gelling process occurs due to the formation of an infinite network of 3D agarose fibers, and at temperatures above 85 C the networks of agarose hydrogel disassemble. In solid state, agarose is brittle, but maintains its shape for a long period of time at a broad range of temperatures. Moreover, agarose is an attractive natural polymer for optical
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applications, since it is possible to tune the optical refractive index of agarose hydrogels according to concentration (Shan et al. 2018; Gao et al. 2014; Giuseppe et al. 2019). Recently, Fujiwara et al. used agarose to develop an optical fiber able to be used in sensing and imaging applications. This fiber was produced by pouring food-grade agar into a mold with stacked rods, forming a solid core surrounded by air holes, in which the fiber geometry and refractive index can be tuned through the mold design and agarose solution composition, respectively (Fig. 4e). The fiber exhibited practical transmittance at 633 nm, in comparison to other hydrogel waveguides, and can also be used for chemical sensing either by detecting volume changes, due to agar swelling or contraction (dehydration), or by modulation of transmitted light, through the insertion of fluids into the air holes (Fujiwara et al. 2020).
4
Conclusion and Future Perspectives
Natural polymers have been used to establish many important technologies in the application of biophotonics. Biomaterials composed of natural chitosan, cellulose, keratin, melanin, silk, and agarose have demonstrated key advantages including biocompatibility, degradability, and suitable mechanics, leading to the development of several optical biomedical devices. The major advancements of natural polymers in biophotonics have focused on diagnostics and treatments, and are featured in sensing, imaging, drug delivery, and therapeutic technologies. However, despite these great features there are still some limitations, namely, the reproducibility and high spatial resolution regarding the current processing of naturally based materials. Biophotonics is a quickly growing field, driven by the increasing number of patients with chronic diseases in developed countries. According to a report by Grand View Research, San Francisco, CA, it is estimated that the biophotonics market will reach $91.31 billion by 2024 (Grand View Research, San Francisco, C 2016). Currently, there are still are some limitations that hamper market growth, like the high price and complexity of biophotonics technologies and the inherently slow commercialization process of medical devices. However, these developments in optical technologies and the increasing demand for early diagnosis and homebased point-of-care devices is expected to spur the growth of the market. In future, it will be necessary to bring together the current medical techniques with the broad spectrum of possibilities offered by biophotonics. Laser systems and light-emitting nanostructures will be just few of the biophotonic options. Biophotonics will continue to be a highly active research field, and results will lead to new applications in clinical diagnosis and therapies (Krafft 2016). The ability to use several natural polymers in different optical applications leads to enormous opportunities and opens new areas in the use of these materials in photonics and in the biomedical field. For that, a deeper understanding of the optical properties of natural polymers will be required for scale-up procedures, therefore allowing reproducibility and lower manufacturing costs. With that, biphotonic natural polymers demonstrate that personalized medicine with green renewable materials can be achieved.
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Acknowledgments R. Rebelo would like to acknowledge BREAST-IT PTDC/BTM-ORG/28168/ 2017 project. M. Caldas would like to acknowledge PD/BD/150476/2019 FCT Grant and V.M. Correlo would like to acknowledge SofTE- PTDC/EMD-EMD/31590/2017 project.
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Part VI Tissue Engineering Applications
Polysaccharides-Based Biomaterials for Surgical Applications
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Plant-Origin Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Animal-Origin Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Microbe-Origin Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Processing and Modifications of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Bacterial Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Gellan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Carrageenan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Xanthan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Glucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Surgical Applications of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Orthopedic Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Neural Surgery and Plastic Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Wound Healing and Miscellaneous Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Polysaccharides are highly abundant natural polymers and can be obtained from three mainly different renewable resources such as plants, animal, and microorganisms. These polysaccharides have shown a great potential in biomedical applications, especially tissue engineering due to their high availability in nature, cost-effective production, ease of processing, nontoxicity, excellent G. Agrawal School of Basic Sciences, Indian Institute of Technology Mandi, Kamand, India, A. Kumar (*) School of Chemical Engineering, Yeungnam University, Gyeongsan, South Korea © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_51
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biocompatibility, and intrinsic immunogenic ability. Among them, microbial polysaccharides have received a great attention in tissue engineering or surgical applications. In this chapter, an introduction of surgical tissue regeneration and used biomaterials are briefly discussed. Further, an overview of different polysaccharides, their processing and modifications (especially microbial polysaccharides with other polysaccharides) as well as their applications in various surgical procedures are reviewed and discussed precisely. Keywords
Polysaccharides · Biomaterials · Hydrogels · Surgical applications · Tissue adhesives · Sealants
1
Introduction
In the last decade, tremendous efforts have been made to explore various natural and synthetic polymers to design remarkable biomaterials for targeted biomedical applications (Agrawal et al. 2017, 2019; Birkholz et al. 2016; Wiemer et al. 2017; Doermbach et al. 2014). In this regard, natural polymers such as polysaccharides are particularly interesting owing to their inherent biocompatibility. Hence, increasing attention has been given to develop polysaccharides-based biomaterials that can perform the desired task under complex biological environment that is encountered especially during or post-surgery. The biggest challenge of surgical process is the proper closure of surgical wounds by efficient stabilization of wound margins in their desired position and achieving required mechanical support during surgical wound healing. Sutures, surgical tape, staple, and clips have been frequently explored so far for wound healing (Pillai and Sharma 2010; Taira et al. 2010; Shantz et al. 2013). However, time-consuming procedure, leakage, tissue scarring, and infections are some of the disadvantages here (Jain and Wairkar 2019). For example, lung tissue wound closed by sutures might encounter gaseous leakage while surgery of traumatic injury with sutures might lead to lesions agglomeration (Foster et al. 2010). Hence, significant efforts are being made for developing surgical glues to address the abovementioned issues and achieve minimally invasive surgery. Surgical glues are often used to seal the injured tissue or wounds and maintain stabilization of wound margins in required position, thus influencing the success of any surgical procedure. To achieve the desired outcome, these surgical glues should have high binding affinity, elasticity, biocompatibility, and biodegradability along with being economical and easy to use. Surgical glues can be classified into three categories based on their mode of action: (1) hemostatic agents which form clot in blood; (2) sealants are used to seal the wound opening to prevent leakage and blood loss; and (3) adhesives which merge the margins of wound site together (Jain and Wairkar 2019). A variety of surgical glues are being developed as their market is growing fast globally from $4 billion in 2012 to $7 billion in 2017 (Annabi et al. 2015). Despite significant efforts for fabricating novel surgical glues, most of the
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developed systems still require a unique combination of elasticity and adhesion for being suitable for both hard and soft tissue. Recently, an extensive research has been done to develop surgical glues based on both natural (e.g., fibrin, collagen, gelatin, albumin, chitosan, dextran, etc.) and synthetic (e.g., polyurethanes, polyethylene glycols, polycyanoacrylates, etc.) polymers (Chao and Torchiana 2003; GruberBlum et al. 2010; Hidas et al. 2006; Jeon et al. 2017; Berger et al. 2001; Thorn et al. 2004; Ates et al. 2014; Osbun et al. 2012; Leggat et al. 2007). Tissucol ®, Beriplast ®, Bolheal ®, and Biocol ® are some of the examples of commercially available fibrin-based tissue adhesives (Janmey et al. 2009). However, their widespread use is limited due to poor adhesion efficiency and lower strength. Histoacryl ® is an example of cyanoacrylate-based glue having good bonding strength but it suffers with cell toxicity, inflammation, and foreign body type giant cell reaction after usage (Shivamurthy et al. 2010; Mizrahi et al. 2011). On the other hand, GRF glue and BioGlue ® suffer from the toxicity associated with their components formaldehyde and glutaraldehyde, respectively (Chao and Torchiana 2003; Shaffer and Belsito 2000). In quest to explore other options, different polysaccharides-based (e.g., chitosan and dextran) hemostatic agents such as Surgicel®, Celox™, HemCon® Bandage, and mRDH have been developed and are currently available in the market (Valeri and Vournakis 2011; Pozza and Millner 2011; Matsumura et al. 2014; Wang et al. 2012; Li et al. 2011; Hyon et al. 2014). In this chapter, we provide the readers with a brief overview of different polysaccharides-based biomaterials that have been reported in the literature for surgical applications. We will also focus on different types of polysaccharides, their processing and modification (especially microbial polysaccharides), and their use for various surgical procedures.
2
Polysaccharides
Polysaccharides represent an important class of natural polymers that have been widely explored for biomedical applications (Sauraj et al. 2020). These natural polymers consist of different monosaccharide units connected via o-glycosidic linkages. Cellulose, starch, chitin/chitosan (CS), chondroitin sulfate, hyaluronic acid (HA), dextran, and xanthan gum are some of the examples of widely used polysaccharides (Fig. 1). These polysaccharides exhibit a wide range of physicochemical properties based on different monosaccharide units present in the polymer chain, chemical composition, and source of extraction. In general, polysaccharides are obtained from renewable sources including plants, animals, and microorganisms.
2.1
Plant-Origin Polysaccharides
Plant cell wall is one of the major sources of polysaccharides, namely cellulose and starch, and their extraction mainly depends on the structure of the cell wall. Starch is
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Fig. 1 Schematic representation of chemical structures of various natural polysaccharides
considered as the storage polysaccharide, while cellulose is considered as structural polysaccharide. Cellulose is the most abundant natural polymer found in the cell wall of plants and can also be produced from microorganisms. It is a linear polysaccharide consisting of D-glucose units linked via β(1!4) glycosidic linkages. In general, oxidized cellulose obtained by partial oxidation of hydroxyl groups is used as surgical glue. Surgicel® consisting of oxidized regenerated cellulose (ORC) is widely used to achieve hemostasis during surgery (Breech and Laufer 2000). It has been successfully used in different surgeries due to its nonadherence to medical equipment, and possibility to crop it as required based on the wound site. Although ORC-based hemostatic agents are broadly used, yet they encounter the problem of relatively poor hemostasis, acidity, and low biodegradability (Cheng et al. 2018). It is believed that the hemostatic action of oxidized regenerated cellulose is due to the incorporation of negative charge caused by carboxylic group leading to thrombus formation via platelets activation (Ryšavá et al. 2003). Platelet activation makes the platelet glycoprotein (GPIIb/IIIa) receptor active for binding soluble fibrinogen and thus releasing clotting factors (Cheng et al. 2013).
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Starch is another naturally occurring polysaccharide obtained from plants such as potato, cereal grain, chickpeas, etc. Similar to cellulose, starch is also a polymer of glucose molecules. However, starch consists of only α-glucose while cellulose is made up of only β-glucose. Starch contains both (1!4) and (1!6) glycosidic linkages. Starch is formed by photosynthesis in plants and it functions as an energy storage unit. Arista AH and Quickclean are commercially available hemostatic agents prepared by physical/chemical modification of starch (Murat et al. 2006; Zhu et al. 2019). They are made up of microporous polysaccharide hemosphere which can absorb blood components leading to concentration of clotting proteins and thus enhancing blood clotting (Lewis et al. 2015).
2.2
Animal-Origin Polysaccharides
Polysaccharides such as chitin-derived chitosan, chondroitin sulfate, and hyaluronic acid are found in various body parts of different animals. Among polysaccharides, chitin holds the position of second most abundant natural polymer after cellulose (Min et al. 2004). It is found in the exoskeleton and endoskeleton of crustaceans and molluscs, respectively (Hamed et al. 2016). It is obtained from arthropods such as crabs, lobsters, and shrimps along with cell wall of fungi. As the native chitin form is insoluble in water and difficult to process, it is converted into chitosan (CS) by deacetylation of chitin. Chitosan is a linear copolymer of D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) linked via β(1!4) glycosidic linkages (Li and Hsieh 2006). The generated free amino groups not only impart solubility to chitosan in water and various organic solvent but also make it suitable for further chemical modification. Chitosan undergoes degradation in the presence of enzymes such as lysozyme, chitotriodidase, di-N-acetylchitobiase, and N-acetyl-β-D-glucosaminidase without generating harmful by-products, making it a suitable candidate for surgical applications (Lim et al. 2008). Amino group present in the polymer chain forms bonds with tissue collagen via electrostatic interaction, thus facilitating blood coagulation and showing great potential as tissue adhesives (Jain and Wairkar 2019). It has also been reported that chitosan-based biomaterials help in wound healing by releasing important growth factors (Okamoto et al. 2003). Chondroitin sulfate is a sulfated glycosaminoglycan which is mainly found as a structural component of cartilage around joints in the body (Garnjanagoonchorn et al. 2007). This unbranched polysaccharide consists of alternating N-acetylgalactosamine and glucuronic acid bound together in a polymeric chain (Meziane-Tani et al. 2006). It is generally extracted from shark and cow cartilage. It contains several functional groups such as –OH, –COOH, sulfate, etc. which can be further modified into methacrylate-, –CHO-, and NHS-activated ester groups, thus providing enormous possibilities of functionalization. Based on its association with cartilage and other tissues, it has been explored for developing surgical glues for tissue regeneration and wound healing (Rainer et al. 2005; Strehin et al. 2010).
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Hyaluronic acid (or hyaluronan) is a linear, nonsulfated glycosaminoglycan and it is a major component of extracellular matrix. It is composed of N-acetyl-d-glucosamine and d-glucuronic acid and it degrades in the presence of hyaluronidases (Flynn et al. 2011). It is also an important part of skin and plays a crucial role in tissue repair. It is generally extracted from rooster combs. It is biocompatible and has plenty of functional groups which make it a potential candidate for designing novel biomaterials for surgical applications (An et al. 2019; Liu et al. 2018; Luo et al. 2019).
2.3
Microbe-Origin Polysaccharides
Microbial origin polysaccharides are an important class of polysaccharides due to the possibility of producing them in high yield. Hence, different microbe-based polysaccharides such as dextran and xanthan have been widely used for designing functional biomaterials for biomedical applications. Dextran is a complex polysaccharide made up of glucose units connected via α(1!4) glycosidic linkages and having branches from α-1,3 linkages. As compared to chitosan, no amine functional groups are present in dextran. Hence, dextran is chemically modified to incorporate aldehyde or methacrylate functional groups. Dextran having a wide range of molecular weights is synthesized from sucrose mainly by the action of Leuconostoc bacteroides and Streptococcus mutans (Sarwat et al. 2008). The enzymes glucosyltransferase and dextran sucrase are involved in the synthesis of dextran (Devulapalle et al. 2004). These enzymes polymerize the glucose fraction of sucrose releasing fructose. It has been found that dextran exhibits enzymatic degradation in the spleen, liver, and colon (Khalikova et al. 2005). On the other hand, xanthan is a high-molecular-weight polysaccharide which is commercially prepared by fermentation process using microorganism Xanthomonas campestris. Xanthan consists of glucose, mannose, and glucuronic acid. It is a good viscosity modifier and used as a stabilizer to avoid the segregation of components in a mixture (Franceschini et al. 2012; Bassetto et al. 2008; Kumar et al. 2018).
3
Processing and Modifications of Polysaccharides
Polysaccharides are emerged as potential and ideal candidate toward biomimicry of the native extracellular matrix (ECM). Particularly, microbial polysaccharides are considered better candidate due to their economical and sustainable benefits in fast and high-yield production processes. Therefore, microbial polysaccharides show an increasing potential in addition to plant- and animal-origin polysaccharide-based biomaterials in tissue engineering or surgical applications. However, they do not interact with human tissues biologically that limits their translation into model scaffold for in vitro/in vivo tissue regeneration. To overcome this, these polysaccharides should undergo modification through physical or chemical or in combination, enzymatic, and biological methods to improve the gelation behavior or functional
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properties of the hydrogels/scaffolds. Further, the mixing of two or more polysaccharides/polymers facilitates in different characteristics and functional properties as compared to the properties of single polysaccharide/polymer in hydrogel network (Ng et al. 2020; Ahmad et al. 2015; Karaki et al. 2016). Therefore, the proper processing, modification, and characterization of polysaccharides are important factors in fabricating functional biomaterials for successful surgical applications. Microbial polysaccharides are gaining research interests in surgery due to their renewable nature, cost stability in production, and reproducible physicochemical properties of the microbial polysaccharides have shown a great potential as compared to the polysaccharides from plants (Sutherland 2001; Vanhaverbeke et al. 2003) and found applications in diverse fields in industrial and biomedical applications. Microbial polysaccharides can be categorized into three ways according to their particular morphological existence as: (1) intracellular, (2) cell wall, and (3) extracellular polysaccharides. Further, these polysaccharides can also be classified in two ways: (1) homo-polysaccharides and (2) hetero-polysaccharides (Ahmad et al. 2015; Mollet 1996). Homo-polysaccharides possess only one sugar unit, whereas hetero-polysaccharides possess two or more sugar units (Mollet 1996). These polysaccharides can have various lengths of side chains in some structures along with the existing linear molecular chains and lead to a broad range of possible complex shapes and architectures. In addition, the association of these polymeric chains having high molecular weight leads to the chain entanglements in complex network and thereby the physical properties in double-stranded (e.g., xanthan gum, gellan gum, etc.) or triple-stranded (e.g., schizophyllan) helical form (Atkins 1986; Sutherland 1994; Nishinari and Takahashi 2003). Therefore, depending on chemical and physical properties, the processing and the modification of polysaccharides can be decided for surgical applications. Among microbial polysaccharides, gellan gum, xanthan gum, dextran, pullulan, and bacterial cellulose are considered and used extensively in biomedical applications. Further, polysaccharides-based hydrogels lack of flexibility in their main molecular chains that lead to the higher viscosities (Ahmad et al. 2015; Morrison et al. 1999). Moreover, in surgical applications, hydrogels in the form of adhesive or glues or in other forms are expected to deliver desired components for proper tissue regeneration without affecting other properties. Here, some microbial polysaccharides, including their combinations with other polysaccharides, are discussed for their processing and modifications to achieve improved properties for tissue regeneration in surgical applications.
3.1
Bacterial Cellulose
Bacterial cellulose (BC), prepared by aerobic bacteria, has shown a great potential in tissue engineering applications due to its high values of purity, porosity, high water holding capacity, and mechanical strength, including excellent biocompatibility. With this objective, various processing and modification approaches have been used for different focused surgical applications (Pang et al. 2020a).
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Fig. 2 Digital images of freshly prepared BC (a), dried BC (b), hydrated BC (c), PD-coated BC (d), BC-PDAg nanocomposite (e), and dried BC-PDAg nanocomposite (f). (Reproduced with permission from Elsevier, Jiji et al. 2020)
In a study, BC graft myringoplasty showed good results and can provide a rapid and safe surgery under local anesthesia in outpatient clinic within shorter time of surgery with better hearing and healing as compared to fat graft myringoplasty and temporalis fascia graft myringoplasty (Mandour et al. 2019). In another study for hernia repair surgery, the modification of BC with CS provided improved results as surgical meshes (BCS) for facilitating better absorption in native tissue with reduced risk of mesh-related infections. To analyze immunological responses due to hypersensitivity to the implants in rats, three types of meshes were used as (a) propylene mesh, (b) modified with BC only, and (c) BCS mesh for 1 and 3 months after intramuscular implantation. Here, BCS showed lowest immune response and highest degree of fibroplasia for 1 and 3 months of implantation. Moreover, toxicological analyses for BCS demonstrated negligible inflammation with no signs of sensitization or allergic responses (Piasecka-Zelga et al. 2018). Jiji et al. developed polydopamine (PD)-coated BC with silver nanoparticles (AgNPs) for third burn wound healing (Fig. 2). Here, AgNPs were successfully synthesized into BC matrix through catecholic redox method. This BC-PDAg nanocomposite exhibited an excellent biocompatibility, antibacterial activity against both gram-positive and gram-negative bacteria, and fast in vivo burn wound healing without scar formation in rat. Here, nanocomposite promoted re-epithelization and COLL deposition and gene expression analyses exhibited the upregulation of IL-10, VEGF-A, VEGF-B, and bFGF, while suppression of IL-1α and IL-3 transcripts. In addition, expression of TGF-β3 and SMAD-3 revealed the promotion of scarless wound healing (Jiji et al. 2020). Enhancement in moisture-holding capacity or moist environment is a key factor in applying and economically preferable extended wear time. With this objective,
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Fig. 3 Preparation method of BC/SA composite dressings (a) and digital image of BC/SA composite dressing showing flexibility (b). (Reproduced with permission from Elsevier, Sulaeva et al. 2020)
BC dressing was impregnated with sodium alginate as second hydrophilic domain (see Fig. 3). The developed crosslinked BC-SA composite dressing exhibited enhanced water retention properties through facilitating smooth dressing exchange in a wound-imitating model (Sulaeva et al. 2020).
3.2
Dextran
As demanding needs, an injectable gel or surgical adhesive to be used in different situation, in stopping bleeding and fastening tissues well together, is a major challenge. Further, the developed adhesive should also be biocompatible, biodegradable, nontoxic, economically viable, and easy to use in complex surgical circumstances or locations. With this objective, an injectable enzymatically crosslinked dextran tyramine (Dex-TA)/heparin tyramine (Hep-TA) hydrogels were prepared and showed good cell viability and proliferation followed by the improvement in the production of chondroitin sulfate and significant collagen as compared to only Dex-TA hydrogel (Jin et al. 2011). In another approach, Dex was oxidized by sodium periodate (NaIO4) and then developed a DDA sponge with appropriate absorption capacity of blood (47.7 g/g) and strong tissue adhesion property (~100 kPa) for hemorrhage control (Liu et al. 2019).
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With this DDA, Araki et al. mixed DDA and poly(l-lysine) (PLL) to prepare a DDA/PLL hydrogel glue as surgical sealant and compared with a conventional fibrin glue. The obtained DDA/PLL glue (mean bursting pressure: 38.4 4.6 cm H2O) was found to be more effective in sealing of pulmonary air leakage than fibrin glue (mean bursting pressure: 32.1 4.5 cm H2O). Further, no adhesions or infections were observed in the application areas of the glue and degraded nearly 90% within 3 months, but complete degradation was not observed till 6 months as the fibrin glue. The product exhibited a strong sealing behavior and good biocompatibility to be used in lung surgery (Araki et al. 2007). In addition to this study, PLL (-NH2) was modified by acylation and prepared DDA/a-PLL bioadhesive via Schiff base reaction. Further, the mechanical (from 120 Pa to 20 kPa) and degradation properties of bioadhesive DDA/ a-PLL were controlled by the oxidation degree of Dex and the concentration or type of anhydride species in the a-PLL (Matsumura et al. 2014). It is worth to note that self-crosslinking (Schiff base reaction) in hydrogels is more advantageous over the external chemical crosslinker, but the mechanical properties of the hydrogels can be enhanced further by incorporating nanoparticles. In a study by Pang et al., for self-crosslinking reaction, dextran as microbial polysaccharide was oxidized by using sodium periodate (NaIO4) to incorporate dialdehyde functional groups as dextran dialdehyde (DDA) and chitosan was modified with carboxymethylation by using chloroacetic acid. Further, chitin nanowhiskers (ChtNWs) were prepared by sulfuric acid and incorporated into selfcrosslinked carboxymethyl chitosan (CMCS)/oxidized dextran through Schiff base reaction that led to a mechanically strong tissue adhesive (Fig. 4). The optimal composition of complexed CMCS-DDA/ChtNWs hydrogel with ChtNWs showed 1.87-fold higher compressive strength as compared to noncomplexed hydrogel and 1.51 times higher adhesive strength on porcine skin. Also, this hydrogel system showed negligible cytotoxicity, optimum antibacterial and hemostatic abilities, in vivo degradability without long-term inflammatory responses, and could prevent severe postoperative adhesion and necrosis in case of wound repair (Pang et al. 2020b). Similarly, in another study, N-carboxyethyl CS (CECS) was prepared by the modification with acrylic acid. Then this CECS and DDA were used to prepare in situ gelable hydrogels at physiological pH and body temperature (37 C). The developed DDA/CECS gels showed improved wound healing when applied to mice full-thickness transcutaneous wound models (Weng et al. 2008). Du et al. designed and prepared multifunctional injectable and self-healable hydrophobically modified CS (hmCS)/DDA hydrogels with excellent hemostatic, antibacterial, and accelerated wound healing performances. Here, CS was hydrophobically modified with dodecyl aldehyde through Schiff base reaction and then prepared a tissue adhesive hmCS/DDA hydrogel that showed good potential for hemorrhagic and infected wound healing. The self-healing process, rheological properties, injectability, and in vivo gelation of the hydrogel are shown in Fig. 5 (Du et al. 2019). In another study, photocrosslinkable tissue adhesive based on Dex was prepared. Here, first urethane dextran derivative was prepared by incorporating
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Fig. 4 Modification of CS to CMCS (a) and Dex to DDA (b), TEM image of CtNWs (c), schematic and digital image of complexed CMCS-DDA/CtNWs hydrogel formed by the extrusion by using double-barrel syringe (d). (Reproduced with permission from Elsevier, Pang et al. 2020b)
Fig. 5 (a–d) Digital images of self-healing process, (e) rheological properties, (f) injectability, and (g) in vivo gelation behavior of the hmCS/DDA hydrogel. (Reproduced with permission from Elsevier, Du et al. 2019)
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2-isocyanatoethyl methacrylate (IEMA) onto dextran (Dex-U) that could be photocrosslinked under UV irradiation and progressively adhere to the surface of gelatin and mouse skin that encourage development of the human tissue. The highest adhesive strength (2.99 MPa) and burst pressure (35 mmHg) were observed, where adhesive strength was found to be higher than that of fibrin glue (Li et al. 2011). In addition to this study, a photocrosslinkable tissue adhesive composed of oxidized urethane dextran (Dex-U-AD) and gelatin was developed. Here, Dex-U was oxidized by using NaIO4 that led to the final polymer as Dex-U-AD. The obtained Dex-U-AD was processed with gelatin to form hydrogel through Schiff base reaction followed by photocrosslinking under ultraviolet (UV) irradiation. The results showed good adhesive property to the surface of gelatin that simulated the human tissue in a control manner and maximum adhesion strength could be observed to 4.16 0.72 MPa that was higher than that of fibrin glue significantly. Moreover, Dex-U-AD/gelatin gels were observed to be nontoxic and could be used as good tissue adhesive biomaterial (Wang et al. 2012). Further, a facile in situ approach was presented to prepare anionic inter-polymeric complex polysaccharide biomaterial, where sodium alginate (SA) and kappa-carrageenan (k-CG) were treated with silver salt and formed in soft nano-floral inter-polymeric complex with selfstability. The obtained polymeric complexes exhibited good resistance toward S. aureus and E. coli bacterium, and improved exudate absorption, thereby promoting effective proliferation and evolving the compact fibrous network and hair follicles (Zia et al. 2020). In another study, amine-modified succinyl CS was prepared and used with DDA for preparing a series of hydrogels. The properties and rate of preparation of the hydrogel were dependent on the level of both amine and aldehyde-based precursors. By monitoring the reaction conditions, these levels could readily be altered and allowed good control over the gel properties. These gels exhibited excellent hemostatic properties and adhesion reduction in animal models (Liu et al. 2009). In another study, thiolated-Dex (Dex-SH) was modified by thiolation reaction and prepared an in situ crosslinked hydrogel composed of Dex-SH and vinyl sulfone modified Pluronic 127 (PL-VS) or acrylated Pluronic 127 (PL-Ar) through Michaeltype addition reaction. The results showed rapid formation of hydrogel in situ and exhibited a wide range of storage moduli (from 0.3 to 80 kPa) and thermosensitive behavior (from 10 to 37 C), when increasing concentration of PL from 5% to 20 w/v %. Moreover, Dex-SH/PL-Ar hydrogels were observed to be degradable at physiological conditions and exhibited lower cytotoxicity compared to Dex-SH/PL-VS hydrogels (Lin et al. 2010).
3.3
Gellan Gum
Learmonth et al. developed a dopamine-modified gellan gum hydrogel (DGG) with enhanced physicochemical and biological properties, suitable for minimally invasive cell delivery and retention in terms of cartilage repair. The developed DGG hydrogels underwent rapid ionic crosslinking under physiologically relevant
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mono- or divalent cations to prepare stable 3D hydrogels with excellent tissue adhesiveness. In addition, DGG hydrogels maintained mammalian cell viability and promoted upregulation of the expression of healthy chondrogenic ECM markers upon stimulation (Learmonth et al. 2020). In tissue regeneration, macrophages serve a critical role in regulating the host response to implanted materials. The macrophage phenotype tends to be dynamic throughout the host response and this phenotype should be balanced for favorable progression from injury to actual wound healing. Therefore, methacrylated GG (MGG) hydrogel was prepared and demonstrated the effect of hydrogel on macrophage phenotype and proliferation. Here, MGG hydrogels were prepared with various thiol-ene (SH/C¼C) and different crosslinking mechanisms (crosslinked with chain growth or step growth or in combination) (Li and Bratlie 2019). In another study, GG was grafted with cinnamate (Cin) to prepare photocrosslinkable polymer (GG-Cin) under UV irradiation, where 14.7% of D-galacturonic residues of GG were reacted with Cin and exhibited maximum absorption at 254 nm. GG-Cin showed 82% crosslinking efficiency after 16 min of UV irradiation in preparing antiadhesion GG-Cin films. The prepared films showed high gel contents (88 2%), appropriate mechanical properties, and highly promising antiadhesion activity, when implanted into rats, in two rats from ten rats without forming any tissue adhesion. Also, film could inhibit inflammatory response significantly in rats (Lee et al. 2012).
3.4
Carrageenan
To eliminate wound infection and accelerated wound healing on wound area, kcarrageenan (k-CG)-based sprayable adhesives with multifunctional properties was developed. Here, k-CG was modified by the methacrylation reaction to prepare methacrylated k-CG (k-CGMA). Further, zinc oxide nanoparticles (ZnO NPs) were synthesized by using aloe vera leaf extract and then modified with polydopamine to prepare ZnOPD NPs. Finally, visible-light crosslinked hydrogel was prepared by using k-CGMA and ZnOPD NPs. Furthermore, L-glutamic acid was added to this nanocomposite hydrogel network to accelerate wound healing. The results showed significant improvement in tensile strength (from 64.1 10 to 80.3 8 kPa) and elongation (from 20 4% to 61 5%) of nanocomposite hydrogel with the addition of 1 wt.% ZnOPD NPs. In addition, effectual blood clotting capacity and biocompatibility (more than 95% cell viability) was observed after 3 days of cell culture. L-glutamic acid in hydrogel exhibited significant acceleration in wound healing with superior granulation tissue thickness as compared to control in a full-thickness skin defect model (Tavakoli et al. 2020). In another study, an injectable hydrogel composed of k-CG and C-phycocyanin was developed through ionic crosslinking. Here, a pigmented protein as C-phycocyanin was used with k-CG due to its antimicrobial, antioxidant, anti-inflammatory, wound healing properties as well as fluorescence imaging ability in vivo. The developed injectable hydrogel exhibited good hydrophilic nature, mechanical stiffness, and improved in vitro proliferation of dermal fibroblasts without
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creating inflammation, including reduction in blood clotting time with no hemolysis. Moreover, hydrogel showed superior hemostatic abilities in traumatic injury situation and rapid wound healing process (Dev et al. 2020). Tytgat et al. prepared 3D printed scaffolds composed of methacrylated k-CG and methacrylated gelatin (GelMA). Here, k-CG and Gel were modified with methacrylic anhydride according to their alcohol and amine moieties. Then both modified polymers were blended to prepare hydrogel inks and printed hydrogel structures by using extrusion-based 3D printing. The developed only GelMA and Gel-MA/k-CGMA printed scaffolds were observed to be stable over time for 21 days, absorb high amounts of water, and showed mechanical performances comparable to that of native breast tissue (2 kPa). Further, both hydrogel scaffolds showed a similar adipose tissue–derived stem cell viability and proliferation rate after 14 days of incubation. Moreover, cells were able to be differentiated into adipogenic lineage on hydrogel scaffolds, but lower on Gel-MA/k-CGMA printed scaffold compared to only GelMa printed scaffold (Tytgat et al. 2019). In another study, dopamine-modified graphene oxide (GOPD) was used with k-CGMA and prepared injectable nanohybrid hydrogels. Here, shear-thinning property and injectability through the interaction of active catechol functional groups of PD with other moieties in hydrogel structure further promoted the mechanical properties of hydrogels and significant improvement in compressive strength (8 times), toughness (6 times), and recovery of the hydrogels was observed in case of 20 wt.% GOPD reinforcement. Furthermore, k-CGMA/GOPD20% hydrogel improved fibroblast cell proliferation and spreading was observed after 5 days of cell culture (Mokhtari et al. 2019).
3.5
Pullulan
PLN hydrogel (10%) was used in sutureless wounds in rats for analyzing its healing efficacy. Here, a PLN hydrogel-treated incision wound in rats was healed within 6 days, while wounds in positive control and control rats took 11 and 15 days, respectively, in complete healing (Fig. 6). Also, two times increase in tensile strength (3.63 MPa) was observed in PLN hydrogel-treated wounds as compared to positive control (1.34 MPa) or control (1.17 MPa) wounds. Further, more than 25% increase in shrinkage temperature was also evaluated as compared to control. Moreover, improved fibroblast cell proliferation followed by faster epithelialization of wounds in PLN hydrogel-treated rats was demonstrated (Priya et al. 2016). To improve the properties of PLN, Choudhary et al prepared a composite hydrogel composed of GG and pullulan (PLN), without any chemical functionalization, and the developed GG/PLN hydrogel exhibited fast setting and maintained its elasticity at high frequency and temperature (Choudhury 2019). In another study, PLN was oxidized by using NaIO4 and chondroitin sulfate (CHS) was modified with adipic dihydrazide, and both were used for self-crosslinked and injectable oxPLN/ CHS-ADH hydrogels. This oxPLN/CHS-ADH hydrogel with 7/3 weight ratio could provide a best tissue-mimetic 3D microenvironment and maintained chondrocyte cell phenotype and improving chondrogenesis (Li et al. 2018). To prevent or reduce
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Fig. 6 Digital images showing healing rate in sutureless wounds. (Reproduced with permission from Elsevier, Priya et al. 2016)
postoperative abdominal adhesions is a major concern, where adhesion causes serious complications (e.g., postoperative pain, intestinal obstruction, and infertility). In recent times, various form of tissue adhesion barriers such as sprays, films, membranes, hydrogels, and knits have been developed. Among them, hydrogels possess various advantages and especially injectable hydrogels can fill and cover all spaces of any shape and do not need a surgical process for implantation. In this way, Bang et al. modified PLN with the reaction of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) to incorporate carboxyl groups followed by the addition of tyramine on carboxylated PLN with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) and N-hydroxyl-succinimide (NHS) to incorporate phenyl functional groups as crosslinking sites. Further, tyramine-grafted PLN-based hydrogel was prepared by an enzymatic reaction through horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). PLN-Tyr hydrogel showed significant inhibition of cell proliferation and exhibited proper prevention of abdominal tissue adhesion (in an animal model) (Bang et al. 2016). The control over resolution of hydrogel features, modification, and mechanical performances is very beneficial for providing possible natural tissue niches on cell growth and differentiation. With this aim, PLN-based cell adhesive hydrogel with
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3D printable in different dimensions and tunable mechanical properties was processed through stereolithography technique. Here, methacrylated PLN (PLN-MA) was synthesized and printed by using multiscale light-assisted 3D printing techniques as visible stereolithography (SL) and two-photon lithography (TPL). Further, complex 3D shapes through spatially controlled irradiation (SL) were printed and mechanical properties were controlled by incorporating a bifunctional crosslinker and also enabled water absorption capacity of PLN-MA hydrogels (Della Giustina et al. 2019).
3.6
Xanthan Gum
Xanthan gum (XG) has widely been applied in different industrial (e.g., food and food packaging, paints, and cosmetics) and biomedical (drug delivery and tissue engineering) applications. Due to some disadvantages in processing and mechanical properties of only XG, it needs appropriate modification or processing to be used efficiently in surgical applications (Kumar et al. 2018). XG-based bilayered mucoadhesive buccal patch with zolmitriptan drug was developed. Here, hydroxypropyl methylcellulose E-15 to improve film-forming ability and polyvinyl alcohol (PVA) to enhance the tensile strength of the mucoadhesive patches were used. The results exhibited modified rate of drug release and good bioadhesion due to XG and bilayered patches may prevent hepatic metabolism in a large extent possible (Shiledar et al. 2014). Generally, the resulting adhesion between surgical tendon and synovial sheath tends to cause poor functional repairing after tendon repair surgery. Seprafilm, a commercial product composed of hyaluronan (HA) and CMC hydrogels, has been used to prevent this adhesion during surgery. With this issue, Kuo et al. developed a new composite membrane composed of XG, GG, and HA and analyzed in a rat model (Kuo et al. 2014). Membranes of different formulations of XG/GG/HA (XGH) as well as Seprafilm were wrapped around repaired tendons and evaluated grossly and histologically after healing for 3 weeks. Certain designed XGH hydrogel membranes decreased tendon adhesion with equal ability without reducing the tendon strength as compared to Seprafilm. These membranes rapidly swelled and more readily and closely blanketed onto tendon tissue with slow degradation than that of Seprafilm (see Fig. 7) (Kuo et al. 2014). After abdominal surgery, postoperative adhesion is a significant challenge. With this objective, various polymeric barriers have been applied to prevent adhesions, but complete prevention of adhesion was not observed in all situations. Therefore, Song et al. developed new XG-based biomaterial and investigated the estimation of its potential as injectable tissue adhesion barrier with various concentrations (0.5–2.0% w/v) and molecular weight (2.5 106 – 6.9 106 Da). Here, XG provides an antiadhesion property in the rat abdominal cavity. One percent of XG gel having high molecular weight (6.9 106 Da) was observed to be more effective to prevent adhesions as compared to commercial product (1.2% HA). In addition, XG gel exhibited no cytotoxicity in vitro to L929 cells and no side effect during
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Fig. 7 Animal model: (a) Achilles tendon exposed by sharp dissection, (b) transected Achilles tendon (midpoint), (c) tendon treated with 5-0 Prolene sutures, (d) postrepaired tendon wrapped with XGH membrane (smooth fitting on tendon), and (e) tendon wrapped with Seprafilm (irregular deformation). (Reproduced with permission from Elsevier, Kuo et al. 2014)
wound healing. Moreover, XG showed good potential in preventing intra-abdominal adhesion. In this study, intra-abdominal adhesions were divided into two groups as insubstantial (grade 0 and 1) and substantial (grade 2–4) adhesions (Fig. 8). Here, substantial adhesions were difficult to separate and considered clinically significant high-grade adhesions (Song et al. 2019).
3.7
Glucan
Intra-abdominal infections are associated with the deposition of fibrin, which may result significant abscess formation and adhesions clinically. In this way, several agents have been applied in experimental and clinical studies to prevent postoperative adhesions. Further, nonspecific immune-stimulant beta-glucan was used for the reduction of intra-abdominal abscesses and adhesions. Here, beta-glucan did not have significant effect on mortality and abscess formation. However, it was capable in reducing the adhesion frequency (Bedirli et al. 2003). In another study, a new functional wound dressing composed of CS-glucan complex (GC) hollow fibers reinforced collagen (COLL) embedded with aloe vera (AV) to be used in especially infected chronic wound and ulcers for skin regeneration. Here, CSGC hollow fibers were prepared from mycelium beads and then AV aqueous solution was mixed with CSGC hollow fibers under high-speed homogenizer (12,000 rpm) to prepare CSGC@AV and further was mixed with COLL to prepare CSGC@AV/COLL suspensions, which were homogenized, centrifuged, and freeze-dried to prepare functional wound dressing (Abdel-Mohsen et al. 2020).
4
Surgical Applications of Polysaccharides
4.1
Orthopedic Surgery
Different surgical sealants having higher adhesion and bonding strength have been used for joining two surfaces in orthopedic surgery. An ideal sealant for orthopedic surgery should promote bone healing, should not hamper blood circulation in
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Fig. 8 Digital images of intra-abdominal adhesions with adhesion severity stages on postoperative day 8: stage 0 (a), stage 4 (b), stage 1 (c and d), stage 2 (e and f), and grade 3 (g and h). (Reproduced with permission from Elsevier, Song et al. 2019)
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musculoskeletal tissue, and prevent gap formation in bones. Strehin et al. prepared chondroitin sulfate/polyethylene glycol (CS/PEG)-based tissue adhesive for cartilage surgery (Strehin et al. 2010). Here, chondroitin sulfate succinimidyl succinate (CS-NHS) was reacted with six arm polyethylene glycol amine (PEG–(NH2)6). The properties of the developed gel could be tuned by altering the polymer ratio and degree of NHS functionalization (Strehin et al. 2009, 2010). The gels showed ten times higher adhesive strength than fibrin glue and their stiffness and swelling properties could be adjusted by tuning the pH of starting materials. The gels displayed good sealing properties when used for treating collagen membranes in rats. Wang et al. synthesized methacrylate and aldehyde functionalized chondroitin sulfate which was used as a sealant (Wang et al. 2007). This sealant could effectively form chemical bonds with tissue proteins and the incorporated photoinitiator led to the hydrogel formation. It was reported that the developed sealant could effectively achieve strong tissue adhesion and proteoglycan production when tested in vivo in rat model for cartilage tissue repair. Furthermore, Simson et al. fabricated bone marrow and chondroitin sulfate–based adhesives for meniscus tissue repair (Simson et al. 2013). Different adhesives samples were prepared by varying the ratio of bone marrow to chondroitin sulfate. It was found that bone marrow component helped in achieving fibrochondrocyte viability and its migration. On the other hand, chondroitin sulfate was useful for achieving a wide range of adhesive strength (60 17 to 335 88 kPa). The adhesives were tested in athymic rats and were found suitable for fusion of adhered meniscus. Li et al. synthesized chondroitin sulfate modified with glycidyl methacrylate to develop photocrosslinkable gels showing their high potential for cartilage tissue repair (Li et al. 2004). Chou et al. synthesized gelatin/hyaluronic acid/chondroitin-6sulfate (GHC6S) tri-copolymer particles which were subsequently added to fibrin glue (Chou et al. 2007). The addition of these particles helped in achieving higher mechanical strength, efficient cell seeding, and distribution. The developed glues were tested to repair a damaged articular cartilage showing improved cartilage regeneration in vitro. For orthopedic and maxillofacial surgical applications, Fricain et al. prepared PLN/Dex scaffolds with or without nanohydroxyapatite (nHAp) (Fricain et al. 2013). In this study, in vitro analyses exhibited the formation of multicellular aggregates and expression of early and late bone-specific markers with human bone cells in the medium with deficient of osteoinductive factors. Further, only without cell-seeded PLN/Dex-nHAp scaffold showed maintained subcutaneously local growth factors, induced deposition of biological apatite formation, and favorable subcutaneously formation of a dense mineralized tissue in mice and well osteoid tissue after intramuscular implantation in goat. Further, the developed scaffold was implanted in three orthopedic preclinical models of critical size defects (e.g., small and large animals) in rat and goat. This scaffold induced highly mineralized tissue in all three models whatever the site of implantation, including osteoid tissue and regeneration of bone tissue (Fricain et al. 2013).
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Neural Surgery and Plastic Surgery
Recently, polysaccharide-based biomaterials are gaining the attention for being used as surgical glue in neural and plastic surgery. Ereth et al. performed a comparative study to determine the safety and hemostatic efficiency of different commercially available formulations in neurosurgery (Ereth et al. 2008). In this study, a brain tissue defect was created in 228 rats followed by application of different hemostatic agents such as starch-based Arista AH, oxidized cellulose-based Surgicel, collagen-based Avitene, gelatin-based FloSeal, and kaolin. The rats were sacrificed and investigated at different time intervals and presence of residual material and foreign body reaction was determined. It was reported that Arista AH was not only able to stop bleeding efficiently buy also to degrade quickly without causing any inflammation. Franceschini et al. used ORC (Tabotamp ®) for breast conservative oncoplastic surgery (Franceschini et al. 2012). It was observed that ORC applied after oncoplastic surgery allowed not only to reduce the bleeding but also to lower the rate of infection at the surgical site. It also helped in optimizing cosmetic defects especially in small–medium size breast. Bassetto et al. reported the use of ORC (Tabotamp®) for hemostasis while performing a facelift procedure (Bassetto et al. 2008). It was showed that no local pain or skin damage was observed post-operation.
4.3
Wound Healing and Miscellaneous Applications
Wound healing requirements and challenges have led to the preparation of various tissue scaffolds or adhesives to resolve the problems associated with conventional sutures and staples for their time-consuming, tissue damage, immunological response, and insufficient reduction of fluid leakage from the wounds (Balakrishnan et al. 2017). With this objective, MGG hydrogels were developed and good cytocompatibility and an improved anti-inflammatory production of nitrites from naïve and classically activated macrophages by stiffer biomaterials as compared to the softer biomaterials. Further, arginine and CD206 expression markers for alternatively activated macrophages were inhibited by higher thiol content. Moreover, the introduced ionic crosslinking by using calcium ions (Ca2+) had no influence on the proliferation or polarization for any of three macrophage phenotypes (Li and Bratlie 2019). Further, tyramine-modified carboxymethyl cellulose (CMC) (CMC-Tyr) was prepared and then injectable hydrogel was synthesized in situ by using an enzyme-mediated reaction of CMC-tyr with HRP and H2O2. Further, PLN was added to CMC-Tyr hydrogel solution to enhance adhesiveness to the wound area and acceleration of biodegradation. Moreover, in vivo analysis demonstrated significant reduction in postoperative tissue adhesion (Bang et al. 2017). In another study, Abdel-Mohsen et al. developed a functional CSGC@AV/COLL wound dressing that exhibited high hydrolytic stability with improved swelling properties, enhanced hemostatic effect, and excellent biocompatibility on human dermal fibroblast cells as compared to native COLL. Further, high antibacterial behavior against various bacteria (gram-positive/gram-negative) was observed by this functional
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CSGC@AV/COLL wound dressing. Moreover, an improved percentage of wound closure was observed after 1 week treatment. This developed wound dressing showed good potential to be used in especially infected chronic wound and ulcers for skin regeneration (Abdel-Mohsen et al. 2020). In recent years, different polysaccharides and their derivatives have been explored as surgical glues for various types of surgeries. Di Lello et al. used ORC for sutureless epicardial fixation of long aortocoronary saphenous vein grafts and compared its efficiency with fibrin glue (Di Lello et al. 1989). It was reported that (a) ORC gauze readily adheres to the surface wet with blood; (b) it has antibacterial properties; and (c) ORC gauze is more economical than fibrin glue. Chakoli et al. prepared ORC-based composites by reinforcing with multiwalled carbon nanotubes (MWCNTs) (Chakoli et al. 2014). Cellulose films and fibers were oxidized by using nitrogen dioxide and carbon tetrachloride. Further, ORC fibers and films were reacted with amine functionalized MWCNTs using 1-ethyl-3(3-dimethylamino-propyl)-carbodiimide (EDC)/N-hydroxyl-succinimide (NHS) system. The developed composites were crosslinked using glutamic acid. The incorporation of MWCNTs led to enhanced hydrophilicity and hemostatic efficiency of ORC gauze. Cheng et al. developed hemostatic composite gauze consisting of carboxymethyl chitosan and ORC (Cheng et al. 2016). It was reported that these composite gauzes not only show enhanced hemostatic behavior in rabbit liver injury model but also good antibacterial activity against both gram-positive and gramnegative bacteria. The animal experiments indicated that the composite gauze could stop the bleeding in 90 s showing rapid coagulation as compared to pure ORC which took 4 min to achieve efficient hemostasis. These composite gauzes are also beneficial for postoperative adhesion prevention. Hoffmann et al. investigated the peritoneal adhesion after the use of different hemostatic agents in intra-abdominal surgeries (Hoffmann et al. 2009). It was reported that starch (Arista AH) and polyethylene glycol (CoSeal)-based hemostatic agents could effectively reduce the peritoneal adhesion. Arista AH did not leave any residual agent and showed no difference in acute inflammation when compared to other commercially available hemostatic agents such as FloSeal, Bioglue, and Tisseel. Zhu et al. synthesized Ca2+-modified crosslinked porous starch microparticles (CPSM) which were used as a hemostatic agent in the mouse tail amputation model (Zhu et al. 2019). The fast release of Ca2+ from porous particles led to enhanced platelet adhesion resulting into faster clot formation. It was reported that incorporation of Ca2+ played a pivotal role in blood clot formation via facilitating conversion of prothrombin into thrombin and crosslinking of fibrin (Zhu et al. 2019). Sharma et al. investigated the efficiency and safety of surgicel for the creation of neovagina in 10 patients (Sharma et al. 2007). It was observed that the use of surgicel led to minimum complications, morbidity, and discomfort with 8 out of 10 patients showing satisfactory results. Finley et al. reported the use of fibrin glue–oxidized cellulose sandwich for hemostasis during laparoscopic wedge resection of small renal tumors (Finley et al. 2005). Excellent hemostasis was achieved in all the 15 cases during May 2002–December 2003 omitting the requirement of blood transfusion.
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Ono et al. developed chitosan-based photocrosslinked gels to be used as a biological adhesive for soft tissues (Ono et al. 2000). In that work, solubility of chitosan was enhanced by treatment with lactobionic acid which introduced the lactose moieties in the polymer structure. Further, azide moieties were introduced by reacting it with 4-azidobenzoic acid, thus enabling photocrosslinking. The binding efficiency of developed gel was investigated against air leakage from the pinholes on trachea in rabbits showing their potential as tissue adhesive. The efficiency of developed system was compared with fibrin glue and it was observed that chitosan-based adhesives could be applied more easily without any spill (Ono et al. 2000). Similarly, Ishihara et al. reacted chitosan with p-azidebenzoic acid and lactobionic acid to develop sample that could be converted into gel under UV light (Ishihara et al. 2002). These gels could not only stop bleeding from mouse tail within 30s of UV irradiation but also attach two skin parts with each other in mouse model showing their great potential as tissue adhesive and hemostatic agent. Furthermore, Dowling et al. reported the formulation of sprayable foams (Fig. 9a) based on hydrophobically modified chitosan (hmC) for treating noncompressible hemorrhage (Fig. 9b) (Dowling et al. 2015). Here, hemostatic action was achieved by converting the blood into self-supporting gel where hydrophobic chains are inserted into the blood cells bilayer thus creating a network of cell clusters. The developed foam was tested in vivo to treat the liver injury in pig model. It was observed that hemostasis could be sustained for upto 60 min without any compression and total blood loss was 90% lower as compared to the control samples. Development of the sprayable foam able to work without compression paves a new pathway for treating traumatic injury caused to soldiers in combat as well as to mankind in serious accidents (Dowling et al. 2015). Lih et al. designed tyramine-modified polyethylene glycol which was further grafted on chitosan by reacting with amine groups (Lih et al. 2012). As a next step, tyramine moieties were crosslinked with the help of horseradish peroxidase and H2O2 resulting into hydrogels which showed excellent tissue adhesive properties when tested in rat skin model. Hyaluronic acid has been often reacted with methacrylic anhydride for introducing methacrylic groups which can be further polymerized under the presence of light (Smeds et al. 1999). Photopolymerization of methacrylate functionalized hyaluronic acid results into hydrogel formation. Various mechanical properties (e.g., swelling, compression, and creep resistance) of these hydrogels can be tuned by adjusting the degree of methacrylation which in turn affects the extent of covalent bonding in the hydrogel system (Smeds and Grinstaff 2001). Miki et al. reported that these methacylated hyaluronic acid–based hydrogels can be used for sealing a 3 mm corneal laceration in rabbits (Miki et al. 2002). It was found that the hydrogel patch helped in proper sealing of corneal perforations and normal intraocular pressure was achieved within 7 days. The applied hydrogel showed minimal inflammation, higher proliferation of stromal cells, and caused tight adherence of extracellular matrix. Further, sodium salt of hyaluronic acid (Seprafilm ®adhesion barrier) has been used to prevent abdominal adhesion after surgery in hernia treatment and intestinal resection (Kramer et al. 2002; Fazio et al. 2006).
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Fig. 9 Schematic representation of hemostatic action of sprayable foam (a); hemostatic foam consisting of hydrophobically modified chitosan (hmC) and AB-46 propellent (b). (Reproduced with permission from American Chemical Society, Dowling et al. 2015)
Reyes et al. reacted chondroitin sulfate aldehyde with polyvinyl alcohol-co-vinyl amine to develop tissue adhesives for repairing penetrating corneal wounds (Reyes et al. 2005). As the repair of such wounds requires watertight sealing, the efficiency of developed adhesives was compared with sutures. The adhesive was tested ex vivo in enucleated rabbit eyes for treating corneal incision. It was reported that maximum intraocular pressure (104.7 mm Hg) could be achieved by using the adhesive. It showed no leakage of the balanced salt solution that was used to fill the globes and was reported to be much more efficient in avoiding the leakage than sutures. Furthermore, Pirouzmanesh et al. used chondroitin sulfate–based tissue adhesive and sutures for investigating comparative graft stability of donor eyes and astigmatic changes (Pirouzmanesh et al. 2006). Here, less blurred vision was reported in case of
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chondroitin sulfate–based tissue adhesive making them an attractive alternative for surgery of corneal endothelial disorders. Further, dextran is oxidized by reacting with sodium periodate resulting into the formation of dextran aldehyde which in turn can be reacted with amines to get degradable imine bonds. Bhatia et al. developed dextran aldehyde–based surgical glue and investigated the influence of different degree of oxidation (Bhatia et al. 2007a) It was reported that the surgical glues formed by dextran aldehyde having degree of oxidation higher than 60% exhibited very rapid crosslinking leading to inefficient tissue binding. Fifty percent degree of oxidation was reported to be the most optimum with leakage pressure as high as 500 mm Hg. This natural polymerbased biomaterial degraded within 3 days during in vitro study. Here, the faster degradability was credited to the hydrolysis of imine bonds ensuring the clearance of by-products from the body. However, it is to be noted that due to the fast degradation higher amount of material was required for proper healing of corneal incision resulting into foreign body response. To address this issue, Chenault et al. designed a novel tissue adhesive based on dextran aldehyde (MW 10 kDa) which was reacted with PEG 8-arm star polymer (MW 10 kDa) having two amine groups per arm (Chenault et al. 2011). In vitro testing showed no cytotoxicity, higher bonding strength, and the leak pressure was found to be approximately 141 mm Hg. The efficiency of developed tissue adhesive was tested in vivo to seal the corneal incisions in New Zealand White rabbits. It was reported that the corneal incision was healed after 5 days without complete degradation of the adhesive during this time. This tissue adhesive is on its way to be available as commercial material Actamax™ by DSM and DuPont in a joint effort (Bouten et al. 2014). In an effort for increasing its shelf life, aldehyde-functionalized polyglycerol is being used as an alternative for dextran aldehyde (Chenault 2013). Postsurgical bleeding and undesired tissue adhesion are the two major challenges encountered after endoscopic sinus surgery. In this regard, Athanasiadis et al. investigated the potential of chitosan-/dextran-based hybrid gel in preventing unwanted tissue adhesion and supporting the mucosal wound healing (Athanasiadis et al. 2008). The investigation was performed in sheep model having chronic sinusitis. Severity of adhesion and healing was monitored at regular time intervals exhibiting chitosan/dextran gel as a potential alternative. Aziz et al. further explored the antimicrobial activity of chitosan-/dextran-based hydrogel for surgical application (Aziz et al. 2012). The bacteria killing efficiency of the hydrogel was tested against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Clostridium perfringens. The surgical concentration of 50,000 mg/L of hydrogel was found to be effective. Here, dextran aldehyde was found to be responsible for antimicrobial activity by disrupting cell walls resulting into loss of cytosolic components. Balakrishnan et al. fabricated dextran-based injectable tissue adhesives that can stop bleeding, bond the tissue effectively, and can be easily applied while performing a surgery (Balakrishnan et al. 2017). Here, dextran was treated with periodate to generate aldehyde groups which in turn could spontaneously react with chitosan hydrochloride via Schiff’s base formation. It was reported that the strength of the developed tissue adhesive is five times higher than that of commercially available
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fibrin glue. It was tested to seal the bleeding in rabbit liver injury and was found to be more tissue compliant than BioGlue ®. Further, Liu et al. reported dextran aldehyde– based adhesive sponge (pore size 30–50 μm) with high tissue adhesion properties to control hemorrhage (Liu et al. 2019). The sponge exhibited remarkable reduction in blood loss when tested for ear vein, femoral artery, and liver injuries in rats and rabbits. It was reported that the dextran-based sponge induced RBCs and platelets aggregation promoting blood coagulation along with sealing the wound, thus controlling massive blood loss. These sponges were biocompatible, biodegradable, and showed almost no skin inflammation. In another study, DDA-/PLL-based degradable glue via Schiff base formation prevents the air leakage in lung surgery (Araki et al. 2007). The efficiency of this glue was found to be higher than fibrin glue in preventing air leakage from large pleuroparenchymal defects. It was observed that almost 90% glue degraded within 3 months and the normal lung structure could be achieved within 6 months. Therefore, the product exhibited a strong sealing behavior and good biocompatibility to be used in lung surgery. Pang et al. synthesized dextran aldehyde (DDA)/carboxymethyl chitosan (CMCS) gels loaded with chitosan nanowhisker (CtNWs) (Pang et al. 2020b) The developed gel exhibited approximately two times higher mechanical strength and tissue adhesive strength. It displayed good hemostatic performance while simultaneously avoiding inflammation and postoperative adhesion when tested for rat liver injury, showing higher efficiency than commercially available 3M™ vetbond™ tissue adhesive. Liu et al. prepared a library of hydrogels with tunable hemostatic and adhesive properties by reacting amine functionalized succinyl chitosan and dextran aldehyde to generate imine linkage (Liu et al. 2009). Succinyl chitosan with enhanced water solubility was synthesized by reacting chitosan with succinic anhydride. The properties of developed gels could be tuned by varying the crosslinking degree showing their great potential for surgical use. Bhatia et al. synthesized DDA which was further reacted with eight-arm amine functional group containing polyethylene glycol crosslinker (Bhatia et al. 2007b) In vitro experimental results with 3T3 fibroblast cells and J774 macrophage cells showed that the developed tissue adhesive was biocompatible and did not cause inflammatory response. Similarly, Nie et al. created CS-/PLL-based gels which showed excellent adhesive and hemostatic properties (Nie et al. 2013). These gels exhibited four times higher adhesive properties than commercially available fibrin glue.
5
Conclusions
Polysaccharides are highly abundant natural polymers and can be obtained from three mainly different renewable resources such as plants, animal, and microorganisms. These polysaccharides provide more beneficial properties in surgical applications as compared to synthetic polymers due to their high availability and renewable nature, easy processing, excellent biocompatibility, and intrinsic immunogenic ability. Among them, microbial polysaccharides or their combination with other plant-/ animal-origin polysaccharides, including natural and/or synthetic nanomaterials,
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have received a great attention in surgical applications. However, the blending of microbial polysaccharides with other polysaccharides provides better properties as compared to single polysaccharide. Therefore, several modification approaches and processing techniques have been used for getting desired properties similar to that of native tissues. But, their successful use in surgical applications as native-like tissue regeneration is still challenging and further research is needed through a wide range of new processing and modifications. Therefore, in this chapter, a brief review of polysaccharides, especially focusing on microbial polysaccharides and their processing and modifications are presented in their promising application for surgical applications. Recently, these polysaccharides are extensively used in additive manufacturing methods (i.e., 3D printing or bioprinting) to produce tissue hydrogel scaffolds and/or tissue models for future surgical applications (Kumar et al. 2020; Zidarič et al. 2020; McCarthy et al. 2019). Here, the optimization of print-related challenges associated with polysaccharides, cells, printing techniques, and processing parameters is very critical to develop biomaterials with desired properties. Therefore, a comprehensive understanding of polysaccharides, cells, and printing techniques (e.g., 3D, 4D, and 5D printing) is needed to create more complex 3D bioprinted architectures and their clinical translation in surgical applications (Kumar et al. 2019). Acknowledgments The writing of this chapter was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (Grant No. 2017R1D1A3B03036276). Also, DST Inspire Faculty Award of the Department of Science and Technology, Government of India (DST/INSPIRE/04/2015/003220) is highly acknowledged.
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Smeds KA, Grinstaff MW. Photocrosslinkable polysaccharides for in situ hydrogel formation. J Biomed Mater Res. 2001;54:115. Smeds KA, Pfister-Serres A, Hatchell DL, Grinstaff MW. Synthesis of a novel polysaccharide hydrogel. J Macromol Sci – Pure Appl Chem. 1999;36:981. Song Z, Zhang Y, Shao H, Ying Y, Mei L, Ma X, Chen L, Ling P, Liu F. Effect of xanthan gum on the prevention of intra-abdominal adhesion in rats. Int J Biol Macromol. 2019;126:531. Strehin I, Ambrose WM, Schein O, Salahuddin A, Elisseeff J. Synthesis and characterization of a chondroitin sulfate-polyethylene glycol corneal adhesive. J Cataract Refract Surg. 2009;35:567. Strehin I, Nahas Z, Arora K, Nguyen T, Elisseeff J. A versatile pH sensitive chondroitin sulfate– PEG tissue adhesive and hydrogel. Biomaterials. 2010;31:2788. Sulaeva I, Hettegger H, Bergen A, Rohrer C, Kostic M, Konnerth J, Rosenau T, Potthast A. Fabrication of bacterial cellulose-based wound dressings with improved performance by impregnation with alginate. Mater Sci Eng C. 2020;110:110619. Sutherland IW. Structure-function relationships in microbial exopolysaccharides. Biotechnol Adv. 1994;12:393. Sutherland IW. Microbial polysaccharides from Gram-negative bacteria. Int Dairy J. 2001;11:663. Taira BR, Singer AJ, Rooney J, Steinhauff NT, Zimmerman T. An in-vivo study of the woundbursting strengths of octyl-cyanoacrylate, butyl-cyanoacrylate, and surgical tape in rats. J Emerg Med. 2010;38:546. Tavakoli S, Mokhtari H, Kharaziha M, Kermanpour A, Talebi A, Moshtaghian J. A multifunctional nanocomposite spray dressing of Kappa-carrageenan-polydopamine modified ZnO/L-glutamic acid for diabetic wounds. Mater Sci Eng C. 2020;111:110837. Thorn J, Sørensen H, Weis-Fogh U, Andersen M. Autologous fibrin glue with growth factors in reconstructive maxillofacial surgery. Int J Oral Maxillofac Surg. 2004;33:95. Tytgat L, Van Damme L, Arevalo M d PO, Declercq H, Thienpont H, Otteveare H, Blondeel P, Dubruel P, Van Vlierberghe S. Extrusion-based 3D printing of photo-crosslinkable gelatin and κ-carrageenan hydrogel blends for adipose tissue regeneration. Int J Biol Macromol. 2019;140:929. Valeri CR, Vournakis JN. mRDH bandage for surgery and trauma: data summary and comparative review. J Trauma Acute Care Surg. 2011;71:S162. Vanhaverbeke C, Heyraud A, Mazeau K. Conformational analysis of the exopolysaccharide from Burkholderia caribensis strain MWAP71: impact on the interaction with soils. Biopolymers: Orig Res Biomol. 2003;69:480. Wang D-A, Varghese S, Sharma B, Strehin I, Fermanian S, Gorham J, Fairbrother DH, Cascio B, Elisseeff JH. Multifunctional chondroitin sulphate for cartilage tissue–biomaterial integration. Nat Mater. 2007;6:385. Wang T, Nie J, Yang D. Dextran and gelatin based photocrosslinkable tissue adhesive. Carbohydr Polym. 2012;90:1428. Weng L, Romanov A, Rooney J, Chen W. Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran. Biomaterials. 2008;29:3905. Wiemer K, Dörmbach K, Slabu I, Agrawal G, Schrader F, Caumanns T, Bourone S, Mayer J, Steitz J, Simon U. Hydrophobic superparamagnetic FePt nanoparticles in hydrophilic poly (N-vinylcaprolactam) microgels: a new multifunctional hybrid system. J Mater Chem B. 2017;5:1284. Zhu J, Sun Y, Sun W, Meng Z, Shi Q, Zhu X, Gan H, Gu R, Wu Z, Dou G. Calcium ion–exchange cross-linked porous starch microparticles with improved hemostatic properties. Int J Biol Macromol. 2019;134:435. Zia T, Usman M, Sabir A, Shafiq M, Khan RU. Development of inter-polymeric complex of anionic polysaccharides, alginate/k-carrageenan bio-platform for burn dressing. Int J Biol Macromol. 2020;157:83. Zidarič T, Milojević M, Gradišnik L, Stana Kleinschek K, Maver U, Maver T. Polysaccharide-based bioink formulation for 3d bioprinting of an in vitro model of the human dermis. Nano. 2020;10:733.
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Susana Correia, Joaquim Miguel Oliveira, Hajer Radhouani, and Rui L. Reis
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Kefiran Potential for TERM Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Kefiran Physicochemical and Biological Properties of Interest for TERM Applications . . . 3.1 Kefiran Structural Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Kefiran Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Kefiran Thermal Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Kefiran Mechanical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Kefiran Biological Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
A wide variety of exopolysaccharides (EPS) are synthesized by microorganisms, and kefiran is an example of EPS produced by the microflora of kefir grains, an ancient culture used to ferment milk to produce the kefir beverage. Kefiran is the main polysaccharide in kefir grains and consists of a water-soluble-branched glucogalactan heteropolysaccharide containing approximately equal amounts of glucose and galactose, mostly produced by Lactobacillus kefiranofaciens. Kefiran has attracted a lot of attention due to its unique features and beneficial properties, being explored for numerous applications in diverse areas, mostly in the food industry and biomedical fields. However, kefiran has only recently been more extensively investigated for its potential for tissue engineering and S. Correia (*) · J. M. Oliveira · H. Radhouani · R. L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected]; [email protected]; [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_52
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regenerative medicine. Herein, we describe the different kefiran applications and properties that have shown that this exopolysaccharide can indeed represent a promising alternative to already existing treatments and gold standard materials in regenerative medicine and tissue engineering. Keywords
Biopolymer · Kefiran · Regenerative medicine · Scaffold · Tissue engineering
1
Introduction
Microorganisms synthesize a wide variety of exopolysaccharides (EPS) that are mostly composed of carbohydrates such as glucose, galactose, mannose, and fructose, but also uronic acid and other noncarbohydrate moieties such as acetate, phosphate, pyruvate, and succinate (Cottet et al. 2020). Kefiran is an example of EPS produced by the microflora of kefir grains that are used to ferment milk to produce the kefir beverage. It is the main polysaccharide in kefir grains, which are composed by lactic acid bacteria, acetic acid bacteria, and yeasts, together with casein and complex sugars in a polysaccharide matrix (Arslan 2015; Cottet et al. 2020). Kefiran was first studied and named by La Riviere and Kooiman (1967) and consists of a water-soluble-branched glucogalactan heteropolysaccharide containing approximately equal amounts of glucose and galactose, mainly produced by Lactobacillus kefiranofaciens (Moradi and Kalanpour 2019; Radhouani et al. 2018b). This EPS has been receiving increasing interest due to its unique features, such as rheological behavior, biodegradability, biocompatibility, safety, emulsifier effect, stabilizing effect, resistance against hydrolysis, barrier and mechanical properties, and water vapor permeability (Moradi and Kalanpour 2019). Moreover, numerous beneficial properties have been associated to kefiran including antimicrobial, antioxidant, antitumor, and anti-inflammatory properties (Cottet et al. 2020; Moradi and Kalanpour 2019). The unique features and properties of kefiran have been explored for numerous applications in diverse areas, mostly in the food industry and in biomedical fields. It is one of the biopolymers whose film making capabilities have been studied in detail and has also attracted considerable attention as a new biological and medical material, due to its biocompatibility and water sensitivity (Ahmed and Ahmad 2017; Moradi and Kalanpour 2019). Kefiran was found to be effective in decreasing blood pressure, in cancer prevention, and in inhibiting and healing allergic disorders and inflammatory diseases. It has also showed prebiotic behavior and antitumor, hypocholesterolemic, hypotensive, atherosclerosis, and immunomodulatory effects by oral administration. These and other healing effects and health benefits reveal its great potential to be used in medical applications (Moradi and Kalanpour 2019). However, the unique features and properties of kefiran have only recently been more extensively explored for tissue engineering and regenerative medicine (TERM) applications.
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Kefiran Potential for TERM Applications
The field of regenerative medicine and tissue engineering has emerged due to the promising possibility to promote tissue repair and regeneration, being regenerative biology focused on using degradable biomaterials to either release bioactive factors to promote healing and/or provide scaffolds to seed therapeutic cells that can repair and regenerate tissues (Radhouani et al. 2018b). Natural biomaterials have received an increasing interest for tissue-engineering scaffolding due to their unique structure and composition similarities with the natural extracellular matrix, having the additional advantage of interacting with cells and cellular enzymes, being remodeled and/or degraded as space for growing tissue is needed (Moradi and Kalanpour 2019; Radhouani et al. 2018b). Kefiran has already shown great potential to form proper scaffolds for TERM applications. Montesanto et al. (2016) obtained dense films and porous scaffolds from 2% (w/v) kefiran-aqueous solutions and characterized them to evaluate their potential application for tissue engineering. Freeze-dried scaffolds with high porosity and interconnected pore structure were obtained (Fig. 1a) allowing good cellular penetration and the diffusion of both waste products out of the scaffold and supply of nutrients into the tissue (Montesanto et al. 2016). The dense films obtained (Fig. 1b) were proposed to have potential as support for submerged culture; the authors propose that the obtained results provide new insights into the foaming methods and fabrication parameters for kefiran scaffolding that could address new applications of bioabsorbable scaffolds in TERM (Montesanto et al. 2016). Later, Radhouani and collaborators also developed kefiran cryogels from 2% (w/v) kefiran-aqueous solutions to be evaluated as proper scaffolds for TERM and controlled drug delivery (Fig. 2). The developed scaffoldds were more extensively characterized showing stability, elastic behavior, and high porosity, being capable of controlled release of diclofenac for 2 weeks; the cryogels also showed to be biocompatible, sustaining human adipose-derived Stem Cells (hASCs) metabolic activity for 72 h, which is a fundamental feature for TERM (Radhouani et al. 2019; Radhouani et al. 2017).
Fig. 1 Morphology of kefiran cryogels obtained by Montesanto et al. (2016). SEM microstructure of (a) dense films obtained via solvent casting (left, outer surface; right, cross-section) and (b) porous scaffolds obtained via direct quenching (left, outer surface; right, cross-section). (Adapted with permission from Montesanto et al. (2016). Copyright (2016) AIDIC Servizi S.r.l.)
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Fig. 2 Morphology of kefiran cryogels obtained by Radhouani et al. (2019): (a) macroscopic image; (b) SEM surface microstructure at different magnifications: (i) x50 and (ii) x300; and (c) 3D reconstructed kefiran scaffolds by microcomputed tomography: (i) cross-sectional, (ii) longitudinal, and (iii) fully reconstructed views. Scale bar: 250 μm. (Reprinted with permission from Radhouani et al. (2019). Copyright (2019) Elsevier)
More recently, Sabatino et al. (2020) developed injectable, in situ forming kefiran/propylene glycol gels to vary their potential as scaffolds for tissue regeneration or implantable drug delivery devices, showing prospects of enabling the diffusion of small molecules (e.g., nutrients) and the entrapment of large biomolecules (e.g., growth factors). Kefiran-electrospun nanofibers with distilled water as solvent were first reported by Esnaashari et al. (2014) showing promising properties for further application as a biomaterial for cell-growth scaffolds, wound dressing, and also as drug carriers. The authors reported an increase in the diameter of the developed kefiran nanofibers with the increase of applied voltage, kefiran concentration, and biopolymer feeding rate (Esnaashari et al. 2014). Jenab and collaborators encapsulated platelets in kefiran and tested the system for bioavailability as a new drug for surface bleeding, showing it may have potential as a model in a range of biomedical applications, including local and sustained drug delivery, versatile artificial blood, and immune protection of artificial tissues (Jenab et al. 2015a). Afterward, the same team developed electrospun kefiran/polyethylene oxide (PEO) nanofibers to evaluate their antimicrobial activity as a biocontrol agent for food packaging and food preservation (Jenab et al. 2017) and very recently developed and characterized different electrospun pure poly-acrylonitrile (PAN)kefiran nanofibrous scaffolds (Fig. 3) with suggested superior properties for neural stem cell culture (especially for spinal cord injury repair) and tissue engineering (Jenab et al. 2020). This last study also showed that pure kefiran could be used for the enhancement of peripheral blood mononuclear cells (PBMC) growth and for reducing the growth of MCF7 cancerous cells, highlighting again the promising use of kefiran for regenerative medicine (Jenab et al. 2020).
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Fig. 3 SEM images of kefiran nanofibers obtained by Jenab et al. (2020) at different magnifications (B1: x1000; B2: x5000; B3: x10,000; B4: x20,000). (Re-used with permission from Jenab et al. (2020). Copyright (2020) Dove Medical Press)
Hyaluronic acid (HA) and HA-based materials are extensively used for the preparation of gels, hydrogels, and scaffolds for a variety of tissues, cartilage being the most intensively studied (Chircov et al. 2018). HA is among the most widely used biomaterials in the biomedical field, being considered the gold standard treatment for viscosupplementation and currently used as a commercial injectable biomaterial for osteoarthritis (OA) treatment (Radhouani et al. 2018a). Hence, Radhouani et al. (2018a) performed a comparative study regarding the biological performance of kefiran and its potential for regenerative medicine applications when compared with HA. The antioxidant activity of kefiran and HA was assessed through their reducing power activity (ascorbic acid equivalent reducing capacity), metal chelating activity (ferrous ion-chelating capacity), and through their hydroxyl and superoxide radical scavenging activity. The high antioxidant potential of kefiran against reactive oxygen species was demonstrated, highlighting its ability to protect cells in oxidative stress environments, having in fact outperformed HA regarding reducing power performance. Kefiran and HA anti-inflammatory activity was evaluated through their capacity to scavenge nitric oxide radicals generated in a cell-free system where again kefiran outperformed HA. Kefiran also proved to be an interesting immunostimulant by considerably reducing the concentration of NO, which is
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a significant mediator of various physiologic and pathologic processes and a powerful inflammatory mediator by strongly reacting with oxygen, superoxide, and ironcontaining compounds. Additionally, the incorporation of kefiran in the culture media of human adipose derived stem cells (hASC) improved their viability and metabolic activity, representing a promising finding as these stem cells are able to differentiate into several lineages, displaying great potential for TERM applications. The results obtained by Radhouani et al. (2018a) encourage the use of kefiran as an alternative or adjunct treatment to promote tissue repair and regeneration while reducing inflammation, particularly in OA contexts.
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Kefiran Physicochemical and Biological Properties of Interest for TERM Applications
The development of biodegradable polymeric materials for biomedical applications has advanced significantly in the last half century. Such materials are preferred for the development of therapeutic devices, including gels and three-dimensional scaffolds for tissue engineering, and the potential role of polysaccharide-based materials in clinical applications of tissue-engineered medical products has been increasing (Oliveira and Reis 2011; Song et al. 2018). Due to their physicochemical behavior and interesting structural similarities with biological molecules, great potential has been envisioned for the future application of polysaccharide-based materials in the biomedical field, especially in cartilage regeneration (Oliveira and Reis 2011). Analyzing the physico-chemical properties of polysaccharide-based scaffolds and how they influence their suitability for the desired biological setting is critical in defining the appropriate design needed to achieve the necessary bioactivity for a particular TERM application (Tchobanian et al. 2019). Moreover, evaluation of the biocompatibility and biomimetic efficiency of a material is essential when evaluating its potential for biomedical applications (Pradhan et al. 2017; Sousa et al. 2017). Characterization of the physicochemical and biological properties of polysaccharide-based TE products is often overlooked but is essential to improve the design of these materials, and crucial in improving their ability to attain the intended biological response and activity (Tchobanian et al. 2019). The physicochemical and biological properties of kefiran have been vastly studied for applications in the food industry; however, few studies reported these kefiran properties envisioning TERM applications. Radhouani and collaborators specifically investigated kefiran’s physicochemical and biological properties in order to demonstrate its competence and clarify its high application potential in TERM. Kefiran properties were assessed for (i) articular cartilage or bone defect applications by modulating its structure (Radhouani et al. 2018b); (ii) as a promising viscosupplementation alternative or adjunct treatment to promote tissue repair and regeneration while reducing the inflammation in OA (Radhouani et al. 2018a); and (iii) as scaffolds for TE and controlled drug delivery (Radhouani et al. 2019).
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Kefiran Structural Properties
The interaction of a polymeric biomaterial with the surrounding biological environment and the respective biological response determines its biocompatibility; hence, before using a polymeric biomaterial in therapeutic applications, it is critical to acquire detailed structural and chemical information to attain an accurate characterization and to evaluate its biomimetic efficiency (Pradhan et al. 2017). Physical properties are also governed by the molecular structure of polymers, being instrumental in the study of polysaccharides. The applicability of a polysaccharide for tissue engineering is greatly dependent on its physical behavior, and the presence of reactive functional groups makes a polysaccharide easily flexible to modifications, which are of high importance for medical applications. The molecular structure and component dynamics of materials are commonly examined by nuclear magnetic resonance (NMR) spectroscopy and Fourier-transform infrared spectroscopy (FTIR). Quantitative NMR has become a well-recognized and widely applied analytical tool for the quantification of diversified classes of compounds in a great variety of samples, representing an essential tool when studying the chemistry of polysaccharides, being used over decades for the structural characterization of polysaccharides from different origins (de Souza 2017; Radhouani et al. 2018b). 1H-NMR spectroscopy is characterized by some interesting advantages upon chromatography, such as easy sample preparation, easy equipment calibration, and fast obtained results (de Souza 2017; Radhouani et al. 2018b). The 1H-NMR spectrum of kefiran extracted from kefir grains in D2O at 60 C was obtained by Radhouani et al. (2018b) and allowed the identification of the molecule structure of the extracted kefiran polysaccharide, revealing a peak at 5.15 ppm for an anomeric β hydrogen and six signals at the chemical shifts of 4.85, 4.83, 4.78, 4.76, 4.67, and 4.62 ppm for several anomeric α hydrogens, assigned to a sugar on a lateral branch (Fig. 4a). Similar results were previously obtained by Micheli et al. (1999) in kefiran polysaccharides obtained from both a ropy Lactobacillus strain and kefir grains which were also analyzed by 1H-NMR in D2O but at 80 C; peaks at 5.1 ppm for an anomeric β hydrogen and at 4.82, 4.77, 4.52, 4.50, 4.45, and 4.65 ppm for several anomeric α hydrogens were described. The 1H-NMR spectrum of kefiran produced by L. kefiranofaciens has also been obtained by Maeda et al. (2004) in D2O at 80 C (Fig. 4b), where the anomeric region (δ 4.4–5.5) contained seven signals corresponding to a minor peak at δ 4.61, assigned to (1!6)-β-D-Galp (corresponding to a small proportion of 2,3,4-tri-O-methyl D-galactose), and other six peaks containing three well-resolved signals and three overlapping signals corresponding to the hexasaccharide repeating unit; the residue at 5.14 ppm (c1, Fig. 4b) was assigned to an α-hexapyranosyl residue, and the residues at 4.82, 4.68, 4.53, 4.53, and 4.49 ppm were assigned to a pyranose ring formed in a β anomeric configuration (respectively f1, b1, e1, d1, and a1, Fig. 4b). The heteropolysaccharide structure of kefiran isolated from kefir grains grown in cheese whey has also been analyzed by Ghasemlou et al. (2012) who described in the anomeric
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Fig. 4 1H-NMR spectrum of the kefiran polysaccharide obtained by (a) Radhouani et al. (2018b) (D2O, 60 C); (b) Maeda et al. (2004) (D2O, 80 C); and (c) Ghasemlou et al. (2012) (D2O, 27 C). (Adapted/reprinted with permission from the referenced sources. Copyright (2018) SAGE Publications. Copyright (2004) American Chemical Society. Copyright (2012) Elsevier)
region of the 1H-NMR spectrum obtained in D2O at 27 C six signals at 4.80, 4.69, 4.62, 4.53, 4.52, and 4.48 ppm, assigned to anomeric protons of β-D-Glcp-(1!, !2,6)-β-D-Galp (1!, !6)-β-D-Galp-(1!, !4)-β-D-Glcp-(1!, !3)-β-D-Galp(1!, and !6)- β-D-Glcp-(1!, respectively, and a signal at 5.16 ppm that was assigned to anomeric protons of !4)-α-D-Galp-(1! (Fig. 4c). Later, Jenab et al.
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(2017) obtained the 1H-NMR spectrum of the kefiran used to produce the abovementioned kefiran/PEO nanofibers for food packaging; deproteinized kefiran was assayed in D2O at 25 C revealing signals on the anomeric region at 4.31, 4.12, 3.79, 3.63, 3.60, 3.52, 3.15, 2.50, 2.12, 1.24, and 1.56 ppm. FTIR analysis allows functional group determination in polymeric biomaterials, which is necessary to evaluate their biological response, biocompatibility, and biodegradability; moreover, drug-polymer interaction studies can be confirmed by observing the loss or appearance of FTIR peaks from the drug and polymer conjugate (Pradhan et al. 2017). FTIR has been performed to identify the fundamental groups present in the kefiran structure, representing a useful tool to investigate structural changes in biopolymers (Jenab et al. 2015b). IR analysis is also very important to identify the reactive functional groups in kefiran which make it more flexible to many modifications (Moradi and Kalanpour 2019). Envisioning TERM applications, Radhouani et al. (2018b) studied the FTIR spectrum of kefiran extracted from kefir grains (Fig. 5a) reporting a band at 3430 cm–1 corresponding to the hydroxyl groups (region attributed to the stretching vibration O–H in the constituent sugar residues), a band at 2930 cm–1 linked with the stretching vibration of C–H in the sugar ring which was assigned to methyl and methylene groups, a band at 1700 cm–1 associated to the stretching vibration of O–H, and a band around 1400 cm–1 ascribed to CH2 and OH groups. Moreover, the region of 1100–1150 cm–1 showed intense absorptions, characteristic of C–O–C stretches and alcohol groups in carbohydrates, and the presence of a band at 900 cm–1 indicated a β-configuration and vibration modes of glucose and galactose (Radhouani et al. 2018b). Wang et al. (2008) reported the FTIR spectra of kefiran produced by L. kefiranofaciens ZW3 isolated from Tibet kefir (Fig. 5b) revealing the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide (broad-stretching hydroxyl group at 3405 cm1; weak C–H stretching peak of methyl group at 2924 cm1; broad stretch of C–O–C, C–O at 1000–1200 cm1 related to carbohydrates; absorption at 1643 cm1 attributed to amide I > C ¼ O str and C–N bending of protein and peptide amines; and peak at 1378 cm1 assigned to C ¼ O str of the COO and C–O bond from COO). The FTIR spectra of the EPS accumulated during milk fermentation by Tibetan kefir was also obtained by Chen et al. (2015) (Fig. 5c), allowing the identification of carboxyl, hydroxyl, and carbonyl functional groups, and also from the EPS obtained from fermented kefir grains of liquid soymilk (Fig. 5d) by Botelho et al. (2014). The FTIR spectra of kefiran isolated from kefir grains by different extraction conditions were obtained by Pop et al. (2016), identifying four major absorption zones (Fig. 5e): 3700–3310 cm–1 (water and hydroxyl groups); 2720–2900 cm–1 (C–H stretching vibration zone; bands at 2850 and 2933 cm–1 were attributed to C–H, and peak intensity reduction was related to disruption of the kefiran solution structure due to absorbed water molecules whose presence masked the C–H bond of the carbohydrate rings, thus reducing the contribution of C–H absorbance bands); 1650–1413 cm–1 (specific for water molecules, assigned to the bending mode of O–H; provides relevant information for industrial applications, since the relative
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Fig. 5 Infrared spectrum of kefiran obtained by (a) Radhouani et al. (2018b); (b) Wang et al. (2008); (c) Chen et al. (2015); (d) Botelho et al. (2014); (e) Pop et al. (2016); (f) Jenab et al. (2015b); and (g) Jenab et al. (2020). (Adapted/reprinted with permission from the referenced sources. Copyright (2018) SAGE Publications. Copyright (2008) Elsevier. Copyright (2015) Elsevier. Copyright (2014) Elsevier. Copyright (2016) National Agricultural and Food Centre, Slovakia. Copyright (2020) Dove Medical Press)
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absorption intensities of bands assigned to water molecules depend on the kefiran solution quality); and 1200–852 cm–1 (distinct for each polysaccharide, being this region dominated by ring vibrations overlapped with stretching vibrations of C–OH side groups and C–O–C glycosidic band vibrations; absorptions at 1035, 1080, and 1153 cm–1 indicated a pyranose form of carbohydrates, associated with the presence of the glucose and galactose components of the purified kefiran structure). The results obtained by Pop et al. (2016) suggest that kefiran was composed of α- and β-configurations of saccharides in pyranose form, and the disruption of the polymer chain was related to the high temperature of extraction. Jenab and collaborators have also investigated the platelet-kefiran interaction by FTIR (Fig. 5f) to assess platelet encapsulation in kefiran and to detect the bioavailability of immobilized platelets in kefiran as a possible new probiotic drug for surface bleeding (Jenab et al. 2015b). FTIR of kefiran revealed different peaks including 3224 cm–1 (O–H group, C–H, N–H), 3036 cm–1 (O–H, ¼CH), 2846 cm–1 (C–H, aldehyde, O–H), 2053 cm–1 (triple), 1414 cm–1 (O–H, COC), 1092 cm–1 (C–C, COC, CO stretch), and 615 cm–1 (C–CL), with peaks 1092 and 1414 cm–1 containing bands of carbohydrates and peaks 3224, 3036, 2846, and 2053 cm–1 associated to the kefiran chemical structure. The mixture of encapsulated platelet-polysaccharide and platelets was shown as the amide I and II did not change, but peak 1140 cm–1 in the polysaccharide region changed; the fingerprint region changes showed to be less than in the polysaccharide region, all changes being almost in the polysaccharide region (900–1200 cm–1) of the encapsulated plateletpolysaccharide. Although envisioning applications in the food industry, the same authors (Jenab et al. 2017) have also investigated the composition and in vitro biodegradation of kefiran and electrospun kefiran/PEO nanofibers through FTIR. The specific wavenumbers of 3201, 3367, and 3435 cm1 identified O–H bands in the oxidizing functional groups in kefiran/PEO nanofibers, but only one peak at 3415 cm1 belonging to this group was observed in kefiran, these groups being more obviously presented in the electrospun kefiran/PEO nanofibers than in the neat kefiran. Biodegradability was shown by the lower number of peaks detected in the spectrum of the nanofibers after the biodegradation period and higher functional group concentration (Jenab et al. 2017). More recently, Jenab and collaborators characterized electrospun kefiran/PAN nanofibers manufactured to be used for regenerative medicine through attenuated total reflectance FTIR spectroscopy (Jenab et al. 2020), identifying in the FTIR spectra of PAN-kefiran scaffolds two peaks (1038 and 1118 cm1) related to the polysaccharide region and one peak (1576 cm1) related to NH2 (Fig. 5g). While FTIR allows analysis of the overall availability of different functional groups in the kefiran chemical structure, X-ray photoelectron spectroscopy (XPS) supports the identification of surface functional groups, FTIR and XPS results being complementary (Radhouani et al. 2018b). XPS allows verification of surface viability, meaning that it can, among other aspects, demonstrate the suitability of a polymer to be functionalized and also the study of a range of properties that include
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polymer biocompatibility (Yahia and Mireles 2017). The XPS spectra of kefiran extracted from kefir grains were obtained by Radhouani et al. (2018b); the C1s XPS spectra revealed the presence of the C–OH bond at 286.6 eV and O–C–O bond at 287.8 eV, and the oxygen O1s peak was decomposed in two components attributed mainly to the O–C bond at 533 eV and to the oxygen bond to hydrogen [H–O–C] at 534.6 eV. The XPS results obtained by Radhouani et al. (2018b) also revealed a C/O atomic ratio of 1.46.
3.2
Kefiran Molecular Weight
Molecular weight and molecular weight distribution are important parameters which define the behavior and physicochemical properties of polymers such as degradation, solubility, mechanical strength and performance (e.g., viscosity, rheological behavior), flexibility, and resistance to deformation, among others, and the particular applications of a specific polymer are closely related to these properties (Dragostin and Profire 2017; Radhouani et al. 2018b). Analysis and characterization of polymeric structures is usually accomplished through spectroscopic techniques (e.g., IR spectroscopy, NMR spectroscopy), which contribute to common functional group identification; however, these traditional methods were subsequently complemented with specific techniques for the investigation of molecular weight and molecular weight distribution such as gel permeation chromatography (GPC) and size exclusion chromatography (SEC) (Dragostin and Profire 2017). To determine the suitability of the kefiran polysaccharide for TERM applications, Radhouani et al. (2018b) obtained through SEC a number-average molecular weight (Mn) of 357 kDa and weight-average molecular weight (Mw) of 534 kDa (polydispersity index (PDI) of 1.49) for kefiran extracted from kefir grains, highlighting the interest and desirability of low-molecular weight biomaterials in modern medicine due to shorter degradation rates. Maeda et al. (2004) studied the molecular weight of kefiran produced by L. kefiranofaciens through GPC, reporting a Mw of 7.6 105 Da and an estimated z-average radius of gyration (Rgz) of 39.9 nm. Ahmed et al. (2013) reported a Mw of 5.5 104 Da after GPC analysis of kefiran produced by L. kefiranofaciens ZW3 isolated from Tibet kefir. Exarhopoulos et al. (2018a) reported a Mw of 6.144 105 Da, a PDI of 1.978 and a Rgz of 35.84 0.1 nm after SEC analysis of kefiran produced from kefir grains. Ghasemlou et al. (2012) obtained kefiran with a Mw of 1.35 106 Da from kefir grains grown in cheese whey, and Piermaria et al. (2008) reported a Mw of 107 Da in kefiran from kefir grains. Pop et al. (2016) investigated the molecular weight of kefiran extracted from kefir grains under different conditions reporting values ranging between 2.4 106 Da and 1.5 107 Da. More recently, Sabatino et al. (2020) reported a Mw of 4.32 0.44 MDa (Rgz ¼ 88.5 8.9 nm) through static light scattering (SLS) and a Mn of 2.17 MDa and Mw of 4.38 MDa (PDI ¼ 2) through gel filtration chromatography (GFC) for kefiran extracted from commercial kefir grains for the development of injectable in situ forming gels for TERM and drug delivery applications.
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Kefiran Thermal Properties
The applicability of a polysaccharide is greatly determined by its thermal performance (Chen et al. 2015). Thermal analysis delivers fundamental information for research and development and quality control of new materials/products such as transition temperatures, crystallinity, heat, and thermal expansion; a variety of techniques in which the physical property of a material is continuously measured as a function of temperature are used in polymer research (Tanzi 2017). Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) are among the most widely used techniques to obtain quantitative calorimetric measurements, being effective analytical tools to characterize the thermal properties and transitions of a polymer (Radhouani et al. 2018b; Tanzi 2017). In order to characterize kefiran for TERM applications, Radhouani et al. (2018b) subjected samples obtained from kefir grains to DSC and TGA (Fig. 6a, b) obtaining agreeable results. The kefiran thermogram obtained through DSC (endothermic heat flow) revealed a marked endothermic peak at 98.7 0.2 C, being the transition (approx. 99 C) discussed to be linked to the polysaccharide melting point, justified by the hydrophilic nature of kefiran functional groups and the existence of a water bound. TGA analysis revealed two events, one with approximately 9% mass loss during temperature increase from 40 to 106 C, which could be associated with the loss of moisture, and a second one with most weight loss (12–65%) during increase from 264 to 350 C, being related to major degradation of the kefiran structure and associated with the extensive enthalpy change observed in the DSC curve (Radhouani et al. 2018b). The thermal stability of kefiran (degradation at 352 C) was also reported by Botelho et al. (2014) in the EPS obtained from the fermented kefir grains of liquid soymilk (Fig. 6c). Montesanto et al. (2016) analyzed kefiran dense films and porous scaffolds by DSC (Fig. 6d) in order to evaluate their potential application for tissue engineering, reporting melting peaks of 102.4 and 107.3 C and melting enthalpies of about 355.2 and 294.8 and J/g, respectively. Thermal analysis has also been performed by DSC for kefiran produced by L. kefiranofaciens ZW3 from Tibet kefir by Wang et al. (2008) (melting at about 93.38 C, endothermic enthalpy change of 249.7 J/g) and by Ahmed et al. (2013) through TGA (melting at 93.38 C and degradation at 299.62 C, Fig. 6e). A melting point of 131.46 C was reported by Chen et al. (2015) for kefiran obtained from Tibetan kefir grains being the enthalpy change 209.6 J/g (Fig. 6f). During DSC, heat absorption and emission are associated with physical changes in the polymeric material, such as structure deformations or melting of crystals (Wang et al. 2008). Sabatino et al. (2020) also used TGA to evaluate the purity and molecular structure of kefiran obtained from commercial kefir grains for the development of injectable, in situ forming kefiran gels to use as potential scaffolds for tissue regeneration or implantable drug delivery devices. The thermogravimetric curve revealed an initial polymer weight loss (55–150 C) that was attributed to moisture (free and bound water representing approx. 10%w), and a second inflection point at 280 C (weight loss of about 3%w) was proposed to reflect residual proteins in the system; thermal degradation of the biopolymer was observed at 300 C, as reported by other authors, where the main inflection point reflected the most prominent mass loss (Sabatino et al. 2020).
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Fig. 6 Thermal properties of kefiran obtained by Radhouani et al. (2018b) through (a) DSC and (b) TGA, (c) Botelho et al. (2014) by TGA, (d) Montesanto et al. (2016) by DSC, (e) Ahmed et al. (2013) by TGA, and (f) Chen et al. (2015) by DSC. (Adapted/reprinted with permission from the referenced sources. Copyright (2018) SAGE Publications. Copyright (2014) Elsevier. Copyright (2016) AIDIC Servizi S.r.l. Copyright (2013) Elsevier. Copyright (2015) Elsevier)
3.4
Kefiran Mechanical Properties
Rheological characterization plays a major role in the evaluation and modeling of the macroscopic behavior exhibited by complex systems under flow and deformation conditions; polymeric and disperse systems are complex systems composed of both viscous and elastic component and therefore show intermediate mechanical responses between those of solids and liquids. Among polymeric biomaterials,
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hydrogels and polymer solutions or suspensions represent complex materials being rheological characterization of major importance to investigate their structural features that are essential to evaluate their potential use and performance in different applications (Borzacchiello et al. 2017). The rheological behavior of kefiran has been investigated in detail for a range of applications, mostly in the food industry (Exarhopoulos et al. 2018b; Moradi and Kalanpour 2019). Radhouani and collaborators investigated the rheological properties of both kefiran aqueous solutions (Radhouani et al. 2018b) and cryogels (Radhouani et al. 2019) in order to evaluate their potential for drug delivery and TERM applications. Flow behavior of kefiran 1% and 10% w/v solutions was analyzed through rotational experiments where shear viscosity was shown to decrease with the increment of shear rate at 37 C (Fig. 7a). The pseudoplastic behavior and gelation ability reported by Radhouani et al. (2018b) emphasize that kefiran may be exploited as matrix environments of various therapeutic agents such as stem cells, genetically engineered cells, or proteins. From curves of shear stress in function of shear rate, kefiran solutions revealed an infinite viscosity until a sufficiently high stress is applied to initiate flow (Fig. 7b) (Radhouani et al. 2018b). The viscoelastic properties demonstrated through frequency sweep curves obtained from oscillatory
Fig. 7 Kefiran rheological properties. Shear viscosity (a) and shear stress (b) versus shear rate of 1% (w/v) (open symbol) and 10% (w/v) (filled symbol) samples; frequency dependence of G00 (open symbol) and G0 (filled symbol) of 1% (w/v) (c) and 10% (w/v) (d) samples at 37 C (Radhouani et al. 2018b). G00 and G0 (open and filled symbols) versus frequency of 2% (w/v) kefiran scaffolds (e) at 37 C (Radhouani et al. 2019). G0 , storage modulus; G00 , loss modulus. (Adapted/reprinted with permission from the referenced sources. Copyright (2018) SAGE Publications. Copyright (2019) Elsevier)
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experiments (Fig. 7c, d) highlighted the applicability of the studied kefiran solutions in TERM applications, namely as viscosupplement in OA to restore the joint synovial fluid viscoelastic properties; their applicability as an economically and environmentally improved alternative to the gold-standard hyaluronic acid is also proposed (Radhouani et al. 2018b). The adhesive performance of kefiran was shown through preliminary pull-away experiments, this effect being much higher for 10% solutions (1.159 0.018 N/s) than for 1% solutions which presented adhesion values similar to water (0.135 0.049 N/s) (Radhouani et al. 2018b). The elastic/gel character, which is an important feature for TERM, was also observed in kefiran scaffolds obtained through freeze-drying (Fig. 7e) where the elastic behavior was quantified by a constant average phase angle of 16 0.7 (Radhouani et al. 2019). The mechanical properties of scaffolds are of major importance in many tissue-engineering applications. For regeneration, the energy stimulus (stored and dissipated energy) depends on scaffold viscoelasticity, and promising mechanical properties were obtained with only 2% (w/v) kefiran concentrations, revealing that this type of information can be used to improve design strategies to create kefiran scaffolds with specific properties for biomedical applications (Radhouani et al. 2019). From an application point of view for injectable biomaterials, the knowledge of the dependence of viscosity upon shear rate is essential as it is highly desirable that these materials show fast decreasing viscosity when subjected to increasing shear rate in order to have low resistance during injection (Borzacchiello et al. 2017). The injectability of kefiran has been investigated by Radhouani et al. (2018b) by analyzing the pressure or force required for injection, evenness of flow, and freedom from clogging (no blockage of the syringe needle). When measured at room temperature and a rate of 1 mL/min by an injectable measurement device (syringe pump with a 1 mL plastic syringe and a needle gauge of 21), kefiran showed an extrusion force of 1 N, a much lower value than HA (11.3 N). This highlighted the interest of using kefiran formulations as viscosupplementation products, for instance over HA intra-articular injections, as they allow smoother injectability by low extrusionforces (Radhouani et al. 2018b). Injectable, in situ forming kefiran gels have very recently been developed by Sabatino et al. (2020) for potential applications as implantable drug delivery devices or scaffolds for tissue regeneration. Kefiran aqueous solutions at 4, 5, and 6% (w/v) were assessed for their capacity to undergo gelation when mixed with different alcohols, and for viscosity and injectability through G26 syringe needles (Sabatino et al. 2020). The injection time to extrude 1 mL of solution at 25 C ranged from approximately 10 to 20 s/mL, being within the range of commonly recommended injections rates. The viscoelastic properties of the different kefiran solutions at 25 C were presented as the apparent viscosity as a function of shear rate, and as storage (G0 ) and loss (G00 ) moduli as a function of frequency. At higher concentrations, kefiran exhibited decreasing viscosity typical of pseudoplastic behavior in the whole shear rate range; however, at the lower concentration, a Newtonian plateau was observed at low shear rates. In the mechanical spectra, obtained in small amplitude oscillation mode, all solutions showed typical behavior of viscoelastic liquids (G00 higher than G0 ) at low frequencies; however, at
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higher frequencies, there was not enough time for polymer chain disentanglement (G0 curve approached G00 curve). Sabatino et al. (2020) also investigated the gelation of aqueous kefiran with propylene glycol (PG) by rheological analysis where oscillatory tests were performed to measure time to gel and gel strength (varying frequency with constant deformation amplitude). The viscoelastic properties of all kefiran-PG gels were plotted as G0 , G00 versus time (values extracted at 1 Hz from consecutive frequency sweep runs at 25 C) and versus frequency (48 h room temperature-stored gels). Kefiran at 4% (w/v) with PG required a longer time to set into gel and formed a relatively weak gel, leading to the rejection of this system rejected. G0 , G00 versus frequency plots were then obtained for the highest kefiran/PG gel formulation incubated for 1, 2, 6, 12, 24, 36, and 48 h at 25 and 37 C. An increased gel time and reduced gel strength at 37 C when compared to 25 C were observed and discussed to be due to weakened hydrogen bonding interactions at higher temperatures, with reduced driving force for gelation (Sabatino et al. 2020).
3.5
Kefiran Biological Characterization
Evaluation of the biocompatibility of a material is essential when evaluating its potential for biomedical applications and the use of cell culture models represents the gold standard for the in vitro testing of biomaterials. Biocompatibility has been defined in many different ways in the past decades; however, following from its numerous definitions, determining the biocompatibility of a biomaterial must rely not only on the potential damaging effects associated with biosafety but also on the desirable properties it may present in the context of its intended application and function (Sousa et al. 2017). For instance, regarding TERM applications, the biocompatibility of a scaffold also denotes its performance as an adequate substrate to support and/or promote appropriate cellular activities (cell growth, extracellular matrix deposition, and induction of desired gene expression patterns) for optimal tissue repair and/or regeneration, being in fact currently designed to be cell instructive (i.e., capable of guiding cell behavior and of prompting specific cell responses) rather than bioinert (Sousa et al. 2017). Hence, after assessing the cytotoxicity of a biomaterial, other traits regarding cellular behavior and cell-material interactions are usually evaluated including metabolic activity, adhesion, proliferation, motility and migration, morphology, and protein/gene expression, among others (Sousa et al. 2017). Envisioning the applicability of kefiran for TERM, Radhouani and collaborators have assessed the cytotoxicity of kefiran extracts and scaffolds over the mouse L929 fibroblastic cell line and hASCs (Radhouani et al. 2018a, b, 2019). Kefiran extracts showed a lack of cytotoxic response over L929 cells by supporting cell-metabolic activity and cell proliferation, having no effect on cell morphology (Radhouani et al. 2018b), and were shown to improve both the viability and metabolic activity of hASCs (Radhouani et al. 2018a). Also, hASCs seeded on kefiran scaffolds were metabolically active during 72 h, showing their biocompatibility (Radhouani et al. 2019); considering that this an essential feature in TERM and the fact that hASCs are
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able to differentiate into several lineages, kefiran revealed great potential for TERM applications. The biocompatibility of the before-mentioned kefiran/PG scaffolds has also been tested over preosteoblastic MC3T3-E1 cells; no toxic effects were observed on cells placed in direct contact with the kefiran gel, encouraging further biological evaluation of kefiran/PG-water formulations as injectable, in situ forming scaffolds or implantable delivery devices (Sabatino et al. 2020). Kefiran cytotoxicity over PC12 and MCF7 cells (neural stem cell culture and breast cancer cell line, respectively), and morphological changes of PC12 cells, has also been evaluated for the already mentioned electrospun PAN/Kefiran nanofibers revealing their potential as an antimicrobial, antitumor, biocompatible, and costeffective system that is exploitable for regenerative medicine and promising for neural stem cell culture (Jenab et al. 2017, 2020). Kefiran has also exhibited a better resistance to hyaluronidase degradation as compared to HA, highlighting its potential as a promising alternative viscosupplement to the gold standard HA (Radhouani et al. 2018b). Moreover, in a comparative study with HA, kefiran has demonstrated great antioxidant performance against reactive oxygen species, showing stronger reducing power activity and superoxide and nitric oxide radical scavenging capacity over HA. This study highlighted not only the immunostimulatory potential of kefiran but also its potential to protect cells in oxidative stress environments; this substantiated the knowledge regarding its biological performance, such as antioxidant and anti-inflammatory properties, encouraging its application as an alternative or adjunct treatment to promote tissue repair and regeneration while reducing inflammation, for instance, in the osteoarthritis context (Radhouani et al. 2018a). Given the major medical burden that antimicrobial infections represent worldwide, and the rising concerns regarding antimicrobial resistance, the antimicrobial properties of kefiran represent one of its most interesting biological characteristics. Anti-infective biodegradable materials are highly demanded for clinical applications as they could represent a solution to tackle both problematics by preventing possible infections and reducing the use of antimicrobial agents. Kefiran antimicrobial properties have been vastly reported for a variety of applications (Moradi and Kalanpour 2019). However, studies investigating how these antimicrobial properties prove beneficial in specific contexts of TERM are scarce and of utmost importance.
4
Conclusions and Future Perspectives
Kefiran has been explored for numerous applications in food, medicine, health, and film making. It has been reported to act as an anticancer, antioxidant, and antimicrobial agent, decrease cholesterol levels, reduce bleeding, heal wounds, and support drug delivery. Kefiran has also been used for encapsulation of either bionutrients or immobilized platelets that can be applied to treat surface bleeding. However, only recently, kefiran has been more explored specifically for TERM applications, and the recent studies described herein have shown that this exopolysaccharide can indeed
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represent a promising alternative to already existing treatments and gold standard materials. Nonetheless, despite the remaining drawback of the low-kefiran production yields reported so far, the therapeutic attributes and technological applications of kefiran, together with its generally recognized as safe status and numerous health benefits, make this an ideal candidate for solutions in the tissue engineering and regenerative medicine fields. Acknowledgments S. Correia is supported by the Portuguese Foundation for Science and Technology (FCT) though the R&D Project KOAT – Kefiran exopolysaccharide: Promising biopolymer for use in regenerative medicine and tissue engineering, with reference PTDC/BTMMAT/29760/ 2017 (POCI-01-0145-FEDER-029760), financed by FCT and cofinanced by European Regional Development Fund (FEDER) and Operational Programme for Competitiveness and Internationalisation (POCI). H. Radhouani is supported by the FCT (CEECIND/00111/2017), and J. M. Oliveira would like to thank the FCT for funding provided under the program Investigador FCT 2015 (IF/01285/2015).
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Gums for Tissue Engineering Applications
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Pritisha S. Khillar and Amit Kumar Jaiswal
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Natural Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Classification of Natural Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Tissue engineering involves the replacement and renewal of desired cells, tissues, and organs to restore the normal biological functions of specific cells or tissue types. Recently, the field is growing actively at both the laboratory and clinical levels contributing to research and medicines. The increase in population, changes in lifestyle, and better life expectancy have increased the chances of disease and trauma causing tissue damage which in turn leads to the need for advancement in the field of tissue engineering. The regeneration of the damaged tissues requires a supporting material known as a scaffold. The scaffolds are used for cell adhesion, proliferation, and differentiation for proper tissue regeneration and also help in vitro and in vivo drug and gene delivery to the biological system. The fabrication of the scaffolds is achieved by combining some biologically active materials known as biomaterials with some specific cell types that are needed for the desired tissue repair process. The natural polymers used as the biomaterials in the fabrication of scaffolds are especially gums that show excellent biological response and mimic the nature of the native extracellular matrix. This chapter briefly explains the gums used in tissue engineering, from where they can be derived, their classifications, properties, and applications. P. S. Khillar · A. K. Jaiswal (*) Centre for Biomaterials, Cellular and Molecular Theranostics, Vellore Institute of Technology, Vellore, Tamil Nadu, India e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_53
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Keywords
Tissue engineering · Biopolymers · Gums · Polysaccharides
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Introduction
Every year millions of patients are facing tissue damage, organ failure issues, and problems associated with the same. Conventionally it can be solved by transplantation of the organ from a donor either as an autograft or allograft. But sometimes it leads to donor shortage along with immunological rejection to the donated body part. Tissue engineering and regenerative medicine have the potential to overcome the shortage of tissues and organs, and it can mimic the nature of the specific tissue and organ with some promising results (Dzobo et al. 2018). Tissue engineering combines bioactive molecules, cells, and scaffolds with biomaterials (Fig. 1) that encourage tissue formation and integration in the host environment. Biomaterials
Biopolymers
Cells
Tissue Engineering
3 dimensional Bioprinting
Drug Delivery
Wound Healing
Fig. 1 The design and applications of tissue engineering
Tissue Development
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provide structural support implicative of the native extracellular matrix (ECM) to stimulate cell growth and serve as an integral component ensuing tissue regeneration. Biomaterials offer several choices in regulating the frame, morphology, and chemistry that help in mimicking the ECM functions. The biomaterials are biodegradable, biocompatible, and should be clinically compliant. They need to be produced, purified, and processed for the delivery of the cells and growth factors to the biological system. They help to provide a specific niche for the cells and tissues for better growth, development, and differentiation. The materials are two types such as natural and synthetic. Both natural and synthetic polymers are extensively used in tissue engineering and regenerative medicine field applications. Natural polymers are typically obtained from nature. They are especially proteins, polysaccharides, and polypeptides, including collagen, alginate, gelatin, actin, albumin, chitosan, keratin, chitin, cellulose, silk, gellan, and hyaluronic acid. Biological systems easily accept these polymers, support cell adhesion, and function, but sometimes there is a lack of desirable immunogenicity, structural complexity, and biomechanical properties (Tang et al. 2014). Synthetic polymers are biodegradable polymers used for tissue engineering and drug delivery applications. It can mimic the nature of macromolecular components of the biological system. It has a well-defined structure with fewer immunological concerns. The synthetic polymers which are actively employed in tissue engineering include polyesters, polyphosphazene, poly(glycerol sebacate), polyanhydrides, and polyurethane (BaoLin and Ma 2014). Natural gums are polysaccharides, hydrophilic compounds with a high molecular weight, and possess numerous biological applications. These gums are used in several industries such as food, environment, medical, and biotechnology, including research areas and tissue engineering (Ahmed et al. 2019). Besides, compared to synthetic polymers, natural polymers are easily available, low in cost, biodegradable, biocompatible, nontoxic, and eco-friendly. These gums have achieved lots of curiosity in the field of tissue engineering and three-dimensional (3D) cell culture because of their effectiveness in tissue regeneration and development in several areas like bone tissue engineering, cartilage repair, skin and wound repair, as well as retinal, neural, and also in three-dimensional bioprinting studies. These are used as binders, crosslinkers, thickening agents, and gelling agents, and also used as hydrogels because of their hydrophilic nature (Mohammadinejad et al. 2020). Besides, the gums can be manipulated to act in several forms such as scaffolds, hydrogels, emulsion, film-forming, and encapsulating agents (Fig. 2). Natural gums are also known as hydrocolloids. These hydrocolloids are divided into several groups depending upon their structure, function, applications, and also according to the charge, viz. ionic and nonionic, according to the source where they are derived from, viz. marine, plant, and animal, according to their shape, viz. linear and branched, and according to monomeric units present in it, viz. homoglycans, di-, tri-, tetra-, and penta-heteroglycans. So, here in this chapter, we are going to briefly understand some natural gums used in tissue engineering, their application, advantages, and disadvantages along with the source and the class where they are derived from.
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Fig. 2 Different forms that can be made out of gums
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Natural Gums
Natural gums are polysaccharides usually obtained from natural sources like plant origin, marine origin, and microbial origin, and they are highly effective, biodegradable, biocompatible, nontoxic, chemically inert, easily available, environment friendly, and less expensive along with a high molecular weight. They are commonly used as a binder, stabilizer, thickening agent, gelling agent, disintegrant in tablets, protective colloids in suspensions and diluents, emulsifier, etc. in several fields like pharmaceutical industries, biotech industries, food industry, agriculture, cosmetics, textiles, paper, personal care products, paint industries, biomedical and tissue engineering, etc. (Ahmed et al. 2019; Bhosale et al. 2014; Mohammadinejad et al. 2020).
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Classification of Natural Gums
As directed before the natural gums are usually polysaccharides derived from natural sources; the classification of these gums depends upon the source from which they are derived. They are further classified into several other groups depending upon their charge, shape, structure, and functional groups present in them. As described below (Fig. 3): Generally, the gums are derived from plants, animals, marine sources, and bacteria (Fig. 3). The plants that produce gums are mainly herbaceous and woody and usually belong to the families of Fabaceae, Sterculiaceae, and Combretaceae. The gums are generally secreted by some physical injury or pathological attack on the plant surface or due to some adverse circumstances such as cell wall breakdown or drought forming gummosis, an extracellular formation. The gums are extruded from seeds, tree exudate, tubers, and roots. Most of the gums are water soluble and highly viscous (Mano et al. 2007; Mohammadinejad et al. 2020; Ahmed et al. 2019; Bhosale et al. 2014; Silva et al. 2017) (Table 1). Here we are going to briefly discuss some of the gums that are widely used in tissue engineering applications.
2.1.1 Gum Arabica Gum arabica is a heteropolysaccharide consisting of complex polysaccharides, oligosaccharides, and glycoproteins with the main chain that has galactan with lateral multibranched chains that consist of galactose/arabinose (Rad et al. 2018).
Fig. 3 Classification of the gums based on derived sources
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Table 1 Classification of the gums Gums Plant derived
Animal derived
Microbial source derived
Marine source derived
Others (cellulose derived)
Arabica gum Ghatti gum Karaya gum Tragacanth gum Guar gum Pectin Acemannan Konjac glucomannan Xylan Cellulose Chitin Chitosan Chondroitin sulfate Hyaluronic acid Xanthan gum Gellan gum Dextran gum Scleroglucan Pullulan Alginate Agar Agarose Carrageenan Ulvan Fucoidan Laminarin Carboxy methyl cellulose
Charge Anionic Anionic Anionic Anionic Nonionic Anionic Anionic Anionic Nonionic Anionic Cationic Cationic Anionic Anionic Anionic Anionic Nonionic Nonionic Nonionic Anionic Anionic Anionic Anionic Anionic Anionic Anionic Anionic
Shape Branched Branched Branched Branched Branched Linear Linear Branched Branched Linear Linear Linear Linear Linear Branched Linear Branched Branched Linear Linear Linear Linear Linear Branched Branched Branched Linear
Units Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Homogeneous Heterogeneous Heterogeneous Homogeneous Homogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Homogeneous Homogeneous Homogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Heterogeneous Homogeneous Homogeneous
It is a naturally existing biomaterial and derived from the exudate of Acacia senegal and Acacia seyal tree that belongs to the family Leguminosae (Silva et al. 2010). It has β-D-glucopyranuronic acid, α-L-rhamnopyranose, β-D-galactopyranose, β-D-4glucopyranuronic acid, α-L-arabinose furanose, and α-D-galactopyranose constituent units (Ahmed et al. 2019). The main chain of the polysaccharide contains β-(1!3) and (1!6)-linked D-galactose units in addition to β-(1!6)-linked D-glucopyranosyl uronic acid units (Mano et al. 2007). This gum is highly branched. It is highly water soluble and has a lower viscosity and is used as a suspending agent, binding agent, demulcent, emulsifier, thickener, stabilizer, and crosslinker (Izydorczyk et al. 2005; Bhosale et al. 2014). In tissue engineering, it is used in the fabrication of several scaffolds, and it has bone healing properties. The gum possesses biocompatibility and antioxidant and antibacterial properties. It is nontoxic and because of its wound healing and hydrophilic properties, it can be used as a convenient material
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in skin tissue engineering (Ahmed 2018). The addition of gum arabica in a scaffold with other biomaterials increases the water uptake activity of the composite scaffold. This gum also results in the formation of flexible films (Silva et al. 2010). Gum arabica–based scaffold was taken in order to produce a porous scaffold that would mimic the ECM of the skin. The final findings showed that the scaffold containing gum arabica increased the hydrophilicity of the scaffold; the scaffold showed antibacterial property, cell viability, and excellent cell proliferation in vitro proving its potential toward the skin tissue engineering field (Rad et al. 2018).
2.1.2 Gum Ghatti Gum ghatti is one of the effective polysaccharides among the natural plant-based gums that is actively used in the research field because of its biodegradability, sustainability, and safe nature (Lett et al. 2020). It is a complex nonstarch polysaccharide obtained from the bark of Anogeissus latifolia trees. It is a promising biomaterial having the chemical structure of polysaccharides and has β-D-glucopyranuronic acid, β-D-galactopyranose, α-L-arabinosefuranose, α-L-arabinopyranose, and β-D-mannopyranose constituent units in addition to several functional groups present, viz. OH, -NH2, -CONH2, -COOH, and -SO3 (Sharma et al. 2014; Izydorczyk et al. 2005). It is used as an emulsifier, binder, stabilizer, thickener, flocculating agent, suspending agent, macromolecular surfactants, drug delivery carriers, and superabsorbent materials. It is biodegradable, biocompatible, bioabsorbable, nontoxic, and renewable. It has a controlled swelling property, permeability, mechanical strength, and pH stability along with a higher solubility. In tissue engineering, this gum is used in the fabrication of scaffolds and hydrogels (Sharma et al. 2015; Mohammadinejad et al. 2020). In bone tissue engineering gum ghatti has been used as a binder for the preparation of porous scaffolds along with other biomaterials like hydroxyapatite (HAp). The concentration of gum ghatti affects the rheology and helps to bring down a favorable rheological property for the fabrication of scaffold. The presence of gum ghatti improves the porosity of the scaffold. Moreover, the gum ghatti–based scaffolds support cell growth and proliferation, thus it can be said that this gum serves as an excellent binder for the production of more useful scaffolds that would contribute to bone tissue regeneration applications (Lett et al. 2020). 2.1.3 Karaya Gum Karaya gum is a polysaccharide extracted from Sterculia urens plant. It is a complex, highly branched heteropolysaccharide having α-L-rhamnopyranose, β-Dgalactopyranose, α-D-galacturonic acid, and β-D-glucopyranuronic acid constituents units. It is anionic, hydrophilic, and acidic (Ahmed et al. 2019). The gum is made up of rhamnogalacturonan, has α-1,4-linked D-galacturonic acid and α-1,2-linked-Lrhamnosyl residues as the backbone, and the side chain contains 1,3-linked β-D-glucuronic acid or 1,2-linked β-D-galactose on a galacturonic acid unit where half of its unit is partly substituted by 1,4-linked β-D-galactose (Reddy et al. 2019; Izydorczyk et al. 2005). It is biocompatible with high gel forming and adhesion abilities. It has a high viscosity, pH-dependent solubility, good swelling, and water
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retention properties. It is nontoxic and used as suspending agent, emulsifying agent, sustaining agent, binder, crosslinker and dental adhesive (Bhosale et al. 2014). In the biomedical field, it is used in controlled drug release in drug delivery systems. The gums are also used in soft and hard tissue engineering in hydrogels and scaffold fabrication along with other biopolymers (Mohammadinejad et al. 2020; Patra et al. 2013). In tissue engineering, a scaffold made of gum karaya and silk fibroin was used for a study and the scaffold was found to exhibit remarkable cytocompatibility, cell adhesion, and proliferation property showing its potential toward skin tissue regeneration (Patra et al. 2013).
2.1.4 Gum Tragacanth Gum tragacanth is a branched exudate polysaccharide found in several plants belonging to the genus Astragalus. It comprises several constituent units such as D-galacturonic acid, D-galactose, D-glucose, D-xylose, L-fucose, L-arabinose, and L-rhamnose (Ahmed et al. 2019). It is biocompatible and biodegradable with excellent rheological properties (Ghani et al. 2016). It acts as an emulsifier, demulcent, adhesive, and suspending agent (Bhosale et al. 2014). Thus, it is involved in several applications in biomedicals, tissue engineering, and controlled drug delivery (Ahmed et al. 2019). Its functionality depends upon its molecular weight and viscosity. It has low toxicity, better cell adhesion ability, and is also shown to have greater cell viability and osteogenic differentiation. Besides gum tragacanth is reported as one of the novel materials used for osteogenic purposes. So, this polymer is likely to be actively used in bone tissue engineering. Gum tragacanth can be said as an appropriate agent for skin tissue engineering as the scaffolds containing tragacanth gum may produce an ultrathin highly porous scaffold with high stability and flexibility with wound healing properties. That can enhance attachment and proliferation of skin cells and leads to skin tissue regeneration (Ghani et al. 2016). 2.1.5 Guar Gum Guar gum is a natural polysaccharide procured from Cyamompsis tetraganoloba seeds. This is nonionic and water soluble with a high molecular weight (Bhosale et al. 2014). It can form gels by changing its chemical and physical properties. The gum is comprised of β-D-mannopyranose and α-D-galactopyranose with a backbone consisting of a long linear chain of β-(1,4)-linked mannose residues with α-(1,6)-Dgalactose (Ahmed et al. 2019; Lett et al. 2018). It is highly hydrophilic, biodegradable, nontoxic, viscous, and used as an emulsifier, binder, stabilizer, crosslinker, disintegrating, and thickening agent (Bhosale et al. 2014). Therefore, they are tremendously used in several fields like pharmaceuticals, food industries, cosmetics, and biomedical genetics. The gum has good water blocking, swelling, and gelling properties. In the tissue engineering field, it is considered to have a high potential to develop artificial bone scaffolds as well. These are used as gelling agents in hydrogels (Bhosale et al. 2014; Thankam et al. 2018). It can be used as wound care material. In a bone tissue engineering study guar gum was used as a binder for the preparation of porous scaffolds of HAp. The use of guar gum helped in a
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successful scaffold preparation with the desired density, porosity, and mechanical property (Lett et al. 2018).
2.1.6 Pectin Pectin is a linear anionic plant polysaccharide used as a stabilizer (Bhosale et al. 2014). Pectin can be found in most of the plant cell walls. It is a biodegradable, biocompatible, and water-soluble polysaccharide that promotes cell adhesion and proliferation results to show a better osteogenic activity. It also possesses antiulcer, anti-inflammatory, and antitumor properties. So, the pectin-based scaffolds are applied in wound dressing, bone, and skin tissue engineering. It is comprised of (1,4)-linked α-D-galacturonic acid residues, containing L-rhamnopyranose units (Zhao et al. 2016). Pectin can be used as a stabilizer and crosslinking agent (Bhosale et al. 2014). As crosslinker pectin can improve the resistance and compressibility capability of a scaffold. Pectin can also be believed to overcome low mechanical strength to lead the material to show better characteristics with no toxic effect. Pectin is used in skin tissue engineering as it is hydrophilic and can promote wound healing and cellular interaction. Pectin-based scaffolds have been reported to promote skin regeneration in a promising time limit. Apart from that pectins are believed to manage chemical modifications. This polymer is applicable in both conventional freeze-drying and modern 3D bioprinting technologies (Turkkan et al. 2017). 2.1.7 Acemannan Acemannan is a plant polysaccharide extracted from Aloe vera. It consists of β-(1,4)acetylated polymannose (Silva et al. 2017; Liu et al. 2019). Being a medicinal plant, it has numerous biological properties. It is proven to have antiviral, antibacterial, anticancerous, anti-inflammatory, and antioxidant activity. It shows immunoregulation and immunomodulatory properties (Liu et al. 2019). This has wound healing properties and is used in pharmaceutical, cardiovascular disease, and dental applications. Acemannan stimulates the immune system, vitalizes tissue regeneration, promotes wound healing, reduces skin tissue damage, and helps in increasing the proliferation of dermal tissues (Liu et al. 2019). In bone tissue engineering acemannan can increase dental pulp tissue proliferation and bone morphogenic protein activity. It is also reported to be biocompatible and promotes bone mineralization, wound healing, and tissue organization. Acemannan scaffolds were studied in periodontal tissue applications and it has been suggested as a useful biomaterial in the case of periodontal tissue regeneration. As periodontium is the soft and hard tissue construct, acemannan can be considered valuable to both soft and hard tissue engineering. Acemannan is also reported to show osteogenesis, bone tissue regeneration, and periodontal tissue regeneration (Liu et al. 2019). 2.1.8 Konjac Glucomannan Konjac glucomannan is a plant polysaccharide derived from Amorphophallus konjac that belongs to the family Araceae. It is composed of β-(1-4)-linked D-glucose and Dmannose with branched of β-1, 6-glucosyl units (Kondo et al. 2009). It is highly viscous (Kondo et al. 2009). It is biodegradable, biocompatible, and behaves as a
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gelling and thickening agent. It has excellent viscosity and water retention properties. It has several health-related applications such as it can reduce cholesterol level, promote wound dressing, immune function, and be involved in heart disease treatment, diabetes, hyperthyroidism, and wound dressing. It forms thermally stable gels and applied in several fields such as drug delivery, forming biodegradable materials and materials for enzyme encapsulation (Behera and Ray 2016). Konjac glucomannan is used in several tissue engineering applications such as bone, skin, cartilage, and vascular tissue engineering. In bone tissue engineering, konjac glucomannan results in producing a mechanically strong porous scaffold with extensive interconnectivity. The scaffolds are nontoxic and exhibit osteointegration properties (Kanniyappan et al. 2018). Konjac glucomannan–based scaffolds are also involved in cartilage tissue engineering and have been reportedly provide a promising platform for the viability and culture of chondrocytes that can result in cartilage tissue regeneration (Kondo et al. 2009). Besides, as previously said, it helps in wound healing and wound dressing, so konjac glucomannan composites may further be applied in skin injury treatments.
2.1.9 Xylan Xylan is a plant polysaccharide, generally produced during the xylem differentiation mechanism. It consists of β-(1-4) glycosidic bonds linked to D-xylopyranose residues as its backbone. It is a heterogeneous polymer having glucuronic acid, arabinose, and acetate as its substitute units. Xylan-based materials are nontoxic, biodegradable, and biocompatible and can be employed in the biomedical field and tissue engineering (Venugopal et al. 2013). Xylan affects the immune system and has been shown to have immunomodulatory properties. In bone tissue engineering the xylan-based composites are reported to show bone repair mechanism. A study revealed that xylan-based composite helps in healing and remodeling a fractured bone along with the regeneration of bone tissues (Bush et al. 2016). Besides, xylan-based scaffolds are also used in cardiac tissue engineering. In a study, an electrospun xylan-based scaffold was investigated for its property toward myocardial infarction. Xylan was taken for the study for its functional properties toward myocardial infarction and the outcomes proved that the scaffold had a suitable mechanical strength and Young’s modulus. The scaffolds were found to provide a satisfactory platform for cell culture and proliferation and the cultured cells performed normal cell morphology (Venugopal et al. 2013). These functional properties of the polymer xylan prove the potential of the polysaccharide toward its application in tissue engineering. 2.1.10 Cellulose Cellulose is a natural gum derived from plants. Those are sustainable, low cost, renewable, biocompatible, bioactive, and nontoxic with high mechanical and thermal stability (Latour et al. 2020; Hickey and Pelling 2019). It is one of the major polysaccharides naturally available on earth that consists of a linear chain of Dglucose units linked to β-(1,4) glycosidic bonds (Silva et al. 2017; Mano et al. 2007). Cellulose is considered as one of the ideal biomaterials because of its tunable
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physical, chemical, and mechanical properties; apart from this, it has a highly porous structure with adjustable stability (Hickey and Pelling 2019). Due to these satisfactory properties, cellulose has vital applications in several areas like bone tissue engineering in hydrogel and scaffold fabrications, bone regeneration, skin tissue engineering, artificial skin making, wound healing purposes, drug delivery system, promotes neuron regeneration, scaffolds for regenerative medicine, 3D cell culture, etc. (Hickey and Pelling 2019; Silva et al. 2017). Cellulose is also used in cardiac tissue engineering. The scaffold made of cellulose is uniformly fibrous porous composite with high molecular strength and it supports cell growth and proliferation. Besides, as stated before it takes part in bone tissue engineering; a study was accomplished by selecting apple-derived cellulose scaffolds for bone tissue engineering. The prepared scaffold showed satisfying porosity, osteoblast cell adhesion, proliferation, and also promoted mineralization. These properties confirm the capabilities of cellulose toward bone tissue engineering (Latour et al. 2020).
2.1.11 Chitin Chitin is a linear polysaccharide generally extracted from exoskeletons of crabs, shrimps, insects, and fungi (Silva et al. 2017; Mano et al. 2007). Chitin has been reported as the second largest available natural polysaccharide after cellulose. It comprises a repeated oxygen-containing hexose ring. Besides it has an acetamido group tied unitedly by β-glycosidic bonds at the second carbon (C2) position and referred to (1,4)-2-acetamido-2-deoxy-β-D-glucan (Wan and Tai 2013). Chitin is a nitrogenous polysaccharide with several excellent properties such as biocompatibility, biodegradability, antibacterial activity, mechanical integrity, and nontoxicity for which it is highly useful in tissue engineering (Jayakumar et al. 2011). Also, it has been shown to enhance some biological activities like immunogenicity, better drug delivery, and wound healing properties with greater compatibility in humans (Jayakumar et al. 2011). Chitin-based materials are broadly used in biomedical fields. Chitin is applied as fibrous scaffolds, porous sponges, and hydrogels in tissue engineering (Venkatesan et al. 2014a). Chitin-based materials stimulate wound healing, skin tissue repair, bone regeneration, and construction of engineered scaffolds for skin tissue engineering (Silva et al. 2017). Chitin can maintain the morphology of chondrocytes; scaffolds composed of pure β-chitin showed the ability to support chondrocytes and can be used in cartilage tissue engineering as spongy scaffolds (Jayakumar et al. 2011). Apart from this chitin-based scaffolds are used in nerve tissue engineering. 2.1.12 Chitosan Chitosan is an unbranched amino polysaccharide consisting of D-glucosamine and N-acetyl D-glucosamine bonds with β-(1,4) bonds and repeating units of glucosamine. The polysaccharide is nontoxic, cationic, soluble in dilute acid solution, and biocompatible (Venkatesan et al. 2015a; Mano et al. 2007). It is obtained from the deacetylation of chitin, from the insect, fungi, and yeast (Pandey et al. 2017). Chitosan is considered an essential biomaterial because of its cationic and polyelectrolyte nature, biodegradability, mucoadhesion, film-forming ability, antimicrobial
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action, hemostatic action, and bioactive nature (Silva et al. 2017). It is widely used in the biomedical and tissue engineering field in bone skin and cartilage tissue engineering (Silva et al. 2017). It plays a vital role in wound repair and wound healing for burn injuries and surgeries. Due to its biodegradability, biocompatibility, low cost, and wound healing properties it gained a lot of interest in the skin tissue engineering field and used in scaffold fabrication for skin graft applications. The pure chitosan was found to show cell adhesion, proliferation, and tissue regeneration. But chitosan-based scaffolds fabricated along with other natural polymers showed better stability, mechanical property, and biological activity and were recognized as a favorable material for skin grafting and skin tissue engineering applications (Pandey et al. 2017). Chitosan in bone tissue engineering serves as an ideal biomaterial. Chitosan in scaffolds performs surface modification making the scaffold eligible for bone regeneration and believed to promote bone bioactivity (Venkatesan et al. 2015a). Chitosan helps improve neovascularization, thus supports heart function. In a study chitosan-based scaffold was modified to a conductive scaffold when doped with carbon nanofibers. The resulted scaffold showed to have better elasticity showing satisfying viable cardiac cells with greater metabolic activity than the pure chitosan scaffold producing a better tissue construct for cardiac tissue engineering (Martins et al. 2014). In cartilage tissue engineering chitosan-based scaffolds combined with other biomaterials are being used in an articular cartilage repair application. The involvement of chitosan was notable as it showed an effect on the deacetylation degree and molecular weight of the scaffold. The scaffold was porous like the articular cartilage tissue and it was effective for proliferation and differentiation of chondrocyte showing an adequate capacity toward cartilage tissue repair (Li et al. 2018).
2.1.13 Chondroitin Sulfate Chondroitin sulfate (CS) is a linear unbranched polysaccharide generally as part of a proteoglycan that remains attached to proteins. It is generally sulfated glycosaminoglycan with proteoglycan attachments composed of variable monosaccharide chains such as D-glucuronic and N-acetyl-D-galactosamine (Pal and Saha 2019; Kwon and Han 2016). It is biodegradable, biocompatible, easily available, and highly versatile. Chondroitin sulfate is used in cartilage tissue-specific applications and cartilage tissue engineering (Kwon and Han 2016). It supports osteogenic differentiation with the increase of bone anabolic growth factors effectiveness, bone formation, and also involved in the fabrication of hydrogels in bone tissue engineering. CS-based materials are also involved in skin tissue engineering and dermal tissue engineering; it promotes wound healing and stimulates regeneration in skin defects. CS is also been involved in nerve tissue engineering by supporting nerve roots growth in vitro and resulting in nerve tissue damage repair (Kwon and Han 2016). 2.1.14 Hyaluronic Acid Hyaluronic acid (HA) is a linear, anionic polysaccharide. It is comprised of repeated β-(1,4)-D-glucuronic acid and β-(1,3)-N-acetyl-D-glucosamide disaccharides units (Silva et al. 2017; Mohammadinejad et al. 2020). It is usually present in the body
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at specific matrices, in specialized tissues like vitreous humor of eyes, cartilage, and several bodily fluids (Falcone et al. 2016). It has numerous biological properties, viz. biodegradability, biocompatibility, and nontoxicity. It often lacks mechanical strength and immunogenicity, but when combined with crosslinkers and other polymers the mechanical stability can be improved (Zhu et al. 2017). It is hydrophilic and used in the tissue engineering field in processing techniques, scaffolds, hydrogels, cryogels, sponges, and injectable hydrogels. In bone tissue engineering HA is used in the craniofacial as well in dental fields; HA-incorporated scaffolds help in bone regeneration. These scaffolds also have great potential for the improvement of the formation and mineralization of bones. Thus, it is considered a favorable material in bone regeneration (Zhai et al. 2020). It promotes cell migration, proliferation, wound healing and tissue regeneration. HA hydrogels are also used in cartilage regeneration in cartilage tissue engineering (Zhu et al. 2017).
2.1.15 Xanthan Gum Xanthan gum is a branched heteropolysaccharide extracted from the bacteria Xanthomonas campestris (Mano et al. 2007). It is comprised of repeating units of (1,4)-linked β-D-glucose residues, with a side chain of trisaccharide β-D-mannose β-D-glucuronic acid α-D-mannose attached to D-glucose alternate units (Mano et al. 2007; Ahmed et al. 2019). It is anionic, soluble in water, highly biocompatible, immunogenic, thermally stable with modified plasticity, and high molecular weight (Kumar et al. 2017). This gum is nontoxic, bioadhesive, and biodegradable. As it possesses all these properties, it has several applications in tissue engineering. It is usually applied as an emulsifier, stabilizer, and suspending agent (Bhosale et al. 2014). Xanthan gum in combination with other polymers is used in hydrogel and scaffolds preparation for skin tissue engineering and it also has wound healing properties. Xanthan gum reportedly can protect cartilage at joints and helps reduce papain-induced osteoarthritis (Kumar et al. 2017). Xanthan gum–based scaffolds exhibit improved mechanical properties, swelling behavior, and cell proliferation showing promising distribution toward bone tissue engineering (Kumar et al. 2017). 2.1.16 Gellan Gum Gellan gum, an unbranched anionic high molecular exopolysaccharide achieved by the fermentation of Sphingomonas paucimobilis bacteria. This has repeating (1!3)-β-D-glucose, (1!4)-β-D-glucuronic acid, (1!4)-β-D-glucose, and (1!4)-α-L-rhamnose units with a COOH side group (Muthukumar et al. 2019; Mano et al. 2007). It is used as a disintegrating, gelling, stabilizing, suspending, and thickening agent. It is gel forming, elastic, thermally stable, nonangiogenic, nonimmunogenic, and low cost with excellent rheological property. The polymer shows promising applications toward cell adhesion, dental care, bone regeneration, and wound healing (Muthukumar et al. 2019). Gellan gum–based materials are biocompatible, they are involved in the fabrication of injectable hydrogels in tissue engineering applications (Spera et al. 2018). Gellan gum can assist support in cartilage regeneration applications (Muthukumar et al. 2019). In bone tissue engineering gellan gum is considered a promising agent to provide support to
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cartilaginous and promoting bone regeneration; also the improved gellan gum–based scaffolds with some other biomaterials can result in highly porous scaffolds with high mechanical strength and better swelling properties (Spera et al. 2018). The gum has been studied for soft tissue engineering and angiogenesis enhancement by Gorustovich et al. (2010) A novel skin graft was proposed with the gellan gum and studied in vitro. The result showed that this gum has the potential for applications in skin regeneration.
2.1.17 Dextran Dextran is a branched exopolysaccharide derived from bacterial sources synthesized by the activity of an enzyme called dextransucrase (Mano et al. 2007). These are the homopolysaccharides that contain repeated glucose units (Silva et al. 2017). It has a linear backbone of α-(1,6)-linked D-glucopyranose residues linked with α-(1,2), α-(1,3), and α-(1,4)-linked small side chains. It is a nontoxic, biodegradable, and biocompatible polysaccharide with a less inflammatory response (Mohammadinejad et al. 2020). This polymer is directed to form scaffolds, hydrogels, gelling agents, and encapsulation/coating material. Based on these properties and applications, the polymer is considered a promising material in the areas of biomedical and tissue engineering. In cardiac tissue engineering, a study showed that dextran was used as a protective agent to maintain the bioactivity of the encapsulated material in the scaffold resulting in more sustainable growth factor release helping in cardiovascular tissue regeneration (Tian et al. 2012). A dextran-based hydrogel was examined and found to be suitable for cell viability and cartilaginous tissue generation, proving its potential for cartilage tissue engineering strategies. Dextran hydrogel was used as a wound dressing layer and the result showed that it can also promote wound healing with the complete regeneration of dermal tissue and skin appendages. In another study, a dextran-based scaffold was found to show a high swelling property, good compressive strength, and supported cell proliferation showing its promising applications toward the field (Pan et al. 2014). 2.1.18 Scleroglucan Scleroglucan is a microbial source–derived polysaccharide consisting of β-1,3-linked glucopyranosyl residue as the backbone substituted by the β-1,6-glucose at every third unit (Corrente et al. 2013). This is a natural polymer used in pharmaceuticals and tissue engineering applications. They are used as hydrogels in tissue engineering as well in drug delivery systems (Corrente et al. 2013; Coviello et al. 2005). It is also known as sclerogum. It could be used as a crosslinker, laxative, stabilizer, and thickening agent with an amazing rheological property. It possesses antimicrobial and antitumor properties and is able to stimulate the immune system (Coviello et al. 2005). Scleroglucan along with other biomaterials results in the formation of stronger hydrogels and can be modified to improve the mechanical properties of the hydrogels. The concentration of scleroglucan in the mixture affects the hardness and cohesiveness of the hydrogel. Scleroglucan-based materials are more viscous and in tissue engineering applications they show natural
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tissue-like behavior by showing swelling properties when associated with biological fluids (Corrente et al. 2013).
2.1.19 Pullulan Pullulan is a microbial polysaccharide found in the exoskeleton of Aureobasidium pullulans. It is linear having α-(1,4) and α-(1,6)-linked maltotriose with glycoside linkages (Singh et al. 2016). It is biocompatible and nontoxic with extensive mechanical strength. The polymer is soluble in water and has no taste or smell. Pullulan is thermally stable, biodegradable, nontoxic, nonhygroscopic, noncarcinogenic, and elastic with structural flexibility (Rekha and Sharma 2007). Due to all these properties, it is involved in several biomedical applications. It encourages cell adhesion, proliferation, and also bone tissue formation in bone tissue engineering. The polymer is involved in vascular and dermal tissue engineering and regeneration. Pullulan scaffolds are yet a suggesting material in skin tissue engineering as involved in full-thickness skin graft and wound healing with better water uptake properties (Singh et al. 2016). Pullulan is nonimmunogenic and can act as a cytoadhesive material for cell-mediated cartilage repair if applied in cartilage tissue engineering. These biomaterials also result in cartilage tissue regeneration (Singh et al. 2016). It can be modified to improve several properties like degradability, solubility, colloidal and structural stability, and can also be applied in drug and gene delivery for liver targeting therapies. Heparin-pullulan-based materials used in scaffolds inhibit smooth muscle cell generation, hence can be used for vascular endothelial cell regeneration (Rekha and Sharma 2007). In tissue engineering, pullulan can be used as hydrogels, scaffolding material, nanocomposites, and injectable beads. Pullulan-based scaffolds can result in advanced calcification, bone tissue formation, differentiation, regeneration, and mineralization. These scaffolds are also included in trabecular tissue-related applications. Pullulan provides a proper environment for wound healing and dressing, so it might be a satisfactory material in skin tissue engineering (Singh et al. 2016). 2.1.20 Alginate Alginate is one of the linear natural polysaccharides made of β(1,4)-linked D-mannuronic acid and α(1,4)-linked L-guluronic acid; the blocks of this polymer consist of consecutive and alternative G and M residue as (GGGGGG), (MMMMMM) in consecutive residue, and (GMGMGM) in alternative residue, respectively (Mano et al. 2007; Venkatesan et al. 2014b). It is procured from brown algae. It has anti-inflammatory and antioxidant activities. It has tunable chemical and physical properties, including biodegradability, biocompatibility, mechanical strength, and gelation property (Sun and Tan 2013). It is used as a suspending and sustained release agent (Bhosale et al. 2014). It is easily available, low cost, and nontoxic. It has several biomedical applications such as drug delivery, skin, bone, cartilage repair, and wound healing. In tissue engineering, it can be used as hydrogels, microcapsules, and 3D scaffolding materials (Sun and Tan 2013; Venkatesan et al. 2015b). Gelatin is also used in cardiovascular tissue engineering and hepatic tissue engineering.
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2.1.21 Agar Agar is obtained from specific marine red seaweeds and algae. The polysaccharide is unbranched, composed of agarose and agaropectin units (Mano et al. 2007). It behaves like a gel, thickener, and stabilizer. It has the property that it can be prepared easily and can be given any kind of shape. Due to its porous structure, it behaves like a sponge. The gel of a particular shape can be dried as can regain its shape upon rehydration, which can be a promising technique in tissue engineering (Verma et al. 2006). It is biocompatible, biodegradable, and antimicrobial in nature. Because of its favorable compatibility, elasticity, controllable size, and adjustable mechanical properties, it can be regarded as a promising biological agent in tissue engineering applications (Thu-Hien et al. 2018). Agar-based scaffolds exhibit excellent cytocompatibility and swelling properties, so they can be also used as a suitable polymer in skin tissue engineering (Verma et al. 2006). Agar-based materials are also used in bone tissue engineering and regeneration. 2.1.22 Agarose Agarose is a natural linear polysaccharide extracted from red algae (Zarrintaj et al. 2018). Agarose is comprised of a repeated (1!3)-linked β-D-galactose and (1!4)linked 3,6-anhydro-α-L-galactose units (Mano et al. 2007). It resembles ECM, behaves as a gel, exquisitely biocompatible, thermoreversible, and has physicochemical properties that support cell growth and drug delivery in biological systems with a low rejection rate to the immune system (Zarrintaj et al. 2018). It behaves as a gel and used in the formation of hydrogels and sponges (Mano et al. 2007). It is involved in cartilage tissue engineering. In neural tissue engineering it has been used in an electroactive scaffold with some other conductive polymers; because of its cell adhesion potential, biodegradability, and charged behavior it can also be a favorable material in neural tissue engineering (Zarrintaj et al. 2018). Agarose is used in bone tissue engineering as it shows better porosity distribution over the scaffold, supports biomineralization, and plays a promising role. Apart from these, the agarose-based scaffolds are also used in skin tissue engineering as they show noncytotoxic and antiinflammatory effects and reportedly capable of helping in the regeneration of the histological structure of human skin (Zarrintaj et al. 2018). 2.1.23 Carrageenan Carrageenan is an unbranched polysaccharide derived from red algae and consists of 1,3-linked β-D-galactose and 1,4-linked α-D-galactose chains (Mano et al. 2007). The polymer possesses sulfate groups, depending upon the sulfated groups present carrageenans are divided into three separate classes named κ-carrageenan, ι-carrageenan, and λ-carrageenan. κ-Carrageenan contains one sulfate group, ι-carrageenan has two, and λ-carrageenan has three sulfate groups. κ-carrageenans are generally involved in bone tissue engineering and regeneration of bone tissues (Silva et al. 2017). They are nontoxic, biocompatible, and low cost (Mohammadinejad et al. 2020). κ-carrageenan and ι-carrageenan form gels in the presence of some specific ions such as potassium ion (K+) and calcium ion (Ca2+) and λ-carrageenan is viscous (Mano et al. 2007).
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Carrageenan supports both conventional freeze-drying and 3D scaffolding technology. Carrageenans can produce scaffolds with excellent mechanical behavior. They provide a niche for cell proliferation proving its ability toward tissue engineering applications (Sharma et al. 2013). k-carrageenan hydrogels are also said to be as an active material with satisfying properties toward the application in cartilage tissue engineering.
2.1.24 Ulvan Ulvan is a marine-based polysaccharide found in green algae. Rhamnose, uronic acid, xylose, and sulfate are the major constituent units of this polymer (Silva et al. 2017). It is hydrophilic and anionic. It shows antiviral, antibacterial, and anticoagulant activity (Kogelenberg 2017; Tziveleka et al. 2019). The polymer shows osteoconductivity and ulvan-based scaffolds are biocompatible and applicable for tissue regeneration applications with improved stability and mechanical strength. The addition of this material to the scaffold can result in better water uptake capacity and a highly porous scaffold. Besides, the scaffolds are shown to possess satisfying cell adhesion, proliferation, and viability (Tziveleka et al. 2019). Because of all these satisfying properties, the polymer has gathered a lot of recognition in biomedical fields for the involvement in the preparation of hydrogels, nanofibres, 3D porous scaffolds, wound healing, cells, and drug delivery (Silva et al. 2017). Ulvan is utilized in the fabrication of scaffolds for skin tissue engineering as it affects wound repair and wound healing mechanism. Apart from all these ulvan is a satisfying material for 3D bioprinting and can be used as a bioink (Kogelenberg 2017). 2.1.25 Fucoidan Fucoidan is a marine-based polysaccharide derived from brown algae. It is heterogeneous comprising several constituent units, viz. D-galactose, D-mannose, D-fucose, D-glucose, and D-xylose (Pajovich and Banerjee 2017). It is biodegradable, biocompatible, and noncytotoxic (Venkatesan et al. 2014c). The polymer is soluble in water. It is anti-inflammatory, antitumor, antithrombin, and anticoagulant in nature, thus applicable in wound healing, biomineralization, tissue formation, and regeneration in bone and skin tissue engineering (Silva et al. 2017; Pajovich and Banerjee 2017). The polymer also has potential toward skin tissue engineering, and composites having fucoidan help wound healing, skin tissue formation, and regeneration by replacing damaged tissue. Scaffolds having other biomaterials combined with fucoidan are shown to have better cytocompatibility and helps in bone tissue regeneration (Pajovich and Banerjee 2017). Fucoidan-based scaffolds are observed to exhibit higher water uptake ability. The presence of fucoidan in the scaffold results in an increment of the free functional group, thus resulting in a higher swelling degree (Venkatesan et al. 2014c). Fucoidan exhibits calcium ion binding properties that help in calcium deposition and biomineralization. The polymer enhances alkaline phosphatase activity and osteocalcin production promoting bone tissue formation and differentiation (Pajovich and Banerjee 2017; Venkatesan et al. 2014c). It has
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been also experimentally proved that the addition of fucoidan to the composite scaffold can enhance bioactivity and porosity of the scaffold leading to bone regeneration and mineralization. Thus, it can act as a suitable agent in areas of bone tissue engineering (Venkatesan et al. 2014c).
2.1.26 Laminarin Laminarin is a marine-based natural polysaccharide derived from brown algae. It behaves like a rich source of carbon in the marine ecosystem. It is comprised of repeating units of β-(1-3)-linked glucose with irregular β-(1-6) branches (Custódio et al. 2016). It is less viscous having a lower molecular weight that supports the material to be involved in microfabrication techniques (Martins et al. 2018). The polymer has immune-stimulatory and antibacterial properties (Custódio et al. 2016). Laminarin assists in cell adhesion and proliferation (Martins et al. 2018; Larguech et al. 2017). Laminarin has an abundant hydroxyl group that helps its molecules to bind to the bioactive agent, thus making the polymer a bioactive material. Modified laminarin-based materials such as methacrylate laminarin particles provide a suitable platform for cell culture and cell growth and can enhance cell proliferation, differentiation, and migration. With these properties, laminarin has been suggested as a useful material for tissue engineering applications (Martins et al. 2018). Though there is no complete evidence of the utilization of laminarin in cartilage tissue engineering, still this polymer has shown some impact on chondrogenic differentiation that can be applicable in mesenchymal stem cells therapies. As mesenchymal stem cells are considered as one of the relevant agents in cartilage tissue repair and regeneration process, this role of laminarin can allow itself to be involved in cartilage tissue engineering application (Larguech et al. 2017). 2.1.27 Carboxymethyl Cellulose Carboxymethyl cellulose (CMC) is cellulose-derived unbranched polysaccharide. It is achieved by the alkalization of cellulose with sodium monochloroacetate. It is used in pharmaceutical, food, and adhesive industries as a gum. It is white in colur with no taste and odor (Alizadeh et al. 2017). It is a low cost, biodegradable, biocompatible, and nontoxic gum that is actively used in tissue engineering applications and supports cell growth. In coordination with metal ions, carboxymethyl cellulose can produce hydrogels of improved mechanical strength. CMC-based composite in combination with other biomaterials like HAp in bone tissue engineering promotes osteoconductivity, swelling degree, stability, compressive strength, and mechanical properties of the scaffold and supports bone tissue regeneration (He et al. 2019). Carboxymethyl cellulose is also used in cartilage tissue engineering that results in cartilage tissue regeneration and healing. In Table 2 we have given a concise overview of all the gums described in this chapter in order to provide some instantaneous information.
Guar gum
Gum tragacanth
acid, Dgalactose, D-glucose, D-xylose, L-fucose, L-arabinose, and Lrhamnose β-D-mannopyranose, α-Dgalactopyranose
D-galacturonic
α-L-rhamnopyranose, β-Dgalactopyranose, α-Dgalacturonic acid, β-Dglucopyranuronic acid
Gum karaya
Gum ghatti
Structural constituents β-D-glucopyranuronic acid, α-L-rhamnopyranose, β-Dgalactopyranose, β-D-4glucopyranuronic acid, α-Larabinose furanose, α-Dgalactopyranose β-D-glucopyranuronic acid, β-D-galactopyranose, α-Larabinosefuranose, α-Larabinopyranose, β-Dmannopyranose
Gums Gum arabica
Table 2 Overview of the gums
Emulsifier, binder, stabilizer, crosslinker, disintegrating, and thickening agent
Emulsifier, demulcent, adhesive, and suspending agent
Emulsifier, binder, stabilizer, thickener, flocculating agent, suspending agent, macromolecular surfactants, drug delivery carriers, and superabsorbent materials Suspending agent, emulsifying agent, sustaining agent, binder, crosslinker, and dental adhesive
Biodegradable, bioabsorbable, nontoxic, renewable, controlled swelling property, permeability, mechanical strength, and pH stability Biocompatibile, gel forming, adhesive, highly viscous, pH-dependent solubility, nontoxic, good swelling, and water retention properties Biocompatible biodegradable, excellent rheological properties, low toxicity, and better cell adhesion ability Nonionic, water soluble, high molecular weight, highly hydrophilic, biodegradable, nontoxic, viscous, good water blocking, swelling, and gel forming
Applications Suspending agent, binding agent, demulcent, emulsifier, thickener, stabilizer, and crosslinker
Properties Water soluble, lower viscosity, biocompatible, antioxidant, antibacterial, nontoxic, and hydrophilic
Gums for Tissue Engineering Applications (continued)
Ahmed et al. (2019), Lett et al. (2018), Bhosale et al. (2014), Thankam et al. (2018)
Ahmed et al. (2019), Ghani et al. (2016), Bhosale et al. (2014)
Reddy et al. (2019), Izydorczyk et al. (2005), Ahmed et al. 2019, Bhosale et al. (2014)
Sharma et al. (2015), Mohammadinejad et al. (2020), Sharma et al. (2014), Izydorczyk et al. (2005)
References Izydorczyk et al. (2005), Bhosale et al. (2014), Ahmed (2018)
42 1015
Antiviral, antibacterial, anticancerous, antiinflammatory, antioxidant, immunoregulatory, and immunomodulatory Highly viscous, biodegradable, biocompatible, and water retention ability Nontoxic, biodegradable, biocompatible, and immunomodulatory Low cost, renewable, biocompatible, bioactive, and nontoxic, mechanically and thermally stable, tunable physical, chemical, and mechanical properties Biocompatibile, biodegradabile, nontoxic, antibacterial, mechanical integrity, and nonantigenic
Β-acetylated polymannose
Glucose, mannose and glucosyl units
Xylopyranose, glucuronic acid, arabinose, and acetate
D-glucose
units linked to β-(1,4) glycosidic bonds
Oxygen-containing hexose ring, acetamido group, β-Dglucan
Glucosamine with and N-acetyl
Acemannan
Konjac glucomannan
Xylan
Cellulose
Chitin
Chitosan
D-glucosamine
Properties Biodegradable, biocompatible, water soluble, nontoxic, and hydrophilic
Structural constituents α-D-galacturonic acid and Lrhamnopyranose
Gums Pectin
Table 2 (continued)
Latour et al. (2020), Hickey and Pelling (2019), Silva et al. (2017), Mano et al. (2007)
Wan and Tai (2013), Jayakumar et al. (2011)
Promotes immunogenicity, drug delivery, wound healing, supports tissue growth, and regeneration Wound healing, wound repair, tissue regeneration and
Kondo et al. (2009), Behera and Ray (2016), Kanniyappan et al. (2018) Venugopal et al. (2013), Bush et al. (2016)
Silva et al. (2017), Liu et al. (2019)
References Zhao et al. (2016), Bhosale et al. (2014)
Tissue regeneration, wound healing, and 3D cell culture
Gelling, thickening agent, promotes osteointegration, and tissue regeneration Tissue healing, repair, and regeneration mechanism
Applications Stabilizer, crosslinking agent, promotes wound healing, and manages chemical modifications Stimulates the immune system, tissue regeneration, and tissue proliferation
1016 P. S. Khillar and A. K. Jaiswal
β-D-glucose, β-D-glucuronic acid and α-L-rhamnose units
with α-(1,2), α-(1,3) and α-(1,4)-linked small side chains
Gellan gum
Dextran gum
D-glucopyranose
β-(1,4)-D-glucuronic acid and β-(1,3)-N-acetyl-Dglucosamide disaccharides units β-D-glucose, β-D-mannose β-Dglucuronic acid-α-D-mannose
Hyaluronic acid
Xanthan gum
Glycosaminoglycan, proteoglycan, D-glucuronic, and N-acetyl-D-galactosamine
Chondroitin sulfate
Gel forming, elastic, thermally stable, biocompatible nonangiogenic, nonimmunogenic, low cost, and excellent rheological property Nontoxic, biodegradable, biocompatible, and less inflammatory response
Soluble in water, highly biocompatible, thermally stable with modified plasticity, and high molecular weight
Biodegradable, biocompatible, nontoxic, and hydrophilic
Biocompatible, biodegradable, antimicrobial, better stability, and mechanical property Biodegradable, biocompatible, easily available, and highly versatile Tissue-specific application, osteogenic differentiation, wound healing, and stimulates tissue regeneration Bone tissue development and regeneration, promotes cell adhesion, proliferation, and wound healing Emulsifier, stabilizer, suspending agent, wound healing, and enhances the mechanical properties and cell proliferation Disintegrating agent, multifunctional, gelling agent, stabilizing agent, suspending agent, thickening agent, promotes cell adhesion and wound healing Encapsulation material, drug delivery, cell therapy, cell viability, promotes wound healing, dermal, and cardiovascular tissue regeneration
development, and promotes osteogenesis
(continued)
Mohammadinejad et al. (2020), Tian et al. (2012), Pan et al. (2014)
Muthukumar et al. (2019), Mano et al. (2007), Bhosale et al. (2014)
Ahmed et al. (2019), Kumar et al. (2017), Bhosale et al. (2014)
Silva et al. (2017), Mohammadinejad et al. (2020), Zhu et al. (2017)
Venkatesan et al. (2015a), Silva et al. (2017), Pandey et al. (2017), Mano et al. (2007) Pal and Saha (2019), Kwon and Han (2016)
42 Gums for Tissue Engineering Applications 1017
D-mannuronic
acid and -guluronic acid
Agarose and agaropectin
β-D-galactose and 3,6-anhydro-α-L-galactose
Agar
Agarose
Biocompatible, nontoxic, thermally stable, biodegradable, nontoxic, nonhygroscopic, noncarcinogenic, elastic with structural flexibility, and water uptake properties Anti-inflammatory, antioxidant, tunable chemical and physical properties, biodegradable, biocompatibile, high mechanical strength, gel forming and nontoxic properties Gel forming, water holding capacity, biocompatible, biodegradable, and antimicrobial Gel forming, biocompatible, thermo-reversible, biodegradable, noncytotoxic, and anti-inflammatory
α-(1,4) and α-(1,6)- maltotriose with glycoside linkages
Pullulan
Alginate
Properties Rheological property, antimicrobial, antitumor, and viscous
Structural constituents Β- glucopyranosyl residue substituted with β-1,6-glucose
Gums Scleroglucan
Table 2 (continued)
Support cell growth and tissue repair
Thickener, stabilizer, promotes excellent cytocompatibility and bone tissue regeneration
Suspending and sustained release agent, drug delivery, 3D fabrication, tissue repair, and encapsulating agent
Applications Crosslinker, laxative, stabilizer, thickener, stimulates the immune system, and develops mechanical properties Cell adhesion, proliferation, tissue formation, and cellmediated tissue repair applications
Mano et al. (2007), Zarrintaj et al. (2018)
Mano et al. (2007), Verma et al. (2006)
Mano et al. (2007), Venkatesan et al. (2014b), Sun and Tan (2013)
Singh et al. (2016), Rekha and Sharma (2007)
References Corrente et al. (2013), Coviello et al. (2005)
1018 P. S. Khillar and A. K. Jaiswal
D-galactose, D-mannose, D-
Fucoidan
Carboxymethyl cellulose
Laminarin
Rhamnose, uronic acid, xylose, and sulfate
Ulvan
unit with sodium monochloroacetate
D-glucose
Glucose residues with β-(1–3) and β-(1–6) branches
fucose, D-glucose, and D-xylose
α-D-galactose, β-D-galactose
Carrageenan
Low cost, biodegradable, biocompatible, and nontoxic
Water soluble, biodegradable, biocompatible, and noncytotoxic, antiinflammatory, antitumor, antithrombin, anticoagulant, higher water uptake ability, higher swelling degree, and calcium ion binding properties Less viscous, lower molecular weight, immune stimulatory, and antibacterial
Nontoxic, biocompatible, low cost, gel forming, viscous, excellent mechanical behavior, thickener, and emulsifier Hydrophilic, antiviral, antibacterial, anticoagulant, and better water uptake capacity
Supports microfabrication techniques, assists cell adhesion, proliferation, and migration Supports cell growth, improves mechanical strength, promotes osteoconductive and tissue regeneration
Promotes osteoconductivity, tissue regeneration, improves stability, mechanical strength, cell adhesion, cell viability, wound healing, and drug delivery Wound healing, tissue formation and regeneration, and replaces damaged tissue, and promotes cytocompatibility
2D and 3D culture, promotes cell proliferation and tissue regeneration
Alizadeh et al. (2017), He et al. (2019)
Custódio et al. (2016), Larguech et al. (2017), Martins et al. (2018)
Pajovich and Banerjee (2017), Venkatesan et al. (2014c)
Silva et al. (2017), Kogelenberg (2017), Tziveleka et al. (2019)
Mano et al. (2007), Silva et al. (2017), Mohammadinejad et al. (2020)
42 Gums for Tissue Engineering Applications 1019
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Conclusion
In this chapter, we have reviewed some of the natural gums that are biodegradable, biocompatible, nontoxic, easily available, and low cost with tuneable mechanical properties and mostly used in soft and hard tissue engineering and tissue regeneration applications. The gums are commonly derived from some natural sources like plants, animals, marine, and bacteria. They are further distinguished according to their charge (ionic and nonionic), shape (linear and branched), and unit (homogenous and heterogeneous). The natural gums are likely to provide a suitable environment to mimic the native ECM, support cell attachment, and new tissue formation. It displays improved biological activity compared to other synthetic biomaterials. This chapter outlines the properties and application of the natural gums or gum-based materials in the fabrication of scaffolds and hydrogels as well as their use in the form of crosslinkers, binders gelling, thickening, and emulsifying agents in bone, skin, cartilage, cardiac, retinal, and neural tissue engineering applications. These gums sometimes show superior performance when combined with other biomaterials, some of their properties can be modified and improved with the addition of other natural and synthetic polymers and biomolecules (e.g., growth factors) to perform toward specific tissue engineering applications. Either in pure form or as a modified polysaccharide, these gums can be used as bioinks in 3D bioprinting that appears to be a promising approach in tissue engineering applications. Apart from tissue engineering, these natural polysaccharides have numerous applications in drug delivery, nanomedicines, and therapeutic research as shown in Fig. 4. Due to these valuable contributions, these gums are being considered promising materials in biomedical research.
Fig. 4 Different applications of natural gums
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References Ahmed AA. Health benefits of gum Arabic and medical use. Gum Arabic. 2018. https://doi.org/10. 1016/b978-0-12-812002-6.00016-6. Ahmed S, Ahmed M, Manzoor K, Purwar R, Ikram S. A review on latest innovations in natural gums based hydrogels: preparation and applications. Int J Biol Macromol. 2019;136:870–90. Alizadeh AS, Mousavi M, Labbafi M. Synthesis and characterization of carboxymethyl cellulose from sugarcane bagasse. J Food Process Technol. 2017. https://doi.org/10.4172/2157-7110. 1000687. BaoLin G, Ma PX. Synthetic biodegradable functional polymers for tissue engineering: a brief review. Science China Chem. 2014;57(4):490–500. https://doi.org/10.1007/s11426-014-5086-y. Behera SS, Ray RC. Konjac Glucomannan, a promising polysaccharide of Amorphophallus konjac K. Koch in health care. Int J Biol Macromol. 2016. https://doi.org/10.1016/j.ijbiomac.2016.07.098. Bhosale RR, Osmani RA, Moin A. Natural gums and mucilages: a review on multifaceted excipients in pharmaceutical science and research. Int J Pharma Phytochem. 2014; 6(4):901–12. Bush JR, Liang H, Dickinson M, Botchwey EA. Xylan hemicellulose improves chitosan hydrogel for bone tissue regeneration. Polym Adv Technol. 2016;27(8):1050–5. https://doi.org/10.1002/ pat.3767. Corrente F, Amara AHM, Pacelli S, Paolicelli P, Casadei MA. Novel injectable and in situ crosslinkable hydrogels of dextran methacrylate and scleroglucan derivatives: preparation and characterization. Carbohydr Polym. 2013;92(2):1033–9. https://doi.org/10.1016/j.carbpol. 2012.10.018. Coviello T, Palleschi A, Grassi M, Matricardi P, Bocchinfuso G, Alhaiqu F. Scleroglucan: a versatile polysaccharide for modified drug delivery. Molecules. 2005;10(1):6–33. Custódio CA, Reis RL, Mano JF. Photocrosslinked Laminarin based hydrogels for biomedical applications. Biomacromolecules. 2016. https://doi.org/10.1021/acs.biomac.5b01736. Dzobo K, Thomford NE, Senthebane DA, Shipanga H, Rowe A, Dandara C, Pillay M, Motaung KSCM. Advances in regenerative medicine and tissue engineering: innovation and transformation of medicine. Stem Cells Int. 2018. https://doi.org/10.1155/2018/2495848. Falcone SJ, Palmeri D, Berg RA. Biomedical applications of hyaluronic acid. In: Zhu XX, Ravenelle F, editors. Polysaccharides for drug delivery and pharmaceutical applications. San Luis Obispo: FzioMed Inc., Bonetti Drive; 2016. p. 155–74. Ghani M, Mak WC, Cheung KY, Montazer M, Rezaei B, Griffith M. Cross-linked superfine electrospun tragacanth-based biomaterial as scaffolds for tissue engineering. Eur Cells Mater. 2016;21(1):204. Gorustovich AA, Roether JA, Boccaccini AR. Effect of bioactive glasses on angiogenesis: a review of in vitro and in vivo evidences. Tissue Eng Part B. 2010;16(2):199–207. https://doi.org/10. 1089/ten.TEB.2009.0416. He X, Tang K, Li X, Wang F, Liu J, Zou F, Yang M, Li M. A porous collagen-carboxymethyl cellulose/hydroxyapatite composite for bone tissue engineering by bi-molecular template method. Int J Biol Macromol. 2019;137:45–53. Hickey RJ, Pelling AE. Cellulose biomaterials for tissue engineering. Front Bioeng Biotechnol. 2019;7:2296–4185. https://doi.org/10.3389/fbioe.2019.00045. Izydorczyk M, Cui SW, Wang Q. Polysaccharide gums: structures, functional properties, and applications. In: Cui SW, editor. Food carbohydrates: chemistry, physical properties, and applications. Boca Raton: CRC Press; 2005. p. 43. Jayakumar R, Chennazhi KP, Srinivasan S, Nair SV, Furuike T, Tamura H. Chitin scaffolds in tissue engineering. Int J Mol Sci. 2011;12(3):1876–87. https://doi.org/10.3390/ijms12031876. Kanniyappan H, Thangavel P, Chakraborty S, Arige V, Muthuvijayan V. Design and evaluation of Konjac glucomannan-based bioactive interpenetrating network (IPN) scaffolds for engineering vascularized bone tissues. Int J Biol Macromol. 2018. https://doi.org/10.1016/j.ijbiomac.2019. 12.012.
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Microbial Polysaccharides as Cell/Drug Delivery Systems
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Microbial Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Fungal Polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Xanthan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cell Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Dextran-Based Cryogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Dextran-Based Colon Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Gellan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Salecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Heparosan Polysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Pullulan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Schizophyllan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Curdlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Polysaccharides are considered to be excellent materials when compared with other polymers, due to their ease in tailoring, biocompatibility, bioactivity, homogeneity, bioadhesive nature, and ability to mimic the natural extracellular matrix microenvironment. Polysaccharides show great potentials for drug delivery and M. Ramesh (*) Department of Mechanical Engineering, KIT-Kalaignarkarunanidhi Institute of Technology, Coimbatore, Tamil Nadu, India K. Sakthishobana · S. B. Suriya Department of Biotechnology, KIT-Kalaignarkarunanidhi Institute of Technology, Coimbatore, Tamil Nadu, India © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_54
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tissue engineering applications. The main aim of this chapter is to spotlight the new advancements and challenges concerned with microbial polysaccharides used for cell/drug delivery applications. In this chapter, the essential microbial polysaccharides including cellulose, Dextran, Pullulan, Xanthan, Salecan, Gellan, etc., which are sourced from plants, microbes, and animals, respectively, are reviewed. From the chapter it is concluded that the series of biocompatible microbial polysaccharides were promising candidates as smart drug delivery systems in future biomedical field. Keywords
Microbial · Polysaccharides · Biocompatible · Drug delivery systems · Dextran · Cellulose
1
Introduction
From the middle of twentieth century, microbial polysaccharides contributed to applications in various fields including biomedical field where dextran solution is used as plasma expanders for first clinical trial. Many other microbial polysaccharides are utilized as pharmaceutical excipients like xanthan gum as suspension stabilizer or pullulan in capsules and enteral products. They originate from plant or animal sources but are mostly produced from microbial fermentation process. In current modern biomedical field, bacterial cellulose is used in wound dressings and as scaffolds. Control release drug systems of both micro- and nano-particulate materials were emerging as a new application for biopolymers in biomedical field. Polysaccharide based nano-particulate systems plays major role as drugtargeting and carriers since they display biocompatibility and nontoxicity (Moscovici 2015). Treatments for many noncommunicative diseases include tablets, capsules, and syrups. They cause adverse effects like microbial resistance, cytotoxicity, and hypersensitivity reactions. In search for the novel methods of therapy without producing adverse effects, researchers designed novel drug delivery system, microbe-based delivery systems, and gene delivery systems. Microbes, especially whole bacteria, and its toxin, viruses or fungi express some desired results in drug delivery system. Drug delivery systems that use microbes are considered safe, nontoxic, with minimal side effects and site-specific targeted action. They serve as a drug carrier and as a therapeutic agent in current research. From the results gathered, microbe-based drug delivery system has become one of the most hopeful drug delivery systems for many diseases like cancer, inflammatory bowel diseases, and also in cancer therapy. Viruses and bacteria can aid in anticancer therapy by accumulating in the cancer cells, which will be helpful in tumor and tumor imaging and it will be the futuristic advanced system for dynamic improvement in patient’s health (Shende and Basarkar 2019).
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Polysaccharides
2.1
Microbial Polysaccharides
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Polysaccharides are natural polymers produced by microorganisms that are secreted extracellularly. They are a large group of biopolymers used in various applications, namely, food, pharmaceutical, and medical field (Giavasis 2013). They pose prodigious range of variety in both structural and functional properties and are commercially available. Specifically, curdlan, cyanobacteria, dextran, pullulan, schizophyllan, xanthan, and xylinan polysaccharides are commercially available microbial polysaccharides (Morris and Harding 2009). The key producers of microbial polysaccharides are fungi of the Basidiomycetes family and several gramnegative bacteria such as Xanthomonas, Pseudomonas, Alcaligenes, etc., and gram-positive bacteria such as lactic acid bacteria. Yeast belonging to Saccharomyces genus also synthesizes polysaccharides (Giavasis 2013). Presently, marine microbial extracellular polysaccharides have novel properties in structure and chemical composition that have specific applications in adhesives, textiles, pharmaceuticals, and medicine fields as anticancer drugs, food additives, oil recovery, and metal removal (Manivasagan and Kim 2014).
2.2
Fungal Polysaccharide
One of the common and well-studied fungal polysaccharide is pullulan. Pullulan is a white, tasteless, water-soluble homo-polymer of glucose. Genus of mushroom Ganoderma is rich in bioactive compounds whose polysaccharides are greatly consumed in Asia. It reveals most distinctive biological properties, as well as antiinflammatory, antitumor and immunomodulatory activities (Ren et al. 2021). It is used as traditional medicine in China for health and longevity. Presently, compounds such as polysaccharides, alkaloids, amino acids, triterpenes, steroids, ergosterols, furan derivatives, nucleosides, fatty acids, and lipids from Ganoderma have been isolated and identified. In that mainly polysaccharides and triterpenoids are accountable for the pharmacological activities of Ganoderma. They are responsible for mitogen-activated protein kinase (MAPK) pathways in immune cells and also for cancer immunotherapy through nuclear factor-κB (NF-κB) (Ren et al. 2021). Even though most of the microbial polysaccharides are derived from fungi and bacteria, yeast such as Saccharomyces cerevisiae produces polysaccharide glycan, which is extracted from yeast cells walls (Giavasis 2013). In the fruiting body of Flammulina velutipes, a novel water-soluble polysaccharide (Flammulina velutipes Polysaccharide1 – FVP1) is seen, which aids in Inflammatory Bowel Diseases. This polysaccharide is the active component showing bioactivity like immune regulation, antimicrobial activity, antineoplastic. The novel water-soluble polysaccharide has the potential to act as a functional food ingredient or a preventive drug for Inflammatory Bowel Diseases (IBD) (Zhang et al. 2020).
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Cellulose
Cellulose, a bacterial-derived natural polymer produces 3D nano-fibril networks, which are biocompatible. It forms very narrow pore size and this obstructs cell adhesion and gas/fluid transportation. The meticulous designing of biomaterial with an appropriate pore size and pore interconnectivity can lead to cell adhesion, cell infiltration, migration, vascularization, and host integration therefore mimicking the natural extracellular matrix (ECM) (Dubey et al. 2021). The drug should provide a specific targeted therapy than developing a generalized effect. Nanotechnology in biomedical field involves enhanced drug targeting, imaging, and diagnosis. Thus, the use of nanoparticles has become the latest trend since it provides result (Unnikrishnan et al. 2020). Bacterial cellulose was made into scaffolds using desired shape molds and achieved a porous network of scaffold. This scaffold showed very slow degradation behavior due to lack of cellulase enzymes in human and it is found that even after 60 days there was no change in its structure/decomposed. Thus, it can be employed for medical conditions that require long-term healing like large bone fractures. Growth factors are crucial for cellular process and tissue regeneration. Consequently preconditioning the cells before depositing in scaffolds plays a role in cellular repair and regeneration (Dubey et al. 2021).
2.4
Xanthan Gum
Xanthan gum is an external polysaccharide derived from fermentation of glucose and sucrose by the bacteria Xanthomonas campestris (Fig. 1). It is mainly utilized as food additive as stabilizer and thickening agent. Recently, xanthan gum was used along with other polysaccharides as drug delivery molecules. Ngwabebhoh et al. (2021) reported a crosslinking between chitosan, xanthan gum, and hydroxypropyl methylcellulose using Schiff’s base reaction for controlled antibiotic drug delivery of minocycline, rifampicin, and ampicillin to inhibit microbial growth. They showed cumulative sustained release up to 80%.
3
Cell Delivery
Currently, nanoemulsions have been widely functional in the various areas of targeted drug delivery, cosmetic industry and food industry. They are non -equilibrated, kinetically stable systems containing oil, water and an emulsifier. One of the main uses of nanoemulsion is the delivery of drugs and bioactive materials to the specific target site. Nanoemulsions involve two synthesis steps: macro-emulsion and conversion to a nanoemulsion. The techniques involve high energy methods like high pressure homogenization, ultrasonication, and low energy method like phase inversion temperature, emulsion inversion point. Richa and Choudhury (2019) proposed a nanoemulsion with fucoid and κ-carrageenan polysaccharide, which
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CH2OH
CH2OH O O
O
OH
COOH H3C
O
OH
H3C
R6
O
R
n
O
O
4
OH
O OH OH
6 R O
O OH 4 R O
O
COOH
OH
O O
OH OH
Fig. 1 Structure of xanthan gum (Giavasis 2013)
showed potential biological safety, stability, and biodegradability compared to synthetic emulsifiers. Microbe-based drug delivery systems are responsible for safe, nontoxic, site-specific targeted action with fewer side effects taken by conventional drug delivery system. Microbes, especially whole bacteria, bacterial toxin, viruses, or fungi express some validated results in drug delivery system for cancer and inflammatory bowel disease. Due to accumulation in tumor and tumor imaging, both bacteria and viruses are equally shown to be an interesting candidate for drug delivery. It will be helpful in futuristic advanced system for dynamic improvement in patient’s health (Shende and Basarkar 2019). Probiotics are beneficial microbes which promote gut health and are recommended as treatment for Crohn’s disease, colorectal cancer therapy, and antibiotic-associated diarrhea. Lactobacilli and Bifidobacterium sp. are the most used probiotics and keeping them viable during formulation and storage is the crucial part in its preparation. So a novel microbial polysaccharide and sunflower oil-based emulsion gel formulation was developed to simultaneously deliver these anaerobes as well as a lipophilic antimicrobial drug metronidazole (Pandey et al. 2016). The use of microbial polysaccharide has many advantages. It promotes targeted delivery since they are indigestible in stomach or small intestine and digested only in colon. The sugars in polysaccharides provide a dual role (i) an anaerobic condition and (ii) as a nutrient to maintain its viability. The polysaccharides used are xanthan gum and guar beans-derived guar gum. The study showed
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stability in oil-in-water type emulsion gels and their viability retained after 30 days of refrigeration. It also helped in controlled release of metronidazole drug in colon (Pandey et al. 2016).
3.1
Dextran
The dextran is a complex microbial polysaccharide made of branched α 1, 6 linked d-glucopyranoside residues, which has lots of medicinal use (Fig. 2). The most prevalent lactic acid bacteria like Leuconostoc mesenteroides and Streptococcus mutans produce dextran. This polysaccharide cannot be digested by human since we lack dextranase enzyme produced by colon residing bacteria. The degradation product of dextran is a biocompatible molecule, thus nontoxic in nature. This polymer protects the drug delivery particle from cell and protein adhesion thus avoiding any immune response from the system. It is an approved plasma substitution and plasma volume expansion molecule (Yao et al. 2014). One way of administering is by covalently binding the drug with dextran macromolecule, forming a prodrug. Alternatively, nanoparticles can be synthesized. Colorectal cancer is the third most common cancer type (Tiryaki et al. 2020) affecting people and the therapy does not provide permanent relief from the disease. This was addressed by Tiryaki et al. by designing an enzyme triggered controlled drug delivery system. The drug encapsulated silica gel has dextran in the outer coating and 5-fluorouracil is used as the anticancer drug. 5-Fluorouracil inhibits the nucleotide pathway by controlling thymidylate synthase activity (Tiryaki et al. 2020). But it can also affect the normal cells and thus it is essential to deliver drug only at the intended site. This drug carrying silica gel was prepared by sol-gel method and dextran outer coating provides the enzyme triggered control release in colon. Fig. 2 Structure of dextran (Giavasis 2013)
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The drug release pattern in gastric-, intestinal- and colon-simulated regions showed that silica aerosols released 85–89% of drug in all the regions within 24 h. At the same time silica aerosol coated with dextran and dextran-CHO released around 1.7% and 3.4%, in gastric and intestinal fluid, respectively, and 4.2% and 0.97% in dextranase free colon fluid medium. In presence of dextranase enzyme the release was increased to 24% and 13.4% for dextran and dextran-CHO-coated silica aerosols. This confirms the controlled drug release triggered by enzyme for the cancer therapy. MTT assay showed the nontoxic nature of the produced biomolecule in vitro (Tiryaki et al. 2020). Dang and Guan concluded that image-guided drug delivery is the best example in which it incorporates magnetic resonance imaging (MRI) with drug delivery nanoparticles to monitor the bio-distribution, circulation, and targeting the behavior of nanoparticles because they are not only required to deliver drugs but also to offer diagnosis and drug monitoring during cancer treatment (Dang and Guan 2020).
3.2
Dextran-Based Cryogels
One more dextran-based biomaterial involves the fabrication of cryogels. Cryogels are hydrogels differing in the production temperature that is below the freezing point of the solvent giving rise to high elastic and macroporous biomaterial (Pacelli et al. 2021). Interconnectivity between the pores can help in the movement of molecules and cell adhesion. They are prepared using natural or synthetic polymers or in combination to achieve the expected result. The solvent used is mostly water and thus the cryogels are formed at 15 C associated with immediate freeze drying to get spongy like porosity. Since many parameters are involved in the making process, variation in any of the parameters may produce the desired result. The dextran methacrylate with the cross-linking agent as synthetic polymer polyethylene glycol dimethacrylate and water as solvent were used to produce cryogels. The polymer concentration and rate of reaction contributes to the main parameter for proper formation of cryogels. Unlike a hydrogel, the cryogel can withstand heavy mechanical stress and can resist deformation. It did not compromise on the biomolecule release profile due to its high porosity and it exhibited no cytotoxicity (Pacelli et al. 2021).
3.3
Dextran-Based Colon Cancer Therapy
The on-off kind of controlled drug release can be formed using a hydrogel with addition of electro-active aniline trimer with dextran polysaccharide and hexamethylene diisocyanate. They are external stimuli-based functional hydrogels, which can respond to electric stimuli. The external stimuli generally used are pH, temperature, light, enzymes, magnetic field, and electrostatic potential (Qu et al. 2019). In these, the electrostatic stimulation can be a great advantage since it can regulate the drug release based on its oxidation/reduction characteristics. The use of
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aniline is biocompatible since it can be excreted through renal filtration and thus can act as smart biomaterial. The electric potential driven release of the drug showed higher cumulative effect in a 120 min duration at 3 V than the 35% released drug in case of no external electric stimuli. Instead of continuous electric potential, intermittent voltage potential of 3 V per 30 min was given, which released drug in the same effect. The in vitro and in vivo cell culture showed biocompatibility. The release effect is due to the movement of charged molecules in the electric field and the changes in net charge of hydrogel due to oxidation/reduction reactions. This kind of controlled stimulibased release helps in localized sustained drug release therapy (Qu et al. 2019). Dextran polysaccharide can assist in prolonging the circulation time of drug in the blood stream that is usually not stable in vivo. An antiviral drug zidovudine (AZT) is the first approved drug for AIDS-HIV disease. But its effect is diminished due to its short half-life in blood circulation as it can easily dissolve in most of the pH. To increase the life span and to control its release a suitable vehicle needs to be designed. An encapsulation method of core- and shell-based nanoparticles was prepared with AZT present in the core. Dextran and stearic acid-coated polyethylene glycol (PEG) forms the outer shell and AZT is presented in the core. This nanoparticle preparation promotes circulation time, protects from phagocytosis, and controlled release. The delivery of anticancer drug and its cellular uptake also proved to be efficient in normal and cancer cells. Thus, a novel dextran using hybrid nanoparticle can be used in the positional delivery of the drug (Joshy et al. 2018). Drug delivery system formulated using liposomes and natural polysaccharide conjugates have great potential to improve drug-like properties and curative efficacy improving oral absorption, controlling the release, enhancing the in vivo retention ability, targeting the delivery, and exerting synergistic effects (Li et al. 2017). Targeting cancer cells can be done by its characteristic lower pH, higher temperature, and high concentration of glutathione in the cells. The various therapies that make use of this property can be designed to produce external stimuli-based controlled drug delivery system. A system that uses pH as external stimuli was performed by hydrophobic camptothecin drug encapsulated by dextran with POGMEA through atom transfer radical polymerization (Bai et al. 2018), histidine cross-linked cholesterol–dextran micelles (Yao et al. 2014), and vicinal diol modified with hydrophobic acid-labile phenylboronic ester groups-based dextran B-Dex polymer, which is shown in Fig. 3 (Li et al. 2013). These micelles can be designed to target specific cellular or subcellular locations for drug delivery and making it tumor-specific. They can enter the targeted tumor cell plasma membrane through enhanced permeability and retention effect (EPR) and it unloads its cargo when it encounters endosomes or lysosomes thereby lysing the cell (Bai et al. 2018). The disulfide cross-linking between dextran and the drug increased the tumor cell apoptosis. These micelles were found to possess high drug loading capacity, stable micelle structure in circulation fluid, rapid cellular internalization, and targeted drug delivery (Bai et al. 2018). The disulfide cross-linking is desired for stability and targeted drug delivery. It readily degrades in reducing environment making it subcellular-specific lysosomes (Lian et al. 2017). pH and
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Fig. 3 Schematic of B-Dex polymer production and its pH stimuli-based doxorubicin drug delivery in lysosomes (Li et al. 2013)
reductive environment-based external stimuli provides dual targeting. Histidine cross-linked with cholesterol dextran conjugate micelles Doxorubicin (DOX) was the drug used in this study and it was greatly internalized and released its drug load inside the cell thus killing it (Lian et al. 2017). Polyethylene glycol (PEG) is a FDA-approved molecule for biomedical use. In spite of its biocompatibility, opsonization, and longer half-life, it was found that use of PEG gives rise to hypersensitivity reaction, Anti-PEG antibody production, and activation of complement system. So it is desired to use an alternative for PEG, and research on dextran is becoming popular for the production of nanoparticles. So a drug delivery for the specific colorectal cancer cells was designed to use dextran with
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poly-o-nitrobenzyl alcohol (PNBA), a light-sensitive polymer. The ester bond in this polymer absorbs 2 photons from UV light and converts into carboxylic acid functional group, which modifies its polymeric structure. The structure modification causes the nanoparticle to spill its contents at the specific site. The photo-radiation study revealed the disappearance of nanoparticles in the solution and the disappearance depends on the irradiation time and nanoparticle chemical composition (El Founi et al. 2018). Multifunctionality nanoparticle combinations like diagnostic and therapy together in a single micelle can contribute to advanced cancer therapy. One such research provides anticancer drug doxicillin DOX and diagnostic super-paramagnetic iron oxide (SPIO) for magnetic resonance imaging (MRI) in an encapsulated nanoparticle for cancer therapy. The application of manganese ferrite MnOFe2O3 (Mn-SPIO) aids in the precise MRI scans, since it possesses superior MR transverse relaxation (T2) shortening effects. The nanoparticle is micellar in structure, which is made by dextran coated with stearic acid. This kind of micelle can carry hydrophobic drug in its core and can help in imaging and therapy of cancer cells simultaneously. Cellular internalization when compared between free and nanoparticle DOX, free DOX displayed the concentration with 24 h incubation while nanoparticle-based DOX achieved the same within 2 h of incubation in breast cancer MCF-7/Adr cells. The MRI signal provided a darkening effect than the control sample without Mn-SPIO when studied under clinical 3.0-T MRI scanner (Lin et al. 2015).
3.4
Hydrogels
Hydrogels are water insoluble matrix made up of cross-linked hydrophilic polymers, which are termed as smart biomaterials (Chandra et al. 2020; Qi et al. 2020). Hydrogels imbibe a lot of liquid and as a result it expands in volume and size. The hydrogels due to its swelling nature lack mechanical strength and collapse easily. To improve its stability cross-linking with either natural or synthetic polymers are used. Many polysaccharides derived from microbes, plants, and animals are used as a green technology to achieve biocompatible, biodegradable materials for tissue engineering application (Qi et al. 2020).
3.5
Gellan Gum
Gellan gum is a negatively charged linear polysaccharide produced by the bacteria Pseudomonas/Sphingomonas elodea (Lee et al. 2012). It consists of repetitive units of sugars like D-glucose, D-glucuronic acid, D-glucose, and L-rhamnose (Fig. 4). It is chiefly used in the food industry as a stabilizer and gelling agent. Currently, it is exploited in biomedicine field owing to its biocompatibility and biodegradation profile and it has been approved as an excipient in pharmaceutical preparations. Cross-linking gellan gum with cations or chemical molecules generate hydrogels with malleable property. One such nontoxic cross-linker used is an endogen
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CH3 C
O
O CH2
0.5 -
+
COO M O
CH2OH
O O
O OH
OH
O C
O O
OH OH
CH2OH
OH OH
O
CH3
OH
O
OH n
O
Fig. 4 Structure of gellan gum (Giavasis 2013)
spermidine, and hydrogels are constructed by ionotropic gelation technique, which promotes tissue regeneration and biocompatibility (Psimadas et al. 2012). This hydrogel can be used as a vehicle for supplying growth factors for cellular repair and regeneration. When a growth factor BMP-2 was supplied through the gellan gum-based hydrogel in a slow release manner to a healthy muscle cells, it developed ectopic bone formation (López-Cebral et al. 2017). Another research employed a photo-dimerization method to create hydrogel using cinnamate as a cross-linker with gellan gum. Cinnamate occurs naturally in plants like Erythroxylum coca and is a natural tropane alkaloid, which is nontoxic and exhibits anti-inflammatory properties. This research aimed at producing an anti-adhesion film to reduce postoperative inflammation, adhesion fibrosis, and to escalate general well-being. The antiadhesion film should possess softness, flexibility, ease of application, transparency, be nonreactive and fixed in position, and have resolvable properties. The prepared film possessed high tensile strength, owing to the extent of cross-linking, which is an important property of anti-adhesion film (Lee et al. 2012). The visual examination of postoperative anti-adhesion film showed nil to mild adhesion in a group of operated rats, and experiment on number of inflammatory neutrophil cells showed less than the control group of cells. Thus, cinnamate also provides its anti-inflammatory property to the healing tissues (Lee et al. 2012). A separate research used Chlorine 6 drug in the form of Chlorine 6 (Ce6)fucoidan/alginates@gellam gum (Ce6-Fu/AL@GG)-based hydrogel as the anticancer drug in treating early colorectal cancer by way of photodynamic therapy. This hydrogel constitutes few naturally occurring polysaccharides like fucoidan, alginate, and gellen gum and used as a vehicle to carry the photosensitizer chlorine 6 to the tumor cells. Irradiation of laser light to this photosensitizer develops reactive oxygen species (ROS) and free radicals. This ROS can destroy the tumor cells through apoptosis and is believed to effectively heal wounds through vascularization and
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cellular regeneration. The MTT assay with the HT-29 cells reveled exceptional cell death through augmented drug delivering hydrogel and laser irradiation. The hydrogels without photosensitizer drug showed no toxicity. Thus, it was concluded that the produced hydrogel can be effectively used as a carrier for chlorine 6 while it showed no cytotoxicity on its own (Karuppusamy et al. 2019). By using gellan gum as a chief polymer and polyvinyl alcohol (PVA) as supporting polymer by a process of facile electro-spinning, gellan gum-based nano-fibrous transdermal substitute carrying antimicrobial drug amoxicillin was fabricated to combat microbial infection at wound site. The gellan/PVA nano-fibers have more potential as growth scaffold compared to PVA nano-fibers (Vashisth et al. 2016).
3.6
Salecan
Salecan, a linear polysaccharide belonging to the family of β-glucan, is a novel molecule isolated from Agrobacterium sp. ZX09. It is made up of repetitive glucopyranosyl units and has the tendency to absorb a great deal of water. This polysaccharide was found to possess antioxidant activity, nontoxicity, and is mostly used in food and biomedical field. Poly (N-(3-dimethylaminopropyl) acrylamide-coacrylamide) is cross-linked with salecan polysaccharide by semi-interpenetrating polymer network to derive a biocompatible and a durable hydrogel. Incorporation of salecan resulted in less pores with bigger pore sizes. The drug delivery of model drug amoxycilin showed increased drug release due to large pore size (Qi et al. 2017). A novel method of salecan and gellan gum mixture produced a biocompatible hydrogel scaffold by physical method of cross-linking through heating-cooling. Different ratios of the polymer gave rise to varying mechanical strength and swelling properties, which help in desirable design flexibility and biocompatibility with dense cell population (Qi et al. 2020).
3.7
Heparosan Polysaccharide
Heparosan polysaccharide, a precursor to heparin and heparin sulfate, is derived from Escherichia coli. It is metabolized in lysosomes into simpler monosaccharides, thus constituting its low toxicity and good biocompatibility. It can be used in the cancer treatment by synthesizing heparosan-based micelles carrying the anticancer drug. The anticancer drug doxorubicin was employed in a study and the highest cellular uptake of the drug was observed in B16 cells, greatest cytotoxicity took place in MGC80-3 cells (Qiu et al. 2019). This diversifying effect is related to its drug delivery pathway into cells like phagocytosis, pinocytosis, clatherin receptor-mediated endocytosis, and caveolae-mediated endocytosis. The antitumor drug after internalization into cell enters into either mitochondria or nucleus to induce apoptosis (Qiu et al. 2019).
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Pullulan
Pullulan is a fungal extracellular polysaccharide Aureobasidium pullulans consisting of repetitive maltotriose units connected through α-1, 6 and α-1, 4 glycosidic bonds. The Pullulan polymer does not cause immunogenicity in humans and thus it can act as a plasma expander. It has an innate affinity for uptake by liver cell (Constantin et al. 2020). This FDA-approved polymer exhibits enhanced water solubility, biocompatibility, hemocompatibility, oil resistance, flexibility, ability to form fibers, adhesion property, edible nature, and oxygen impermeable film formation (Su et al. 2020). Since pullulan is a macromolecule containing many surface hydrophilic groups, in association with a hydrophobic molecule can be fabricated to create a nanoparticle micelles for drug delivery. The shell and core type nanoparticle can accommodate the drug in core while the shell can have pullalan and derivatives (Constantin et al. 2020). A study on the use of pullulan for drug delivery involved the synthesis of dibutylaminopropyl carbamate pullulan octanoate, an amphiphilic molecule. Nanoparticle was synthesized in two steps and loaded with model hydrophobic drug diclofenac by core-shell assembly. The diclofenac drug release profile of synthesized nanoparticle was extremely slow and it followed biphasic pattern. It took more than 100 h when kept under pH 7.4 and 5.4 at physiological temperature of 37 C, on the contrary, free diclofenac was entirely released within 4 h of incubation in phosphate buffer (Constantin et al. 2020). This may be contributed to the hydrophobic nature of the drug and its dissipation in the reduced environment. They showed sustained and systemic release of drug in the specific pH. Cytotoxicity studies revealed that administering low concentration of drugloaded and drug-free nanoparticle does not produce cell death; rather it increased the cell viability. Around 80% cytotoxicity was observed when the drug containing nanoparticle was above 62.5 μl/ml concentration. Free polymer also showed similar results and it developed cytotoxicity above 62.5 μl/ml concentration (Constantin et al. 2020). Pullulan in combination with chitosan is used in electro-spinning of nano-fibers for preparing fast dissolving oral film (FDOF). FDOF is a rapid dissolving thin film containing a drug molecule for absorption of drug in buccal cavity. The drugs that cause bowel irritation; GI tract susceptible drugs and saliva activated drugs are formulated for absorption in the abundant buccal vasculature. Electro-spinning utilizes the surface charges in polymer to conduct high electricity, and jet expulsion of the polymers by spinneret at room temperature produces nanofibers once solvent is evaporated. Chitosan and pullulan combined nano-fibrils are formed by electro-spinning and without use of any nonedible substances. The diameter of the nano-fiber altered based on the chitosan concentration. At first the diameter increased, then reduced with increase in chitosan content. A model drug aspirin was encapsulated in it and its dissolving capability showed it has FDOF nature (Qin et al. 2019).
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Schizophyllan
Schizophyllan is a microbial β-glucan containing exo-polysaccharide produced by Schizophyllum commune which possesses medicinal property and bioactivity. It shows many properties including hepatoprotective immune stimulation, antimicrobial, anti-inflammatory, antitumoral, cholesterol- lowering, anti-fibrotic, antidiabetic, and hypoglycemic activities (Negahban et al. 2021). It is believed that the collaborative effect of schizophyllan carrier and the drug promotes the efficacy of the treatment. Pirzadeh-Naeen et al. (2020) studied about ellagic acid, a natural antioxidant and anticancer agent used in the treatment of breast cancer. This drug was encapsulated separately in chitin (EA/Ch-NP) and schizophyllan (EA/SPG-NP). The nanoparticle was synthesized, and its size was around an average of 39.82 nm and 217.8 nm, respectively. Before investigating the treatment of breast cancer MCF-7 cells, the ellagic acid release was considered in 95% ethanol and in various pH mediums (range from 1.5 to 7.4) portraying digestive environment. The study revealed that schizophyllan-based nanoparticle showcased enhanced encapsulation efficiency and drug-loading capacity than chitin-based nanoparticle. The release study showed high drug release in 95% ethanol than in other pH mediums. The antioxidant activity determined by DPPH assay showed higher scavenging activity for free ellagic acid than both schizophyllan and chitin drug-loaded nanoparticle. But interestingly the chitin nanoparticle at a pH of 7.4 produced higher antioxidant activity than the free drug. The MTT assay was carried to comprehend the cell viability via mitochondrial dehydrogenase assay. The IC50 value obtained for schizophyllan and chitin were 60 and 115 μg/mL, respectively. Thus, targeting the breast cancer through ellagic acid-schizophyllan-based nanoparticle can be used in a potential cancer therapy (Pirzadeh-Naeeni et al. 2020). A novel schizophyllan-based micelle system carrying the encapsulated drug paclitaxel (PTX) was developed to deliver highly hydrophobic drug to a target site. Negahban et al. studied the method of producing nano-micelles using schizophyllan and stearic acid for encapsulating hydrophobic drugs in tumor therapy. The produced nano-micelles showed self-assembly into a micelle. An esterification reaction of steric acid with schizophyllan converted it into an amphipathic molecule and they were selfassembled by simple ultrasound method. The synthesized nano-micelle displayed proper size in nm range and provided high PTX loading capacity. This drug carrying nano-micelles revealed high cytotoxicity against cancer cells. The schizophyllan by itself has anti-inflammatory and immune boosting property, along with the anticancer drug can produce synergistic effect in tumor therapy. Thus, the produced nano-micelle has potential as a new drug carrier for hydrophobic drugs (Negahban et al. 2021).
3.10
Curdlan
Curdlan is a linear polysaccharide produced by Alcaligenes faecalis and Rhizobium, which consist of β-1, 3 glucans. Curdlan epitope is recognized and internalized by macrophages and dendritic cells. This can activate the secretion of cytokines from
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activated macrophages and dendritic cells, which successively help in differentiation of CD4+ cells to TH9 cells. This TH9 cells release IL9 and many granzymes when activated and all these will attack tumor cells. Injection of curdlan intratumorally enhances tumor cell necrosis and inhibits its progression by reprogramming the dendritic cells to provoke differentiation of CD8+ cells expressing CD103, a ligand for cancer cells. Herein, we report, for the first time, the peptide aptamer functionalization of curdlan and the tumor cell-targeted siRNA delivery by the curdlan derivative (Ganbold et al. 2019). Natural carbohydrate polymer-based nanoparticles have great biocompatibility that is required for the safe delivery of various drugs including nucleic acid therapeutics. Herein, we designed curdlan-based nanoparticles for cancer cell-targeted delivery of short interfering RNA (siRNA). Ganbold et al. suggested that iRGD functionalized curdlan may provide a biocompatible carrier for siRNA delivery. Curdlan was chemically modified in order to complex with siRNA and specifically enter cancer cells. The first, chemical modification of 6-amination, the second, modification in peptide bearing cationic molecules. The cellular uptake of 6AC-iRGD/siRNA complex was inhibited by chloroquine suggesting that the complex enters the cells through clathrin-dependent endocytosis mechanism. The iRGD-functionalized curdlan was not only taken up by integrin-presenting cells but also carried and released siRNA in the cytoplasm, inducing significant silencing of a disease-related gene Plk1. Collectively, data demonstrated that the curdlan-based nanoparticle may provide a siRNA carrier with the ability of cancer cell-specific delivery (Ganbold et al. 2019). Mycobacterium tuberculosis is the causative agent for tuberculosis disease, which has the tendency to replicate inside macrophages. The drugs used for its therapy cannot be internalized by the macrophage and due to the sub-therapeutical level of drug inside the immune cell can cause microbial resistance. Macrophage receptor dectin-1 recognizes curdlan and internalizes it. During the drug delivery of rifampicin, polysaccharide is conjugated to poly (D, L-lactide-co-glycolide) nanoparticles to bestow immunostimulatory activity (Gopinath et al. 2018). A nanoparticle carrying antituberculosis drugs such as rifampicin and levofloxacin was prepared through conjugated cyclodextrin for targeting macrophage. It is a straightforward method for the production of curdlan-based nanoparticles. The drugs encapsulated in cyclodextrin remained inactive after conjugation with curdlan nanoparticles and got transformed into a sustained release of the drugs over a prolonged time. The minimal inhibitory concentration showed as good as free drug release. These nanoparticles affect the macrophages, but not the fibroblast cells because they are noncytotoxic to RAW 264.7 and L929 cells (Fig. 5) (Yunus Basha et al. 2019). The cellular internalization of antituberculosis drugs was 1.8 times higher in macrophages via dectin-1 receptor than the fibroblast cells. They could kill more than 95% of a tuberculosis model organism Mycobacterium smegmatis residing inside macrophages within 4 h. Basha et al. concluded that the drug-loaded nanoparticles targeting Mycobacterium smegmatis-infected macrophages provide effective bactericidal activity as it delivered the drugs more effectively than free drugs. Thus, curdlan-cyclodextrin conjugated nanoparticles are a potential nano-carrier for targeted drug delivery to macrophages (Yunus Basha et al. 2019).
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Fig. 5 Microscopic image of RAW264.7 macrophages (a, b, e, and f) and L929 fibroblast (c, d, g, and h) (Yunus Basha et al. 2019)
4
Conclusion
Microbial polysaccharides are a large group of extracellular polymers produced from microbial origin. They have various uses in various fields and currently it is developing into a promising field of research in biomedicine. They contain numerous hydrophilic functional groups in its surface, which can be utilized for preparation
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of hydrogels or nanoparticles. The conjugation of this polymer with a hydrophobic fatty acid molecule can create a micelle offering microenvironment for stable transportation of hydrophobic drug. The hydrogels and nanoparticles derived from the wide selection of polysaccharides can help in controlled/sustained release of drug during cancer therapy. This fabrication can not only simply discharge the drug at target site but can also be devised to release the drug under external stimuli. Many parameters such as temperature, pH, electrostatic potential, concentration of a certain molecule, magnetic particles, etc., can act as the external stimuli for releasing the drug molecules from its carriers. Recently, dual stimuli-based nanoparticle/ hydrogels were prepared, which could unload the drug when both conditions were met, for example, pH and reducing environment, pH and temperature. Even an electrostatic-based stimulus can be an advantage for the treatment. Recently, in tumor therapy, a combination of diagnostic imaging and drug delivery was performed for simultaneous characterization. The drugs and cells can be made to deliver to the respective sites with the help of microbial polysaccharides.
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Unnikrishnan BS, et al. Self-assembled drug loaded glycosyl-protein metal nanoconstruct: detailed synthetic procedure and therapeutic effect in solid tumor treatment. Colloids Surf B: Biointerfaces. 2020;193(April):111082. Vashisth P, et al. Drug functionalized microbial polysaccharide based nanofibers as transdermal substitute. Nanomed Nanotechnol Biol Med. 2016;12(5):1375–85. Yao X, Chen L, Chen X, He C, Zheng H, Chen X. Intercellular pH-responsive histidine modified dextran-g-cholesterol micelle for anticancer drug delivery. Colloids Surf B: Biointerfaces. 2014;121:36–43. Yunus Basha R, Sampath SK, Doble M. Dual delivery of tuberculosis drugs via cyclodextrin conjugated curdlan nanoparticles to infected macrophages. Carbohydr Polym. 2019;218 (January):53–62. Zhang R, et al. Polysaccharide from flammuliana velutipes improves colitis via regulation of colonic microbial dysbiosis and inflammatory responses. Int J Biol Macromol. 2020;149: 1252–61.
Injectable Polymeric System Based on Polysaccharides for Therapy
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Guy Decante, Joaquim Miguel Oliveira, Rui L. Reis, and Joana Silva-Correia
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Injectable Polymeric Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Applications of Injectable Materials Based on Microbial Polysaccharides . . . . . . . . . . . . . . . 3.1 Drug and Cell Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Tissue Engineering and Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusion and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Microbial polysaccharides are inexpensive natural polymers synthesized by various microorganisms such as bacteria, fungi, and microalgae. They have been generally used in the food industry as emulsifier, gelling agents, and thickeners. Nowadays, their favorable biological properties, similarity to native ECM, and wide range of molecular weights, and functional groups have sparked the interest of researchers for their use as injectable scaffolds used in drug delivery, tissue engineering, and regenerative medicine. Notable applications of microbial
G. Decante · J. M. Oliveira · R. L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, GMR, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected]; [email protected]; [email protected] J. Silva-Correia (*) 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, GMR, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_55
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polysaccharides are vaccine preparations and intra-articular injections of hydrogels in osteoarthritis therapy. These systems provide minimally invasive routes of implantation in vivo to locally deliver drugs, cells, and other therapeutics in a controlled manner. They may also provide a stable environment to support cell differentiation and migration to enhance tissue regeneration. The various uses of microbial polysaccharides and their derivatives as injectable scaffolds used in therapy are reviewed herein. Keywords
Hydrogels · Polysaccharides · Bacteria · Drug delivery · Tissue engineering
1
Introduction
Polysaccharides extracted from microorganisms have generated a vivid interest in the last three decades due to their wide range of structural and functional properties, and generally low costs. As natural-based carbohydrate macromolecules formed by repeated monosaccharide units linked by glycosidic bonds, they are intrinsically biocompatible and biodegradable, and possess functional groups that allow for their easy chemical modification. Additionally, polysaccharides have similar structures to those of the glycans which constitute the native extracellular matrix (ECM). Monosaccharide units can also mediate cellular processes at molecular level thanks to their biological role in cell signaling. These substances have been used in numerous fields, including the food industry, and in pharmaceutical research. As an example, microbial polysaccharides have been extensively used to form the integral component of many vaccines (Lee et al. 2001b; Jennings and Pon 2010; Patel and Patel 2011). Microbial polysaccharides are classified according to their morphological localization: Cell wall, intercellular and exocellular polysaccharides. Their morphological localization will influence their properties and subsequent applications. Cell wall polysaccharides, such as chitin, mainly contribute to the structural stability of cells. Intracellular polysaccharides are concentrated to the cytoplasm of cells and act as carbon and energy reserves. Finally, exocellular polysaccharides are secreted by cells and can be subdivided into capsular polysaccharides and exopolysaccharides. Capsular polysaccharides are defined as polymers covalently bound to the cells surface, producing capsules around the cells that act as physical barriers against bacteriophages. Exopolysaccharides produce a slime loosely bond to the surface of cells, allowing cells to adhere to other surfaces, while acting as carbon or water reserves (Patel and Patel 2011; Freitas et al. 2021). Fig. 1 shows the basic anatomy of bacteria and where each of these polysaccharides are located within. Microbial polysaccharides gained prominence in biomedical applications thanks to their properties such as their capability to form intermolecular
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Fig. 1 Basic anatomy of bacteria. Bacteria may secrete capsular polysaccharides or exopolysaccharides as external layers
interactions to create polymeric matrices, which can easily be loaded with inorganic materials to allow for drug encapsulation, or to create composite hydrogels with enhanced properties (Freitas et al. 2021). Herein, the important advances dealing with injectable polymeric materials based on polysaccharides, and their applications in pharmaceutic and medical fields are overviewed.
2
Injectable Polymeric Systems
Injectable polymeric systems cover a broad range of materials which can be inserted into tissues using percutaneous procedures for various medical applications. These systems, which including injectable hydrogels, porous hydrogels, and micelles, are implanted in the body through minimally invasive procedures to create structures that will fill complex tissue defects, and/or deliver drugs, cells and/or other therapeutics in a sustained manner (Fig. 2). These injectable polymeric systems are extensively used in tissue engineering, regenerative medicine, and in drug and cell delivery. Although these systems have similar polymeric structures, their properties and characteristics differ, allowing for their use in different applications. An injectable hydrogel is a polymeric structure which can be extruded as a solution directly into the desired location in vivo, where it will polymerize to
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Fig. 2 Principle of injectable polymeric systems. Polymer precursors loaded with cells, drugs, nanoparticles and/or other therapeutics are injected into the patient’s body. The polymer precursor will polymerize in situ through different chemical and/or physical processes to create polymeric structures that will fill tissue defects and/or deliver therapeutics. This figure was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (https://smart.servier. com/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)
transition from a solution to a gel. As it is well known, hydrogels have high water contents which grant them with good biocompatibility and low cytotoxicity, making them ideal to reproduce the ECM in applications such as tissue engineering, regenerative medicine, or drug and cell delivery. Such hydrogels meet great success as carrier matrices for biologics, thanks to their ability to provide a controlled release at targeted areas. Compared to polymeric scaffolds produced ex vivo and subsequently implanted, injectable hydrogels can alleviate the need for surgical interventions, which reduces the patient discomfort, risk of infection, recovery time while offering better aesthetic outcomes. They also have better handling properties and a better adaptability to the irregular shapes of the defects. Hydrogels possess a degree of high customizability which allows them to be tailored to better suit their intended applications. Controlling the viscosity of such systems for example, is critical to ensure that they can be injected into the desired area without clogging the injection device, damaging their potential loaded biologics, or leaking into surrounding tissues. These systems possess selfassembling or stimuli-responsive gelation mechanisms for their networks to polymerize in situ, resulting in stronger and more stable hydrogels (Yang et al. 2014). These chemistries must be carefully selected to not display any toxicity during polymerization. Photo-crosslinking for example, is a widely used crosslinking methodology for in situ polymerization. These chemistries rely on photo-
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crosslinkers which may present toxicity issues if they are left unreacted (Yang et al. 2014). In tissue engineering and regenerative medicine for example, it is essential that the scaffolds produced in situ offer a stable environment for cells to grow, differentiate and proliferate. For biomedical applications, these crosslinking mechanisms must occur under mild conditions, and not generate damages to surrounding tissues or the possible biologics embedded within them. Porous hydrogels cover a broad variety of materials used in biomedical applications, especially in drug and cell delivery. Also referred to as micro/nanogels, or granular hydrogels, these materials are defined as hydrogels produced from micro- to nano-sized particles of polymeric networks. Regular hydrogels consist of polymeric materials crosslinked in swollen meshes, which mesh size range from the tens to hundreds of nanometers. Because the size of most cells ranges in micrometers, regular hydrogels must be degraded before cellular infiltration can occur. Due to their particulate structures, porous hydrogels possess intrinsic porosities, relative to their particle size, which improve the diffusion of loaded cells and drugs. These particles can be dispersed in solvent or larger polymeric networks, and possess tunable sizes, geometries, properties, and characteristics. The literature on these systems is extremely diverse, and there are already many excellent articles that thoroughly cover this subject such as the review from Chiriac et al. (Chiriac et al. 2019). Summarily, due to their particulate structures, these gels also possess very large surface-to-volume ratios that allow for high loading of therapeutics and multivalent bioconjugation, high-exchange rate, and a fast response to environmental cues. Their characteristics such as their sizes and degradation mechanisms can be tuned, allowing for the controlled delivery of loaded therapeutics at targeted sites. Additionally, micro- and nano-gels possess attractive properties for localized surgical injections, such as large viscosities, shear-thinning behavior, and yield stresses (Riederer et al. 2016). Moreover, these systems meet great success for drug and cell delivery applications thanks to their ability to protect their loaded biologics from host immune response, degradation, and shear stress, while also being able to contain them at the implantation sites. However, nanogels are generally expensive to produce, and limited at experimental production, as they are currently produced using organic solvents and additives which may remain in the medium and cause toxicity issues. Non-harsh reagents should be developed to offer safer processing routes (Wang et al. 2017). Micelles are self-assembled colloidal nanostructures composed of a hydrophilic shell surrounding a hydrophobic core (Torchilin 2007). They are widely used as carriers for delivering drugs with poor water solubilities. Thanks to their small size and hydrophilic shells, micelles remain undetected by macrophages and the reticuloendothelial system, which enhances the circulation time of their encapsulated drug. Moreover, encapsulating these drugs in hydrophilic micelles can allow increasing their bioavailability, which lower the doses required and potential side effects (Amin et al. 2017). Micelles possess tunable sizes which allows to direct the loaded biologics to tissues where permeability is enhanced, particularly tumor and
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inflammatory tissue. Furthermore, micelles can be functionalized to recognize specific molecular signals associated with diseased sites, which allows for the targeted delivery of their encapsulated drugs (Xiong et al. 2011). Micelles are extensively used in drug delivery as they enable to entrap and deliver hydrophobic drugs and proteins thanks to enhanced solubility and stability. For drug delivery applications, micelles should have high stability, high biocompatibility, biodegradations properties and low immunogenicity (Zhang et al. 2013). Microbial polysaccharides then appear as good materials to develop micelles since they possess such properties. Microbial polysaccharides can also possess natural positive, negative, or neutral charges, or they may be modified to. Despite the great diversity of polymers and their properties, modifications are often required for hydrogels to have better suiting characteristics for their applications. Microbial polysaccharides are no exception to that matter. Many applications of microbial polysaccharides imply their modification by direct immobilization of molecular functional groups, or their grafting with other polymers (Bacelar et al. 2016; Hu et al. 2017; Kirschning et al. 2018). In addition, hydrogels based on microbial polysaccharides can easily be loaded with nanoparticles, microspheres, etc. to enhance their mechanical and/or biological properties (Ng et al. 2020).
3
Applications of Injectable Materials Based on Microbial Polysaccharides
Microbial polysaccharides can create polymeric structures with a wide range of properties depending on their types, the species they have been extracted from, their possible modifications, the materials they are combined with, etc. All microbial polysaccharides possess natural biocompatibilities, low toxicities and the capacity to gel under mild, physiologically relevant, conditions (Velasco et al. 2012) which make them attractive to create injectable in situ gelling hydrogels to reproduce the ECM. They may also possess immunoregulatory, anticancer, antiviral, anticoagulant, antithrombotic, and many other properties (Filomena et al. n.d.; Matsuda et al. 1999; Patel and Patel 2011; Moscovici 2015; Yu et al. 2018; Carvalhal et al. 2019; Khan et al. 2019; Ng et al. 2020; Zhou et al. 2020; Freitas et al. 2021), making them very interesting for various applications in tissue engineering, regenerative medicine, and controlled local delivery of drugs, genes, cells, or other therapeutics.
3.1
Drug and Cell Delivery
Microbial polysaccharides have met great success in delivering therapeutics in vivo through injectable polymeric systems (Zhang et al. 2013; Huh et al. 2017; Suner et al. 2018; Cadinoiu et al. 2019; Kumari and Badwaik 2019). These systems represent a substantial share of the global pharmaceutical market, only preceded by oral medication. They offer the possibility to rapidly target specific areas by minimally invasive routes such as intravenous, subcutaneous, intradermal,
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intramuscular, intracutaneous, intraperitoneal, intrathecal, intraarterial, intraspinal, and intracardiac. Generally, these delivery systems are preferred if drugs have short half-lives and/or poor absorption by conventional routes of administration (Cadinoiu et al. 2019). Additionally, using injectable hydrogels for delivery allows to bypass first-pass metabolism, which mainly occurs with the oral administration of drugs which then enter the portal circulation before entering the systemic circulation, resulting in rapid decreases in their concentrations (Mathew et al. 2018). Microbial polysaccharides are attractive materials for delivery systems thanks to their multiple functional groups which allow for their bioconjugation with various therapeutic agents, and the wide range of their molecular weights which allows to tune the biodegradation kinetics and the therapeutic release of subsequent hydrogels (Thambi et al. 2016). Thanks to their carbohydrate structures, hydrogels produced from microbial polysaccharides can undergo total degradation within the body, avoiding additional surgeries to remove residual material (Mathew et al. 2018). Moreover, microbial polysaccharides possess attractive immunoregulatory properties for delivery applications. For instance, microbial polysaccharides have been extensively used as antigens for vaccine preparations (Lee et al. 2001a). However, they can only induce the production of low-affinity antibodies by themselves, therefore immunization with bacterial polysaccharides only leads to T cell-independent immune responses (Hütter and Lepenies 2015). Bacterial polysaccharides are often conjugated with proteins to promote long-term antibodies and memory response (Robbins et al. 1983; Finn 2004). Many currently commercialized vaccines are based on conjugated capsular polysaccharides and have proved to be very efficient in preventing a large range of human diseases (Mettu et al. 2020). In situ gelling hydrogels are attractive systems for delivery applications. Therapeutic agents can be mixed with the precursor polymer solutions before injection, resulting in their entrapment within the hydrogel networks which can provide their sustained release at the target sites. Local and controlled delivery of therapeutics is associated with lowered dosing frequencies and side effects (Singh and Hari Kumar 2012). However, since polymer networks may act as diffusion barriers for therapeutics, particulate structures with intrinsic porosities are preferable to allow for their diffusion through the interior of polymeric matrices. Nanogels, microgels, and micelles based on microbial polysaccharides have been extensively studied as delivery vehicles (Zhang et al. 2013; Suner et al. 2018; Chiriac et al. 2019; Kumari and Badwaik 2019). Numerous currently available vaccines systems are based on chitosan micro- and nano-particles, for example, and can increase the efficacy and response to immunization with model protein antigens, viral antigens, bacterialderived antigens and toxins, and DNA plasmids (Chua et al. 2012). Such structures possess attractive advantages for drug delivery. Their small scale allows for the penetration of tissues by paracellular or transcellular pathways, and their fast environmental responses allow for a highly controlled release of their loaded therapeutics (Suner et al. 2018). As aforementioned, microbial polysaccharides are also particularly attractive to develop micelles used in drug delivery thanks to their natural properties and ease of modification. Micelles can be tailored to have adequate size, charge, and shape for delivery, but they can also be further modified with peptides,
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molecules or other polysaccharides to improve site accumulation and uptake (Zhang et al. 2013). They possess a number of benefits over other drug delivery carriers, in particular: (1) they allow to encapsulate and deliver hydrophobic drugs, which allows to reduce the required doses and therefore minimizes the side effects; (2) their small size and hydrophilic shells render them invisible to macrophages and the reticuloendothelial system, which increases the circulation times of their embedded therapeutics; (3) micelles can take advantage of the enhanced permeability and retention effect to deliver drugs through passive targeting; and (4) micelles are also easy and rapid to produce, allowing them to be readily prepared in large quantities owing. However, micelles are also generally unstable due to their dilution by body fluids, and require modification to alleviate this issue (Amin et al. 2017). Moreover, due to their size, micro-, nanogels and micelles have short diffusional paths which results in a rapid release of their encapsulated therapeutic compared to larger carriers (Chiriac et al. 2019). Furthermore, nanoscale materials are often subjected to fast clearance by body defense mechanisms due to their size. Injectable hydrogels have also met great success in gene delivery thanks to their ability to prevent genes from aggregation and extracellular degradation, which allows for their long-term release in a controlled and sustained manner (Mathew et al. 2018). Chitosan for example, has been used as a non-viral vector for gene delivery thanks to its natural cationic charges able to make complexes with negatively charged DNA, siRNA and oligonucleotides (Riederer et al. 2016; Huh et al. 2017). Despite their numerous functional groups, polysaccharides generally require modification to increase their affinity with cells, genes, proteins, etc. Resulting nanoparticles have demonstrated improved delivery capacities by effectively targeting the implantation sites, while protecting the different genes from degradation, and with efficient gene transfection (Huh et al. 2017). Microbial polysaccharides can be used alone or combined with other materials to better tailor the subsequent hydrogels to the intended applications (Alves et al. 2016; Ng et al. 2020). Especially, thanks to their multiple functional groups, microbial polysaccharides can be modified or supplemented with other polymers to create “smart” or “stimuli-responsive” hydrogels (Thambi et al. 2016). These hydrogels are designed to react various stimuli such as temperature, pH, ionic concentration, enzymes, and magnetic or electric field, which can be externally applied or specific to the targeted site. They will then respond to these stimuli with structural changes such as shape morphing, or the modification of their properties like their solubilities and sol-gel transitions. Such types of changes can be reversible or irreversible. Stimuli-responsive hydrogels are very attractive to create delivery vehicles capable of providing a controlled release of therapeutics in vivo which may lead to considerable advantages over traditional medications such as greater drug efficiency, rapid drug absorption, reduced side effects (Bhardwaj et al. 2015). Although they may be based on similar materials, in situ gelling hydrogels, micro- and nanogels, and micelles have their strengths and weaknesses that researchers must consider when developing a novel injectable system to deliver drugs, cells or other therapeutics. Table 1 summarizes these pros & cons of injectable polymeric structures for applications in drug and cell delivery.
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Table 1 Strengths and weaknesses of injectable polymeric systems for drug and cell delivery applications (Torchilin, 2007; Xiong et al. 2011; Chua et al. 2012; Zhang, Wardwell and Bader 2013; Yang et al. 2014; Riederer et al. 2016; Amin et al. 2017; Wang, Qian and Ding 2017; Chen et al. 2017b; Mathew et al. 2018; Suner et al. 2018; Chiriac et al. 2019; Kumari and Badwaik 2019; Ng et al. 2020; Wang et al. 2020) Injectable polymeric system In situ gelling hydrogels
Description Polymer precursors are dispersed in a solution which is injected into the body and undergoes a sol-gel transition in situ
Microgels
Microscale particles of polymeric networks
Nanogels
Nanoscale particles of polymeric networks
Advantages Ease of modification: Can be modified with other materials, and/or loaded with particles. Control over crosslinking also provides tunable mechanical and drug release properties High loading capacities, they can protect encapsulated biologics from external factors, possess attractive properties for localized surgical injections, such as large viscosities, shear-thinning behavior, and yield stresses, they also have fast environment response, easy functionalization, active and passive targeting abilities, intrinsic microscale porosity to allow for the diffusion of cells High loading capacities, they can protect encapsulated biologics from external factors, possess attractive
Disadvantages The nanosized mesh of the polymeric network acts as a diffusion barrier to larger cells and therapeutics, unreacted crosslinkers are often toxic
References Yang et al. (2014), Chen et al. (2017b), Mathew et al. (2018), Ng et al. 2020 and Wang et al. (2020))
Micro-sized carriers have short diffusional paths which result in rapid release of the loaded cargo
Chua et al. (2012), Riederer et al. (2016), Wang et al. (2017), Chen et al. (2017b), Suner et al. (2018), Chiriac et al. (2019) and Wang et al. (2020)).
Their porosity is too fine for cells to diffuse, nanosized materials often present a fast clearance due to
Chua et al. (2012), Riederer et al. (2016), Wang et al. (2017), Suner et al. (2018), Chiriac et al. (continued)
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Table 1 (continued) Injectable polymeric system
Micelles
3.2
Description
Hydrophilic polymer aggregates with a hydrophobic core
Advantages
Disadvantages
References
properties for localized surgical injections, such as large viscosities, shear-thinning behavior, and yield stresses, they also have fast environment response, easy functionalization, active and passive targeting abilities, intrinsic porosity which allows for the diffusion of oxygen and nutrients. Their nanoscale allows them to penetrate tissues by paracellular or transcellular pathways Can encapsulate and deliver hydrophobic drugs, easy functionalization, active and passive targeting abilities. Their nanoscale allows them to penetrate tissues by paracellular or transcellular pathways
body defense mechanisms, nanosized carriers also have short diffusional paths which results in a rapid release of the loaded cargo. Nanogels are expensive to produce, and their production methods generally require organic solvents and additives which may remain in the medium
(2019) and Kumari and Badwaik (2019)
Nanosized materials often present a fast clearance due to body defense mechanisms, nanosized carriers also have short diffusional paths which results in a rapid release of the loaded cargo, subject to dilution by body fluids
Torchilin (2007), Xiong et al. (2011), Zhang et al. (2013), Amin et al. (2017), Chiriac et al. (2019) and Kumari and Badwaik (2019)
Tissue Engineering and Regenerative Medicine
Hydrogels produced from microbial polysaccharides possess numerous attractive properties to be used for injectable systems in tissue engineering. Hydrogels are biocompatible and can crosslink in situ through mild processes to create soft structures that resemble that of natural ECM. These structures are permeable to allow for the diffusion of nutrients and the clearance of waste products, their
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mechanical properties can be tailored to match those of targeted tissues, and they are able encapsulate bioactive molecules and/or cells during injection while providing the latter with suitable environments for their proliferation and differentiation. However, microbial polysaccharides generally display limited performance in these aspects, therefore they are often modified and/or supplemented with other materials to better comply to the requirements of tissue engineering (Ng et al. 2020). Injectable nanocomposites made from microbial polysaccharides and mineral nanoparticles are an effective way to create biomimetic polymeric scaffolds with suitable mechanical properties and bioactivity. Bones for example, are nanocomposite materials consisting of minerals (60%), organic materials (30%) and cellular components (10%) (Tran et al. 2011). The inorganic components of bones provide them with the required strength to withstand their dynamic environment while still being able to maintain cellular function. It also provides them with cellular binding sites for integrin and/or growth factors, which promotes osteoconductivity, osteoinductivity, and osseointegration that polysaccharides generally lack (Tran et al. 2011; Ng et al. 2020). So far, injectable hydrogels based on polysaccharides have been used for numerous applications of tissue engineering including wound healing (Li et al. 2015), soft tissue augmentation (Silva et al. 2015), bone defects repair (Jung et al. 2018), and cartilage regeneration through intra-articular injections to treat osteoarthritis (Chen et al. 2017a). Osteoarthritis is a degenerative joint disease characterized by the wear and tear of joint cartilage, leading to their erosion and the inflammation of underlying bone. This disease can affect any joint and results in pain and stiffness. Intraarticular injections of hydrogels (or viscosupplementation) are considered effective treatments against osteoarthritis. A hydrogel is injected between the cartilages to relieve pain, improve joint lubrication, and delay further cartilage damage (Fig. 3). Microbial polysaccharides met great success in this latter application thanks to their compositions and structures which resemble to that of chondrocyte ECM, providing a biomimetic environment to maintain chondrocytes function, and which degradation can provide elements required for new tissue synthesis (Chen et al. 2017a). Moreover, some polysaccharides present high rheological and water-retention characteristics, improving joint lubrication and protecting the cartilage from mechanical degradation (Elmorsy et al. 2014). Hyaluronic acid (HA) is a natural component of synovial fluid and has been considered as an effective treatment for osteoarthritis. However, HA is unstable and prone to degradation by hydrolytic or enzymatic reactions in vivo (Zhong et al. 1994), leading to its short-term efficacy ( 13) above the pKa value of the hydroxyl groups (approximately 10), almost all those functional groups are deprotonated, and therefore their nucleophilic character increases to higher than the deprotonated carboxyl groups. The epoxides therefore react preferentially with the hydroxyl groups to form ether bonds. However, when the pH is lower than the pKa value of the hydroxyl group, only a small quantity of hydroxyl groups is deprotonated. Therefore, the reaction through the carboxyl group is predominant.
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Fig. 9 Chemical modification of HA by diglycidyl ether compounds
Fig. 10 Chemical modification of HA by DVS
The use of divinyl sulfone (DVS) has been established as an efficient method to crosslink HA for many authors (Collins and Birkinshaw 2007; Ramamurthi and Vesely 2002). Typically, the reaction is driven at high pH values (higher than 13) and creates sulfonyl bis-ethyl linkages between the hydroxyl groups of HA (Fig. 10). The advantage of this method is that it occurs at room temperature, which decreases the degradation of HA at high pH compared to higher temperatures, and it is a fast reaction. Another type of strategies that can be used for ether group formation in HA through reaction of the hydroxyl group is via reaction with ethylene sulfide (Serban, Yang, and Prestwich (2008)] or using glutaraldehyde for HA crosslinking (Crescenzi et al. 2003a; Collins and Birkinshaw 2007; Tomihata and Ikada 1997).
2.2.2 Ester Formation Ester formation using alkyl succinic anhydrides has been described by several authors (Eenschooten et al. 2010; Tommeraas and Eenschooten 2009). As an example, the reaction between HA and octenyl succinic anhydride (OSA) is illustrated in Fig. 11. Under alkaline conditions, the hydroxyl groups of HA react with the anhydride to form ester bonds. Acyl-chloride activated carboxylate compounds can be also used to form ester bonds through reaction with the hydroxyl group of HA. This method can be carried out by reaction of the carboxyl groups of the compound to be grafted (first activated by chloroacylation with thionyl chloride) and HA at room temperature in an organic solvent (Pravata et al. 2008) (Fig. 12). Esterification with methacrylic anhydride is also possible in order to obtain a photocrosslinked version of HA. Several authors have reported this reaction
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Fig. 11 Chemical modification of HA by OSA
Fig. 12 Chemical modification of HA by acyl-chloride-activated carboxylate compounds
Fig. 13 Chemical modification of HA by acyl-chloride-activated carboxylate compounds
(Burdick et al. 2005; Seidlits et al. 2010; Smeds and Grinstaff 2001). Typically, the reaction is performed in ice cold water for 12 h at pH 8–10 (Fig. 13).
2.3
Modification of the –NHCOCH3
N-acetyl group of HA can be deacetylated in order to recover the amino group which can then react with an acid following the amidation strategies described previously. Deacetylation is usually performed using hydrazine over a period of 5 days at 55 C, which induces severe chain fragmentation (Schanté et al. 2011). Then, the deacetylated HA can react with one carboimide-activated acid in order to form a new amide compound based on HA (Fig. 14).
2.4
Other HA-Crosslinking Strategies for Biomedical Applications
For photo-initiated systems, hydrogels result mainly from the polymerization of water-soluble monomers in the presence of a multifunctional crosslinker by UV radiation (photo-crosslinking) or by the reaction between complementary groups as shown in Fig. 15 (Bae et al. 2014; Choi et al. 2015). Although the rationale behind
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Fig. 14 Deacetylation of HA by hydrazine
Fig. 15 A photo-curable HA system. (Reprinted with permission from Bae et al. 2014)
the development of these hydrogels is very simple, the resulting hydrogels commonly have an increased structural stability, longer degradation, and high swelling profiles (Collins and Birkinshaw 2008). However, photo-polymerized hydrogels bring a few concerns relating cell viability for cell encapsulation. Cell exposure to UV radiation increases the cellular production of ROS [e.g., superoxide (O2) and hydrogen peroxide (H2O2)], which may cause lipoperoxidation and damage to DNA (Markovitsi et al. 2010) and increase the oxidative degradation of HA (Valachova et al. 2016; Valachova et al. 2015). The use of photo initiators (such as Irgacure 2959, 2,2-dimethoxy-2phenylacetophenone – DMPA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate – LAP, and eosin Y) in HA gelation has been well described (Poldervaart et al. 2017; Jha et al. 2010; Gwon et al. 2017; Rosales et al. 2017). However, it is known that these photo-initiators are cytotoxic in a time- and concentration-dependent manner, although LAP is shown to be the most benign (Kessler et al. 2017). In relation to the use of photo-initiators in HA gelation processes for cell encapsulation strategies, the cell type being encapsulated can influence the construct outcome. For instance, different cell types respond differently to photo-initiator’s toxicity which is
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thought to be due to the variable intracellular antioxidative machinery that different cell types use to quench ROS (Williams et al. 2005). For example, pancreatic β-cells are particularly sensitive to oxidative stress because of their low-antioxidant capacity (Drews et al. 2010). Therefore, when using photo-polymerization in HA gelation processes for cell encapsulation applications, additional protective measures from oxidative stress, such as antioxidants and scavenger enzymes, should be put in place to ensure the maintenance of β-cell viability (Weaver and Stabler 2015; Asami et al. 2013). Smart hydrogel systems which were reported to behave in a stimuli-responsive manner, where polymerization is triggered by environmental changes, such as temperature and pH, have been reviewed (Lim et al. 2014). They are mainly crosslinked via weak, noncovalent interactions, such as hydrophobic and electrostatic interactions between oppositely charged biomolecules (Wu and Gong 2011). For example, HA conjugated to Pluronic ® F127 exhibited thermo-sensitive sol-gel transitions and is being envisaged as a suitable candidate for cell delivery systems (Fig. 16) (Lee and Park 2009). Interpenetrating polymer networks (IPN) consisting of two or more networks of different components self-assemble into entangled arrangements, displaying synergistic effects from the simultaneous operation of the two networks (Kheirabadi et al. 2015). HA-based semi-IPN hydrogels have been reported for bioprinting
Fig. 16 A thermosensitive HA grafted with Pluronic ® F127. (Adapted with permission from Lee and Park 2009)
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Fig. 17 In situ formation of double IPN of HA with fibrin (Reprinted with permission from Zhang et al. 2016)
(Pescosolido et al. 2011) and cell encapsulation (Zhang et al. 2016; Chen et al. 2016a) as shown in Fig. 17. Recently, increasing attention is being directed toward guest-host assemblies (Fig. 18a). HA has been conjugated to cyclodextrin (host) and adamantane or azobenzene (guest), curcubit(6)uril (host) and polyamine (guest), such as spermine and 1,6-diaminohexane, to self-assemble into an IPN by reversible guest-host interactions (Rodell et al. 2013; Park et al. 2012a; Rosales et al. 2018). The noncovalent character of guest-host networks confers to hydrogels full reversibility under shear, allowing association or self-healing when shear is removed. These materials offer exciting possibilities for in situ minimally invasive injectable procedures. In addition, their use in bioprinting strategies is also appealing as these hydrogels are easily printed, i.e., show an increase of flowability when passing through the nozzle and fast recovering after that without requiring UV curing, which can be more beneficial to cells. Double network (DN) hydrogels, a subset of IPN, with a combination of physical, covalent, or ionic bonds are also gaining importance (Fig. 18b) (Highley et al. 2016). DN hydrogels exhibit resistance to mechanical failure because they combine a shearthinning self-assembly hydrogel network and a crosslinked hydrogel network that provides self-adhesive properties (Lu et al. 2013). HA DN hydrogels were developed for cell encapsulation and drug delivery systems (Mealy et al. 2015; Rodell et al. 2015a). It is possible to tune their degradation through modification of amino acid tethers (Rodell et al. 2015a), and porosity is controlled by HA concentration/ modification (Rodell et al. 2016).
Fig. 18 Hydrogels produced by: (a) guest-host crosslinking. (Adapted from Rodell et al. 2015); (b) DN crosslinking. (Adapted from Rodell et al. 2015); (c) click chemistry crosslinking. (Reprinted with permission from Takahashi et al. 2013); and (d) enzymatic crosslinking. (Adapted from Schante et al. 2011)
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Fig. 19 Schematic reaction between HA and BIED. (Reproduced with permission from Zamboni et al. 2020)
Other smart hydrogels also under the spotlight are polymers with complementary functional groups which can avail of click chemistries (Fig. 18c) or enzymatic crosslinking under physiological conditions (Fig. 18d) while displaying minimal toxicity. In situ click chemistry crosslinking was reported for azide-modified HA and cyclooctyne-modified HA and for tyramine-conjugated HA hydrogels formed by the oxidative coupling of tyramines catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP) (Takahashi et al. 2013; Ni et al. 2015; Abu-Hakmeh et al. 2016). Labile crosslinking strategies of hyaluronic acid via urethane formation using bis(β-isocyanatoethyl) (BIED) disulfide have been recently developed (Zamboni et al. 2020). The alkyl-based isocyanate reacts readily with hydroxyl groups on the HA to form stable urethane crosslinks (Fig. 19). A centrally located disulfide bond allows versatility with respect to reversible crosslinking by disulfide bond hydrolysis, and this allows potentially controlled layer by layer deposition of the HA for tailored pharmaceutical applications. The study further shows how these materials can modulate the immune system. With cellular grafts of LMW HA-BIED showing decreased fibroblast production of GM-CSF, indicating these grafts could be transplanted into tissues abundant in fibroblasts. On the other hand, cellular grafts of HMW HA-BIED show no monocyte activation and TNF-alpha production, while protecting LPS induction, thus showing the potential of being transplanted in tissues with direct blood contact.
3
Potential HA-Based Scaffold-Processing Methods
Several approaches to the fabrication of porous degradable polymer scaffolds have been developed. Some commonly used techniques are discussed here.
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Phase Separation
Phase separation is a swift, controllable, and scalable approach to fabricate two- and three-dimensional scaffolds with interconnected porous structures. Phase separation has been classified as nonsolvent-induced phase separation (NIPS) (Wang et al. 2019a), chemically induced phase separation (CIPS), and thermally induced phase separation (TIPS) (Jeon et al. 2018). The TIPS method is composed of two components, polymer and solvent. Phase separation is induced by removing the thermal energy from the dope solution. Upon the removal of solvent by means of extraction, evaporation, or sublimation, the residual polymer solidifies into the skeleton, while the space previously occupied by the solvent becomes pores (Jung et al. 2016). The TIPS method is typically employed to fabricate highly porous membranes, which are inherently more reproducible and less prone to defects (Kim et al. 2016). The pore morphology varies depending on the polymer, solvent, concentration of the polymer solution and the phase separation temperature. Recently, polymerizationinduced phase separation was used to formulate poly(ethylene glycol)/hyaluronic acid (PEGdA/HA) semi-IPNs that could provide dynamic microenvironments with improved mechanical properties to support cell survival, spreading and sustained migration, showing great potential in orthopedic tissue engineering (Lee et al. 2015).
3.2
Supercritical Fluid Technology
Supercritical fluid (SCF) technology provides a cost-effective and eco-friendly alternative to conventional solvent-based methodologies for polymer synthesis in pharmaceutical and biomedical applications, such as drug delivery and tissue engineering. As the use of organic solvents can be drastically reduced or eliminated, it is regarded as a nontoxic process. In this process, an SCF is used, which turns into a homogeneous phase where the vapor/liquid boundary disappears above its critical point in terms of temperature and pressure. This supercritical phase shows an intermediate behavior between that of a liquid and a gas and induces the formation of small gas bubbles homogeneously throughout the polymer in response to the thermodynamic driving force (Collins and Birkinshaw 2013a). Carbon dioxide (CO2) is the most widely used SCF due to its low toxicity, chemical inertness, and nonflammability, as well as its readily availability at high purities and low cost. The SC-CO2 polymerization can be operated at rather mild conditions due to its low critical point, i.e., at temperature of 31.1 C and pressure of 7.38 MPa, as shown in Fig. 20 (Kankala et al. 2017). The latest progress of SCF technology in biomedical engineering can be read in this review, while further information on engineering scaffold materials utilizing gas-foaming technologies can be read in the following articles: Diaz-Gomez et al. 2016; Costantini and Barbetta 2018; Poursamar et al. 2016; Maniglio et al. 2018; Manavitehrani et al. 2019; and Rao et al. 2019.
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Fig. 20 Schematic showing the mechanism of SCF polymerization. (Reproduced with permission from Tsai and Wang 2019)
3.3
Porogen Leaching
Porogen leaching is known as an expedient and economical technique to process scaffolds with a homogeneous porous structure, as pore shape, size, and total porosity can be tailored (Yin et al. 2016). Recently, this method has been combined with other processing methods to engineer various porous scaffolds, such as polycaprolactone/graphene oxide (PCL/GO) nanocomposite scaffolds through a SC-CO2 technology/porogen leaching route (Yildirim et al. 2018); poly(lactic acid) (PLA) with high mechanical performance assisted via a solid state extrusion/ porogen leaching approach (Yin et al. 2016); nanoporous polyacrylonitrile/calcium carbonate (PAN/CaCO3) nanofiber through a solvent casting/porogen leaching technique (Mahmoodi and Mokhtari-Shourijeh 2016); and porous polyetheretherketone (PEEK) and bioactive hydroxyapatite (HA)-reinforced PEEK (HA-PEEK) by a compression molding/porogen leaching method (Conrad and Roeder 2020). As shown in Fig. 21, Thomas et al. have developed HA-based scaffolds with complex 3D architecture using cheap sacrificial crystals (potassium dihydrogen phosphate or urea) that act as porogen (Thomas et al. 2017). This offers a route for the synthesis of biomimetic 3D tissue scaffolds for in vitro studies of pore architecture influences on cellular behavior, as well as to engineer implantable biomaterials for specific niches in the body, such as regeneration of the nervous system.
3.4
Electrospinning
Microfibers and nanofibers processed by electrospinning provide extremely high surface-to-volume ratios, complex porous structures, and diverse fibrous morphologies, which are coherent with the demands of advanced biomedical applications such as tissue engineering scaffolds and wound healing. The recent progress and potential developments of electrospinning in biomaterial engineering have been
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Fig. 21 Schematic showing the preparation process of porous HA-based scaffolds through porogen leaching. (Reproduced with permission from Thomas et al. 2017)
reviewed (Ding et al. 2019; Miguel et al. 2018). A range of nanofibrous (Sun et al. 2019; Chanda et al. 2018; Figueira et al. 2016; Seon-Lutz et al. 2019) HA-based scaffolds have been successfully produced by electrospinning, for potential wound healing and tissue regeneration applications. A distinct advantage of this method is the capability of producing core-shell nanofibers through coaxial electrospinning which exhibit desired properties from each of the ingredients. For example, coreshell-structured polyurethane/starch (HA) PU/St (HA) nanofibers have been fabricated by coaxial electrospinning (Movahedi et al. 2020). This unique core-shell structure represents excellent biological properties from starch, superb water absorption ability, and flexibility from HA which promotes cell adhesion, as well as mechanical strength from PU, which make it an ideal scaffold biomaterial for wound healing and skin tissue engineering. Another example worth noting is the development of a HA-based silk fibroin/zinc oxide (HA–SF/ZO) core-shell electrospun dressing, where the antibacterial agent ZO was loaded as the core to improve the drug release as well as to maintain its bioactivity, which is critical in burn wound management. In vivo studies indicate that the wound healing procedure was substantially improved while the inflammatory response at the wound site was significantly reduced by this ZO-loaded wound dressing.
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Freeze Drying
One of the most common processes to prepare porous scaffolds in tissue engineering is freeze drying, also known as lyophilization. The material in hydrogel form is first frozen, while the water content forms nuclei of ice, which act as porogen, that are homogeneously distributed throughout the polymer. The frozen water is then removed through sublimation by reducing the pressure and adding heat, and a porous spongy structure is left behind. The pore size echoes with the nuclei size, which can be controlled by exposing the hydrogel to different temperatures as freezing conditions. Freeze drying is widely used in processing the spongy HA-based scaffolds that can provide the support required in tissue engineering and regenerative medicine (Kalam 2016; Lu et al. 2017; Yin et al. 2019; Zhang et al. 2017; Zapotocky et al. 2017; Kaczmarek et al. 2018).
3.6
Centrifugal Casting
The extensive existence of tubular tissues in the human body, such as capillaries, bones, kidney tubules, and genitourinary structures, suggests the potential of centrifugal casting in tissue engineering. Lee et al. have used a centrifugal casting technique to fabricate silk fibroin (SF) film for corneal tissue engineering. Compared with SF films prepared by dry casting methods, SF films fabricated with the aid of centrifugal force demonstrated superior surface roughness, better tensile strength, as well as better cell proliferation, which was speculated to be helpful in the regeneration of the corneal layer (Lee et al. 2016). The same research team later combined centrifugal casting with a digital light processing (DLP) 3D printer and enabled rapid, easy, and low-cost fabrication of sophisticated fixation systems, such as screws and plates, based on tailor-made geometric design (Kim et al. 2018). Nanofibrous porous tubular scaffolds can be fabricated by centrifugal casting combined with phase separation, as shown in Fig. 22, to provide an advantageous environment for cell adhesion and viability, which has significant potential for bone tissue regeneration (Chen et al. 2016a).
3.7
Scaffold-Templating Techniques
During the process of scaffold templating, HA-based solution is vacuum injected into templates made from sintered beads. Then the HA is crosslinked around beads, which are subsequently removed by leaching. Afterward, lyophilization is carried out, leaving adequate porosity for cell and tissue invasion. Shape and size of the beads can be controlled to obtain scaffolds with desired microporous morphology. Rodriguez-Perez et al. have adopted this methodology to fabricate a novel biomaterial with interpenetrating polymeric networks (IPNs) based on HA and poly(ethyl acrylate) (PEA). These scaffolds display a homogeneous interconnected morphology consisting of pores with size around 182 μm, and they show good cell response
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Fig. 22 Schematic illustrating the fabrication of nanofibrous tubular scaffolds: (1) mold, (2) polymer solution, (3) porogen addition, (4) motor assembly, (5) centrifuge and phase separation, (6) mold removal, solvent exchange, (7) freeze drying, and (8) acquisition. (Reproduced with permission from Chen et al. 2016a)
in in vitro studies (Rodriguez-Perez et al. 2016). As shown in Fig. 23, Singh et al. reported a magnetic templating technique, which used dissolvable magnetic alginate microparticles (MAMs) to form highly aligned, 3D, tubular microarchitecture under an applied magnetic field. Scaffolds based on glycidyl methacrylate hyaluronic acid (GMHA) and collagen I were tailored to mimic the microarchitecture of the native nerve, with the ability to guide cellular migration and potentially aid in peripheral nerve regeneration after injury (Singh et al. 2020).
3.8
Micropatterning Techniques
Micropatterning, initially a miniaturization technique used in electronics, has recently gained some momentum in biomaterial engineering and is applied in cellular biology (Ahmadian et al. 2020). HA-based micropatterns (HAP) can be fabricated using spatially controlled light exposure (Hui et al. 2019; Porras et al. 2016) and capillary force lithography (CFL) (Li et al. 2013; Han et al. 2019). These micropatterns are used to regulate the cells’ behaviors, including their attachment, proliferation, distribution, morphology, and even cytokine secretion on the HA-based hydrogel scaffolds with submicrometer resolution. Credi et al. have
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Fig. 23 Schematic illustrating magnetic templating: (a) mold is loaded with a mixture of MAMs and hydrogel precursor solution; (b) the hydrogel precursor is aligned under a static, uniform magnetic field; (c) the hydrogel is UV cured around the aligned MAMs; (d) MAMs are dissolved; and (e) hydrogel scaffolds patterned with aligned porous channels are formed. (Reproduced with permission from Singh et al. 2020)
optimized a single-step photolithographic process for selectively micropatterning glycidyl methacrylate-modified HA onto UV-curable perfluoropolyether (FPE)based surfaces. HA micropatterns with complex geometrical features can be rapidly and cost-effectively designed, in order to affect cell adhesion efficiency, which can be utilized to selectively capture and immobilize cancer cells (Credi et al. 2016).
3.9
Rapid Prototyping: Solid Freeform Fabrication (SFF) and Bioprinting
Bioprinting is the most suitable technique to produce well-defined 3D structures with highly complex and patient specific geometries (Zhang et al. 2019; Gleadall et al. 2018). As shown in Fig. 24, this technique is based on a bottom-up approach, where hydrogel materials and cells are combined to form a bioink which is then deposited layer-by-layer in a controlled manner, making up a 3D complex structure (Pati et al. 2014). There are three common printing methods, i.e., inkjet, microextrusion, and laser-assisted bioprinting, while the selection of method should be based on the biomaterial to be used, cell viability to be achieved, and desired resolution (Sundaramurthi et al. 2016; Murphy and Atala 2014). In order to reproduce the complex and heterogeneous 3D architecture of functional organs, 3D printing and bioprinting techniques coupled to imaging modalities such as computer tomography (CT) and magnetic resonance imagining (MRI) provide unique spatially accurate
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Fig. 24 Schematic elucidating bioprinting of a tissue-engineering construct using bioink. (Reproduced with permission from Pati et al. 2014)
models in regenerative medicine for the direct printing of tissue constructs (Cahill et al. 2019). The application of pristine HA as bioink is limited by its high hydrophilicity, but this can be mitigated using chemical techniques described above or through a combination with other biomaterials. For example, semi-IPN of HA and modified dextran (Zoratto and Matricardi 2018), thiolated HA crosslinked with PEG (Godesky 2020), gelatin-methacryloyl (gelMA), gellan and methacrylated HA (HAMA) (Mouser et al. 2020), HA, hydroxyethyl acrylate (HEA) and gelatinmethacryloyl (Noh et al. 2019), HA and alginate (Antich et al. 2020), and methacrylated collagen I, thiolated HA, and gelatin nanoparticles (Clark et al. 2019) have been reported as successful bioinks. Recently, increasing attention is drawn to the development of bioinks encapsulated with stem cells. For example, Sakai et al. have demonstrated the utility of the bioprinting technique to create cell-laden hydrogel constructs using HA-gelatinbased bioink crosslinked through irradiation by visible light. The hydrogelation rate and mechanical properties of the hydrogel can be controlled by adjusting the crosslinking condition. After printing, the human adipose stem cells (hADSCs) enclosed in the hydrogel elongated and proliferated while maintaining their differentiation potential, which demonstrated the great potential of the bioprinting technique in applications of tissue engineering and regenerative medicine (Sakai et al. 2018). For further information on the fabrication techniques of tissue engineering scaffolds, the reader is referred to a review (Hokmabad et al. 2017) and a book chapter (Rey and St-Pierre 2019).
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HA in Scaffold Vascularization Strategies
Natural tissues and organs exhibit a 3D architecture which allows cell-to-cell and cell-to-ECM interactions. The survival of large 3D architectures relies on blood perfusion within an intricate vascular network, delivering oxygen and nutrients while removing carbon dioxide and metabolites. The adequate vascularization of 3D tissue-engineered substitutes is a major engineering hurdle in creating artificial organs. Posttransplantation success of these tissue substitutes is highly dependent on the ability to promote rapid and stable neovascularization (formation of new blood vessels) to support growth, function, and viability. Researchers rely on increasing knowledge of angiogenic and vasculogenic processes to stimulate vascular network formation within 3D tissue constructs, such as the incorporation of proangiogenic materials or growth factors in the development of cellular scaffolds. The influence of HA on the angiogenic process was reported in the early 1980s (West et al. 1985). While native HMW-HA was reported as an angiogenic inhibitor, LMW-HAs are highly bioactive and stimulate the angiogenesis process (Gao et al. 2019). The size of these HA oligomers also influences the proangiogenic nature of the fragments. Oligomers with 6–10 saccharide units were shown to be proangiogenic (Wang et al. 2019a) while fragments with only 4 saccharide units were unable to prompt a proangiogenic response (Cui et al. 2009). In the endothelium, HA interacts with endothelial cells that line the interior surface of blood and lymphatic vessels. This angiogenic capacity comes from receptor-mediated interactions of HA oligomers with the CD44 and hyaluronanmediated motility receptor (RHAMM) of endothelial cells, which triggers endothelial cell proliferation, migration, collagen synthesis, and cell sprouting (Pardue et al. 2008). Park et al. suggested a new mechanism of HA-promoted angiogenesis, where plasminogen activator-inhibitor-1 (PAI-1) is inducted through RHAMM and transforming growth factor β receptor I (TGFβR1) signaling (Park et al. 2012a). As a result, HA oligomers have been used to increase the angiogenic capacity, and consequent vascularization, of other biomaterials (Silva et al. 2016; Perng et al. 2011), and also to enhance wound-healing strategies (Wang et al. 2016). Recently, a clinical trial demonstrated that HA hydrogels enabled the vascularization of free gingival grafts and functioned as a scaffold between the recipient’s transplantation bed and the gingival graft, reducing graft shrinkage (Cankaya et al. 2020). In a lab-on-a-chip model for a functional blood-brain barrier system, a fibrin 3D-lumenized vasculature together with perivascular cells showed better astrocyteendothelium interaction when the ECM of the lumen was modified to contain HA (Lee et al. 2020). In another microfluidic study, an in vitro assessment of biomaterialbased angiogenesis showed that a HA matrix was able to sequester growth factors and enabled endothelial cells derived from human-induced pluripotent stem cells to form stable, capillary-like networks (Natividad-Diaz et al. 2019). In addition, HA-based matrices were also reported to selectively bind to vascular endothelium growth factor (VEGF) (Lim et al. 2016) and release VEGF by mediated MMP degradation (Jha et al. 2016). Endothelial cells encapsulated in HA-dextran
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hydrogels containing VEGF were able to form a functional vascular network integrated to the host-vascular system (Portalska et al. 2014). HA hydrogels also upregulated the expression of angiogenic markers such as VEGF and FGF-2 in fibroblast and endothelial cells, resulting in a neovascular response in vitro (Ciccone et al. 2019). Scaffolds made of a microfibrous blend of poly(L-lactide-co-ε-caprolactone) and HA also increased CD34 expression of endothelial cells, resulting in the formation of small vessels (Kenar et al. 2019). Endothelial cells also increased their CD31 expression [also called platelet endothelial cell adhesion molecule (PECAM-1)], a marker for endothelial cell junctional integrity and vascular permeability barrier, when cultured on HA-modified collagen nanofibers for a vascular tissue-engineered scaffold (Kang et al. 2019). Furthermore, higher expression of CD31 by endothelial cells, when exposed to HA, shows immunomodulatory properties, as it inhibits the circulation of leukocytes (Lertkiatmongkol et al. 2016). HA-modified collagen nanofibers also promoted lymphatic endothelium formation by increasing the expression of lymph vessel endothelial hyaluronan receptor-1 (LYVE-1) (Gao et al. 2019). Additionally, other strategies can be used to tackle hypoxia thus providing early oxygenation to the graft. For example, HA hydrogels functionalized with perfluorocarbon moieties (fluorinated oxadiazole) improve cell oxygenation and promote fibroblast growth under hypoxic conditions (Palumbo et al. 2014).
5
Immunomodulatory Properties of HA-Based Scaffolds
Combinatorial libraries of biomaterials and chemical modification offer a good strategy to determine optimum material selection for mitigation of the immune response while allowing the diffusion of nutrients, therapeutic agents, and cell wastes; see Fig. 25 (Vegas et al. 2016). The immune system is a defense mechanism against environmental threats and pathogens; it can be divided in the innate and adaptive immune system. When the immune system experiences a biological imbalance, several chronic and acute inflammatory conditions can be triggered, as well as the development of several autoimmune conditions (Zamboni et al. 2018). The immune system also plays an important role in the success of cell and organ transplantation. Normally, when an injured or failing organ needs to be repaired or replaced using allogenic (stem or somatic cells) and xenogeneic sources available, these foreign cellular sources are recognized by the host immune system leading to its rejection. However, the immune system is not the only player during an inflammation process; the remodeling of the ECM plays an important role during inflammation by promoting immune cell activation, tissue invasion, and destruction (Hallmann et al. 2015; Sorokin 2010). The ECM is the noncellular component of all tissues, responsible for the provision of physical support to cells and for the regulation of diverse cell functions through biochemical and biomechanical cues. There are two major types of ECM, each with distinct architecture and composition: (i) interstitial matrices that surround
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Fig. 25 Schematic representation of an encapsulation system for cells delivery, immunomodulation, and immunoprotection. (a) Cells are delivered by a carrier (e.g., hydrogel, microparticles, and microcapsules) and then are able to migrate to the diseased tissue/organ under controlled or stimuli-responsive release, and (b) cells are embedded within a semipermeable membrane that protects the biological material from immunological recognition while permitting the inflow of ant outflow of key elements for cell survival and function
cells, providing mechanical support; and (ii) pericellular matrices, which are in closer contact with cells (e.g., basement membranes), protecting them from rupture (Theocharis et al. 2016). Essentially, ECMs are composed of water and fibrousforming proteins, such as collagens, elastin, and fibronectin; glycoproteins; proteoglycans (PGs); and glycosaminoglycans (GAGs) (Theocharis et al. 2016). ECM remodeling plays an important regulatory role over the immune system. The degradation of ECM components by proteases fine tunes inflammatory responses by the generation of bioactive fragments called matrikines. Matrikines act as damage-associated molecular patterns (DAMPs) which are responsible for the activation of the immune system (Zamboni and Collins 2017). In order to avoid transplant rejection, the development of biomaterials, which are capable of intervening in immunological processes to achieve therapeutic results, is of critical importance in the overall aim to prevent or reverse the development of immune-mediated cell attack. During HA remodeling, HMW-HA is degraded into LMW-HA; these HA fragments promote leukocyte recruitment by the activation of toll-like receptors (TLRs). However, when hyaladherin proteins crosslink HA into stable structures, they enhance CD44 binding on lymphocytes. Tumor necrosis factor stimulated gene
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6 (TSG-6) is a hyaladherin that has an important function in preventing HA degradation by inhibiting HYALs and enhancing HA binding to the cell surface receptor CD44 on lymphocytic cell lines (Baranova et al. 2011). The effect of TSG-6 on diabetes development has shown to delay autoimmunity and enhance tolerogenicity of cells (Kota et al. 2013). When HMW-HA alone or in association to hyaladherins binds to CD44 receptors on Tregs, it promotes its immune suppressive capacity by increasing the transcription factor Foxp3 expression and increasing the production of anti-inflammatory cytokines, such as IL-10 (Ruppert et al. 2014). Fibroblasts are predominantly present in connective tissues, and they elicit immunological responses by producing proinflammatory cytokines and chemokines that aid the immune system to induce and recruit inflammatory cells (BautistaHernandez et al. 2017). Scaffolds of porous freeze-dried poly(lactide-co-glycolic) acid (PLGA) and 3D printed poly lactide acid (PLA) showed enhanced fibroblast proliferation when coated using HMW-HA hydrogels (Zamboni et al. 2017; Souness et al. 2018). Fibroblasts when activated secrete cytokines that modulate the immune system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine secreted from fibroblasts that induces monocyte differentiation as part of the immune response chain. HAs of varying MW elicit contrasting production of GM-CSF by fibroblasts. LMW-HA shows basal fibroblast production of GM-CSF, while HMW-HA was able to elicit a rapid spike in production of GM-CSF within 24 hours of incubation often associated with an acute inflammatory response (Zamboni et al. 2020). Hyaluronidase administration for in vivo acute inflammation and sepsis treatment showed that HA fragmentation suppressed neutrophil infiltration and cytokine production (IL-1β and IL-6) (Pereira et al. 2020). HA oligomers were able to regulate macrophage polarization into the anti-inflammatory M2 subtype (Wang et al. 2019a). However, HMW-HA hydrogels have been reported to increase lymphocyte and T-helper cell proliferation and activation into an anti-inflammatory environment. T-helper cells increased the secretion of IL-10, a known antiinflammatory cytokine, while decreasing the secretion of IL-2 and interferon gamma (IFNγ), known proinflammatory cytokines. In addition, they also increased monocyte differentiation into M2 macrophage subtype (Gomez-Aristizabal et al. 2016). Moreover, the complement system is part of the innate immune system and comprises a series of proteins that are activated in response to a pathogen to enhance the ability of antibodies (adaptive immune system) and phagocytic cells (innate immune system) to fight a threat. Its modulation can be linked to autoimmunity and transplant rejection. Thus, complement modulation can impact transplant rejection. GAGs have the ability to modulate complement activation, which is highly dependent on sulfate and negative charge. For example, it has been observed that highly sulfated GAGs can promote C3 activation while little or no effect was seen with negatively charged GAGs, such as HA (Meri and Pangburn 1994). Interestingly, HA is also reported to modulate platelet activation during inflammation. Platelets are the main cellular effectors for hemostasis, but they also possess a plethora for intracellular mediators and surface receptors (such as integrins, CD44, and TLR) known for
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their involvement in inflammatory processes. Platelets actively degrade HA from the surface of endothelial cells, via HYAL-2; when they are exposed to ECM of a disrupted vessel wall, they initiate inflammatory and angiogenic signaling by stimulating mononuclear leukocytes in the immediate microenvironment to produce cytokines such as IL-6 and IL-8 (Zamboni et al. 2018). The immunological effect that a material can have is also influenced by the intrinsic relationship between structure and function properties, such as polymer molecular weight, concentration, degree of substitution, backbone chemistry, and crosslinking density. Porosity and pore size fine tune the permeability of a 3D scaffold. Permeability will dictate the diffusion and permeation rates of molecules (such as antibodies and cytokines) that impact the immune response. The stability of the scaffold structure can also impact the immune response, as degradation can release DAMPs. Molecular weight cut-off (MWCO) determines the minimum molecular weight of a solute that is completely excluded by this 3D matrix. However, these values can lead to misinterpretation as the size of solutes with the same molecular weight can vary dramatically between polysaccharides and proteins. Hence, other parameters such as diffusion coefficients are considered more accurate to characterize permeation. Permeation is a vital factor to consider for the success of tissue-engineered constructs in order to avoid graft rejection. It will prevent the inflow of molecules such as antibodies as small as IgG (150 kDa), cytokines and chemokines such as IL-1 (17.5 kDa) and CCL-2 (8 kDa), respectively, while allowing the diffusion of oxygen and LMW biological compounds such as glucose (180 Da) and carbon dioxide (44 Da), essential for the cellular survival and function. For example, higher polymer and crosslink concentrations correlate to decreased pore sizes, which creates matrices with smaller MWCO and slower diffusion rates (Weber et al. 2009; Bal et al. 2014). The MW of HA is very important regarding new vaccine delivery systems. For example, studies evaluated the delivery of different HA MW (between 7 and 741 kDa) to the lungs of mice. Regarding the pharmacokinetic parameters of HA, lower HA MW showed more rapid systemic distribution, while 67 and 215 kDa HA showed longer persistence in the lungs. Lung exposure appeared to be optimum in this size range due to the rapid absorption of 215 kDa (Kuehl et al. 2016). Beside MW, the crosslinking degree (CD) can also modulate the physicomechanical properties of HA viscosupplements (Lee et al. 2014). HA crosslinked with divinyl sulfone (DVS) showed that higher CD increased the viscoelasticity and the extrusion force, where HA:DVS 1:1 (CD ¼ 90%) showed extrusion force of 14.9 N and HA:DVS 5:1 (CD ¼ 14.2%) showed an extrusion force of 6.8 N. Futhermore, increased CD showed reduced swelling ratios (SR) (90% CD ¼ 23.6% SR and 14.2% CD ¼ 73% SR). HA hydrogels also showed Newtonian pseudoplastic behavior and were classified as weak (Shimojo et al. 2015). Regarding the relationship between the crosslinking densities and the HA hydrogel degradation, studies have shown that higher crosslinking densities reduce enzymatic degradation, suggesting that the slower degradation rate of the HA is
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associated with limited access of the enzyme to the active sites of the biopolymer chains due to the presence of large amounts of covalently incorporated crosslinker (DVS) (Lai 2014). The increased stability of the matrix, due to the slower degradation profile, benefits immunoprotection and immunomodulation in tissue engineering by decreasing the release of LMW-HA fragments that act like DAMPs to the environment. Studies have documented the effects that HA induces in the immune system by analyzing proinflammatory gene and cytokine expression of IL-1β and IL-6 and nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced macrophages. The results indicate that lower DVS (up to 50%) and 1,4-butanediol diglycidyl ether (BDDE) at concentrations of 0.5 and 1.0 ppm do not induce the expression of genes nor cytokines (Choi et al. 2015). These results are promising for tissue engineering applications, where null immunological response is required.
6
HA in Tissue-Engineering Applications
HA has been used extensively to regenerate many different tissue types. Here, we focus on emerging nerve (peripheral and central) and skin regeneration strategies.
6.1
Peripheral Nerve Regeneration (PNS)
Recent developments on the use of HA for nerve regeneration applications comprise the delivery of neural stem cells embedded in HA matrices. Farrel et al. (2017) reported on injectable uncrosslinked HA as a suitable candidate for in situ delivery of neural stem cells (NSCs). The resulting gel, a blend composed by HA, Type-1 collagen, laminin, and chondroitin sulfate, maintained its integrity after 10 days but was rapidly degraded in the presence of HYAL (between 2 and 6 h). Interestingly, NSCs’ differentiation toward neuronal or glial cells was driven by the relative concentration of the components present in the blend. Anti-FAS-conjugated HA microsphere gels were also reported to deliver NSCs (Shendi et al. 2017), protecting cell microspheres from deleterious cytokines. Moreover, the presence of anti-Fas motif decreased cell viability of inflammatory T cells, inducing their apoptosis. As T cells are the principal responsible for NSCs’ death after transplantation, these HA-based microspheres work as cell vehicle and immunomodulators. In an experimental study for peripheral nerve scarring, the topical application of HA on adult Wistar rats at 4 and 12 weeks displayed significant reduction of neural scar thickness. Histomorphologic analysis and electrophysiological attributed this to the retardation of lymphocyte migration, degranulation, and chemotaxis. HA allowed the regeneration of nerve by inhibiting the epineural and extraneural scar formation at the repair site of the peripheral nerve (Ozgenel 2003). K. Ikeda et al. have conducted an experiment on rabbits to show HA can inhibit the adhesion of peripheral nerve on a rabbit model. At 6 weeks of histological and electrophysiological study, it was shown that HA can effectively reduce scar tissue after neurolysis (Ikeda et al. 2003).
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Studies have shown that electrical stimulation aids the process of nerve repair. With this in mind, HA-based tubular scaffolds were seeded with Schwann cells with 450 μA and 900 μA current intensities applied to study the increase of cellular activity. Results show that 450 μA accelerate the cell number (Ramos et al. 2018). A scaffold composed of HA-collagen composite incorporated with neurotrophin-3 and BrdU-labeled neural stem cells conduit was inserted at the facial nerve ends of rabbits to study peripheral nerve injuries. At 12 weeks, it was found from the electrophysiology study that the composite incorporated with neural stem cells facilitates the reinnervations of the damaged facial nerve (Zhang et al. 2008).
6.2
Central Nervous System (CNS)
The CNS has very limited capacity for regeneration, particularly in the spinal cord, caused primarily by reactive activation of astrocytes and the presence of reactive oxygen species (ROS) microenvironment following injury (Zhang et al. 2020). Spinal cord injury (SCI) is a common CNS injury, often resulting in the paralysis of the patient at or below the injury site, caused by a primary injury to the spinal cord by means of traumatic contusion or laceration. Upregulation of the surrounding cells and inhibitory factors, such as astrocytes and chondroitin sulfate proteoglycans, result in the formation of a glial scar around the periphery of the cyst. This scar acts as a barrier, both physically and chemically, to the reestablishment of axonal and neuronal tracts in the spinal cord (Liu et al. 2013). With limited and often ineffective treatment outputs, TE strategies for SCI repair have been explored by the research community. Among many biomaterials used for this treatment, HA shows promise. HA plays an important role not only as a CNS ECM component by influencing the viscosity of the tissue due to its affinity to bind with water, but also as a cell behavior modulator in the CNS by means of cell surface receptors such as CD44 or RHAMM (Gaudet and Popovich 2014; Burnside and Bradbury 2014; Toole 2004). The molecular weight of HA appears to also play a vital role in the cellular response in the CNS. In SCI studies, the addition of HWM-HA prevents astrocyte proliferation, and LWM-HA promotes differentiation of cells, while degradation of the HA in the ECM by means of hyaluronidase promotes astrocyte proliferation (Struve et al. 2005; Khaing et al. 2011; Seidlits et al. 2019). These results have the potential to be utilized in various CNS repair strategies, especially in the inhibition of glial scar formation. Traditional TE strategies focus on utilizing HA as a scaffold material, often utilizing the lyophilization technique to achieve scaffolds with controlled porosity and orientated channels for guided neuronal regrowth. The morphological aspect of a scaffold is crucial to the survival and proliferation of cells by allowing nutrient diffusion throughout the scaffold matrix. HA and PLLA fiber scaffolds studied by Martinez-Ramos et al. were created by first lyophilizing HA scaffolds with 400 μm poly-ε-caprolactone fibers in order to create channels within the scaffold. After lyophilization, the fibers were removed, and 20-30 μm PLLA fibers were placed in
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the created channels. The scaffold was seeded with spinal cord-derived ependymal progenitor cells (epSPC). In vivo results indicated that the scaffold improved the regrowth of neuronal fibers at the epicenter of rat SCI lesions and reduced astrogliosis, though a longer study is recommended by the authors (MartinezRamos et al. 2019). Though an alternative technique for the fabrication of scaffolds for CNS repair could utilize 3D printing techniques, the use of HA as the printing material for such an application has not been thoroughly explored by the research community to date. In other strategies, HA is often utilized as an injectable hydrogel which gels under physiological conditions by means of chemical modifications (Ho et al. 2019; Gupta et al. 2006; Fuhrmann et al. 2015). Führmann et al. combined HA with methylcellulose, RGD peptide, cell survival and differentiation factor PDGF-A, and humaninduced pluripotent stem cell-derived oligodendrocyte progenitor cells (hiPSCsderived OPCs). Injected into SCI lesions in rat in vivo models with the hydrogel gelling in situ, the transplanted cells in the hydrogel expressed OPCs migration to the lesion site, where the potential myelination of axons could occur. This is attributed to the promotion of cell attachment and hence cell survival by means of the RGD peptide. Also, at 9 weeks postinjury, the transplanted cells expressed markers associated with astrocytes (glial fibrillary acidic protein-GFAP) and OPCs (SOX10), though the presence of a teratoma in a number of the studied rats prevented extensive analysis (Führmann et al. 2016). The HA and methylcellulose hydrogel was further expanded in a study by He et al. A key aspect of any TE material is the retention of the desired shape and geometry in vivo to not induce any additional damage to the surrounding tissue with material swelling. In the study, the injected HA hydrogel swelled to only 15% in situ. In addition, when examining the tissue histology, it was reported that the presence of the hydrogel inhibited the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 and upregulated the levels of anti-inflammatory cytokines, thus promoting the inhibition of glial scar formation (He et al. 2019). The combination of the RGD peptide and HA has also been explored by Zaviskova et al. Hydroxyphenyl derivative of HA combined with RGD, fibrinogen, and Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) was tested in vitro and in vivo in rat SCI models. Observed in vivo results have shown an infiltration of axons and blood vessels into the hydrogel-filled lesion site. GFAP staining showed the migration of astrocytes from the lesion periphery to the hydrogel lesion site, indicating that the material’s presence is permissive toward glial cell infiltration. Though the authors did not observe any hWJ-MSCs cells 8 weeks posthydrogel implantation, it is speculated that neurotrophic and immunomodulatory effects can be observed for a long period of time postinjury due to the release of trophic factors by the initial presence of the transplanted cells at the site of injury (Zaviskova et al. 2018). An addition of growth factors into the HA hydrogel as a combinatory TE strategy has also been explored. Xie et al. combined sodium hyaluronate with ciliary neurotrophic factor (CNTF) and tested the efficacy of this material in vivo in rat SCI lesions. Postinjury injections of BrdU aimed at labeling dividing cells showed
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that Nestin+ and BrdU+ cells were observed in the lesion site of the HA-CNTF tested group, indicating the activation and migration of endogenous neural stem cells (NSCs) into the lesion site. New-born Tuj1+ & BrdU+ immature neurons and new-born NeuN+ & BrdU+ mature neurons were also observed in the lesion area 30 days post-SCI. Interestingly, Dil (a lipid membrane dye which labels ependymal cells) positive cells were observed in the parenchyma outside the central canal, with this result not being replicated in the control lesion SCI model. This indicates that the presence of the HA-CNTF hydrogel prompted the activation and migration of ependymal originating NSCs (Xie et al. 2018). Novel HA-based hydrogels for CNS TE investigate the combination of HA as a biomaterial with novel molecules to further prompt and aid in the regenerative capacity of nerves. One recent study combined HA with 2,6,6tetramethylpiperidinyloxy (TEMPO) with the aim of reducing the ROS environment in the SCI lesion site and promote regeneration. The results showed a positive ratio of NF (neurofilament marker) to GFAP, suggesting that the ROS environment was reduced, further evidenced with an improvement in the Basso, Beattie and Bresnahan (BBB) locomotor-rating scale scores (Zhang et al. 2020). Recent biomedical engineering strategies for CNS repair have focused on the utilization of neural stem cells and biomolecules, though nowadays this strategy alone is not as popular due to the hostile nature of the environment around the lesion site, which prevents stem cell proliferation and differentiation (Mothe and Tator 2012). Therefore, more emphasis is being placed on tissue engineering strategies which support and promote cell growth within this specific stem cell environment. The utilization of HA as a cell encapsulation material has shown promising results in SCI models. A study by Zarei-Kheirabadi et al. utilized a commercially available HA kit (HyStem-C Cell Culture Scaffold Kit) as the hydrogel material to encapsulate human embryonic stem cell derived neural stem cells (hESC-NS). After 1 week post-SCI in rat in vivo models, hESC-NS/HA hydrogel was injected into the lesion site. GFAP expression was lower than in the hESC-NS cell only group, indicating that the utilization of HA reduces the presence of the glial scar around the lesion site by inhibiting astrogenesis. The differentiation of the hESC-NS cells in the presence of the HA into oligodendrocytes was also elevated, suggesting that HA promotes oligogenesis, though maturation of oligodendrocytes appears to be inhibited by the presence of HA by means of TLR2 signaling (Zarei-Kheirabadi et al. 2020; Sloane et al. 2010). Overall, the utilization of HA as a hydrogel material for CNS TE strategies appears to be promising, with HA playing a dual role as both an ECM environment component as well as providing complex cellular cues to the surrounding cells. The more that we study this material in CNS ailments, the more informed we can be as to how we can tailor the body’s response to achieve the desired regenerative result.
6.3
Skin
HA plays a major role in hydration of the surface of the skin (Papakonstantinou et al. 2012). Full epidermal-dermal-based scaffolds have been developed using a porous HA
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scaffold as the dermal component and poly-L-Lysine as the epidermal component utilizing a spray-assisted layer-by-layer assembly technique, with keratinocytes cultured on top of the epidermal layer which assists cell adhesion, and regeneration of skin layers (Monteiro et al. 2015). Hydrogels incorporated with hyaluronic acid have been utilized to treat second-degree burn wounds (Koul et al. 2016). Gelatin hydrogels in combination with hyaluronic acid, chondroitin sulfate, and asiatic acid with nanoparticles like ZnO and CuO were evaluated in Wistar rats with a second-degree burn wound and at 4 weeks, histopathology results revealed better healing of burn wounds in comparison with commercially available wound dressings (Thanusha et al. 2018), and these gels can also function as a skin substitute (Av et al. 2020). In another study with electrospun-based scaffolds composed of cationic gelatin, HA, chondroitin sulfate and sericin were used to coculture human-derived keratinocytes and mesenchymal stem cells (hMSCs). After 5 days of coculture in vitro, results showed that there was epithelial differentiation from hMSCs with expression of dermal protein markers (Bhowmick et al. 2016). An artificial skin-equivalent nanofibrous substitute was developed using HA and collagen with the programmed release of multiple angiogenic growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), and basic fibroblast growth factor (bFGF) either encapsulated in the gelatin nanoparticles or directly embedded in the nanofibers by electrospinning technology. The tensile strength of these Col-HA-GN scaffolds was similar to that of human skin. In vivo studies were conducted on streptozotocin-induced diabetic rats; histopathology and immune-histochemical analysis revealed that faster epithelization rates and enhanced maturation of vessels were associated with scaffolds (Lai et al. 2014). In another study, an artificial nanofibrous skin substitute was developed using mPEG-PCL-grafted gelatin and incorporated with HA, chondroitin sulfate, and sericin to study the in vivo efficacy of second-degree burn wound model on Wistar rats. The results showed that at 21 days, wounds were contracted followed by collagen upregulation (Bhowmick et al. 2018). Thus, HA-based scaffolds play an important role in wound healing and dermal tissueengineering applications.
7
Antimicrobial Properties of HA for Tissue-Engineering Strategies
Medical device-related infections pose a huge financial burden on healthcare services and are associated with increased patient morbidity and mortality (VanEpps and Younger 2016). For patients, infections come alongside pain, inflammation, fever, and long antimicrobial treatment regimens. Medical device-related infections can also lead to more serious conditions such as the risk of sepsis (spread of infection to blood and other organs), with this being potentially fatal. The development of tissue-engineered substitutes that possess intrinsic antimicrobial activity against the three main classes of microbes (bacteria, fungi, and virus) is vital for the success of implantable devices.
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There is a dire need for new diagnostic and therapeutic strategies to combat infectious diseases. As a multidisciplinary science, tissue engineering focuses on the application of engineering principles to the biological systems for the rapid translation of technologies from the benchtop to the bedside. In the current situation we are all facing with the viral outbreak of COVID-19, tissue-engineering techniques and tools can potentially enable virologists to create infection models that combine the facile manipulation and readouts of three-dimensional tissue cultures relevant for the viral infection complexity of the animal model (Ramanan et al. 2014), as well as the development of new vaccine technologies and small-molecule drug delivery systems (Tatara 2020).
7.1
Antibacterial Activity of HA
Bacteria is a type of prokaryotic unicellular organism, and one of the world’s oldest forms of life. Some bacteria live in communities embedded in a matrix consisting of extracellular polymeric substances (EPS), i.e., protein, DNA, and polysaccharides (Bhattacharya et al. 2015). Biofilm-related infections and contamination of materials are major problems encountered in medicine (Lynch and Robertson 2008). Recent studies indicate that bacterial contamination in open wounds may adversely affect the formation of bone and new connective tissue. Reduction of bacterial burden at the wound site is of importance to improve the clinical outcome of regenerative therapy. In surgical settings associated with implanted biomaterials, Staphylococcus aureus is a leading cause of chronic biofilm infection, and this largely impacts orthopedics, trauma, and cardiology. Treatment of these infections requires long antibiotic administration and in some cases additional surgeries, which often leads to the removal of the infected device (Ibberson et al. 2016). Several investigators have taken an interest on the influence of HA on bacteria and how it relates to invasion and virulence. Among various polymers tested for antibacterial coatings, HA and its derivates have proved promising offering long-term safety with the ability to reduce bacterial adhesion and biofilm formation (Romanò et al. 2017). HA has been shown to exert bacteriostatic, but no bactericidal, dose-dependent effects on different microorganisms in the planktonic phase. Concerning to possible orthopedics applications, the analysis of different coatings on titanium surfaces showed that HA significantly decreased S. aureus adhering and density to these titanium surfaces, unveiling its potential application in osteosynthesis, orthopedics, and dental surgery (Harris and Richards 2004) as well as beyond to tissue-engineering applications. HA-molecular weight (MW) and concentration seem to interact with bacteria and trigger various growth profiles. Three HA MW (low ¼ 0.14 MDa, medium ¼ 0.76 MDa, and high ¼ 1.3 MDa) were tested on S. aureus ATCC 9996, Streptococcus mutans ATCC 10449, and Propionibacterium acnes UD, showing reduction of proliferation in media containing HA; moreover, the medium MW showed the highest bacterial growth inhibition (Pirnazar et al. 1999). Regardless of HA MW and concentration, no bactericidal effects were detected.
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Another study evaluated the antimicrobial property of collagen, hydroxyapatite (HAp), and PLGA and HYAFF-11TM (esterified HA produced by Fidia Advanced Biopolymers) on S. aureus (ATCC 25923), S. epidermidis (ATCC 12228), and β-hemolytic Streptococcus (ATCC 19615) cultures (Rohr and Trepp 1996). In this study, HA suppressed bacteria growth rate in comparison to other materials. The HA effect is due to the saturation of the bacterial hyaluronidase by excess HA, which prevents the bacteria from maintaining elevated levels of tissue permeability (Carlson et al. 2004). Hyaloss ®, a hydrogel matrix composed of HYAFFTM for the treatment of periodontal defects, showed improved bone formation with no bacterial infection due to the bacteriostatic effect of the low MW HA (Ballini et al. 2009). Hyruan Plus ® (linear high MW HA (3 MDa) produced by LG life sciences, Iksan, South Korea) was shown to increase wound healing exhibiting bacteriostatic effects in surgical wounds inoculated with S. aureus (SC 2406) (Park et al. 2017). Seprafilm ® (high MW HA gel and carboxymethylcellulose (CMC) at a 2:1 weight ratio, Genzyme Corporation, Japan) displayed antimicrobial activity at two concentrations in S. aureus ATCC 27217 cultures, as the concentration of HA in the bacteria culture increased the optical density (OD) of the bacteria medium decreased, in accordance with a higher bacteriostatic effect (Uchida et al. 2011). To penetrate the ECM, pathogenic gram-positive bacteria secrete hyaluronidase to cleave HA. S. aureus produces hyaluronidase, encoded in the hysA gene, that is conserved in all S. aureus lineages. HysA encoding is often shown as a virulence factor in a variety of infection models, which is due to its role in facilitating local spread of the infection (Ibberson et al. 2016). In a context, two strains of S. aureus (a wild type hysA positive and a mutant type hysA negative) were cultured in a catheter implanted in a murine model. S. aureus that produced hysA (wild type) was more invasive and increased lesion distribution and severity in comparison to the S. aureus that was unable to produce hysA (mutant type) (Ibberson et al. 2016). Additionally, biofilm formation in vitro was increased in mutant S. aureus when adding HA with MW above 50 kDa to the culture medium (Ibberson et al. 2016). In addition, antimicrobial studies using other gram-positive bacteria from the streptococcus and enterococcus species show that high MW HA (1.8 MDa) against Streptococcus mutans ATCC 25175, Enterococcus faecalis ATCC 29212, and Enterococcus hirae ATCC 10541 possesses dose-dependent bacteriostatic effects (Ardizzoni et al. 2011). Bacillus subtilis ATCC 6633 and S. aureus ATCC 6538 also showed susceptibility to polyethyleneimine-modified HA particles, although these novel particles were more effective against B. subtilis (Sahiner et al. 2017). A porous scaffold made of HA with approximately 125 kDa was produced and tested against S. aureus ATCC 6538 for antimicrobial properties. The results showed that HA has bacteriostatic effects by decreasing the colony formation units (CFU) from 107 (control) to 1.1 103 (HA) (Guzińska et al. 2018). Hyabest ® (S) LF-P, a food supplement containing HA with MW ranging 250– 400 kDa (produced by Kewpie, Tokyo, Japan) was used to synthesize novel hydrogel for microneedle application. Antibacterial activity was observed against S. aureus (ATCC 6538) (Larraneta et al. 2018).
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Escherichia coli is the most thoroughly studied species of bacteria, which has long been the favored organism for investigation of the basic mechanisms of molecular genetics (Blount 2015). Several E. coli strains are pathogenic. Seprafilm® also showed bacteriostatic effect against E. coli ATCC 25922 in a concentration-dependent manner (Uchida et al. 2011). A porous scaffold made of HA with approximately 125 kDa was produced and tested against E. coli ATCC 11229 for antimicrobial properties. The results showed that HA has bacteriostatic effects by decreasing the colony formation units (CFU) from 3.9 107 (control) to 50 (with HA) (Guzińska et al. 2018). Other antibacterial studies using E. coli also showed bacteriostatic effects of high MW HA with approximate 1 MDa (del Hoyo-Gallego et al. 2016; Pérez-Álvarez et al. 2019). Other gram-negative bacteria (Porphyromonas gengivalis ATCC 33277, Prevotella oris ATCC 33573, and Actinobacillus actinomycetemcomitans Y4) were tested against three HA MWs (0.14, 0.76, or 1.3 MDa). Each bacteria showed distinct growth inhibition indexes, where A. actino showed higher growth inhibition in comparison to P. oris and P. gingivalis, respectively (Pirnazar et al. 1999). Another study evaluated the antimicrobial property of collagen, hydroxyapatite (HAp), PLGA, and HYAFF-11TM on Pseudomonas aeruginosas (ATCC 27853), showing that HA significantly suppressed bacteria growth rate in comparison to the other materials (Carlson et al. 2004). Hyabest ® (S) LF-P also showed antibacterial activity against Proteus mirabilis (ATCC 35508) (Larraneta et al. 2018). Mycobacteria invade the lungs through the interaction with GAG, such as HA. Three strains of mycobacteria (M. tuberculosis H37Rv, M. smegmatis mc2155, and M. avium type 4) were used to determine the influence of HA on infection and disease. Interestingly, HA promotes mycobacteria invasion and proliferation (Hirayama et al. 2009). HA has been widely used to encapsulate a wide range of drugs, including antibiotics. Aminoglycoside antibiotics (such as streptomycin) are highly hydrophilic. This pose pharmacological challenges, particularly in the treatment of intracellular bacterial infections. Due to its high hydrophilicity, it has poor penetration within the eukaryotic cell membranes; often high doses of antibiotics still display subtherapeutic concentration inside the cell (Maurin and Raoult 2001). It was speculated that HA could be an antibiotic carrier for the treatment of intracellular bacterial infections. Antibiotics conjugated to HA were able to be phagocytize by infected eukaryotic cells through a CD44-mediated pathway, and reduce bacterial infection burden intracellularly (Qiu et al. 2017).
7.2
Antifungal Activity of HA
Fungi are a type of eukaryote. They can be single-celled, like yeast, or multicellular, like mold. Unlike the antimicrobial activity of HA in bacteria, only a few studies have been conducted to assess the effects of HA on fungi. Intrinsic antifungal properties of HMW HA (1.8 MDa) were demonstrated against Candida glabrata ATCC 90030 and Candida parapsilosis ATCC 22019. The fungistatic activity of HA was reported to be dose dependent (Ardizzoni et al. 2011).
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Antiviral Activity of HA
The dilemma about viruses is if they are living organisms or not. Many feel that viruses are not live organism as they cannot replicate without other cells. Viruses target all domains (prokaryote and eukaryote cell types) containing DNA or RNA as their genetic material. They need the host cell machinery to replicate, leading to hostcellular death. Despite so many studies on the antimicrobial activity of HA in bacteria, only a few studies have been conducted to assess the effects of HA on viruses. In vitro evidence of antiviral activity of HA has been shown with respect to herpes simplex virus (HSV-2) (Tiunnikov et al. 2002), although other reports on HSV-1 showed that HA appears to contribute to HSV-1 infection of both the brain and the skin tissues (Cohen et al. 2011). Other studies evaluated the levels of HA produced by some rheumatoid arthritis (RA) cell lines, some of which were partially or completely resistant to infection with Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and rubella virus (RV). Normal fetal synovial cells lines were susceptible to NDV, VSV, and RV. Once infection-resistant RA cell lines were treated using hyaluronidase, viral infection became possible. Moreover, HA prevented infection of normal fetal synovial cells with VSV (Patterson et al. 1975). Another study has shown that high MW HA (1.800 KD) demonstrated strong antiviral activity against Coxsackievirus B5 (COXB5), mumps virus (MV), and influenza virus A/H1N1; mild antiviral activity was shown against HSV-1 and porcine parvovirus (PPV), and no activity against Adenovirus 5 (ADV-5), human Herpesvirus-6 (HHV-6), and porcine reproductive and respiratory syndrome virus (PRRSV) (Fig. 26). In all cases, no virucidal activity of HA was observed (Cermelli et al. 2011). Recently, a cyclodextrin-conjugated HA was able to encapsulate acyclovir, showing good antiviral activity together with a delayed-release of acyclovir (Piperno et al. 2019).
7.3.1 Highlight on COVID-19 and Other SARS In the first months of 2020, the world was taken by surprise by a fast spreading virus, which put most countries and economies into lockdown for a number of months. The pandemic is still far from being over, and new waves of infection in the coming months are expected. Severe acute respiratory syndrome (SARS)-CoV-2 is the virus responsible for the corona virus disease (COVID-19), which originated in Wuhan, China (Colson et al. 2020). To date, the scientific community is working relentlessly to find suitable drugs to halt the spread of the virus as well as the safe development of a new vaccine. Tissue-engineering skillsets and tools may be leveraged to have an impact on clinical practice in settings of a viral outbreak, such as in recent COVID19 (Fig. 27). A number of antiviral drugs such as lopinavir and ritonavir (normally used in the treatment of human immunodeficiency virus – HIV) and other antiretroviral (such as Favipiravir) and Remdesivir (originally considered for the Ebola virus, Middle East Respiratory Syndrome – MERS, and SARS), in addition to
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Fig. 26 Virus yield of various infected cell lines after exposure to HA in different concentrations. (a) VERO cells infected with COXB5, (b) VERO cells infected with MV, (c) VERO cells infected with HSV-1, (d) VERO cells infected with ADV-5, (e) WSN33, (f) PK15 cell line infected with PPV, (g) JJHAN cell line infected with HHV-6, and H) MARC145 cells infected with PRRSV. [Open access reprinting (Cermelli et al. 2011)]
hydroxychloroquine used in the treatment of malaria, show promise in the treatment of COVID-19 (Cao et al. 2020; Gautret et al. 2020). However, these kinds of drugs show systemic toxicity. Drug modification, by the conjugation of HA, seems
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Fig. 27 Proposed examples of tissue-engineering strategies to fight viral outbreaks (Tatara 2020)
like an alternative in order to reduce toxicity and increase efficacy (Khan and Aroulmoji 2020). Interestingly, HA has been described as a potential cause of fatalities for COVID19 infections. This reasoning is originated from patients with severe conditions accompanied with cytokine release storm (CRS), which induces an hyperactive immune response leading to acute respiratory distress syndrome (ARDS) and lung damage (Mehta et al. 2020). The symptoms of ARDS in patients include short/rapid breathing, and cyanosis. Most of these patients are allocated into intensive care units requiring mechanical ventilators. CT images of the lungs of these patients show characteristic white patches called “ground glass.” Autopsies have confirmed a clear jelly substance lining the lungs of these patients. Although the nature of this gel is not yet confirmed, it is speculated that it contains HA (Shi et al. 2020). On the mucosal surface of the airways of healthy individuals, HA retains bactericidal enzymes in order to protect the mucosa tissue from invading pathogens. However, upon infection, low-MW HA fragments are released to stimulate an immunological response and start inflammation. After resolution of inflammation, local macrophages eliminate HA via CD44-mediated receptor (Hirayama et al. 2009). The assumption that “ground glass” contains HA is originated by the defective production and regulation of HA during COVID-19 and other SARS infections (Fig. 28). Hyaluronidase 3 (HYAL-3) gene is shown to be upregulated in human monocytic cells infected with COVID-19 (Hu et al. 2012). It is well known that the degradation of HA into small fragments induces the immune response milieu (Zamboni et al. 2018). We speculate that the inhibition of upregulated HYAL-3 could potentially improve ARDS. One well-known HYAL inhibitor is hyaluromycin (Kohi et al. 2016). Alternatively, a further potential mechanism to be exploited could be the inhibition of hyaluronic acid synthase (HAS). The production of HA can be
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Fig. 28 Schematic of the progression of COVID-19 infection with HA accumulation in the lungs. (Modified from Mong et al. 2020)
Fig. 29 Mechanism of action of 4-methylumbelliferone on HAS by inhibiting HA production (Nagy et al. 2015)
specifically inhibited by the use of 4-methylumbelliferone (4-MU) (Sukowati et al. 2019). 4-MU inhibits HA production by competing with uridine 50 -diphosphate (UDP) as the substrate for UDP-glucurosyltranferase (UGT), an enzyme involved in the HA synthesis (Fig. 29). 4-MU is a drug already in the market by the name of
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hymecromone; it is a derivative of coumarin (warfarin); however, it does not possess anticoagulant properties. Its typical dose regimen for the treatment of biliary dyskinesia ranges around 300–800 mg three times/day (Nagy et al. 2015). We speculate that the development of a new topic delivery system for 4-MU, directly to the lungs by inhalers, could be of great impact in the treatment of ARDS in COVID-19 patients.
8
Conclusions and Future Outlook
The optimal bioengineered microenvironment is envisaged to mimic the native ECM and integrate different biomaterials and biofunctional molecules to display synergic anti-inflammatory effects thus enhancing cell survival and function. As we have highlighted here, HA is showing promising results as material which is both capable of protecting cells from the immune response and capable of immune modulation. Due to its intrinsic anti-inflammatory and anti-immunogenic functions, HA can protect cells from lymphocyte-mediated cell killing. This coupled with its ease of chemical modification to protract degradation, tailor mechanical response, and couple to other biomolecules or therapeutics is allowing HA to become the material of choice for enabling emerging medical technologies which depend on immune regulation such as vaccines, gene therapy, and drug and cell delivery as well as associated regenerative medicine techniques. Acknowledgments The authors would like to thank the financial support provided by following funding bodies: Irish Research Council (GOIPG/2015/3577); European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 801165, with cofunding through SSPC by its funding body, Science Foundation Ireland, grant no. 112/RC/ 2275_P2.
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Salih Mac¸in
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Extracellular Matrix and Wound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Extracellular Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Wound Formation: Acute and Chronic Wound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Maturation and Restructuring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 The Role of Extracellular Matrix in Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Uses of Natural Polymers in Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Drawbacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 In Vivo and In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
As a result of diseases and accidents, people lose their tissues and organs. Instead of difficult and troublesome methods such as tissue and organ transplantation, biocompatible, nontoxic, antitumor, antimicrobial, and wound healing natural polymers are used for the treatment of these damages. There are glycosaminoglycans as natural polysaccharides in the human body, which act as extracellular matrix and produced from fibroblasts. Wound healing is a dynamic and complex process consisting of successive periods. Tissue healing process is regular and timely in acute wounds. In chronic wounds, healing takes longer time. The use of appropriate dressings plays an important role in the wound healing process. Researches on polymeric dressings used as carriers for S. Maçin (*) Department of Medical Microbiology, Selçuk University, Konya, Turkey © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_57
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local application of active ingredients to the wound surface are increasing. These polymeric systems can be natural, hydrogel-forming materials such as collagen, chitosan, and pectin, or tissue-engineered materials such as alginate. In this section, extracellular matrix, wound formation, wound healing mechanisms, and the function of natural polysaccharides which have an important role in wound healing will be examined. Keywords
Tissue · Extracellular matrix · Glycosaminoglycan · Polymer · Wound healing
1
Introduction
Wound healing is one of the most common health problems and advanced wound management strategies are needed to achieve optimal healing. Definite times cannot be given for wound healing, but the chronic term is used for wounds that cannot heal within a reasonable time. The progression of wound healing depends on many factors, ranging from the patient’s general condition to treatment, to the cause. It is necessary to act according to the characteristics of each wound. Wound healing depends on various factors such as the general health of the person and blood circulation, the reason for the formation of the wound, the location and type of wound in the body, and whether there are infections in the wound. So every wound heals according to its own characteristics (Schreiber et al. 2005). Wounds are divided into two parts as acute and chronic wounds according to the wound healing time. Acute wounds are wounds that we encounter very often, do not cause damage after recovery, and heal quickly. Chronic wounds, on the other hand, are characterized by impaired skin integrity, functional loss, and prolonged recovery. Recovery in chronic wounds takes weeks, months, and years. In this context, appropriate wound treatment is required, which can speed up the healing process and reduce the healing time at the same time. Glycosaminoglycan derivatives, which are mostly involved in the structure of the extracellular matrix (ECM) in wound healing, have been preferred as potential treatments for many years (Tan et al. 2001). Glycosaminoglycans (GAGs), the extracellular matrix molecule, which plays an important role in the acute or chronic wound healing process and supports wound healing, is an effective means of angiogenesis and inflammation and leads to rapid granulation, vascularization, and re-epithelization (Mitchell and Church 2002). Glycosaminoglycans are preferred in many materials due to their function in wound healing and are recommended to reduce the healing time in the chronic wound healing process. In this section, the natural polysaccharides used in wound healing and their mechanisms will be discussed in detail.
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2
Extracellular Matrix and Wound
2.1
Extracellular Matrix
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Connective tissue epithelium, which protects the integrity of the body and keeps all parts of the body together structurally and functionally, provides continuity between the muscle and nerve tissues. Connective tissue covers all the tissues that support the structure of the body, such as ligaments of the joints, beams, bone tissue, cartilage tissue, and adipose tissue. The intercellular substance (extracellular matrix/extracellular matrix) created by connective tissue cells consists of two main structures: 1. Connective tissue fibers. 2. The basic substance that fills the space between connective tissue fibers and connective tissue cells. Basic substance: It is the basic structure consisting of proteoglycan, glycoprotein, and glycosaminoglycans, and forming the extracellular space together with cell fibers. This structure forms the basic environment that meets the actual needs of the cell and tissue (Baum and Arpey 2005). Extracellular matrix (ECM), which plays a role in the connective tissue as an extracellular matrix, is a variety of proteins and polysaccharides that are secreted by some cells in a multicellular organism, which fill cells between cells and act as binding agents in a defined area (Karabekian et al. 2009). There are two main extracellular proteins that make up the matrix. These are fibrous and proteoglycans (Ustunel et al. 2003). Proteoglycans are peptide chains containing covalently linked glycosaminoglycans. They contain 95% carbohydrates and 5% protein in their structure. There are seven types of glycosaminoglycans (GAGs): hyaluronic acid, chondroitin sulfate, keratan sulfate I and II, heparin, heparan sulfate, and dermatan sulfate. Fibrous proteins are two types: structural proteins (collagen and elastin) and adhesive proteins (fibronectin, laminin, tenaskin, vitronectin, and integrin) (Wound repair 2005). Glycosaminoglycans form a gelatinous and hydrated substance in the connective tissue by proteoglycans (PGs) by embedding fibrous proteins. Proteoglycans consist of a central protein called glycosaminoglycans (GAGs) that are bound to one or more polysaccharides (Pelosi et al. 2007). Glycosaminoglycans are heterogeneous polysaccharides containing long, linear, and recurrent disaccharide units. These disaccharide units are galactose, galactosamine, N-acetylgalactosamine-4sulfate, and galacturonic acid. There are two basic types of GAG. The first one is non-sulfated GAG (hyaluronic acid), and the second is sulfated GAG (heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, and keratan sulfate). Except for hyaluronic acid, GAGs are usually covalently attached to a protein nucleus that forms a general structure called proteoglycans (Pelosi et al. 2007; Souza-Fernandes et al. 2006).
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GAGs are usually large complexes consisting of small amounts of protein and negatively charged heteropolysaccharide chains. These complexes form a gel-like matrix called “ground substance” with a large amount of water binding property. They are also strong acidic biopolymers with biomedical importance that prevent the formation of factors such as virus entry into cells and angiogenesis. GAG chains fill most extracellular spaces and provide mechanical support to the tissue, as well as providing rapid diffusion of water-soluble molecules and migration of cells (Ramael et al. 1991).
2.2
Wound Formation: Acute and Chronic Wound
The wound is the disruption of the integrity of the skin or mucosa due to the effect of trauma. Factors such as shock, falling, strong impacts, atmospheric pressure, thermal effects, (burn, freezing) electric shock, and radioactivity cause injuries. As a result of acute or chronic wound, tissue integrity is impaired. Acute wounds heal without problems within the expected time. The time it takes for chronic wounds to heal usually ranges from 5, 10, or 30 days. It may occur as a result of traumatic loss of tissue or surgical procedure (Jackson et al. 1991). Chronic wounds are often characterized by permanent injury and prolonged inflammation, high bacterial biofilm incidence, and excessive proteolysis (Robson et al. 2001). Impairment in macrophage function and angiogenic response, which is often associated with serious wound healing process, is also observed. Due to prolonged inflammation, excessive removal of inflammatory cells into the wound bed by a large number of neutrophils is required. It is known that neutrophils can remove damaged tissue from the temporary matrix of the wound site and prevent microbial infection. On the other hand, as the potential of unmanaged neutrophils to kill pathogens causes degradation of the ECM and growth factors, it can cause excessive production of protease that initiates significant tissue damage to the host that is detrimental to wound healing. In addition, inefficient cell proliferation due to the breakdown of the ECM molecule in the wound leads to angiogenesis, which indicates greater wound bed defacement and healing impairment. Therefore, prevention of prolonged inflammation is a target strategy in the treatment of severe wounds. GAG has been found to bind to neutrophils, macrophages, and lymphocytes, key cells of the inflammatory response. The effect of excessive protease production caused by too many active neutrophils in the wound area can be inhibited by electrostatic binding with some anionic polymers, such as GAG or functionalized dextrans. A high level of anionic GAG will be in ion pairing with cationic neutrophils to interfere with the activity of cationic proteins through charge interactions. Therefore, with this mechanism, it may be possible to reduce excessive neutrophil uptake and move the wound from the inflammatory stage to the next healing stage. However, after severe tissue damage, glycanases and proteases can destroy GAG (Komarcevic 2000). In severe wounds, GAG deficiency is eliminated by adding GAG material, such as a natural polysaccharide, directly to the wound area as a wound dressing. With the rich source of GAG around the wound and a better understanding of the GAG roles in the healing processes, it was possible to formulate therapeutic strategies expected
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to accelerate serious wound healing. Glycosaminoglycans (GAGs) have been shown to play important roles in cell signaling and development, angiogenesis, anticoagulation, and co-receptors for growth factors that belong to control of all wound healing stages, both acute wounds and chronic wounds (Peplow 2005). In chronic wounds, delays occur in the normal stages of healing, the wound cannot be repaired regularly and on time (Jackson et al. 1991). As a result of disruptions caused by various factors in one or more stages of hemostasis, inflammation, proliferation, or remodeling, the healing process cannot be completed completely. This may be caused by increased levels of infection, tissue hypoxia, necrosis, exudate, or inflammatory cytokines. Continued inflammation in the wound causes the tissue to heal response to occur in an uncoordinated and long period, resulting in frequent recurrence of wounds. Chronic wounds can be caused by various causes such as pressure, arterial and venous insufficiency, burns, continuous infection, and vasculitis (Jackson et al. 1991; Komarcevic 2000). Among the various molecules secreted by ECM, GAG has partners that have important roles in controlling all wound healing stages, acute wounds or severe wounds. These molecules participate in cell-cell and cell-matrix interactions, cell proliferation and displacement, cytokine and growth factor signals, thereby locally modulating their biological activities. The ECM functions to guide the organized response characterized by hemostasis, inflammation, proliferation, and restructuring seen in wound healing. The effects of the various ECM components differ according to the wound stages. This dynamic and sequential order occurs as a result of the interaction of cell and growth factors (Ono et al. 1995).
3
Wound Healing
Wound healing is an extremely dynamic process and involves complex interactions of extracellular matrix molecules, soluble mediator molecules, various resident cells, and infiltrating leukocyte subtypes. The main purpose of the repair is to achieve tissue integrity and hemostasis. For this purpose, the healing process takes place in three stages (Negut et al. 2018). 1. Inflammatory phase (hemostasis and inflammation). 2. Proliferation. 3. Maturation and restructuring (remodeling). In one of these phases, delay or negativity results in the wound not closing or healing is prolonged (Muncaster 2001).
3.1
Hemostasis
The first stage of wound healing is devoted to the formation of a hemostasis and a temporary wound matrix. The wound matrix appears immediately after the wound
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and is completed a few hours later. This phase initiates the inflammatory process and is sometimes called the late phase (Robson et al. 2001). The vital agents of hemostasis are fibrin, platelets, and blood vessels. In the first 1 or 2 h after injury, wound repair begins with the formation of a fibrin matrix through proteolytic cleavage of fibrinogen with thrombin, and fibrin binds directly to platelets to produce a clot (Muncaster 2001; Woo et al. 2004). This causes degranulation of alpha granules and dense bodies in the cytoplasm of platelets. In this way, albumin, fibrinogen, fibronectin, IgG, coagulation factor V and VIII, platelet-derived growth factor (PDGF), transforming growth factor alpha and beta, fibroblast growth factor-2 (FGF-2), platelet-derived epidermal growth factor (PDEGF), and endothelial growth factor are secreted into the environment. Among all these factors, PDGF, TGF-beta, and FGF-2 are the most important. Dense bodies, by releasing calcium, serotonin, ADP, and ATP, provide an energy source for inflammatory cells that will come to the environment (Monaco and Lawrence 2003). It also calls and activates PDGF and IGF-1 fibroblasts. GAG and collagen are synthesized to allow cells to migrate and multiply at the wound site. Fibrinolytic enzymes resist clot formation. On the other hand, sprinkles ensure that excessive fibrinolytic activity does not occur. The ECM includes a network of scaffolding proteins that are bound by GAG. GAG, especially heparin sulfate (HS), plays a key role as anticoagulants, which have important actions to manage the regulation of protein networks. HS represents 50– 90% of the total GAG content and is only in contact with blood when an injury occurs. It has been determined that HS binds with more than 100 proteins involved in hemostasis, many growth factors, proteins involved in lipid metabolism, and ECM proteins. In addition, HS maintains hemostasis as an effective mediator of angiogenesis on the surface of endothelial cells (Jacob et al. 2007). In this phase, many cells and factors are activated for the wound healing process occurring in the organism.
3.2
Inflammation
The main purpose of this phase in wound healing is to prevent infections. At this stage, the first response is to neutralize bacteria and foreign particles with phagocytosis or toxic substances released by the neutrophils coming into the scar tissue within 48 h (Lesko and Majka 2008). This phase occurs with symptoms of inflammation, such as redness, body temperature, swelling and pain around the injured place, on average, within 24–48 h. When bleeding is controlled, neutrophils, macrophages, and lymphocytes accumulate in the wound area, simultaneously releasing a large number of active mediators (cytokines and growth factors), thereby stimulating the inflammatory phase (Broughton 2nd et al. 2006; Gosain and DiPietro 2004). Chemotactic agents such as increased vascular permeability caused by inflammation, complement factors, interleukin-1, tumor necrosis factor-alpha (TNF-α), tumor necrosis factor-beta, and platelet factor 4 (PF4) stimulate neutrophil chemotaxis (Broughton 2nd et al. 2006; van Beurden et al. 2005). Neutrophils appear in the
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wound 6 h after trauma and are the cells that prevail for the first 2 days. The main task of neutrophils on the wound surface is to remove the traces of the cell that are damaged by trauma, by the phagocytosis of the foreign body, and the release of proteases (Monaco and Lawrence 2003). Monocyte density begins on the second-third days. As the neutrophil count decreases, the number of monocytes/macrophages increases. In the third-fifth days, macrophages become 12 dominant cells in the wound (Gosain and DiPietro 2004). The presence of active macrophages in the wound area is essential for wound healing. While neutrophil absence does not disrupt the general flow of wound healing, wound healing process stops in the absence of macrophage. The main duties of macrophages in wound healing are outlined: phagocytosis and antimicrobial function, wound debridement, matrix synthesis regulation, cell activation, and angiogenesis (Broughton 2nd et al. 2006). Macrophages not only do phagocytosis but also perform various cytokines, growth factors, and NO synthesis. They increase keratinocyte and fibroblast activation. In addition, while it catalyzes the conversion of plasminogen to plasmid with the help of neutral proteases, it also activates the complement and pre-Hageman factor (Lesko and Majka 2008). Activated macrophages are cells that play a key role in wound healing (Lesko and Majka 2008; van Beurden et al. 2005). Hyaluronic acid (HA), a non-sulfated GAG of ECM, is involved in an important process of the inflammatory phase. At this stage, HA accumulates in the wound bed and regulates early inflammation to modulate inflammatory cell and fibroblast cell migration, pro-inflammatory cytokine synthesis, and phagocytosis of invading microbes. In addition, HA can bind and increase the effectiveness of chemokines to neutrophils. Butler et al. found that the efficiency of HA and neutrophil uptake on the endothelial surface was increased and revealed that HA revealed stimuli to neutrophils (Butler et al. 2009). In the inflammatory phase, neutrophils collagenase and elastase eliminate damaged tissue from the temporary matrix of the wound site, while monocytes inactivate any source of microbial infections through macrophages and the activity of secreted proteases. In inflammation sites, low molecular weight HA fragments (accumulated by the degradation of high molecular weight HA) proliferate IL-6, TNF-α, and IL-1β of Toll-like receptor 2 and Toll-like receptor 4 pro-inflammatory cytokines. Also, growth factors and cytokines released by inflammatory cells induce migration and proliferation of fibroblast and keratinocyte, which synthesize HA levels (Zhong et al. 2010). It was found that the level of HA was significantly high during the reepithelialization process, where epithelial cells migrated through the new tissue to form a barrier between the wound and the environment. The secretion of cytokines such as TGF-β, PDGF, FGF-2, IL-1, and TNF-α modulates collagen accumulation and penetration of new blood vessels into the wound area by fibroblasts. T lymphocytes (especially CD4) migrate to the wound area; it secretes IL-1, IL-2, TNF-alpha, fibroblast activating factor, EGF, and TGF-beta. Inhibition of circulating T lymphocytes delays wound healing. B lymphocytes have not been found to play a role in wound healing (Winter and Scales 1963).
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In summary, in inflammation, leukocytes bind to ECM proteins through integrins. ECM proteins stimulate the activity of monocytes/macrophages and remove neutrophils and wound residues from the wound site (Miller et al. 1998).
3.3
Proliferation
This complex process consists of angiogenesis, granulation tissue formation, collagen deposition, epithelization, and wound closure, and takes about 2 days–2 weeks. A new matrix layer by fibroblasts restores tissue in the wound area. Other mesenchymal cells also enter the inflammatory region of the wound in response to growth factors necessary to stimulate cell proliferation (Lesko and Majka 2008). Also, fibroblasts, endothelial cells, and keratinocytes produce IGF-I, FGF-2, TGF-y, PDGF, and VEGF, which support cell migration and proliferation, matrix synthesis, and angiogenesis. Secreted vascular endothelial growth factor (VEGF) and other cytokines neovascularization, growth factors released from platelets; in particular, the transforming growth factor (TGF-β) and platelet-derived growth factor (PDGF) stimulate the proliferation of fibroblasts. Fibronectin and collagen production occur as the scar tissue becomes rich by fibroblasts. Fibroblasts synthesize collagen and prostaglandins (PG). Both act to create an unstructured connective tissue environment that allows new cells to migrate (Grazul-Bilska et al. 2003). A number of PGs have been presented in the wound area, and their GAG side chains played a role in the stabilization and activation of growth factors. Sulfated PGs with chondrite sulfate (CS) and dermatan sulfate (DS) contribute to collagen polymerization. PGs provide a matrix for cellular attachment, and some PGs form triple complexes that moisten the tissue that promotes hyaluronic acid, cell survival to cover the wound site, and migration on granulation tissue (Brooks et al. 1994). This stage takes place with a dynamic process occurring between the developing granulation tissue formation, fibroblasts, growth factors, and ECM. In this process, macrophages release growth factors that stimulate angiogenesis, collagen synthesis, and fibroblast proliferation after binding to the ECM. Endothelial cells bind ECM proteins through integrins, helping to advance the angiogenesis process (Arora et al. 1999).
3.4
Maturation and Restructuring
Remodeling is the stage characterized by the rearrangement of synthesized collagen up to a year after the initial wound formation. There is a balance between collagen production and destruction, characterized by the wound surface, it is the last stage of wound healing (Miller et al. 2003). At this stage, with the transition of granulation tissue to a mature scar, a new epithelium is formed. This process is accompanied by high mechanical strength of the formed tissue, reduction of capillary amounts by mixing with larger blood vessels, decreasing cell density and metabolic activity of tissue, and lowering GAG content. Inflammatory cells gradually decrease. The early matrix skeleton consists of Type 3 collagen and fibronectin, while the final matrix skeleton is created
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MAJOR CASE
Coagulation Homeostasis
Growth Factor Release
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Collagen Storage
Collagen Crosslinking
Fibroblasts
CELLULAR RESPONSE
Lymphocytes Macrophages Neutrophils
VASCULAR RESPONSE
Vasoconstriction
Vasodilatation
İnflammation
PHASE
Proliferation Remodeling
Injuries
3 Days
7 Days
3 Weeks
1 Years
Fig. 1 Wound healing process Inflammation; the first response is the phase in which bacteria and foreign particles of neutrophils that come to the scar tissue within 48 h are inactivated by phagocytosis or toxic substances secreted. The phase in which proliferation angiogenesis, granulation tissue formation, collagen deposition, epithelization, and wound closure lasting approximately 2 days to 2 weeks, and a new matrix layer by fibroblasts, tissue in the wound area is restored. The remodeling phase creates a balance between collagen production and destruction, characterized by the wound surface, and wound healing takes place in three phases
by Type 1 collagen. The resistance of the scar tissue reaches 80% of the original tissue. The remodeling process continues for 21 days–2 years. In order to achieve optimum wound healing, good wound nutrition should be provided, pain should be reduced, clean wound and wound surface should be created, wounds should be protected from trauma and infection, systemic conditions should be corrected or improved, and expenses should be minimized (Ueno et al. 2001). The mechanical strength of the formed tissue is equal to 25% in relation to the dermis and 80% with unchanged tissue after reconstruction for months (Ko et al. 2014). Considering that GAG activities can reduce inflammatory responses and ECM accumulation in the early stages of wound healing, an appropriate wound treatment at the start of injury of a GAG-rich material is expected to heal closer to normal skin (Mantle et al. 2001) (Fig. 1).
4
The Role of Extracellular Matrix in Wound Healing
Among the various molecules secreted by ECM, GAG has partners that have important roles in controlling all wound healing stages, acute wounds or severe wounds. These molecules participate in cell-cell and cell-matrix interactions, cell
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proliferation and displacement, cytokine and growth factor signals. Thus, it modulates its biological activities locally. The ECM functions to guide the organized response characterized by hemostasis, inflammation, proliferation, and restructuring seen in wound healing. The effects of the various ECM components differ according to the wound stages. This dynamic and sequential order occurs as a result of the interaction of cell and growth factors (Ono et al. 1995). ECM components are involved in every phase of wound healing. By interacting with cells and growth factors, they play a role in a dynamic shopping process that ultimately causes wound closure. More specifically, ECM components play a key role in stimulating cell proliferation and differentiation, directing cell migration, and modulating cellular responses. When the ECM loses its function (for example, in difficult to heal or chronic wounds), wound healing is slowed or stopped (Midwood et al. 2004).
5
Uses of Natural Polymers in Wound Healing
In addition to its presence in bacteria, hyaluronic acid, which is found in extracellular matrix and joint fluids in animal tissues, is important in terms of ensuring the elasticity of cartilage and tendons. However, it has played an important role in synovial fluid, in the vitreous of the eye, cell adhesion, cell mobility, embryo development, and in the living organism as a growth hormone promoter. It is also frequently used in wound healing, cancer metastases, and treatment of diseases such as nervous disorders and arthritis. Since hyaluronic acid polymers are organic substances that are capable of dissolving in water, they are also important because they act as viscosity increasing agents in the liquids they contain. Hyaluronic acid in the skin affects the passage rate of various substances through the skin. Hyaluronidase, which breaks down hyaluronic acid, facilitates the entry of various substances into the tissue. This enzyme, which is found in some microorganisms that cause pathogenic diseases, causes some pathogens to spread in the organism. It is known that the enzyme is also effective during fertilization (Wagener et al. 2017). Heparin is the only GAG with anticoagulant properties. In other words, it inhibits the prothrombin-thrombin conversion and the effect of prothrombin on fibrinogen and prevents blood clotting. It is known to be used in the treatment of many diseases such as allergic rhinitis, asthma, and cancer. It is applied parenterally for the treatment of thrombosis, phlebitis, and embolism. Heparan sulfate oligosaccharide production is associated with the secondary accumulation of GM2 and GM3 ganglia in the brain, the formation of large cytoplasmic content in various brain cell types, in the accumulation of the C subunit of mitochondrial ATP synthesis, and the irregularity of GAP43 mRNA expression in brain tissues. It leads to very serious advanced mental retardation and premature deaths in the early onset of neurological diseases in children (Celebi and Onat 2006). Chondroitin sulfate is involved in adults, part of learning and memory, and the neurohypophysis system in the hypothalamus. It also plays an important role in the damages and diseases in MSS. Chondroitin sulfate is the main stopper in the
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component of glial wounds after damage in CNS. Surgical chondroitin sulfate regulates axonal regeneration and functional gains. It may also affect pathological stages in diseases such as epilepsy and Alzheimer’s (Walker et al. 2005). Dermatan sulfate is also found in the skin, blood vessels, heart valves, tendons, and lungs. It has a heparin-like antithrombic effect. But there are minimal whole blood anticoagulant and blood lipid cleansing activities. Dermatan sulfate is known to show therapeutic properties in coagulation, cardiovascular diseases, infection, recurrent wounds, and fibrosis (Kobayashi et al. 1997).
5.1
Drawbacks
While natural polymers used in wound healing provide advantages for patients, they also create disadvantages. Based on the wound type, healing time and effectiveness of the material used in patients are not the same. According to the researches, it is stated that the traditional cotton gauze allows moisture to evaporate from the wound surface, adheres to the wound bed and causes pain during removal, and therefore it is emphasized that the gauze dressing should be changed frequently. In order to overcome this problem, wound dressings produced with different features, especially modern dressings, meet the need in the medical field to a large extent. However, there are some problems with the dressings used in the treatment of wounds and burns. For example, the fluid amounts of different wound types should vary, and ideal dressings suitable for different wound types should be developed. Therefore, multidisciplinary studies are needed to further improve the existing dressings (Sidhu et al. 1998). Natural polysaccharides have shown significant success in the treatment of chronic wounds for their ability to maintain anti-inflammatory and moist wound environment. However, in people with an allergic disease, natural polysaccharides cause excessive reaction and irritation due to the complex structure of the immune system. Therefore, control of the molecular weight of natural polysaccharides is expected to overcome this limitation. By selecting the desired molecular weight, the portion of the natural polysaccharide that can cause a hypersensitivity reaction should be simplified or removed. In addition, these properties in dry wound can also lead to inefficiency of wound healing process. By causing dehydration of the dry wound, it reduces blood flow and migration ability of epithelial cells around the wound area, thereby interrupting the formation of new tissue (Kumar et al. 1993). Natural polysaccharides have been shown to be a great potential for medical, pharmaceutical, and biomedical applications, including wound dressings, biomaterials, and tissue regeneration, due to their economic, less toxic, and appropriate compatibility profiles. However, these polysaccharides have a lack of protein structures, very poor bio-stability, and difficulty in forming a “matrix” to bridge damaged tissue in the wound healing process, thus facilitating wound contraction and leading to scar formation (Yang et al. 2005). In the light of this information, the most reasonable product should be preferred in natural polysaccharide wound healing.
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6
In Vivo and In Vitro Studies
From the polysaccharides, nanofibrillar structures, which are used in the treatment of wounds and burns, are obtained by the electrostatic spinning method. Among the homoglycans, cellulose, chitin, chitosan, dextran, alpha and beta glucan; among the heteroglycans, alginate, agar, agarose, carrageenan, pectin, gum and glycosaminoglycans (hyaluronic acid, heparin, chondroid sulfate, etc.) are polysaccharides frequently used in wound and burn dressings. Experimental animal wound models are still the most preferred models used to examine wound healing as they provide complex conditions that can best mimic wound formation and tissue repair. In these studies, the effectiveness of many natural polysaccharides has been evaluated. Alginate, chitosan, and hyaluronic acid from natural polysaccharides have been accepted as good candidates for the treatment of wounds for years. Its natural polysaccharide ability reduces scar formation in severe wound injuries due to its rich GAG content, which is known to support wound healing and leads to rapid granulation, vascularization, and reepithelialization, thereby providing absolutely minimal scar formation. Also, when the dressing is combined with the wound, an ion exchange reaction occurs between calcium in alginate and sodium in exudate, which produces a soluble gel that helps maintain a moist wound environment (Beer et al. 1997). Regarding immune system activation, the release of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IFN-γ, and TNF-α after wound injury also plays an important role in the wound healing process. Various important processes have been addressed with these cytokines, such as stimulation of keratinocyte and fibroblast proliferation in the wound site, synthesis and breakdown of ECM proteins, and regulation of the immune response. Expressions during the inflammatory phase of expressions have been shown to be intensely upregulated and strongly reduced after impairment of wound healing. Natural polysaccharide can stimulate human cells to produce cytokines with oligosaccharides (glu-glucan, xyloglucan, chitin, pectin, d-mannuronic, and l-guluronic). In particular, the β-glucan mechanism is mediated by many receptors, including dectin-1 receptor, Toll-like receptors complement receptor 3, scavenger receptor, and lactosylceramide. After binding to dectin-1 as the most important receptor, p-glucan stimulates the production of many cytokines or activates other immune and nonimmune reaction mechanisms (Iwamoto et al. 2005). Chitosan is β-(1–4)-D glycosamino-N-acetyl-D-glycosamine, obtained by deacetylation of chitin. It has very low toxicity after biological degradation. Chitosan is widely used in the treatment of burns and traumatic wounds (33, 102). Chitosan and chitin accelerate wound healing by showing a chemoattractant feature for neutrophils in the early period of wound healing (Ishihara et al. 2006). Ueno et al. (Ueno et al. 2001) and Ishihara et al. (Ishihara et al. 2006) found that chitosan increased the functions of polymorph nuclear neutrophils (PMNs) and macrophages in their studies. In addition to these features, it provides the proliferation and migration of vascular endothelial cells together with fibroblasts, and also prevents the secretion of interleukin-8 (IL-8) from fibroblasts (Ishihara et al. 2006). Cross-linked chitosan containing fibroblast growth factor-2 (FGF-2) has been found to accelerate wound healing in diabetic mice. In the research conducted in second-degree burn injuries with chitosan gel containing epithelial growth factor
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(EGF), it was emphasized that a faster epithelization was achieved compared to the control group. Chitosan stimulates fibroblast proliferation, the collagen needed, and natural hyaluronic acid synthesis from the wound edge (Kim et al. 2002). Chitin and chitosan have been shown to stimulate canine PMNs in vitro to release leukotriene (LTB4), in vivo either directly or through complement activation, affecting canine PMNs by the production of arachidonic acid or cytokine. Glucuronic acid N-acetylglycosamine is one of the most hygroscopic molecules in nature, with a disaccharide structure. This hygroscopic feature of hyaluronan helps weaken the bond between the ECM and the cells, thereby dividing the cells by migration. Due to its high viscous feature, it prevents the contact of viral and bacterial passages into cells by creating pericellular region rich in hyaluronan. Hyaluronan also has antioxidant action as a free radical scavenger. Hyaluronan and derivatives have been used for wound healing, and it has been reported that both hyaluronan and derivatives show a bacteriostatic effect and protect the wound area against microorganisms (Presti and Scott 1994). Martins et al. in their study, they found that a polysaccharide-rich fraction of Agaricus brasiliensis can regulate host response by activating both pro- and antiinflammatory mechanisms, thereby increasing TNF-a and IL-1β production by human monocytes through modulation of Toll-like receptor 4 (Presti and Scott 1994). In addition, even after TLR blockade, these polysaccharides still activate monocytes to produce sufficient levels of IFN-γ, IL-1, and IL-10. TNF-a and IFN-γ are considered important agents of antimycobacterial cytokine cascade, and IL-10 is considered an inhibitory cytokine that is important for adequate balance between inflammatory and immunopathological responses. On the other hand, IL-1β is known as a critical inflammatory agent that plays a role in neutrophil mobilization, endothelial cellular adhesion, and white blood cell infiltration (Dinarello 1996). Zhao et al. in their study, they determined the wound healing effect and mechanism of Astragalus membranaceus polysaccharide treatment through in vitro and in vivo studies. The results showed that this polysaccharide can promote fibroblast spread in the human skin and accelerate the progression of the cell cycle, and also significantly confirms the secretion of TGF-β1, bFGF, and EGF, which substantially confirms accelerated wound closure in the mouse wound. TGF-β1 is an important promoter in fibroblast proliferation and ECM secretion, and while preventing its deterioration, EGF and bFGF are important stimulants in the formation of reepithelialization and keratinocyte migration in wound healing. In addition, pain and mechanism of pain signals, including peripheral and central processing, are related to the modulation of keloids and TGF-mod involved in hypertrophic pathogenesis (Zhu et al. 2012).
7
Conclusion
It is very important for the materials used in wound healing and wound management. Natural polysaccharides used in the treatment of chronic or acute wounds provide many advantages in terms of wound repair and time. Natural polysaccharides are
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more preferred than difficult and laborious methods with their biocompatible, biodegradable, nontoxic, antitumor, antimicrobial, and wound healing properties. With the developing technology, the use of natural polysaccharide should be increased in wound healing and the product with this feature should be developed. Studies have increased the variety of products that protect the wound surface and speed up the healing and reveal new treatment options. However, more studies are needed to create effective evidence.
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Xanthan Gum for Regenerative Medicine
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Renata Francielle Bombaldi de Souza, Fernanda Carla Bombaldi de Souza, Cecília Buzatto Westin, Rafael Maza Barbosa, and Aˆngela Maria Moraes
Contents 1 2 3 4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xanthan Gum Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Properties of Xanthan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modification of Xanthan Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Physical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Enzymatic Treatment and Plasma Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Application of Xanthan Gum in Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Articular Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Tendons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Neuronal Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Bone and Periosteal Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Periodontal Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Delivery of Bioactive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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R. F. Bombaldi de Souza · C. B. Westin · Â. M. Moraes (*) Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil e-mail: [email protected] F. C. Bombaldi de Souza R-Crio Cryogenics S.A., Campinas, Brazil R. M. Barbosa Department of Engineering of Materials and of Bioprocesses, School of Chemical Engineering, University of Campinas, Campinas, Brazil R-Crio Cryogenics S.A., Campinas, Brazil © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_59
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Abstract
Xanthan gum, a branched polysaccharide produced by Xanthomonas bacteria, is traditionally used as an additive in several industrial applications, from food to cosmetics and petroleum, due to its rheological behavior and stability in a wide range of temperature, pH, and ionic strength. These characteristics, along with properties such as biocompatibility and biodegradability, also make this polysaccharide a very attractive material for biomedical applications, including drug delivery and regenerative medicine. The great potential of xanthan gum in tissue engineering and cell therapy fields has been evidenced in the recent years through many studies that show its ability to modulate the release profile of bioactive agents, such as drugs, growth factors, antibacterial agents and cells, and also to tune physicochemical and mechanical properties of biomaterials able to support cell growth. In this chapter, an overview of the microbial polysaccharide production is provided, from the fermentation process to polymer recovery and purification. The structure and conformation of xanthan gum molecule in different conditions is described, as well as its main functional properties, such as viscoelasticity, pH-dependent polyanionic behavior, and gelation capacity. Moreover, methods of functionalization and modification of xanthan gum structure are discussed, including physical, chemical, and chemo-enzymatic treatments to improve polymer processing and properties, such as mechanical performance and bioactivity. Furthermore, examples of the use of xanthan gum-based biomaterials for several targeted applications in soft or hard tissue repair are provided. Finally, current trends are identified and directions on future developments are presented. Keywords
Xanthan gum · Biomaterials · Polysaccharide · Fermentation · Regenerative medicine
1
Introduction
Xanthan gum (XG) is a branched extracellular polysaccharide produced by Xanthomonas bacteria. Since its approval by FDA as a food additive, in 1969, XG has been extensively used in the food industry mostly due to its rheological properties. XG is very efficient as a thickener, shows high viscosity in aqueous solutions even at low concentration, exhibits pseudoplastic behavior in aqueous solutions, is nontoxic, and highly stable in a wide range of pH, temperature, and ionic strength conditions (Kumar et al. 2018). These properties, along with other superior characteristics of this polysaccharide, such as shear resistance, ability to form both physical and chemical networks, as well as biocompatibility, biodegradability, intrinsic activity as an immunological agent, and capacity to mimic the extracellular matrix, make XG very attractive for biomedical applications (tissue engineering, cell therapy, drug delivery) apart from its
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already consolidated uses in industrial applications (e.g., food, cosmetics, petroleum recovery) (Petri 2015; Kumar et al. 2018). XG and its derivatives, in combination or not with other macromolecules and bioactive agents, have been extensively used to produce different types of biomaterials, including micro/nanoparticles, nanofibrous films, foams, and hydrogels, depending on the specific application desired (Kumar et al. 2018). In this chapter, the production process, structure, properties, and methods for the modification of XG with the purpose of further improving its properties are presented. Moreover, several applications that demonstrate the promising future of this polysaccharide in tissue engineering and regenerative medicine, including its use in drug delivery systems related to these fields of application, are discussed.
2
Xanthan Gum Production
Xanthan gum is produced in large scale by fermentation of carbohydrates using bacterial cells belonging to the genus Xanthomonas. Figure 1 depicts the usual process flowchart of XG production, from inoculum build-up to polymer recovery, purification, and packing. XG yield and characteristics, such as acetate and pyruvate content, conformational structure, and molar mass, are affected by several factors related to the fermentation process, e.g., bacterial strain, carbon (C) and nitrogen (N) sources, bioreactor type and operation conditions (e.g., batch or fed-batch), pH, temperature, inoculum concentration, airflow rate, mixing rate, and fermentation duration (Kreyenschulte et al. 2014). Regarding cell strain, despite Xanthomonas campestris being the most used for XG industrial manufacture (Petri 2015), xanthan gum can also be produced by other Gram-negative bacteria from the Xanthomonas genus, such as X. arboricola, X. axonopodis, X. campestris, X. citri, X. fragaria, X. gummisudans, X. juglandis, X. phaseoli, and X. vasculorium. The maximum efficiency of XG production is achieved when glucose, sucrose, maltose, or starch are used as carbon sources. Glucose is the most frequently used carbon source for commercial purposes and product yield using this substrate may reach 2–5%, leading to final XG concentrations of 15 g/L or even higher. The most effective N source for XG production is yeast extract, and by controlling the C:N ratio it is possible to improve XG concentration. However, culture medium formulation issues are normally combined with the choice of fermentation mode (batch, fed-batch, or continuous) to further increase xanthan gum production. Fed-batch fermentation is often more adequate to obtain high XG concentrations. The production of XG during the exponential phase of X. campestris is limited, and higher yields are attained in the stationary phase, which is normally reached after 40 h of fermentation (Kreyenschulte et al. 2014). Not only carbon and nitrogen, but also phosphorus, and sulfur contents in the culture medium affects XG production, while bacterial growth is more influenced by the amount of carbon, nitrogen, phosphorus, and magnesium. An effective medium
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Inoculum preparation
Substrate Antifoam
Air
Acid
Base
Air filter
Alcohol recovery
Distillation
Fermentation
Pasteurization
Salt
Alcohol Centrifugation Cell removal Precipitation and dewatering
Packing Spray drying Washing
Milling
Xanthan gum
Fig. 1 Typical steps observed in processes of xanthan gum production in industrial scale. (Adapted from Kreyenschulte et al. 2014)
formulation combines, for instance, sucrose (40 g/L), citric acid (2.1 g/L), NH4NO3 (1.1 g/L), KH2PO4 (2.9 g/L), MgCl2 (0.507 g/L), Na2SO4 (0.089 g/L), H3BO3 (0.006 g/L), ZnO (0.006 g/L, FeCl3.6H2O (0.020 g/L), CaCO3 (0.020 g/L), and concentrated HCl (0.13 mL/L) (García-Ochoa et al. 2000). However, the formulation can be varied with time to better fulfill process demands. Usually, keeping a low C:N ratio in the cell growth phase and a high C:N ratio in the XG production phase enhances xanthan gum production (Kreyenschulte et al. 2014). The fermentation can then be initiated, as an example, with a medium containing 40 g/L of glucose, and afterward (from 30 to 82 h), continuous supplementation of this carbon source at a flow rate of 1.3 g/(L.h) can be implemented to increase XG concentration (Kreyenschulte et al. 2014). Since the cost of the fermentation broth is a major
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concern in the industrial production of XG, the use of several low-cost raw materials has been considered to provide nutrients to the cells (Kreyenschulte et al. 2014), e.g., sugar beet pulp, olive mill wastewaters, agricultural wastes, unmodified starch, cheese whey, sugarcane molasses, and grape juice concentrate. X. campestris can be successfully cultured at different temperatures, ranging from 25 C to 30 C; however, the optimal growth temperature is usually around 28 C (García-Ochoa et al. 2000). The pH decreases from neutral conditions to values around 5 when XG production starts, due to the acid groups present in the polymer. pH control in the neutral range enhances bacterial growth; however, in this situation, XG production stops once the stationary growth phase is reached. Hence, pH control at such ranges is not recommended (García-Ochoa et al. 2000). XG recovery from the fermentation broth consists in a multistep process and represents up to 70% of all production costs. After fermentation, XG is available in the broth as an extracellular product. This can be considered as an advantage, since there is no need to lyse the cells to access the product. The broth is usually pasteurized to inactivate all microorganisms, the cells are removed by centrifugation, and the polysaccharide is obtained by a precipitation step, followed by washing and drying operations. The pasteurization treatment enhances XG recovery from the cells and decreases the viscosity of the broth, but it should be performed in conditions in which product thermal degradation is avoided. The most used pasteurization conditions are between 80 C and 130 C, for 10–20 min, at a pH of 6.3–6.9 (GarcíaOchoa et al. 2000). The precipitation of the polysaccharide can be done with several water-miscible non-solvents, such as ethanol, isopropanol, and acetone, by the addition of certain salts and by changes in pH. Considering the purification cost, the required volume of alcohol as a precipitating agent is an important parameter. To minimize its need, electrolytes such as potassium or sodium chloride can be combined to the precipitating agent, reducing the amount of alcohol required to about half when compared to the use of alcohol alone (García-Ochoa et al. 2000; Kreyenschulte et al. 2014). The exact sequence of steps for XG recovery and purification depends on the intended use of the purified polysaccharide (García-Ochoa et al. 2000). For various applications, e.g., for uses in the food industry, the steps mentioned above are sufficient to result in a product with adequate quality. Nevertheless, for applications in the biopharmaceutical area, further purification steps are required. In general, the accepted levels of DNA contents derived from bacterial cells in this type of product are extremely low, in the range of 10–100 pg of residual DNA per dose of a parenterally administered product. The processes used to achieve this high degree of purification can include adsorption by diatomite, cake filtration, enzymolysis by addition of alkaline protease, adsorption by active carbon, and a secondary filtration step. In addition, XG can be repeatedly precipitated with alcohol for further purification (Han et al. 2012; Petri 2015). After the polymer is obtained as a wet precipitate, it is dried, milled to a predetermined mesh size, and packed (García-Ochoa et al. 2000). Prior to the use in regenerative medicine purposes, XG sterilization is also required. This can be performed by steam sterilization or by exposure to ethylene or propylene oxide and
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ionizing radiation. Steam sterilization can affect significantly its rheological behavior due to effects on molecular conformation and molar weight distribution, mostly if performed with XG solutions. Furthermore, it is possible to perform filtration sterilization of its dilute solutions. In this case, after the sterilization process, the polymer solution is concentrated by ultrafiltration to obtain the final concentration desired for the product.
3
Properties of Xanthan Gum
As already mentioned, xanthan gum is a branched polysaccharide in which the backbone consists of repeated pentasaccharide blocks. The backbone of XG contains ß-D-glucose units linked at the 1 and 4 positions and the side chain consists of a (1,3) α-D-mannose, a (1,2) ß-D-glucuronic acid, and a terminal (1,4) ß-D-mannose. The side residues are mostly acetylated at position O-6 of the internal α-D-mannose and approximately half of the terminal ß-D-mannose residues are pyruvated, as shown in Fig. 2 (Petri 2015). The trisaccharide branches can be closely associated to the backbone, originating a stiff chain, which can exist as single, double, or triple helices capable of interacting with other polymer molecules and forming complexes (García-Ochoa et al. 2000). The size and content of acetate and pyruvate groups of
Fig. 2 Chemical structure of xanthan gum. Although the presence of pyruvate coupled to the terminal mannose of the side chain is indicated, alternatively this group can be replaced by acetate, since these two molecules compete for the same carbon atom during biosynthesis (Schmid and Sieber 2015)
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XG chains may vary according to the cell strain and process conditions used during the fermentation, as mentioned in the previous section. The structural conformation of XG in aqueous solution can be tuned mainly by changes in temperature, ionic strength, and polymer concentration. Depending on the specific condition, XG chains can be arranged in an ordered or disordered state, represented by helicoidal or coiled conformations, respectively (Milas and Rinaudo 1979; García-Ochoa et al. 2000). The ordered helix conformation of XG chain remains stable due to hydrogen bonds within the molecule. The polysaccharide undergoes conformational transition from ordered to disordered state when the ionic strength is decreased and the temperature is increased. A change from helix to coil occurs as a consequence of chain destabilization due to electrostatic repulsion between charged carboxylic groups (Petri 2015; Kumar et al. 2018). Conformational alterations in the structure of XG caused by changes in environmental conditions may affect the rheological behavior of the polysaccharide (García-Ochoa et al. 2000). The viscosity of a XG solution depends on both the temperature at which the polymer is dissolved and the temperature at which the property is measured (GarcíaOchoa et al. 2000). Usually, the viscosity decreases as the temperature increases, and this behavior is reversible at temperatures between 10 C and 80 C. However, when dissolving XG, the viscosity may increase as the temperature increases from 40 C to 60 C (Milas and Rinaudo 1979; García-Ochoa et al. 2000). This behavior can be associated with conformational changes in the XG molecule that shifts from an ordered state at lower-dissolution temperatures to a disordered conformation when dissolution temperature is higher (García-Ochoa et al. 2000). This transition corresponds to the spreading of the side chains, i.e., they project away from the backbone of the molecule and, as a consequence, the rigidity of the ß-D-glucose main chain is decreased. The spreading of the side chains leads to an increase in the hydrodynamic volume of the molecule and also to an increase in the swelling and hydration of the polymer chain (Milas and Rinaudo 1979; García-Ochoa et al. 2000). The content of acetate and pyruvate in the side chains can also influence the rheological behavior of a XG solution. Usually, high pyruvate content promotes an increased viscosity and gel behavior due to macromolecular interaction within charged carboxylic groups. In turn, high acetyl content decreases solution viscosity and inhibit gel behavior (Petri 2015; Kumar et al. 2018). XG solutions act as non-Newtonian fluids and present a pseudoplastic or shear-thinning behavior, which means that the conformation of polysaccharide chains can be altered with time and shear rate. The apparent viscosity of XG decreases as the shear rate increases (Kumar et al. 2018). This rheological behavior is an important characteristic of XG solutions that make them very attractive for several industrial applications. Despite temperature changes lead to denaturation of the native XG double helix structure, depending on xanthan gum concentration, renaturation may occur by cooling or adding salt to the solution. However, the viscosity of the renatured XG solution is different from the native one, as the global conformation of the chains are no longer the same (Matsuda et al. 2009). As indicated in Fig. 3, in a diluted solution heated up to 80 C, the XG double helix structure can dissociate into two individual
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Fig. 3 Denaturation and renaturation models for dilute and concentrated XG solutions. (Based on Matsuda et al. 2015)
chains and, after a renaturation process, individual helicoidal chains are observed. On the other hand, when concentrated XG solutions are heated, only the final part of the chain is denatured, while the central region remains as a double helix due to interactions of the charged residues of the molecule. In this case, the renaturation process creates complex structures with the denatured portions of other molecules (Matsuda et al. 2009, 2015). When XG concentration is sufficiently high, the renatured polymer solution forms a gel, which is another important property of xanthan in industrial applications (Matsuda et al. 2009). An additional factor that can influence the renaturation process is the molar mass of XG, which can vary between 100 and 20,000 kDa (Matsuda et al. 2015; Petri 2015; Kumar et al. 2018). The stabilization of the ordered structure of XG can originate gels with good mechanical responses, depending on the polymer concentration. The ordered double helix shape (Fig. 3) favors ionic interaction with bivalent cations, resulting in gels with different mechanical properties, such as thermally induced shape-memory and elastic behavior under pressure and tensile modes (Milas and Rinaudo 1979; Petri 2015). The ability of XG to build up physical networks is directly linked to the polymer net charge and mainly to the proportion of acetyl and pyruvil residues. When the pH is above its pKa (4.5), XG assumes an anionic characteristic due to the deprotonation of carboxyl residues. In this case, XG is able to bind to cations, such as Ca2+ and Mg2+, as well as to other positively charged macromolecules, such as proteins below their pKa value (Petri 2015; Kumar et al. 2018). Biodegradability is another important property of XG for application in tissue engineering and regenerative medicine. The main chain of the polysaccharide, comprised of ß-D-glucose, is similar to cellulose and can undergo enzymatic hydrolysis by cellulase. However, it occurs only when the XG molecule presents a disordered conformation, since the side saccharides pose a hysterical hindrance that can block the cleavage of the structure when in ordered form (Milas and Rinaudo 1979; Petri 2015; Kumar et al. 2018). In acidic and neutral conditions, XG hydrogels are stable, but in alkaline conditions, its ester bonds can be hydrolyzed (Petri 2015). In addition, microbial enzymes can also degrade xanthan gum. For these reasons, applications of this polymer in intestine tissues could be of particular interest.
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Several and in vitro studies carried out with XG-based biomaterials with tissue regeneration purposes confirm its non-cytotoxicity (Fabela-Sánchez et al. 2009; Han et al. 2012; Bellini et al. 2015; Liu and Yao 2015; Elizalde-Peña et al. 2017). Besides XG biocompatibility and biodegradability, characteristics such as capacity to biologically mimic the composition and architecture of the extracellular matrix, stability at a wide range of temperatures, resistance to acidic environments, and ability to build up networks via intra- and intermolecular bonds make XG a very versatile and attractive polysaccharide to be used in the biomedical and tissue engineering fields (Kumar et al. 2018).
4
Modification of Xanthan Gum
The use of XG in biomedical or other technological applications often depends on polymer structural modifications and fitting of its physicochemical properties. Indeed, the use of unmodified XG in biomedical formulations presents some drawbacks, such as possibility of microbial contamination, poor bioactivity, and uncontrolled rate of hydration and degradation of XG-based biomaterials. Using the chemically modified form of the polysaccharide can minimize the aforementioned drawbacks and favor processing and mechanical performance, as well as improve the bioactivity of XG-based biomaterials for several targeted applications in tissue engineering and drug delivery (Petri 2015; Kumar et al. 2018). The modification of XG, similarly to what is performed with other polysaccharides, can be carried out using different strategies: (i) by applying metabolic engineering and knowledge about polymer biosynthesis pathways toward improved production of a tailor-made polysaccharide during the fermentation process; or (ii) by in vitro or post-process modification, in which the isolated biopolymer can be modified by subjecting it to a specific treatment. In this section, different approaches for post-process modification of XG are discussed. Due to its branched chain and to the presence of a great number of functional groups, such as hydroxyl and carboxyl, a variety of modification and functionalization methods may be employed to alter XG structure and its ability to build up physical and chemical networks (Petri 2015; Kumar et al. 2018), as mentioned in the previous section. To improve the properties of XG aiming at better biomaterials, the most commonly used methods of functionalization and modification include physical, chemical, and chemo-enzymatic treatments; plasma-assisted chemical grafting; and their combinations (Petri 2015; Kumar et al. 2018).
4.1
Physical Methods
The rheological property of XG solutions correlates with their gel-like behavior and is crucial in the design of biomaterials such as injectable hydrogels used in tissue engineering. The rheological behavior of XG may be altered by physical means,
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such as mechanical treatment, intermolecular cross-linking and network formation, or by blending with other natural polymers (Petri 2015; Kumar et al. 2018). Different methods based on high-pressure processes, such as microfluidization and high-pressure homogenization, represent alternative approaches to control XG rheological behavior without affecting the polymer chemical composition. Mechanical degradation of the polysaccharide occurs when a physical stress is applied, being associated with reduction of its molar weight and increase in polydispersity. It leads to the disorganization of XG structured network and, as consequence, to rheological changes such as reduced viscosity and pseudoplastic behavior. In the previous section, the ability of XG to form physical networks in the presence of specific bivalent cations was discussed. However, several trivalent metallic ions, such as Cr3+, Al3+, Fe3+, have also been used to modify the gelation behavior of XG, improving its rheological properties (Kumar et al. 2018). Electrostatic interactions lead not only to the formation of networks between xanthan carboxylate groups and these ions, but also between XG and positively charged macromolecules, such as proteins or other biopolymers (Petri 2015). Polyelectrolyte complexes resulting from interactions between XG and oppositely charged polymers as well as blends resulting from the physical mixture with other materials may lead to products with enhanced physicochemical properties, such as improved swelling capacity, and biological performance (Bellini et al. 2015; Liu and Yao 2015). Besides, the development of composites by the combination of XG with reinforcement agents or bioactive components, such as hydroxyapatite or magnetite nanoparticles, can be a useful strategy to modulate the characteristics of the biomaterial and tune its mechanical, physicochemical, and biological properties (Bueno et al. 2014; Glaser et al. 2015).
4.2
Chemical Methods
Chemical modification is the most commonly used method to change the structure of polysaccharides, either by removal of an existing functionality or by addition of substituent groups, originating new properties (Kumar et al. 2018). The addition of new functionalities can be carried out by modifying either carboxyl or hydroxyl groups of the XG structure (Kumar et al. 2018). Grafting of molecules on to the polymer chain by covalent bonds to modify the molecular structure of XG is performed through different approaches, such as carboxymethylation, thiolation, esterification, or introduction of aldehyde groups. Table 1 shows how the grafting of different types of molecules on to XG structure affects its properties. The chemical modification of XG by graft copolymerization has also been extensively reported. A graft copolymer is the combination of a macromolecular main chain with a side chain with a different structure. Graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) on to XG was performed to fabricate a XG-g-poly(HEMA-co-AA)-based superporous hydrogel system (Gils et al. 2009). Moreover, graft copolymerization of acrylamide on to XG and carboxymethyl-XG using different methods was also investigated. Using this approach, it is possible to
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Table 1 Effects on XG properties of chemical modification by grafting different types of molecules on to the polysaccharide molecule Modification Introduction of aldehyde groups by periodate-mediated oxidation
Carboxymethylation
Thiol derivatization via esterification with mercaptopropionic acid and thioglycolic acid Thiolation via amide bond formation (covalent attachment of L-cysteine)
Succinic anhydride (SA) functionalization
Esterification with poly (maleic anhydride/1-octadecene)
Effect Schiff base bonding between oxidized XG and amine groups of other molecules (e.g., chitosan) originate self-healing hydrogels or materials that respond to physical, chemical, and biological stimuli Decrease in viscosity; faster release of drug (sodium diclofenac) Better perspective for long-term cell therapies Improved mucoadhesive property; sustained release of drug (metronidazole) over a prolonged period Higher water uptake capacity and stability against erosion; enhanced mucoadhesive strength; faster release of drug (tannic acid) Anionization due to increased presence of carboxylic groups as a strategy to incorporate small cationic molecules (e.g., gentamicin) in XG-based hydrogels; improved elasticity Better performance regarding salt tolerance and temperature resistance; higher viscosity, improved resistance to shear force, and enhanced viscoelastic behavior
References Salazar et al. (2018)
Ahuja et al. (2012) Mendes et al. (2012) Bhatia et al. (2015)
Laffleur and Michalek (2017) Wang et al. (2016a)
Wang et al. (2016b)
obtain materials with increased swelling capacity, faster release of loaded drugs, and improved thermal stability (Badwaik et al. 2013). Further studies also report the graft copolymerization of XG with ethyl acrylate, 2-acrylamidoglycolic acid, and 2-acrylamido-2-methyl-1-propane sulfonic acid aiming to obtain more thermostable polysaccharide-based hydrogels, with improved swelling capacity, metal ion sorption, and resistance to biodegradation (Badwaik et al. 2013). Besides polymer functionalization, cross-linking of functional groups via covalent interactions using chemical agents can be performed to control the kinetics of hydrogel formation, rheological performance, and mechanical stability of XG networks (Kumar et al. 2018). The use of sodium trimetaphosphate (STMP) for the fabrication of XG hydrogels by chemical cross-linking was already demonstrated (Tao et al. 2016). Several other molecules can be used as cross-linkers, such as citric acid, epichlorohydrin, glutaraldehyde, N,N-methylenebisacrylamide, glicerol, polypropylene diglycidyl ether, adipic acid dihydrazide, and adipoyl chloride (Petri 2015). As mentioned before, the removal of an existing functional group from the polymer chain represents another way of modifying the polysaccharide structure.
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The removal of acetyl groups from XG side chains can be performed to improve the viscosity and stability of xanthan solutions. Deacetylation may be performed under alkaline condition or by enzymatic treatment. In the last case, the use of enzymes enables to eliminate targeted acetyl groups, contrary to random removal of the groups and backbone degradation that occurs when the chemical treatment is used (Kool et al. 2014). Other enzymatic treatments are discussed below.
4.3
Enzymatic Treatment and Plasma Irradiation
Besides allowing the targeted elimination of functional groups, the enzymatic treatment may be used for other purposes. Xanthan lyases, for instance, are well characterized xanthan modifying enzymes able to remove the terminal mannosyl residue. In turn, XG molecular weight reduction can be performed via cellulase-mediated hydrolysis (Kool et al. 2014). These treatments can alter the rheological response of XG, as the amount and proportion of acetate and pyruvate groups in the polysaccharide structure, as well as the size of its chain, pose a large influence on the viscosity of XG solutions. Enzymatic treatments affect also the stability of XG solutions toward the addition of salts and changes in temperature and pH conditions (Kool et al. 2014). Finally, the use of plasma irradiation is also described as an efficient route to modify XG structure. Cold-plasma is a nonthermal process used for microbial inactivation and modification of polysaccharides. It can be used to alter the rheological properties of XG solutions by grafting primary amines on to XG molecules, which are, afterward, cross-linked with glutaraldehyde (Kumar et al. 2018).
5
Application of Xanthan Gum in Regenerative Medicine
XG and its derivatives, in combination or not with other macromolecules, bioactive agents, and different types of mammalian cells, have been extensively used to produce materials for biomedical applications with outstanding physical and chemical properties. As already discussed, materials with very different morphologies and purposes can be obtained by the combination of xanthan gum with other compounds, as shown in Figs. 4 and 5. The biocompatibility and biodegradability of XG enable the exploitation of these materials in regenerative medicine and tissue engineering applications as scaffolds for cell adhesion, proliferation, and differentiation. In this section, several examples of the use of XG-based biomaterials for application in soft or hard tissue repair are discussed.
5.1
Skin
Tissue engineered skin substitutes have been developed as alternatives to traditional wound healing strategies, especially in cases of deep injuries and burns. These
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Fig. 4 Different types of XG-based biomaterials. (a) photograph of a XG-chitosan sponge (polymer ratio 1:1); (b) surface morphology of microcapsules constituted of phospholipid (1,2-dioleoyl-sn-glycerophosphoetilamine, DOPE) conjugated with XG, and (c) XG-DOPE microcapsules coated with poly-L-lysine (embedded pictures show the microcapsules); (d) micrograph of the surface of a XG membrane containing 5% (w/w) of hydroxyapatite; (e) cross-sectional morphology of a hydrogel constituted XG and gellan, and (f) the same hydrogel containing chitosan nanoparticles loaded with dexamethasone disodium phosphate. ((a) Reproduced from Ibrahim and Fahmy 2016 by permission of Taylor & Francis Ltd.; (b and c) Reproduced from Mendes et al. 2013 by permission of Elsevier; (d) Kindly provided by M.Sc. Rafael Maza Barbosa; (e and f) Reproduced from Sehgal et al. 2017 by permission of John Wiley & Sons)
Fig. 5 XG-based thermoresponsive hydrogels. (a) 8% (w/v) methylcellulose (MC) hydrogel without XG and (b) 8% (w/v) MC hydrogel with 1% (w/v) XG; (c) photos of the injections of hydrogels containing different proportions of XG and MC into 37 C phosphate-buffered solution: (i) 23 C (MC 10 wt.% solution), (ii) 23 C (XG 2wt.% weak gel) and (iii) 23 C (XG 2wt.%/MC 10wt.% blend solution), as well as (iv) injection of 37 C XG 2wt.%/MC 10wt.% hydrogel using 23G needle. ((a and b) Kindly provided by Dr. Cecília Buzatto Westin; (c) Reproduced from Liu and Yao 2015 by permission of Elsevier)
materials can be either acellular or contain cells such as fibroblasts or stem cells to improve their bioactivity. They are able to provide components from both the dermis and the epidermis that are required for effective wound closure and tissue functions recovery. Most importantly, these materials can act as a protection barrier and are also capable of reducing pain and promoting healing by tissue regeneration (Juris et al. 2011; Bellini et al. 2015).
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XG has been employed in tissue engineered skin substitute’s formulations in the form of solid membranes and, most commonly, topical hydrogels. The topical application of XG-based hydrogels is favored by its high ability to adhere to the lesion bed (Petri 2015). Dense and porous chitosan-xanthan gum membranes can be successfully used to culture multipotent mesenchymal stromal cells for application as bioactive dressings for dermo-epidermal wounds. The membranes are not mutagenic and allow efficient adhesion and proliferation of the cells in vitro. The nonporous membranes, when in combination with the mesenchymal stromal cells, lead to a significant improvement in the healing of skin wounds on Wistar rats (Bellini et al. 2015). Antibacterial polyelectrolyte hydrogels produced by combining XG and chitosan and incorporating silver nanoparticles have demonstrated to be cytocompatible with 3T3 fibroblast cells and hence are also promising for the application as skin substitutes (Rao et al. 2016). XG has been combined with other polysaccharides to prepare hydrogels with a range of mechanical and degradation properties, as well as good biocompatibility with human fibroblast (MeWo) cells. The combination of XG with konjac gum, iotacarrageenan, and kappa-carrageenan originates strong hydrogels that could be explored as epidermis skin scaffolds. While XG and konjac gum may produce amorphous matrices, iota and kappa carrageenans can function as fiber reinforcements for the composite hydrogels (Juris et al. 2011). The hydrogels can be prepared with different elasticity degrees, depending on the softness required (Almeida et al. 2014).
5.2
Articular Cartilage
Joint surfaces are covered with a specialized type of cartilage, called hyaline or articular cartilage (Han et al. 2017). Because the inner cartilage tissues within joints are characterized by the absence of lymphatic vascular system and nerve endings, the treatment of diseases that affect this tissue is difficult. Thus, the use of tissue engineering approaches can represent an attractive solution. XG-based materials have potential to aid in this purpose in different forms, e.g., as solutions, membranes, and hydrogels. XG solutions, for instance, are reported not to cause adverse effects to chondrocytes (the cartilage cell type) in vitro in a wide range of concentrations (from 10 to 2000 μg/mL) (Han et al. 2012). When chondrocytes are cultivated in the presence of XG and interleukin-1β, an inductor of degradation, XG is capable of protecting the cells in a dose-dependent manner, stimulating their growth. A solution of XG ( 10 g/L in phosphate-buffered saline, molar mass from 5100 to 5400 kDa) was reported to be useful for the treatment of joints of Wistar rats with osteoarthritis, a disease that affects the cartilage tissue (Han et al. 2017). The solution was injected into the animal’s joints at a ratio of 0.2 mL/kg and was helpful to reduce the pain caused by the disease, also decreasing cartilage degradation. Porous membranes with high culture medium absorption capacity formulated by combining XG and chitosan (Westin et al. 2017) are also promising for the treatment
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of this condition. This formulation is useful to culture mesenchymal stem cells and if cultivation is performed in the presence of kartogenin, the cells can be successfully differentiated into chondrocytes, what supports the feasibility of this approach for application in cartilage tissue engineering. Different proportions of XG, methylcellulose, and carboxymethyl chitosan can be combined to produce thermoresponsive hydrogels with potential to be used in injectable formulations for noninvasive treatment of cartilage lesions (Westin et al. 2020). These formulations are stable, have high medium absorption capacity, interconnected porous structure, and adequate compressive mechanical characteristics to be used in joint regions. The drugs dexamethasone, diclofenac sodium, and gallic acid, used to treat osteoarthritic joints, can also be incorporated in the XG-based hydrogels, further improving the bioactivity of the formulation.
5.3
Tendons
Tendon tissue engineering has stood out lately in regenerative medicine as an alternative to autograft and allograft treatments, as it overcomes common problems of the grafting techniques, such as pain and donor site morbidity. Besides that, after common tendon-repair surgery, adhesion between the healing tendon and the synovial membrane can occur, resulting in poor functional repair of this tissue. This could be prevented by the use of a simple mechanical barrier implant such as the membranes developed by Kuo et al. (2014), who studied four distinct formulations constituted by different amounts of XG, gellan gum (GG), and hyaluronic acid (HA). The membranes can be wrapped around surgically repaired rat tendons. Evaluation after 3 weeks showed that all formulations of XG/GG/HA hydrogel membranes reduced tendon adhesion with efficacy equivalent to that of Seprafilm ®, a commercially available HA and carboxymethyl cellulose membrane. The developed membranes are capable to quickly swell and can cover the tendon more easily and in closer proximity than Seprafilm ®. These membranes are slowly degraded, which allows them to work as barriers for longer periods.
5.4
Neuronal Tissue
Neurodegenerative diseases and spinal cord injury are the most recurrent conditions that affect the nervous system. Nerve tissue regeneration is a complex biological process (Elizalde-Peña et al. 2017). Depending on the type and site of injury, tissue regeneration does not occur and tissue engineering is an approach that may be adopted. In this sense, biomaterials with composition also based on XG could be promising. Fabela-Sánchez et al. (2009), for instance, combined XG with methacrylated chitosan to produce hydrogels applicable as scaffolds for neural cells from BALB/c mice. These hydrogels were able to provide a favorable environment for cell proliferation. A similar hydrogel, also consisting of (chitosan-g-glycidyl methacrylate)–xanthan gum,
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was introduced into a spinal cord lesion of Wistar rats (Elizalde-Peña et al. 2017). A recovery five times faster was observed when using this biomaterial in comparison to the control. The XG-based hydrogel proved to be biodegradable by enzymatic hydrolysis, which allows the material to act as a scaffold during spinal cord recovery. In the same line, Glaser et al. (2015) demonstrated that hybrid scaffolds made of XG and magnetite nanoparticles at different concentrations and with distinct magnetization profiles are able to promote adhesion and proliferation of neural cells. Cell adhesion is more pronounced on XG-containing magnetic particles than on neat XG scaffolds. However, proliferation is independent from the scaffold type. In turn, the differentiation of embryonic stem cells into neural cells is more expressive when neat XG is used, which was associated to the high density of negative charge present in this case.
5.5
Bone and Periosteal Tissue
Critical-sized bone defects caused by fractures and tumor resections, for example, present very restricted spontaneous regeneration capacity. Such lesions cannot be treated by traditional methods that involve surgical reconstruction with autografts, allografts, or metal implants, since these treatments offer complications as insufficient osseointegration and risk of infection. Bone tissue engineering with polymerceramic composite scaffolds is a promising alternative approach, allowing the modulation of the mechanical and degradation properties of materials to meet the requirements of the defective site (Dyondi et al. 2013; Izawa et al. 2014). XG has been explored for the production of biomaterials in the form of sponges or hydrogels to be used for bone regeneration. XG hydrogels can serve as an organic template for biomimetic mineralization since carboxyl groups in the polymer chain may interact with calcium ions in ceramic materials such as hydroxyapatite (Hap) and other phases of calcium phosphate, the main inorganic component of bone tissue (Dyondi et al. 2013; Izawa et al. 2014). The mineralization of Hap upon a XG hydrogel has already been demonstrated (Izawa et al. 2014), by using an alternate soaking process. The mineralization induces a microstructure change in the XG-matrix from a layered to a porous structure with unique mechanical properties. Xanthan gum chains can also coat the surface of strontium-substituted Hap nanoparticles, significantly increasing colloidal stability (Bueno et al. 2014). These biomaterials are suitable for osteoblasts growth and can induce high alkaline phosphatase activity. A successful approach to enhance the mechanical stability and cytocompatibility of XG scaffolds is the incorporation of silica glass and cellulose nanocrystals to the formulation (Kumar et al. 2017). Through this approach it is possible to produce hybrid scaffolds with good compatibility to osteoblastic MC3T3-E1 cells and mechanical properties appropriate for low load-bearing bone tissue engineering applications. XG has also been explored for the engineering of periosteum, a dense and highly vascularized membrane that covers most of bone surfaces. The periosteum is
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essential for bone healing, as it provides key components for tissue repair, such as osteogenic progenitor cells. Bioactive, porous, and flexible scaffolds can be easily produced by combining XG to other polymeric compounds for periosteum repair. The introduction in the formulation of phosphorylated chitosan, a chemically modified form of chitosan that shows osteoconductive properties, can further improve the bioactivity of these XG-based scaffolds (Bombaldi de Souza et al. 2020). Osteogenic differentiation of adipose-derived stem cells and mineralization on the surface of the materials confirms their potential for osseointegration and bone regeneration. Figure 6 shows the visual aspect and morphology of these scaffolds, constituted by the combination of XG and chitosan or phosphorylated chitosan as dense or porous membranes, and Fig. 7 shows the potential of such material to promote cell attachment and mineralization.
5.6
Periodontal Tissue
Different biomaterials containing XG can be used to aid in the treatment of oral diseases such as periodontitis and loss of alveolar bone. The early detection of the conditions that negatively affect the oral cavity is crucial to choose the adequate After immersion in deionized water
Cross-sectional morphology
PCh-XG (Porous)
PCh-XG (dense)
Ch-XG (porous)
Ch-XG (dense)
Dry scaffolds
Fig. 6 Visual aspect (materials before and after immersion in water) and cross-sectional morphology of dense or porous scaffolds constituted by XG combined with chitosan (Ch) or phosphorylated chitosan (PCh). (Image kindly provided by Dr. Renata F. Bombaldi de Souza)
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Fig. 7 XG-chitosan (Ch) dense scaffolds (membranes). (a) morphology of the surface before cell seeding; (b) surface 96 h after inoculation of mesenchymal stromal cells (from bone marrow of rats); (c and d) surfaces after 3 weeks of adipose-derived stem cells culture in the presence of differentiation medium containing dexamethasone and ascorbic acid. Mineralization is observed due to the presence of calcium phosphate and oxalate crystals. ((a, c, and d) Kindly provided by Dr. Renata F. Bombaldi de Souza; (b) Reproduced from Bellini et al. 2015 with permission from SAGE journals)
technique to address the problem. Depending on the severity of the disease or the tissue affected, different types of biomaterials, in the form of hydrogels or membranes, can be selected for the treatment. The periodontium is the structure responsible for supporting dental elements, being formed by gingiva, ligaments, cementum, and alveolar bone. As the oral cavity supports a heterogeneous microbiota that can exceed 700 different species of microorganisms (Hashim 2018), the periodontal pocket can be particularly vulnerable to microbial attack. In this case, inflammation and degeneration of the tissue adjacent to the dental element are often observed, and, in severe situations, loss of the dental element can occur. XG can be formulated to provide gel-like biomaterials relevant as drug delivery systems for the treatment of diseases that affect the oral cavity. For instance, hydrogel formulations capable to adhere to the oral mucosa can be of particular interest in this sense, since they could increase the retention time of bioactive agents incorporated in their formulations in the affected periodontal pocket. The period in which the biomaterial remains in the oral cavity has a great importance for the effectiveness of the treatment, since an adequate time-response is expected for local drug delivery. With that purpose, Needleman et al. (1997) proposed three
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formulations consisting of chitosan, poly(ethylene oxide), and XG. The XG formulation presented the most prolonged adhesion time in the oral mucosa, approximately 1.7 and 3.6 times higher than the retention time observed for poly(ethylene oxide) and chitosan formulations, respectively. Polyelectrolyte complexes consisting of a XG hydrogel combined with chitosan microspheres are also of relevance concerning this goal, being attractive both for the treatment of acute and chronic periodontitis (Kim et al. 2017). The concentration of XG in the hydrogels can be tuned to control the release of antiseptic molecules. The biomaterial has also shown to be nontoxic to human fibroblasts and able to inhibit the growth of Pseudomonas gingivalis, one of the main causes of periodontitis. The use of XG gels seem indeed to be attractive also as an adjunct therapy with scaling and root planning procedures for the treatment of chronic periodontitis. The clinical effectiveness of a commercial product (Chlosite ®) consisting of a XG hydrogel containing chlorhexidine for the treatment of chronic periodontitis was analyzed in 30 patients, with positive results (Jain et al. 2013). Significant decreases in gingival and plaque index were reported (these dental indices indicate, respectively, the severity of gingiva inflammation and the formation of films or deposits on the edges of the tissues adjacent to the tooth). Moreover, a decrease in bleeding on probing and also on clinical attachment, both considered as criteria to diagnose gingival inflammation loss, were also observed. In severe cases of periodontitis, in which partial or complete absorption of the alveolar bone are detected, guided regeneration techniques that include the use of membranes to aid tissue regeneration can be applied. There are several types of membranes available for the use in guided bone regeneration, most of them constituted by collagen. XG has not yet been exploited for this application; however, materials such as the membranes produced by Bombaldi de Souza et al. (2020), described in the previous section, with intended use in periosteum reconstruction, could be attractive candidates for this purpose.
5.7
Delivery of Bioactive Agents
Delivery of biochemical cues to the body is one of the fundamental pillars of regenerative medicine. Bioactive agents such as growth factors and small molecules can modulate the microenvironment of regenerating tissues, stimulating exogenously added cells or recruiting endogenous cells to act on tissue repair. Cell growth and differentiation, as well as angiogenesis or cicatrization, can be triggered, controlled, or improved. Delivery of drugs with antioxidant, anti-inflammatory, antibacterial, or anticancer properties are also of interest, as they can improve the prognosis of many degenerative diseases. Scaffolds with a variety of morphologies and properties have been used to achieve sustained release of biomolecules and bioactive components. The incorporated bioactive agents can be released at particular phases of tissue regeneration to match specific processes. This controlled release is often achieved by fabricating either multicomponent or core-shell scaffolds. Besides, smart drug delivery systems that respond to external stimuli such as temperature, pH, and physical fields have
Form Injectable hydrogel
Sponge
Coating for ceramic scaffolds
Injectable thermoresponsive hydrogel
Hydrogel
Other components Chitosan nanoparticles
–
Drug loaded chitosan nanoparticles
–
–
Formulation Polymer matrix XG/Gellan gum
XG/Chitosan
XG/Gellan gum (coating)
XG/ Methylcellulose
XG modified with succinic anhydride
Gentamicin (antibacterial agent)
Doxorubicin (anticancer drug)
Bioactive agent Basic fibroblast growth factor (bFGF), and bone morphogenetic protein 7 (BMP7) Rosuvastatin (promotes BMP-2 expression and osteoblast differentiation in vitro) Dexamethasone (osteoinductive drug)
Table 2 Selected examples of XG-based materials for the delivery of bioactive agents
Drug release and tissue engineering
Drug release and tissue engineering
Bone tissue engineering
Bone tissue engineering
Targeted application Bone tissue engineering
Sustained delivered of the drug for 5 days improves the potential for osteogenic differentiation The hydrogel is biocompatible and biodegradable in rat body and releases the drug in a sustained way Antibacterial hydrogels can be used to modify implants and other biomedical devices
Biodegradable sponges bring drug into immediate contact with the fractured bone
Remarks Injectable system with dual growth factors allows bone cells to differentiate
Wang et al. (2016a)
Liu and Yao (2015)
Sehgal et al. (2017)
Ibrahim and Fahmy (2016)
References Dyondi et al. (2013)
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–
Polymethyl methacrylate (PMMA) particles
Dioctadecyldimethylammonium bromide(DODAB) cationic liposomes
AldehydemodifiedXG/ Carboxymethylmodified chitosan
XG/Gellan gum
XG/ Galactomannan (coating)
Coating for liposomes
pH-responsive hydrogel conduits
Injectable hydrogel
Epidermal growth factor (EGF)
Bovine serum albumin (BSA) and diclofenac sodium (model compounds)
Vascular endothelial growth factor, VEGF (angiogenic factor)
Wound treatment/ Skin reconstruction
Peripheral nerve repair
Abdominal wall reconstruction
Controlled drug release in tissues with much excretion or exudation such as digestive tracts and open wounds XG/gellan ratio variability and poreinducing effects of intercalated PMMA yielded a means for fine tuning matrix rigidity and flexibility Association of anionic XG and neutral galactomannan for liposomes coating resulted in nanoparticles capable of improving the release profile of EGF, a low-stability drug Kaminski et al. (2016)
Ramburrun et al. (2017)
Huang et al. (2018a)
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also been developed with this purpose. These stimulus-responsive materials allow to set off or to modulate the delivery of the bioactive molecules using external signals. XG has been employed in the formulation of numerous biomaterials for the delivery of bioactive agents, such as drugs, growth factors, antibacterials, and also cells. These delivery systems are often produced in the form of injectable or topical hydrogels, sponges, pastes, particles, and others. XG is typically combined with other materials, including polymers, ceramics, and metallic particles, to tune the desired properties according to the intended application (Petri 2015). Table 2 comprises examples of XG-based materials developed for the delivery of bioactive molecules in different tissues. The examples show that XG can be successfully used in the formulation of platforms for drug delivery owing to its characteristic functional properties such as viscoelasticity, pH-dependent polyanionic behavior, as well as pH, temperature, and ionic strength-dependent gelation capacity, previously discussed in this chapter. Figure 8 illustrates a drug delivery system developed by Huang et al. (2018a). An injectable hydrogel was formulated by combining solutions of aldehyde-modified xanthan (Xan-CHO) and carboxymethyl-modified chitosan (NOCC), which are
B
A
Origin
Recombined (0h)
Recombined (6h) C
Recombined (24h)
Self-assembly (24h) D
cumulative releasing ratio(%)
PBS
100 90 80 70 60 50 40 30 20 10 0
Fibrin gel XAN-CHO/NOCC hydrogel
0
10 20 30 40 50 60 70 80 Time/hour
Fig. 8 Polysaccharide-based injectable hydrogels via self-cross-linking of aldehyde-modified xanthan (Xan-CHO) and carboxymethyl-modified chitosan (NOCC). (a) recombination process of different parts of the hydrogel; (b) lifting up the hydrogel after recombination; (c) local injection of the hydrogel and fibrin gel in phosphate-buffered saline (PBS) (red arrow: Xan-CHO/NOCC hydrogel, green arrow: fibrin gel); (d) cumulative releasing ratio of protein after local injection in PBS. (Reproduced from Huang et al. 2018a by permission of Elsevier)
Magnetically stimulated system for tissue engineering
Soft tissue engineering
Kolliphor ® P188 (surfactant)/ Silpuran ® 2130A/B (elastomer)
XG/Chitosan
Bioink for the fabrication of 3D scaffolds for tissue engineering
Iron oxide magnetic nanoparticles
Other components –
Targeted application
XG/Chitosan
XG/PEGDA
Formulation Polymer matrix
Table 3 Other applications of xanthan gum in regenerative medicine
Improves water retention/modulates degradation and mechanical properties
Facilitates gelation and improves water retention, mimicking the natural extracellular matrix
Prevents cell sedimentation within the bioink
Role of XG in the formulation
Human dermal fibroblasts/ Adiposederived stem cells
3T3 fibroblasts
3T3 fibroblasts
Cells used
XG + PEGDA behaves as a weak gel due to polymer entanglements. It results in more homogenous cell distribution compared to PEGDA + Alginate The polyelectrolyte complex promotes stabilization of the magnetic nanoparticles. The particles favors cell adhesion and proliferation in a magnetic field Dense and porous XG-based scaffolds present appropriate porosity, liquid uptake capacity, stability, mechanical properties, thrombogenicity and cytotoxicity for applications in soft tissue engineering
Remarks
Xanthan Gum for Regenerative Medicine (continued)
Bombaldi de Souza et al. (2019)
Rao et al. (2018)
Dubbin et al. (2017)
References
47 1155
XG/DOPE(1,2-dioleoylsnglycerophosphoetilamine)
Formulation Glycidyl methacrylatemodified XG (photopolymerizable)
Table 3 (continued)
–
–
Cell-based transplantation therapies
Targeted application Gut repair/ Gastrointestinal fistula closing
When conjugated to DOPE phospholipid, originates an amphiphilic polymer able to form capsular structures by selfassembly
Role of XG in the formulation Performs as an antidigestive polymer due to structural characteristics
Murine ATDC5 chondrocyte cells
Cells used Intestinal epithelial cells-6 (IEC-6)
Remarks IECs-6 cells growth on the surface of the hydrogel and achieved their gut barrier functions. Calcium ions induced a swellingshrinking behavior of the material, resulting in a practical method for removal from the gut wall as tissue regeneration is achieved By using a microfluidic device, stable and sizecontrolled spherical microcapsules were obtained. Cells were encapsulatedwithin the self-assembled XG/DOPE microcapsules and presented enhanced metabolic activity for a prolonged time
Mendes et al. (2013)
References Huang et al. (2018b)
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chemically cross-linked between the two reactant groups via Schiff’s base reaction. A re-gelifying strategy of controlled drug release in liquids was proposed, in which the injected hydrogel is capable of avoiding the influence of tissue exudates so that a loaded drug can be released in a controlled manner. An angiogenic factor (VEGF) was loaded to the hydrogels to improve the effects on abdominal wall reconstruction. The biomaterial had the advantage of providing controlled drug release, especially in tissues with much excretion or exudation such as those of the digestive tract and open wounds. The hydrogel loaded with VEGF resulted in accelerated angiogenesis and was capable of regenerating the abdominal wall tissue.
5.8
Other Applications
XG has been explored for additional biomedical applications. Table 3 shows some examples of the use of XG for regenerative medicine applications not yet discussed in the previous sections. These alternative uses include XG combination with inorganic particles to create smart composite hydrogels for tissue engineering, incorporation in bioinks for the 3D printing of tissues and organs with complex geometries, complexation and blending with other polymers to produce scaffold materials with tunable properties to match the native characteristics of many native tissues, among others.
6
Conclusions
Despite all the recent advances observed in the area of regenerative medicine, defining, designing, and producing biomaterials suitable for this purpose is still a major challenge. Natural polymers, such as xanthan gum, a high molar exopolysaccharide produced by bacteria of the genus Xanthomonas, continue to attract attention due to its structural similarity to the extracellular matrix, its biocompatibility, high availability, and competitive price. The fact that xanthan gum can be obtained on a large scale from fermentative processes increases its attractiveness, since the control of several of the variables associated with the production process makes it possible to obtain a biopolymer with a quality more appropriate for clinical applications than those extracted directly from algae, plants, or animals. The control of the process allows obtaining a more homogeneous product concerning the composition and size of the chains from batch to batch, aspects of paramount importance in the development of clinical and pharmacological biomaterials. Xanthan gum, in addition to not being cytotoxic even at relatively high concentrations, is biodegradable and stable over a wide range of temperature and pH conditions. Besides, XG has peculiar rheological properties and is capable of forming structures in the form of networks stabilized by intra- and intermolecular bonds with a high capacity to absorb physiological solutions. Altogether, these properties make it possible to use xanthan gum to obtain a variety of products applicable in various fields of regenerative medicine.
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Even in situations where xanthan gum alone cannot meet all requirements for a given type of application, this polysaccharide can be successfully combined with other natural or synthetic polymers. Moreover, XG can also have its chemical structure modified by chemical, physical and enzymatic means to better meet specific demands for functionality. The literature already records a vast list of strategies for modifying and combining this molecule with other compounds, not only of the category of polymers, but also of diverse bioactive agents, such as drugs, growth factors, and products with antimicrobial activity. Many other new approaches can still be proposed and explored with the purpose of further expanding the scope of molecular architecture, functionality, and applications of xanthan gum. Another advantage of xanthan gum is its high processability, which makes it possible to obtain biomaterials from it in the form of solutions, hydrogels, particles, films, membranes, and porous monolithic devices, among others. These biomaterials, associated or not with cells, mainly stem cells, have proven to be effective, exhibiting excellent biological response in a wide range of applications in regenerative medicine, both in soft and hard tissues. Examples of its use in this regard include the therapy of lesions occurring in skin, articular cartilage, tendons, bones, neural, periosteal, periodontal, and gastrointestinal tissues. Furthermore, XG can be also used in the release of bioactive agents and to produce bioinks for tissue engineering approaches based on additive manufacturing. Xanthan gum has proved to be much more than a thickening agent, a stabilizer, or an emulsifier additive useful in the food industry. The knowledge acquired in the last decades regarding this polysaccharide shows that it also has solid perspectives of use in tissue repair and regeneration. Acknowledgments The authors would like to acknowledge the financial support by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil – Grant #307829/2018-9), and the Coordination for the Improvement of Higher Educational Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, Brazil – Finance Code 001).
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Bombaldi de Souza RF, Bombaldi de Souza FC, Thorpe A, et al. Phosphorylation of chitosan to improve osteoinduction of chitosan/xanthan-based scaffolds for periosteal tissue engineering. Int J Biol Macromol. 2020;143:619–32. Bueno VB, Bentini R, Catalani LH, et al. Synthesis and characterization of xanthan–hydroxyapatite nanocomposites for cellular uptake. Mater Sci Eng C. 2014;37:195–203. Dubbin K, Tabet A, Heilshorn SC. Quantitative criteria to benchmark new and existing bio-inks for cell compatibility. Biofabrication. 2017;9:044102. Dyondi D, Webster TJ, Banerjee R. A nanoparticulate injectable hydrogel as a tissue engineering scaffold for multiple growth factor delivery for bone regeneration. Int J Nanomedicine. 2013;8: 47–59. Elizalde-Peña EA, Quintero-Ortega IA, Zárate-Triviño DG, et al. (Chitosan-g-glycidyl methacrylate)-xanthan hydrogel implant in Wistar rats for spinal cord regeneration. Mater Sci Eng C Mater Biol Appl. 2017;78:892–900. Fabela-Sánchez O, Zarate-Triviño DG, Elizalde-Peña EA, et al. Mammalian cell culture on a novel chitosan-based biomaterial crosslinked with gluteraldehyde. Macromol Symp. 2009;283–284: 181–90. García-Ochoa F, Santos VE, Casas JA, Gómez E. Xanthan gum: production, recovery, and properties. Biotechnol Adv. 2000;18:549–79. Gils PS, Ray D, Sahoo PK. Characteristics of xanthan gum-based biodegradable superporous hydrogel. Int J Biol Macromol. 2009;45:364–71. Glaser T, Bueno VB, Cornejo DR, et al. Neuronal adhesion, proliferation and differentiation of embryonic stem cells on hybrid scaffolds made of xanthan and magnetite nanoparticles. Biomed Mater. 2015;10:045002. Han G, Shao H, Zhu X, et al. The protective effect of xanthan gum on interleukin-1β induced rabbit chondrocytes. Carbohydr Polym. 2012;89:870–5. Han G, Chen Q, Liu F, et al. Low molecular weight xanthan gum for treating osteoarthritis. Carbohydr Polym. 2017;164:386–95. Hashim NT. Oral microbiology in periodontal health and disease. Oral microbiology in periodontitis; 2018. Huang J, Deng Y, Ren J, et al. Novel in situ forming hydrogel based on xanthan and chitosan re-gelifying in liquids for local drug delivery. Carbohydr Polym. 2018a;186:54–63. Huang J, Li Z, Hu Q, et al. Bioinspired anti-digestive hydrogels selected by a simulated gut microfluidic chip for closing gastrointestinal fistula. iScience. 2018b;8:40–8. Ibrahim HK, Fahmy RH. Localized rosuvastatin via implantable bioerodible sponge and its potential role in augmenting bone healing and regeneration. Drug Deliv. 2016;23:3181–92. Izawa H, Nishino S, Maeda H, et al. Mineralization of hydroxyapatite upon a unique xanthan gum hydrogel by an alternate soaking process. Carbohydr Polym. 2014;102:846–51. Jain M, Dave D, Jain P, et al. Efficacy of xanthan based chlorhexidine gel as an adjunct to scaling and root planing in treatment of the chronic periodontitis. J Indian Soc Periodontol. 2013;17:439. Juris S, Mueller A, Smith B, et al. Biodegradable polysaccharide gels for skin scaffolds. J Biomater Nanobiotechnol. 2011;2:216–25. Kaminski GAT, Sierakowski MR, Pontarolo R, et al. Layer-by-layer polysaccharide-coated liposomes for sustained delivery of epidermal growth factor. Carbohydr Polym. 2016;140:129–35. Kim J, Hwang J, Seo Y, et al. Engineered chitosan–xanthan gum biopolymers effectively adhere to cells and readily release incorporated antiseptic molecules in a sustained manner. J Ind Eng Chem. 2017;46:68–79. Kool MM, Schols HA, Wagenknecht M, et al. Characterization of an acetyl esterase from Myceliophthora thermophila C1 able to deacetylate xanthan. Carbohydr Polym. 2014;111:222–9. Kreyenschulte D, Krull R, Margaritis A. Recent advances in microbial biopolymer production and purification. Crit Rev Biotechnol. 2014;34:1–15. Kumar A, Rao KM, Kwon SE, et al. Xanthan gum/bioactive silica glass hybrid scaffolds reinforced with cellulose nanocrystals: morphological, mechanical and in vitro cytocompatibility study. Mater Lett. 2017;193:274–8.
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Kumar A, Rao KM, Han SS. Application of xanthan gum as polysaccharide in tissue engineering: a review. Carbohydr Polym. 2018;180:128–44. Kuo SM, Chang SJ, Wang H-Y, et al. Evaluation of the ability of xanthan gum/gellan gum/hyaluronan hydrogel membranes to prevent the adhesion of postrepaired tendons. Carbohydr Polym. 2014;114:230–7. Laffleur F, Michalek M. Modified xanthan gum for buccal delivery – a promising approach in treating sialorrhea. Int J Biol Macromol. 2017;102:1250–6. Liu Z, Yao P. Injectable thermo-responsive hydrogel composed of xanthan gum and methylcellulose double networks with shear-thinning property. Carbohydr Polym. 2015;132:490–8. Matsuda Y, Biyajima Y, Sato T. Thermal denaturation, renaturation, and aggregation of a doublehelical polysaccharide xanthan in aqueous solution. Polym J. 2009;41:526–32. Matsuda Y, Sugiura F, Mays JW, Tasaka S. Atomic force microscopy of thermally renatured xanthan with low molar mass. Polym J. 2015;47:282–5. Mendes AC, Baran ET, Pereira RC, et al. Encapsulation and survival of a chondrocyte cell line within xanthan gum derivative. Macromol Biosci. 2012;12:350–9. Mendes AC, Baran ET, Reis RL, Azevedo HS. Fabrication of phospholipid–xanthan microcapsules by combining microfluidics with self-assembly. Acta Biomater. 2013;9:6675–85. Milas M, Rinaudo M. Conformational investigation on the bacterial polysaccharide xanthan. Carbohydr Res. 1979;76:189–96. Needleman IG, Smales FC, Martin GP. An investigation of bioadhesion for periodontal and oral mucosal drug delivery. J Clin Periodontol. 1997;24:394–400. Petri DFS. Xanthan gum: a versatile biopolymer for biomedical and technological applications. J Appl Polym Sci. 2015;132. Ramburrun P, Kumar P, Choonara YE, et al. Design and characterization of neurodurable gellanxanthan pH-responsive hydrogels for controlled drug delivery. Expert Opin Drug Deliv. 2017;14: 291–306. Rao KM, Kumar A, Haider A, Han SS. Polysaccharides based antibacterial polyelectrolyte hydrogels with silver nanoparticles. Mater Lett. 2016;184:189–92. Rao KM, Kumar A, Han SS. Polysaccharide-based magnetically responsive polyelectrolyte hydrogels for tissue engineering applications. J Mater Sci Technol. 2018;34:1371–7. Salazar GM, Sanoh NC, Pernaloza DP Jr. Synthesis and characterization of a novel polysaccharidebased self-healing hydrogel. KIMIKA. 2018;29:44–8. Schmid J, Sieber V. Enzymatic transformations involved in the biosynthesis of microbial exo-polysaccharides based on the assembly of repeat units. Chembiochem. 2015;16:1141–7. Sehgal RR, Roohani-Esfahani SI, Zreiqat H, Banerjee R. Nanostructured gellan and xanthan hydrogel depot integrated within a baghdadite scaffold augments bone regeneration. J Tissue Eng Regen Med. 2017;11:1195–211. Tao Y, Zhang R, Xu W, et al. Rheological behavior and microstructure of release-controlled hydrogels based on xanthan gum crosslinked with sodium trimetaphosphate. Food Hydrocoll. 2016;52:923–33. Wang B, Han Y, Lin Q, et al. In vitro and in vivo evaluation of xanthan gum–succinic anhydride hydrogels for the ionic strength-sensitive release of antibacterial agents. J Mater Chem B. 2016a;4:1853–61. Wang X, Xin H, Zhu Y, et al. Synthesis and characterization of modified xanthan gum using poly (maleic anhydride/1-octadecene). Colloid Polym Sci. 2016b;294:1333–41. Westin CB, Trinca RB, Zuliani C, et al. Differentiation of dental pulp stem cells into chondrocytes upon culture on porous chitosan-xanthan scaffolds in the presence of kartogenin. Mater Sci Eng C Mater Biol Appl. 2017;80:594–602. Westin CB, Nagahara MHT, Decarli MC, et al. Development and characterization of carbohydratebased thermosensitive hydrogels for cartilage tissue engineering. Eur Polym J. 2020;129: 109637.
Cellulose and Tissue Engineering
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Paula Cristina de Sousa Faria-Tischer
Contents 1 Tissue Engineering: Definition and Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Cellulose: Function, Structure, and Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Cellulose: Derivatives and Cellulose Nanostructures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Cellulose Applied to Bone Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Cellulose Applied to Cartilage Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Cellulose for Skin Tissue Engineering and Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Conclusions, Future Directions, and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
The field of tissue engineering is growing fast and at the same time the use of cellulose also has aroused interest of several groups of researchers and companies. The wide possibility of cellulose modification (physical and/or chemical) combined with its biological properties makes this biopolymer an important candidate to produce tissue engineering templates designed to replicate the niche, or microenvironment, of the target cells to produce fully functional tissues. The structural organization and nanofiber three-dimensional (3D) network of this polymer (isolated or biocomposite) have demonstrated fruitful outcome and challenges too. One important factor, of several, to achieve the promising results is to use scientific rationale in each step of development coupling engineering and biology systems. Regulatory aspects, partners (scientific and commercial), ethics, bioprocessing, and financial investment are some of the challenges, in my point of view, that represent opportunities driving the tissue engineering, ensuring the progress toward realizing the clinical and commercial endpoints.
P. C. de Sousa Faria-Tischer (*) Glucom Biotech Ltda., Londrina, Brazil e-mail: [email protected] © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_62
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Keywords
Nanocellulose · Tissue engineering · 3D printing · Wound healing
1
Tissue Engineering: Definition and Requirements
Tissue engineering employs a combination of engineering, biology, and bioactive constructs to improve function by repairing, replacing, or regenerating tissue. The expanded concept of regenerative medicine includes tissue engineering but also incorporates research on the regeneration of tissue directly in vivo, where the body uses its own systems to repair, replace, or regenerate function in damaged or diseased tissue with the help of exogenous cells, scaffolds, or biological factors (Dzobo et al. 2018). The regenerative medicine relies on harnessing the body’s natural ability to self-heal. However, tissue engineering evolved from the field of biomaterials development and refers to the practice of combining scaffolds, cells, and biologically active molecules into functional tissues (Brien 2011). Actually, there are six basic requirements widely accepted for designing polymer scaffolds: high porosity and proper pore size; high surface area; biodegradability and a proper degradation rate to match the rate of neotissue formation; mechanical integrity; not be toxic to cells (i.e., biocompatible); and finally, bioactivity which means interaction with cells, including enhanced cell adhesion, growth, migration, and differentiated function (Ma 2004). The success of the development of tissue engineering is closely related to the conditions for tissue culture that involves the in vitro maintenance and propagation of cells in optimal conditions. The comprehension of interaction between the ligands present in the extracellular matrix and the receptors of the cell allows multiple intracellular signaling processes that can result in the alteration of cellular behaviors, such as growth, migration, and differentiation and several natural biomaterials have been explored recently. Three-dimensional biomaterials with large pore size (greater than 100 μm) carry a high number of functional units essential for the regeneration of various tissues. Pore size greater than 100 μm is essential for the cell adhesion and proliferation. However, to design functional units of tissue, not only are the subcellular and cellular scales required, but also nanostructures, 1–100 nm in size. This type of structural arrangement is essential to control cell behavior, in particular cell– cell interactions, cell–molecular interactions, and the cellular environment (Bhatia and Goli 2018). Cell adhesion, mitosis, and growth are often caused by proteins that have been attached to scaffold material, so bioactive molecules like growth factors or proteins of the extracellular matrix (ECM) have been included in the polymers to support these functions. Bifunctional groups can also be added to the polymer materials for improved spreading of cells. These include glycolipids, oligopeptides, and oligosaccharides. Fibronectin, collagen, laminin, tenascin, vitronectin, and thrombospondin, a glycoprotein that mediates cell-to-cell and cell-to-matrix interactions, are some examples that increase the spreading of cells (Ogueri et al. 2019).
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Particularly the “RGD” arginine-glycine-aspartate sequence in fibronectin is responsible to stimulate the cellular response, in this way several researcher groups are testing only this tripeptide RGD anchored in the polymer surface, instead of the whole molecule (Ruoslahti 1996). Scaffolds coated with polylysine, polyornithine, or lactose and N-acetlyglucosamine and micropaths have been created and a positive effect in the cell attachment and spreading have been observed (Sigma-Aldrich 2008; Lam and Longaker 2012). Actually, the researcher’s centers are looking for materials that mimic natural ECMs in terms of their composition, structural characteristics, and mechanical properties. To find scaffolds capable to transmit a signal to actively construct and degrade their microenvironment, providing cellular adhesion, proteolytic degradation, and growth factor (GF)-binding, as well as space-filing mechanical support capable to behavior as bioactive and dynamic environment to mediate cellular functions is a challenge nowadays and 3D scaffolding materials can represent a new choice to get these properties (Dutta et al. 2019). Cellulose has been widely applied in engineering of blood vessels, reconstruction of urethra and dura mater, liver and adipose tissue, neural tissue, bone, cartilage, repairing connective tissue and congenital heart defects, and constructing protective barriers and contact lenses. This chapter reviewed some of the newest researches in the last 5 years, reporting the development of scaffolds based in cellulose for tissue engineering (bone, cartilage, and skin). The intention is to highlight cellulose structural characteristics that make their application more attractive while tailoring them to tissue regeneration demands improving the methods of repair, replacement, or regeneration of damaged tissues and organs.
2
Cellulose: Function, Structure, and Properties
Structure–property and structure–function relationships have long been considered important explanatory concepts at hierarchical levels and provide insight to design new mimetic tissues able to be employed into tissue engineering. Cellulose is a semicrystalline polymer formed by (1-4)-linked betha-D-glucosyl residues that are alternately rotated by 180 along the polymer axis to form flat ribbon-like chains. Each glucosyl unit bears three hydroxyl groups, one on hydroxymethyl group. It has been long recognized that these hydroxyl groups and their ability to bond via hydrogen bonding not only play a major role in directing how the crystal structure of cellulose forms but also in governing important physical properties of cellulose materials (Brown and Saxena 2000). Cellulose is a polycrystalline material and the crystals are aligned along the microfibrils and present a polymorphism (Delmer and Amor 1995; Atalla and VanderHart 1984). As a result, cellulose has several polymorphs, namely cellulose I, II, III, and IV and their varieties Iα, Ibetha, IIII, IVI, IIIII, and IVII. Most of these polymorphs result from chemical treatments of polymorph (Šturcova et al. 2004; VanderHart and Atalla 1984). The degree of crystallinity, i.e., the quality of the
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Fig. 1 Layers of cellulose microfibril composed by crystalline and amorphous regions surrounded by hemicellulose
cellulose crystal, is another important factor that varies extensively from one cellulosic material to another (Revol 1985). The interaction between water and cellulose is of utmost importance in order to understand and control the properties of cellulosic materials (Sugiyama 1984). Indeed, the unusual physical and chemical properties of cellulose such as highly hydrophilic nature, good mechanical properties, low density and thermal conductivity, and good thermal stability arise from its structural architecture (Chami Khazraji and Robert 2013). Therefore, it is possible to get more assertive manipulation process of cellulose when we have knowledge of its structural crystalline organization. In nature cellulose occurs as a slender rodlike or threadlike entity, called microfibril (collection of cellulose chains); this entity forms the basic structural unit of any “cellulose” independent of its origin (Fig. 1). Each microfibril can be considered as a string of cellulose crystals linked along the chain axis by amorphous domains. The outer regions of wood microfibrils are strongly disordered, mostly due to the direct contact with hemicellulose, which is largely amorphous in nature. These regions can show the form of paracrystalline or fully amorphous cellulose (Fig. 2) (Dufresne 2019; Nishiyama 2009). Not only the microfibril diameter (nm) is different but also the DP (degree of polymerization). Values of DP ranging from hundreds and several tens of thousands have been reported and the DP is heavily dependent on the source of the original cellulose (Habibi et al. 2010). Different protocols focusing in chemical and physical modifications have been applied to cellulose with the purpose to obtain particular morphologies and structures seeking to meet specific properties capable to stimulate specific tissue regeneration.
3
Cellulose: Derivatives and Cellulose Nanostructures
As mentioned above, our body is formed by different tissues and they possess distinct characteristics raised from its structural organization. In this way cellulose usually is obtained and modified in order to provide the specific characteristics
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Fig. 2 Crystalline and paracrystalline regions
necessary to distinct functions. Cellulose can be tailored to exhibit particular physical and chemical properties using different methodologies, one of them is by varying the pattern and degrees of substitution within the cellulose backbone. There is a large distribution in the worldwide market of cellulose ethers and esters, in biomedicine cellulose ethers derivatives are more used (e.g., methylcellulose [MC], ethyl cellulose [EC], hydroxyethylcellulose [HEC], hydroxypropyl cellulose [HPC], hydroxypropyl methyl cellulose [HPMC], and carboxymethyl cellulose [CMC]). Recently, hydroxypropyl methyl cellulose (HPMC) have been evaluated in association with other polymers for tissue engineering, e.g., for cell-based cartilage engineering (Rederstorff et al. 2017), corneal regeneration (Long et al. 2018), and filler material for use in oral and craniofacial fields (Huh et al. 2015), and methylcellulose (MC) was crosslinked to form hydrogels (Niemczyk-Soczynska et al. 2019) for bone regeneration (Kim et al. 2018), thermally reversible hydrogels (Kummala et al. 2020), and more recently aroused interest as a versatile printing material for bio-fabrication of tissues (Law et al. 2018; Ahlfeld et al. 2020; Roushangar Zineh et al. 2018). Carboxymethyl cellulose has been intensively researched for bone regeneration (Singh and Pramanik 2018; Hasan et al. 2018; Matinfar et al. 2019). Due to the reduced structure of the cellulose chain arrangement in the disordered region, it has a lower density and thus exhibits more free volume than the crystalline region (De Souza Lima and Borsali 2004). Therefore, several researches have been focusing to obtain nanostructures of cellulose through preferentially acid hydrolyses which remove the disordered amorphous region leaving the crystalline region largely intact due to their tight packing. The correct timing and hydrolysis conditions enabling the obtention of CNC (cellulose nanocrystals) produced as individualized particles (De Souza Lima and Borsali 2004; Anglès and Dufresne 2001). These nanoscale crystalline structures are isolated from native cellulose mostly via an acid hydrolysis, but enzymatic hydrolysis with cellulases (Siqueira et al. 2010; Henriksson et al. 2007) TEMPO ((2,2,6,6-tetramethylpiperidin-1-oxyl)-mediated oxidation (Saito et al. 2007), and ionic liquids has also been reported to prepare cellulosic nanoparticles (Man et al. 2011). Typically, nanocellulose can be categorized into two major classes: (1) nanostructured materials (cellulose microcrystals and cellulose microfibrils) and (2) nanofibers
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(cellulose nanofibrils, cellulose nanocrystals, and bacterial cellulose) (Trache et al. 2017). Nanocellulose market increased a lot in the last years and the employment of new applications has driven the researchers and the industry to exploit even more its employment. Therefore, International Organization for Standardization (ISO), Technical Association of the Pulp and Paper Industry (TAPPI), and Canadian Standards Association (CSA) standards on cellulose nanocrystals (CNCSs) are being developed and published. In 2011, TAPPI proposed international standards for cellulosebased nanomaterials that could remove the trade barriers and harmonize research and development in nanocellulose-based products (Reid et al. 2017). These standards help to categorize nanocellulose avoiding overcoming the problems related to multiple definitions and ambiguity. The physicochemical and structural properties of nanocellulose are dependent of initial biomass type or microbial source selected (Trache et al. 2017), cellulose polymorphs (Gong et al. 2018), pretreatment process of cellulose extraction and acid hydrolysis, or enzymatic treatment (Mondal 2017), followed for nanocellulose fabrication. Among the standards are ISO/TC 229 – TS 20477:2017: Standard terms and their definition for cellulose nanomaterial and some terms and abbreviations of cellulose nanomaterials were established, e.g., nanocrystalline cellulose (NCC), nanofibrillar cellulose (NFC), cellulose nanocrystals (CNC), cellulose nanowhiskers (CNW), cellulose nanofibrils (CNF), bacterial cellulose (BC), and tempo-oxidized cellulose nanofibrils (TOCN) (Reid et al. 2017). The good choice of biomaterial is the first step to design of scaffold. Scheme 1 shows some important points related to selection and characterization of cellulose materials.
4
Cellulose Applied to Bone Tissue Engineering
Bone acts as the supportive structure of the body, functions as a mineral reservoir, guards vital organs, is the site of blood cell production, and helps maintain acid–base balance in the body (De Witte et al. 2018). The aim of bone tissue engineering is to develop 3D scaffolds that mimic the extracellular matrix (ECM) and provide mechanical support, thereby aiding in the formation of new bone. Scaffolds provide a template for cell attachment and stimulate functional bone tissue formation in vivo through tailored biophysical cues to direct the organization and behavior of cell (Bose et al. 2013). Bone tissue scaffolds present some specific biological requirements between them which are as follows (De Witte et al. 2018): (a) Biodegradable, nontoxic, osteogenic, presence of GFs (growth factors) (b) Mechanical requirements: mechanical properties, compressive strength ~2–12 MPa, and Young’s modulus ~0.1–5 GPa (c) Structural requirements: interconnected porosity, average pores size 300 nm, and nanotopography
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Scheme 1 Steps for selection and characterization of cellulose
Bone tissue engineering requires the presence of a bioactive component like hydroxyapatite Ca5(PO4)3OH (HAp) and tricalcium phosphate (TCP) Ca3(PO4)2. Several researches report different methodologies to achieve the formation of these biocomposites (cellulose and Hap or TCP). Scaffolds were fabricated with silk fibroin (SF) and carboxymethyl chitosan (CMCS) incorporated with strontium
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substituted hydroxyapatite (Sr-HAp) and there were enhanced protein adsorption and ALP (alkaline phosphatase) activity. The expression of osteogenic gene markers such as RUNX2 (Runt-related factor2), ALP, OCN (osteocalcin), OPN (Osteopotin), BSP (Bone Sialoprotein), and COL-1 was also stimulated (Zhang et al. 2019a). A smoother scaffold with better distribution of HA with good interconnectivity, hardness range of scaffolds of 550–640 MPa, compression strength range of 110–180 MPa, an elastic modulus of ~5 GPa, and a fracture toughness value of ~6 MPa1/2 in the range of cortical bone was obtained using the association of (TEMPO)-oxidized cellulose nanofibrils (TCNF) and cellulose nanocrystals (CNC) with hydroxyapatite (HA) (Ingole et al. 2020). The alignment of cellulose nanofibers hydrogels appeared to be a key structural feature in the successful and thorough infiltration of minerals as observed in a study where mineralized hydrogels were fabricated with TEMPO-oxidized cellulose and well-aligned nanofibers using a biomimetic method. The scaffold presented roughly 70 wt.% mineral content in the mineralized cellulose scaffold comparable with the mineral content in natural hard tissues (ranging 70–85 wt.%) (Qi et al. 2019). Biomimetic growth of biphasic ceramics (HA/β-TCP) was used also to produce mineralized tissue with nanocellulose obtained from açaí integument (Euterpe Oleracea Mart.) (HA/β-TCP) (Valentim et al. 2018). Bacterial cellulose (BC) has been an important choice to fabricate mineralized tissues proving to be a template for the ordered formation of calcium-deficient hydroxyapatite (CDHAP) (Hutchens et al. 2006). BC associated to hydroxyapatite (HA) and anti-bone morphogenetic protein antibody (anti-BMP-2) produced a noncytotoxic, genotoxic, and mutagenic biomaterial in MC3T3-E1 cells with increased mineralization nodules and the levels of ALP activity (Coelho et al. 2019). Aligned scaffolds using cellulose nanocrystals loaded with bone morphogenic protein-2 (BMP-2) were produced by electrospinning and cellulose seems aligned human mesenchymal stem cells (BMSCs) growth and mineralized nodules formation in vitro (Zhang et al. 2019b). Cryogels formed upon contact with body fluids, with high porosity and high specific surface area, a rough hydroxyapatite layers and release of ions (Si, Ca, P, and Na) that were produced because there is a synergy between cellulose nanofibrils/bioactive (organic and inorganic) materials. So the cell differentiation is directly affected by the combination of high porosity, hydroxy apatite formation, and ion release and these set of events and conditions increases the release of BMP-2 greatly improving bone formation (Ferreira et al. 2019). Nanocellulose (NC) containing BMP2-VEGF (BNBV) was loaded in porous sponge biphasic calcium phosphate (BCP) scaffold produced by replica method and bone marrow mesenchymal stem cells (RBMSCs) were seeded in these scaffolds. Bone formation increased because BMP2 stimulated the differentiation of stem cells to osteoblasts and the angiogenesis was facilitated by VEGF drawing the attention to the important role of nanocellulose as carrier for growth factors (Sukul et al. 2015). The cellulose modification followed by the incorporation of bioactive molecules expand the possibilities to change its properties and reactivity; it can be achieved
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using ex situ chemical (e.g., periodate oxidation and grafting (Leguy et al. 2018) functionalization through linker (Ribeiro-Viana et al. 2016), or crosslinking reactions (Kirdponpattara et al. 2015)) or physical modifications (physical absorption from solutions or particle suspensions, the homogenization, or dissolving BC mixing with additive material or yet to add the additive material in the culture medium) (Lopes et al. 2014). Recently through controlled nucleation, HA nanocrystals were produced in CNCs functionalized with sulphonic groups and aerogels obtained from nanocellulose with incorporated sulfate and phosphate groups crosslinked with hydrazine increased the cell metabolism of Saos-2 cells on the porous scaffolds. Twelve weeks after implantation in rats the osteoconductivity and bone volume were increased (Osorio et al. 2019). Considering the importance of biodegradability and functionality to regulate the bone regeneration process, injectable bone composed of bisphosphonate-modified nanocellulose (pNC) was prepared with bisphosphonate groups on nanocellulose. The results demonstrated that pNC released under osteoclast microenvironment can control the osteoclast activity and, moreover, pNC-α/β-TCP composites promoted osteoblast differentiation (Nishiguchi and Taguchi 2019). 3D printing has been a recent trend to get the production of hydrogels with biomimetic structures for tissue regeneration and organ reconstruction. For this purpose, the development of bioinks capable to mimetic the properties and morphologies of tissues is fundamental to achieve the success. Bacterial cellulose nanofibers demonstrated a benefic effect improving the shape fidelity and mechanical properties of the 3D printed scaffolds composing silk fibroin and gelatin (Huang et al. 2019). Scaffolds hydrogels were produced using 3D printing through partial crosslinking of TEMPO-oxidized cellulose with alginate and biomimetic mineralization using simulated body fluid was the method used to get hydroxyapatite nucleation using calcium ions (Abouzeid et al. 2018). Other approaches have demonstrated that scaffolds derived from apple hypanthium tissue can act as a bioactive biomaterial (Modulevsky et al. 2014, 2016; Hickey et al. 2018). After removing the native cellular components, the reminiscent structure presented pore size (100 and 200 μm) which was shown to be the optimal pore size for biomaterials used for bone tissue engineering. Additionally, pre-osteoblasts were seeded (MC3T3-E1) and the localized mineralization was proved by the presence of calcium deposits after 4 weeks, specifically on the edge of the pores (Karageorgiou and Kaplan 2005). Table 1 summarizes some representative researches realized in the last 5 years focusing cellulose modification to use in bone tissue engineering.
5
Cellulose Applied to Cartilage Tissue Engineering
Cartilage is a connective tissue which does not spontaneously heal, it is a tough, semitransparent, elastic, flexible connective tissue consisting of cartilage cells scattered through a glucoprotein material that is strengthened by collagen fibers.
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Table 1 Examples of researches with cellulose applied for bone tissue engineering Bone tissue engineering Source of cellulose Silk fibroin (SF) and carboxymethyl chitosan (CMCS) incorporated with strontium Cellulose nanofibers loaded with bone morphogenic protein-2 (BMP-2)
Technique applicated Freeze drying
Electrospinning
TCNF and CNC with HA
Prepared suspensions Ultrasonication Drying overnight in an oven at 60 C
Nanofibrils (CNF) from Eucalyptus grandis and bioctive glass
Freeze-casting
Calcium phosphate (BCP) with BMP2-VEGF incorporated nanocellulose
Sponge replica
TOCN from softwood pulp CaCl2, K2HPO4 and PAA
Biomimetic mineralization process
Bacterial cellulose incubated in solutions of calcium chloride followed by sodium phosphate dibasic
Incubation cycle
Achievements Stimulus of osteogenesis Interconnect porous Mechanical properties
References Zhang et al. (2019a)
Growth of aligned human mesenchymal stem cells (BMSCs) Porous 100–600 nm Stimulus of osteogenesis In vivo formed aligned collagen fibers Hardness range of 550–640 MPa Compression strength range of 110–180 MPa Elastic modulus of ~5 GPa Stimulus of HA crystallites formation High porosity Hight specific surface area Release of ions (Si, Ca, P, and Na) by mineral phase Increase differentiation of BMP-2 Hight cell attachment Hight proliferation No bone formation in ectopic sites BMP2 and VEGF stimulate new bone formation in the orthotopic defects Oriented hybrid nanostructure Cellulose scaffold mineralized with HAp crystals Hard tissues 70 wt.% Incorporated substantial amount of apatite (50–90% of total dry weight) BC provides a template for the ordered formation of CdHAP Composite similar to the physiological biomineralization of bone
Zhang et al. (2019b)
Ingole et al. (2000)
Gadim et al. (2014)
Sukul et al. (2015)
Qi et al. (2019)
Hutchens et al. (2006)
(continued)
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Table 1 (continued) Bone tissue engineering Source of cellulose
Technique applicated
CNC from Açai Synthesis of HA and β-TCP
Biomimetic growth
TOCN from Whatman cotton ashless filter aid
Surface functionalization methods
CNCs from spruce and pine 5–10 nm wide 100–300 nm long
Controlled nucleation with sulphonic and carboxylate groups TEMPO oxidation Casting
TOCN-bleached fibers from Bagasse
Biomimetic mineralization 3D printing
McIntosh apples
Decellularization Scaffolds untreated Scaffolds coated with collagen solution Pre-osteoblasts
Achievements producing apatite crystals of comparable shape and size X-ray diffraction prove nucleation of HA and β-TCP Particle size gauge range of 164.2 10–9–4748 10–9 m Composite with tendencies to flocculation Macropores in the range of 10–950 mm Increased ALP activity over 7 days Nucleation of HA over S-CNC Osteoconductive properties Transparent thin coatings successfully fabricated via evaporation-induced assembly of CNC–HA nanocomposites The c-axes of the CNC and HA in the nanorods aligned parallel to the surface Water-resistant transparent coatings 2–4 μm thick Pastes show highly thixotropic behavior Hydroxyapatite in the mineralized scaffolds was estimated to be 20.1% Best hydrogel with 50% T-CNF and 50% alginate hydrogel (CNF50) Compressive strength of 455 MPa (CNF50) Pre-osteoblasts adhered and proliferated in both scaffolds with and without collagen Mineralization occurred particularly in the edges Increase in the Young’s
References
Valentim et al. (2018)
Osorio et al. (2019)
Ishikawa et al. (2015)
Abouzeid et al. (2018)
Leblanc Latour et al. (2020)
(continued)
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Table 1 (continued) Bone tissue engineering Source of cellulose
Technique applicated MC3T3-E1 Subclone 4 cells seeded
Achievements
References
modulus after mineralization Pore size (100 and 200 μm) Limitation Low Young’s modulus
The main purpose of cartilage is to provide a framework on which bone deposition may begin. Another important purpose of cartilage is to cover the surfaces of joints, allowing bones to slide over one another, thus reducing friction and preventing damage; it also acts as a shock absorber (Zhang et al. 2009). Engineered cartilage regeneration in gels necessitate the control of mechanical properties (strength, rigidity, and elongation) and easy processing into complex shapes (Fu et al. 2017). Hydrogels with nanocellulose also have been extensively explored for cartilage scaffolds, however, to match these requirements. Cellulose-based gels are often combined to other materials and are produced by different methodologies in order to achieve efficient and bioactive scaffolds. As already mentioned, 3D bioprinting is a powerful tool emerged in the last years for the production of highly structured tissue engineering scaffolds, allowing to dispense hydrogels in three dimensions with precision and high resolution (Kang et al. 2016; Mandrycky et al. 2016). The printed material gel is prepared with cells encapsulated with homogeneous density, which permits homogeneous cell distribution and the scaffold can be fully colonized and the combination of crosslinked sodium alginate and NC has been recently explored for cartilage tissue engineering, for articular and nasal reconstruction (Puelacher et al. 1994; Nguyen et al. 2017; Engineering and Wood 2017). Not only for bone tissue but also for human cartilage nanocellulose–alginate hydrogels is a promising combination to obtain scaffolds with 3D printed (Table 2). Recently one of the bionks developed is composed by the induction of pluripotent stem cells (iPSCs) and human chondrocytes printed together with the hydrogel matrix, e.g., nanofibrillar cellulose/alginate (NCF/A) bionk was embedded with human bone marrow–derived stem cells (hBMSCs) and human nasal chondrocytes (hNC) (Möller et al. 2017). The coculture enhanced chondrogenesis and after 60 days chondrocyte cell clusters indicated the ability of embedded cells to proliferate and the formed tissue presented all qualitative features of proper cartilage. Furthermore, cell clusters contained human chromosomes proving their human origin (de Windt et al. 2014). Additionally was observed high cell viabilities of 73% and 86% after 1 and 7 days of 3D culture, respectively, in coculture NFC/A bioinks for bioprinting iPSCs to support cartilage production and formation of cartilaginous tissue by expression of collagen II was observed after 5 weeks (Nguyen et al. 2017). Similarly, human auricular cartilage was obtained from cells cultured for 28 days inside a 3D printed scaffold with 75% of cells were still viable and increased
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Table 2 Examples of researches with cellulose applied for cartilage tissue engineering Cartilage tissue engineering Type of cellulose Technique applied Bacterial nanocellulose Homogeneous Alginate BNC/alginate mixture Phase separation BNC/alginate comoposite scaffolds Freeze-drying process Ionic liquid EMIMAc to join the layers Heating plate at 80 C for 2 min The bilayer BNC scaffolds stabilized in 100 mM CaCl2 in ethanol to precipitate the dissolved cellulose between the layers Simultaneously crosslinking the alginate to bind the BNC in the porous layer Culture-expanded human nasoseptal chondrocytes (NC) combined with human mononuclear cells (MNC) to form cartilage in vitro and in vivo Nanofibrillated 3D bioprinting cellulose (CELLINK technology AB, Gothenburg, Combination of Sweden) nanofibrillated Alginate cellulose/alginate Cellulose/alginate hydrogel, hNCs, and (NFC-A) bioink hBMSCs for 3D bioprinting Crosslinked with 100 mM CaCl2 solution for 5 min at 37 C Human nasal chondrocytes (hNCs) Human bone marrow derived stem cells (hBMSCs) Coculture of hNCs and hBMSCs Subcutaneous implantation of scaffolds in mice
Achievements Bilayer BNC scaffolds Porosity of 75% and mean pore size of 50 25 μm Nonpyrogenic BNC will provide longterm structural integrity after implantation The ability of scaffolds to attract and trap water was enhanced through the production and accumulation of proteoglycans and glycosaminoglycans in the bilayer BNC scaffolds Some limitations: Collagen matrix was not effectively produced in the porous layer Higher cell density is needed to benefit from increased cell–cell contacts signaling chondrogenic
References Martínez Ávila et al. (2015)
Good mechanical properties Keep their structural integrity after 60 days of implantation Increase of GAG deposition The presence of human type II collagen Some limitations: Compression analysis: Difficulties in area determination of the measured constructs Mechanical properties of the neocartilage of this study with native human cartilage was not able to be correlated
Möller et al. (2017)
(continued)
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Table 2 (continued) Cartilage tissue engineering Type of cellulose Technique applied Nanocellulose from Alginate sulfate CELLINK AB synthesis (Gothenburg, Sweden) Nanocellulose water Alginate sulfate dispersion (1.9% dry content) Mixed with either alginate or alginate sulfate
Nanocellulose-based hydrogels combined with sodium alginate Plant-derived nanocellulose (hydrophilic Bioplus ® cellulose nanofibrils gel, hydrophilic Bioplus ® cellulose nanocrystals gel, and hydro philic Bioplus ® blend gel – a blend of fibrils and crystal) provided by American Process, Inc. (Georgia, USA) Produced via the AVAP ® technology
NC-based hydrogels were crosslinked with varying concentrations of CaCl2 Blend nanocellulose produced in situ Hydrogels prepared by mixing nanocellulose with 2.5% (w/v) sodium alginate (80,000–120,000 Da), medium viscosity (2% at 25 C), 1.56 mannuronate/guluronate ratio) Evaluate effect of crosslinking – using calcium chloride (CaCl2) – on the structural and mechanical properties And Exposure of the NCbased hydrogels to different sterilization methods: exposure to ultraviolet (UV) light, autoclaving, and ethanol immersion
Achievements Chondrocytes in alginate sulfate– nanocellulose gel discs were viable, mitogenic and synthesized collagen II Printing conditions greatly affected the behavior of the cells Wide diameter, conical needles providing the best preservation of cell function Biological performance of the cells was highly dependent on the nozzle geometry Increasing concentrations of the crosslinker CaCl2 yielded visible changes architecture, pore size, and porosity Pore size was the most affected by the sterilization method CNC was more affected by the crosslinker concentrations CNF and CNC were affected by all sterilization methods NCB was more resilient to changes when exposed to different sterilization methods and crosslinker concentrations
References Engineering and Wood (2017)
Al-Sabah et al. (2019)
by 20% the expression of ECM proteins (Martínez Ávila et al. 2016). In another study the viability of human chondrocytes within an ear-like structure was 86% after 7 days (Markstedt et al. 2015).
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Bacterial NFC or named BCN (bacterial nanocellulose) was also applied to design layered scaffolds and the cellulose solvent system “ionic liquid EMIMAc,” was used to bond tightly the two layers. Particularly the scaffolds were able to produce and accumulate cartilage-specific ECM components and the chondrogenesis shown by upregulation of the expression of chondrogenic markers. Bone marrow mononuclear cells (MNC) was also loaded and 8 weeks postimplantation had a macroscopically cartilage-like appearance (Martínez Ávila et al. 2015). Alginate acts as a crosslinker to increase gel viscosity. It improves the scaffold structure and guarantees shape to be maintained during the process (Engineering and Wood 2017). Taking into account the importance and potential application of sodium alginate with nanocellulose as cartilage tissue scaffold (nasal and articular cartilage), one of new challenges is to understand how crosslinking and sterilization methods can affect structural and mechanical properties of nanocellulose-based hydrogels, contemplating cellulose nanofibrils, cellulose nanocrystals, or a blend of the two. So, recently the effect of crosslinking – using calcium chloride (CaCl2) – on the structural and mechanical properties of AVAP® produced was evaluated (Al-Sabah et al. 2019). American Process Inc.’s patented AVAP ® technology produces cellulose nanocrystals (CNC); cellulose nanofibrils (CNF); and hydrophobic, lignin-coated varieties of CNC and CNF directly from woody and nonwoody biomass (Nelson 2014). The mechanical properties of the hydrogels were mildly affected by the sterilization method, apart from the chemical sterilization using ethanol that yielded significantly stronger hydrogels, possibly due to the dehydration. Whereas UV and ethanol sterilization have shown roughly similar pore sizes in all NC-based hydrogels not affecting the porosity. All sterilization methods did not significantly affect the stiffness of NCB hydrogels, but in contrast the stiffness of CNC was affected independent of the sterilization method used (Sulaeva et al. 2015). In 2001 bacterial cellulose (BC) was molded into tubular form with diameter 43% branching through α-(1!3) linkages are water insoluble exhibiting non-Newtonian pseudoplastic behavior (Kothari et al. 2014). The molecular weight of dextrans can vary from 3 kDa to 50 105 kDa. Generally, enzymatic synthesis using purified dextransucrase is applied to obtain dextrans with specific molecular mass. Dextrans with Mw > 2 103 kDa behave as an expandable coil and are utilized mostly in food industry as gelling, viscosifying, texturing, and emulsifying agent, also in photo film manufacturing, cosmetic, paper, petroleum, and textile industries. Commonly, dextrans with lower molecular weight
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are more rod-like and used mainly in medicine (as blood plasma substituents, anticoagulants, prebiotics, in treating wounds) (Cote and Ahlgren 2000; Kothari et al. 2014).
3.7
Others Bacterial Polysaccharides
Curdlan is a non-charged extracellular β-(1!3)-D-glucan (Fig. 10), produced mainly from Alcaligenes faecalis, and also from some Rhizobium, Cellulomonas, and Agrobacterium strains (Morris and Harding 2009). It is a high molecular weight polysaccharide (Mw > 2.0 106 Da) with a granule shape, forming a similar to starch structure. Curdlan produces gels at both high and low temperatures, as well as after treatment with alkaline solutions. The formed gels have a texture that combines the resistance of agar and the elasticity of gelatin. Curdlan finds application in various industries: in the food industry as a gelling agent, a texture modifier, a thickener, a stabilizer and fat replacer; in regenerative medicine as a scaffold for bone and tissue regeneration (Zhang et al. 2020). Hyaluronic acid is a linear high molecular weight mucopolysaccharide consisting of alternating N-acetyl-β-(1!3)-D-glucosamine and β-(1!4)-Dglucuronic acid residues. It is a naturally occurring biopolymer, found in skin, joints, and vitreous fluid of animals and humans. However, the production of hyaluronic acid by animal organisms has some disadvantages such as the high cost, the long extraction time, and the high protein contamination. Some biotechnological methods for obtaining hyaluronic acid by Staphylococcus, Pasteurella, and Cryptococcus bacterial strains have been developed, which are preferred for industrial production. Hyaluronic acid is characterized by biodegradability, biocompatibility, viscoelasticity, and low immunogenicity, which makes it a suitable polymer for bioprinting, wound healing, tissue engineering, formulations with modified release, etc. (McCarthy et al. 2019a,b; Morris and Harding 2009). Pullulan is an extracellular, non-charged, linear α-D-glucan found in fungus Aureobasidium pullulans. It is composed of maltotriose units with the following structure: [!6)αDGlcp(1!4)αDGlcp(1!4)αDGlcp(1!]n. Depending on the method of production, the molecular weight of pullulan can vary between 10 kDa and 25 103 kDa (Cote and Ahlgren 2000). Pullulan is a water-soluble polymer, which forms clear and viscous hydrogels. It is approved by FDA as a biocompatible, biodegradable, nontoxic, and non-immunogenic polymer and it is used widely in regenerative medicine, cosmetics, and food industry. Nanocomposites Fig. 10 Chemical structure of curdlan. (Source: Adapted from Zhang et al. 2020)
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with pullulan and hydroxyapatite have been successfully applied for bone tissue regenerations (Della Giustina et al. 2019).
4
Use of Polysaccharides As Bioink
4.1
Cellulose Applications
Three-dimensional bioprinting is an emerging technology that has become an increasingly attractive solution to meet the demand in organ replacement and tissue engineering (Tai et al. 2019). It enables the precise control of composition, spatial distribution, and architecture of biomimetic, volumetric tissues. The additive manufacturing technique requires a selection of multicomponent bioink that exhibits both the rheological properties (i.e., viscosity and shear thinning) and biocompatibility to be able to mimic an extracellular matrix for the cells to grow and thrive (Jiménez et al. 2019; Tai et al. 2019; McCarthy et al. 2019a,b). Bioink composites would exhibit both liquid and solid-like attributes to be able to form hydrogel scaffolds, which could also promote the growth and survival of embedded cells (Tai et al. 2019; Jiménez et al. 2019). One of the most abundant, accessible, affordable, and practical polymers used in bioink fabrication is cellulose. The plant-sourced renewable biopolymer is further processed to form highly ordered cellulose nanomaterial. These nanocellulose-derived polymers have proven to be viable for bioprinting applications due to the high surface area to volume ratio, mechanical strength, durability, and modifiable chemical structure – allowing versatility in the ink application. These nanomaterials vary in origin and hierarchical organization leading to differences in the final form, such as cellulose nanofibers (CNF), cellulose nanocrystals (CNC), and bacterial nanocellulose (BNC) (Moniri et al. 2017; Azeredo et al. 2019). Cellulose fibers treated with mechanical shearing processes such as grinding and high-pressure mixing lead to microfibrils (also termed, nanofibers). These fibers can also be obtained by chemical treatments such as acid hydrolysis, enzymatic reactions, and oxidation reactions on extracted raw pulp (Moniri et al. 2017; Azeredo et al. 2019). This would break the interfibrillated hydrogen bonds and form fibers in micrometer size length and nanometer size width. Cellulose nanocrystals are whisker-shaped or rod-like particles with dominant crystalline regions obtained by acid hydrolysis of all noncrystalline regions, leaving the remaining crystals in nanometer dimension. Finally, bacterial nanocellulose is considered the purest form of cellulose and is synthesized by several microbial genera including the well-known Acetobacter in culture medium (Börjesson and Westman 2014). Cellulose nanocrystals (CNC) has been incorporated into bioink composites to improve the mechanical strength and shear thinning behavior of the ink (Jessop et al. 2019; Müller et al. 2017). In a study by Wu et al. (2018), a hybrid bioink of alginate and cellulose nanocrystals was used to construct a liver-mimicking construct. The polymer composite was mixed with trypsinized fibroblast and human hepatoma cells. The cell-laden ink was then extruded through a nozzle and molded into a
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slab, which was further cross-linked in CaCl2 bath in supplemented CaCl2 medium. Results showed minimal cell death and demonstrated the composite would be a good potential bioink. Certain highly biocompatible and cell-growth enhancing polymers exhibit poor viscosity properties and structural integrity; with the addition of nanocellulose material the overall rheology of the ink would change (Table 1). In another study by Shin et al. (2017), inclusion of CNFs in a gelatin methacrylamide bioink demonstrated an increase in mechanical stability allowing the printing of nose and ear constructs (Fig. 11). The structural integrity of the hydrogel network was maintained for over 7 days while absorbing culture medium. Fixed concentrations of gelatin methacrylamide and variable concentrations of CNF (ranging from 0 to 2.0% w/v) showed that an increase in the compressive modulus of the resulting hydrogels was directly correlated to the increase in the CNF concentration. The incorporation of nanocellulose materials in bioink has not only improved mechanical integrity of the scaffolds but has improved biochemical and biophysical behavior of ECM to promote the interaction of cells and its environment. Cellulose nanofibers have been used in combination with other polymers to form scaffolds that are physically and chemically tuned to be biocompatible, capable of vascularization, and scalable in production. Cytotoxicity tests done in an Table 1 Description of the three types of nanocellulose Cellulose nanofibers (CNF)
Cellulose nanocrystals (CNC)
Fabrication Acidic or enzymatic treatment before or after High pressure homogenization Grinding microfluidization
Microscope images
Dimensions 5–20 nm width, varying length in μm
TEM suspensiona
Acidic hydrolysis of wood pulp Enzymatic hydrolysis
5–70 nm width, 100–250 nm in length (from plant cellulose), 100 nm to several μm from other sources
TEM suspensionb Bacterial nanocellulose (BNC)
20–100 nm width, varying length in μm
Metabolic by-product of microbial strains in the presence of a low-molecular weight sugars, alcohols, oxygen, and low pH SEM surface fibers a
Source: Images adapted from Wu et al. (2018) TEM image of CNF and bChen et al. (2013) TEM image of CNC reprinted with permission of ©SpringerNature
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1.
Viability of hNCs (%)
2.
A 100 80 60 40 20 0
Before Before embedding printing
After printing (day 1)
After printing (day 7)
Fig. 11 (1) Cellulose composite bioink used to print ear constructs that were later cross-linked and B. (before) and C. (after) squeezing; (2) Cell biocompatibility on printed structures with dead (red) and live (green) cells B. (before) and C. (after) hNC (human nasoseptal chondrocyte) cells were cultured for 1 day while D. and E. images depict 4x and 10x magnifications after 7 days of culture. (Source: Adapted images borrowed from Markstedt et al. (2015) reprinted with the permission of © American Chemical Society)
investigation by Markstedt et al. (2015) showed that bioprinting with human nasoseptal cells was successful after the homogeneous distribution of the cells in both nonprinted and 3D bioprinted constructs. The bioink was composed of nanofibrillated cellulose and alginate mixtures, which were cross-linked by CaCl2 after careful deposition. After printing and cross-linking, the printed structures
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maintained their shape and resembled the 3D human printed ear and sheep meniscus. Multiple studies have shown that with the versatility of nanocellulose as a composite in bioink, the application must be considered for the successful proliferation and differentiation of the cells. Factors such as porosity and thickness would influence the diffusion of cells and nutrients throughout the scaffold. Furthermore, the anchoring ability of cells onto the surface of the material is also an important feature. Complex structures bio-inspired by nature allows the subsequent modification of the material for specific applications. Bacterial nanocellulose is recognized for its unique origin and high purity compared to its CNF and CNC counterparts. The microbial nanofibers have demonstrated potential in more specialized applications for biomimetic and stimuliresponsive constructs, which can be used to model drug delivery systems or simulate bacterial resistance. Inclusion of bacterial cellulose in bioink composites is being continuously studied for innate features of the micro/nanofibers that can provide the microporous environment required for cell proliferation and structural reinforcement. Huang et al. (2019) conducted a study testing the bioink potential of extracted silk derivatives together with bacterial cellulose nanofibers (BCNF) synthesized using A. xylinum strains (Fig. 12). Scaffolds were synthesized with predetermined amounts of silk, BCNF, glycerol, and genipin. This study also conducted subcutaneous implantation on mice under the dorsal skin for 4 weeks. Results showed that bioink with incorporated BCNF improved the degradability of the scaffolds and therefore the tissue response as the cells differentiated. This material also showed to
(a) Hydrogel ink preparation & 3D printing Composite SF hydrogel ink
BCNFs
3D printed hydrogel scaffolds
Gelatin Freeze drying
3D printing
(b) Crosslinking Ethanol, 30 minutes
Genipin, 24 hours
Rinsining with water
(c) Subcutaneous implantation
Rinsining with water
Incision
Scaffolds under neath the skin
Fig. 12 Schematic diagram of the composite preparation of SF/gelatin-BCNF mixture to form hydrogel scaffolds followed by subcutaneous implantation of the 3D bioprinted construct. (Source: Huang et al. (2019) reprinted with the permission of © Elsevier)
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trigger moderate acute inflammatory tissue response during the initial implantation mimicking the bio-responsive feature of this material. The resulting porous hydrogel also allowed cells to infiltrate and further accelerate the regeneration of new tissues within the scaffold. This proved that BCNF plays a unique role in providing the structural integrity and mechanical features for cells to successfully differentiate and form tissues that follow biochemical and biophysical cues. Nanocellulose materials have demonstrated to be high quality nanocomposites for the improvement in printing ability, mechanical strength, and cell compatibility. Not only is this material sustainable, environment-friendly, affordable, and nontoxic, but the adaptability of the simple structure of cellulose holds much more potential in further functionality. A range of cross-linking methods can be applied to the glucanmodified units to not only meet the requirements of 3D bioprinting technology, but to cater to the environment-responsive cells. While nanocellulose has been well explored as a material, its potential as a bioink composite in more specialized applications for larger-scale tissue engineering is still being continuously studied.
4.2
Alginate Applications
Alginate is a naturally sourced polymer extracted from brown seaweed, which consists of linear copolymers of α-D-mannuronic acid (M residue) and β-L-guluronic acid (G residue) linked by 1,4-glycosidic bonds (Tai et al. 2019). The distribution and length of the corresponding M and G residues would differ, attributing to the unique physiochemical properties of the alginate. Algal genera such as Laminaria, Macrocystis, Ascophyllum, and Ecklonia to name a few, are responsible for the formation of the unbranched polysaccharides in their cell walls (McCarthy et al. 2019a). This biopolymer is preferred over other polymers for its accessibility, low toxicity, slow biodegradability, and hydrogelation for 3D bioink composites. Alginate is one of the most popular polysaccharides used in 3D bioprinting, which requires a fast gelation process. This is achieved by alginate solutions forming ionic interchain bridges in the presence of multivalent cations. The hydrogel formation of alginate with the addition of calcium or magnesium ions is said to form an “egg box” structure where the ions bind guluronic acid and mannuronic acid blocks (Wan et al. 2009). Researchers have exploited the simple structure of alginate through the extraction and organization of these blocks to engineer alginate for use in specialized applications. Blocks composed of consecutive G residues and M residues intertwine with alternating M and G residues varying in length and sequence. G blocks increase the gel-forming potential while MG and M blocks contribute to the flexibility of the biomaterial (Lee and Mooney 2012). Depending on the bioprinting method used, bioink suitable polymers require a certain rheology and degradative profile for the successful culture and proliferation of seeded cells. Molecular weight of the alginate, alginate concentration, and cell density would contribute to the overall rheology of the composite. An increase in molecular weight can lead to a highly viscous alginate solution, which is undesirable and difficult to process. More importantly, high viscosity can lead to damaging cells or proteins embedded in the solution due to shear forces while extruding through a
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nozzle. A study by Freeman et al. (2017) demonstrated that by maintaining a low viscosity while achieving a desirable elastic modulus can be done by mixing both high and low molecular weight alginate polymers (Fig. 13). Relative to other polymers, alginate has a facile printing and handling method while providing a layer of protection around the cells it encapsulates. Furthermore, a factor that influenced the mechanical properties of the alginate bioink post-print was the young’s modulus where a study found that with an increase in cross-linker used (i.e., CaSO4, CaCO3, CaCl2) a positive correlation was observed (Fig. 13). Varying alginate to cross-linker ratios showed an increasing trend in stiffness and mechanical integrity. This addressed the problem of pure alginate solutions failing to maintain structural integrity after printing layer-by-layer scaleups because of the low phase changing temperature and shear thinning characteristic of the polymer. A plethora of studies have proven the necessity to mix alginate with other polymers to form composites that would meet the prerequisites of bioink status. Table 2 describes the various bioink formulations that catered to specific applications, which allowed for the successful proliferation and viability of cells post-print. Despite alginate hydrogels’ ease of use, the polymer had an additional downside including its limited degradation. The degradation rate of the polymers would contribute to the overall integrity and mechanical strength of the printed 3D constructs. This would also have a factor in the cell spreading, adhesion and proliferation of cells seeded in the constructs. A group of researchers found that when collagen or agarose was incorporated into the alginate solution, the mechanical strength and, therefore, bioactivity of the bioinks improved (Yang et al. 2018). Similarly, this was reaffirmed by Luo et al. (2020) in their study on gelatinalginate composites modified by cellulose nanofiber, resulting in thermal-responsive prints and observable ECM accumulation applicable for cartilage tissue applications. Formulated bioinks are not only applicable to either vascular, bone, or cartilage tissue, but are tunable and versatile in the addition of growth factors and bioactive compounds. Alginate-based materials continuously evolve in complexity by the enhancement of the marine-sourced polymer through chemical modification and conjugation of its structure. Alginate used along with other polymers only widens the different possibilities and resulting effects of the combined features the materials bring. Together with the fast-paced development of 3D bioprinting technologies, the versatile material is said to pave the way for revolutionized bioink-ready polymers.
4.3
Other Examples
Although bioinks composed of cellulose and alginate have proven to be the most popular in recent bioprinting studies – a plethora of naturally occurring polysaccharides have yet to be fully studied in their potential as a bioink component. Polysaccharides such as xanthan, gellan, dextran, and hyaluronic acid are just a few of the many bacterial polymers employed by researchers today. With the larger array of
Fig. 13 Examining the printability of (c) alginate-based bioinks. Graphs comparing ratios of (a) high molecular weight alginate and (b) low molecular weight alginate to cross-linkers. (Source: Reproduced from Freeman et al. (2017))
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Table 2 Alginate-incorporated bioink for 3D bioprinting applications Cross-linking technique
Ionic CaCl2,CaCO3 CaSO4
Covalent
UV
Composition Alginate-gelatin
Cells used Human corneal epithelial cells (hCECs)
Alginate-gelatin mixture with cellulose nanofiber (CNF) Nanofibrillated cellulose with alginate Alginate with collagen (type I) or agarose Alginate with chitosan; double cross-linked with HCl solution Methacrylatedalginate /PEG
Rabbit fibrochondrocyte cells (rFCs)
Norbornene methylamine conjugated alginate/LAP
Effect High printing resolution, cell viability, good mechanical properties Cell viability maintained post print, ECM accumulated High survivability of cells post-print
Reference Wu et al. (2016)
Improved mechanical strength
Yang et al. (2018)
Human corneal epithelial cells (hCECs)
Mechanically strong, higher strain in compression tests
Liu et al. (2018)
(mouse) Calvaraia osteoblast precursor cells (MC3T3) Adriamycinresistant cells (L929)
Enhanced proliferation, cell attachment improved
Samorezov et al. (2015)
Alginate printable at low conc., range of mechanical properties
Ooi et al. (2018)
Induced pluripotent cells (iPS) Chondrocytes
Luo et al. (2020)
Nguyen et al. (2017)
materials to choose from and a diverse set of cross-linking mechanisms, the possible formulations of bioink are exponential. Xanthan gum (XG) is an exopolysaccharide of the microorganism Xanthomonas campestris. Found to be soluble in cold water, xanthan gum solutions exhibit unique pseudoplastic behavior and synergistic interactions with a class of compounds called galactomannans. As described above, XG is made up of a cellobiose backbone with repeating unit and side chains of a trisaccharide: D-mannose (β-1,4), D-glucuronic acid (β-1,2), and D-mannose. These side chains are attached to alternating glucose residues through an α-1,3-linkage. The branched structure of the gum polymer is capable of building not only physical networks, but also chemical networks. XG is a suitable bioink composite due to its range of cross-linkers with itself using adipic acidic dihydrazide or citric acid, for example. These cross-linking mechanisms compensate for the lack of form and shape fidelity in stand-alone solutions. Xanthan gum also offers “softness” and flexibility allowing for the elasticity and spatial requirement for cells to proliferate. Although not as popular as its counterparts, xanthan gum has been increasingly studied due to its interactions with other
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polymers to provide potential microenvironments for cells to grow and differentiate. Not only is this material versatile in its in vivo applications but is also stable under hydrolytic and enzymatic conditions (Petri DFS 2015). Gellan gum (GG) is a water-soluble anionic polysaccharide excreted by the microorganism Sphingomonas elodea also known as Pseudomonas elodea. At an industrial scale, the polymer is fermented and can result in two forms: one with high acyl content and one with low acyl content. Like alginate, gellan is nontoxic and if injected into tissues, immunogenic. Despite its weak structural integrity, it is also used for cell encapsulation and drug delivery systems. In terms of bioprinting, the inclusion of gellan in a bioink composite is found to be more biocompatible and highly favorable for the recognition, adhesion, and eventual proliferation of cells. In a study by Koivisto et al. (2019), a bioink composite made up of gellan gum and gelatin was used to mimic the biochemical attributes of the extracellular matrix. GG can provide the flexibility, structural support, and hydrogelation properties while gelatin allowed for cell attachment to occur. These GG-gelatin hydrogels were found to be successful through the “beating” of the human cardiomyocytes measured over 24 h using a phase contrast video software. Dextran is a bacterial hydrophilic polysaccharide composed of α-1,6-linked D-glucopyranose widely used in the pharmaceutical and biomedical field due to its biocompatible and nontoxic features. Like other polysaccharides, dextran can be chemically modified to improve the degree of networking within a construct. A study by Pescosolido et al. (2011) demonstrated that by combining the viscoelastic and favorable biochemical features of hyaluronic acid and a chemically modified dextran derivative, a bioink with the correct viscosity resulted in a printed scaffold that retained the desired shape and design. The scaffold was also found to have high porosity and tunable fiber spacing/orientation. High porosity would permit the diffusion of nutrients and waste products as cells undergo their usual biochemical pathways to proliferate. Glycosaminoglycans (GAGs) are no strangers to the biomaterial field. This class of naturally sourced polymers is reputable for its role in significantly increasing bioreactivity and biocompatibility as an additive in bioink composites. One GAG is hyaluronic acid (HA) also known as hyaluronan. It is a polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine. Found in the ECM of cells, HA is known to exhibit distinguishable biocompatibility and biodegradability properties. The polymer can also be degraded in the presence of enzymes such as hyaluronidase, β-glucuronidase, and β-N-acetyl-glycosaminidase into smaller molecular weight compounds. HA can be used as an additive in bioprinting to control the viscosity and bioreactivity of the bioink. Although HA is known to be highly water-soluble leading to printed constructs with weak infrastructure, researchers have found ways to make HA more hydrophilic by conjugating hydrophobic moieties along the chain using cross-linking techniques. Another method is to reinforce HA (Fig. 14) by conjugating methacrylate groups, added in the presence of a photo initiator, followed by UV-exposure that would cause for a denser network to form (Poldervaart et al. 2017). Recent studies require even more research on the effects of these anionic polysaccharides due to their valuable inherent characteristics that play important
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Fig. 14 Images of MeHA (methacrylated hyaluronic acid) printed constructs followed by UV-treatment; porous cubes (first and second row) and non-porous vertebrae shapes (third and fourth row) using designs on the left pane (scale bar ¼ 500 μm). (Source: Borrowed from Poldervaart et al. (2017) with the permission of PLOS ONE)
roles in influencing angiogenesis and/or inducing biochemical processes to occur. Despite the shortcomings, studies carried out have proven that with modifications to the side chains of these polymers, the material is built more suitably as a component in a bioink formulation successfully meeting the delicate balance between tolerance of bioprinter shear strains and biochemical adaptiveness for the nurturing of stem cells.
5
Conclusion
The use of microbial polysaccharides in 3D printing applications and notably in regenerative medicine and food engineering revealed their potential in literature but not really in industry where these applications are still primitive. The development of « intelligent polysaccharides » with specific properties such as antibiofilm or other biological activities as that of printing technologies will probably open the way to a commercialization of microbial polysaccharide-based biomaterials in high value markets of tissue engineering and regenerative medicine (Tai et al. 2019). Indeed, the requirements of regenerative medicine for biocompatible hydrogels is probably
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the greatest opportunity for 3D printing of microbial polysaccharides (McCarthy et al. 2019a,b; Tai et al. 2019). Very recently, Tai et al. (2019) described the high potential of anionic microbial exopolysaccharides such as β-(1,4)-polyglucuronic acid (glucuronan) from Sinorhizobium meliloti M5N1CS. This full anionic cellulose mimetic with high molecular weight (more than 500 kDa) has very good gelling properties allowing the production of thermostable hydrogels in the presence of divalent cations (Ca2+, Cu2+, and Ba2+) with potential in skin regeneration (Tai et al. 2019). Consequently, as frequently performed with alginate, this anionic polysaccharide (glucuronan) and derivatives could constitute the new generation of bioink for 3D bioprinting development in regenerative medicine. Moreover the development of the molecular gastronomy always at the research of new textures after physical and chemical transformations of ingredients is a good opportunity for microbial polysaccharides having unique rheological properties and, for some of them such as gellan, curdlan, or xanthan, the status of food additives. Another future opportunity for microbial polysaccharides could be in a next future the emergence of 4D printing technology. Four-dimensional printing was introduced in 2013 in several research areas including biomedical applications and notably tissue and organ regeneration (Ge et al. 2013). It consists of building 3D structures incorporating in them materials able to self-transform after printing depending on external stimuli and physicochemical environment parameters, leading to memory materials. The controlled degradation of 3D printing architectures could be, in this way, classified also as a 4D effect. Recently, a hydrogel composite ink composed of stiff cellulose and acrylamide was successfully printed and led to a biocompatible hydrogel swelling readily in water. The swelling behavior was under the control of cellulose fibrils alignment. This material can be assimilated to a plant cell wall-inspired architecture changing shape depending on immersion in water (Gladman et al. 2016). Native or modified microbial polysaccharides are well known for their ability to be used as smart materials and several studies have been published focusing on this subject. Their implementation in 4D printing strategies could easily lead to the obtaining of controlled biodegradable hydrogels sensitive to external stimulus including magnetic fields, humidity, temperature, pH, light, and others (Tamay et al. 2019). Some of these polysaccharides have been always described and their production is under control. For example, a family of smart water-soluble pullulan, xanthan, and hyaluronan with (thermo)-associative properties influencing by polysaccharide concentration, or salinity, have been described (Hamcerencu et al. 2011; Mocanu et al. 2011; Niang et al. 2016).
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Chitosan-Based Gels for Regenerative Medicine Applications
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Deepti Bharti, Bikash Pradhan, Sarika Verma, Subhas C. Kundu, Joaquim Miguel Oliveira, Indranil Banerjee, and Kunal Pal
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Mechanism of Chitosan Gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Physical Gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Self-Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Radical Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Ionic Gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Cryogelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Application of Chitosan Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Chitosan Nanogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Chitosan Microgel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Chitosan Macrogel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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D. Bharti · B. Pradhan · K. Pal (*) Department of Biotechnology and Medical Engineering, NIT, Rourkela, India S. Verma Council of Scientific and Industrial Research Advanced Materials and Processes Research Institute (CSIR-AMPRI), Bhopal, India S. C. Kundu · J. M. Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, Barco, Guimarães, Portugal ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected]; [email protected] I. Banerjee Department of Bioscience and Bioengineering, IIT, Jodhpur, India © Springer Nature Switzerland AG 2022 J. M. Oliveira et al. (eds.), Polysaccharides of Microbial Origin, https://doi.org/10.1007/978-3-030-42215-8_65
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Abstract
The tissue repair and regeneration process often require implants that can help in the healing of damaged tissue. A naturally obtained polysaccharide chitosan has properties like processability, biocompatibility, biodegradability, and nonimmunogenicity. The presence of an amino group and hydroxyl group provides the scope of many functionalizations to the chitosan. Chitosan gels (nano, micro, and macro) can be prepared either by physical, ionic interactions, or chemical methods. Poor solubility of chitosan is sometimes held responsible for its limited application. Many strategies have been adapted for the fabrication of gels made up of chitosan and its derivative to overcome this limitation. Chitosan gels have been explored by researchers for several applications in the arena of regenerative medicines. The gels of chitosan are having pH sensitivity, temperature sensitivity, and ion-sensitivity behavior. They have been used as a carrier for bioactive molecules, proteins, peptides, and living cells which can aid in the healing of damaged tissues and have been successfully tested in the in vitro and in vivo models. Chitosan gels have the potential to provide an appropriate microenvironment for the living cell to grow and proliferate. A vast number of chitosanbased gels, viz., nanogels, microgels, and macrogels, and their gelation mechanism have been discovered for bone, cartilage, skin, tissue regeneration, which has been discussed in detail in the chapter. Keywords
Chitosan · Crosslinking · Nanogel · Microgel · Macrogel · Regeneration
1
Introduction
The use of gel is extensive and growing rapidly in various filed at the present time. The growing application of gels in the field of regenerative medicine is appreciable. In this regard, the gels can act as a substitute of extracellular matrix (ECM) for a variety of tissue repair and regeneration and can facilitate targeted therapeutic delivery. Based upon the unit size of network, gels can be classified as nano (1–100 nm)-, micro (0.1–700 μm)-, and macro (>1 mm)-gels. Micro- and nanogels have been explored for drug delivery in quite many cases. A few of the crucial requirements of the biomaterials used for any such application will be biocompatibility, biodegradability, and nonimmunogenicity. Since most of the naturally derived macromolecules possess all these features, researchers’ attention in exploring different natural polymers is rising with time. Starch, alginate, cellulose, and chitosan are a few of the commonly utilized polysaccharides obtained from plants, microorganisms, and insects (Takada and Kadokawa 2015). These naturally occurring polysaccharides usually act as an energy provider and structural component for functional bio-based materials. The intra- as well as intermolecular hydrogen bonds present among the hydroxyl group of saccharides are often responsible for their low
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solubility, further assisting in gelation. Chitosan, chemically (1-4)-2-amino-2deoxy-β-d-glucan, is one of the most explored naturally obtained linear polysaccharides. Chitosan can be derived from chitin’s deacetylation, which is an abundant polysaccharide and is extensively found in the exoskeleton of crustaceans. Apart from crustaceans’ shells, chitosan can also be obtained from fungi and insects’ cell walls (Fig. 1). The deacetylation can occur either by enzymatic reaction or under the hot alkali treatment (Pal et al. 2013). Structurally, chitin is made from the glycosidic linkage of N-acetyl-glucosamine (NAG) and N-glucosamine. The period of alkali deacetylation governs the degree of deacetylation plus molecular mass of chitosan. Therefore, chitosan can be defined as a shared term used for de-N-acetylated chitin with diverse degrees of deacetylation, molecular weight, and viscosity. The percentage deacetylation of the commercially available chitosan is nearly 60–100%. The broad scope of functionalization of chitosan comes from the three reactive groups present in them, that is, an amino group, a primary hydroxyl group, and a secondary hydroxyl group (Fig. 2). These groups are located at second, third, and sixth carbon positions, respectively. By these functional groups, chitosan can undergo many modifications like acylation, alkylation, Schiff base formation, carboxymethylation, and others (Shi et al. 2006). Due to the free amino group’s ionization at lower pH, chitosan’s dissolution is possible at the pH value lower than its pKa (~6.5). The presence of an amino group makes
Fig. 1 The schematic diagram indicates the different sources of chitosan in nature
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Fig. 2 Functional group responsible for chitosan modification
chitosan a cationic polyelectrolyte. The possibility for chitosan to interact with polyanions is widely used to develop macro-, micro-, and nanogels. Similarly, the chitosan’s mucoadhesive property is contributed by the free amino group by which they form bonds with mucin(negatively charged), present in the mucosal layer of the humans. The cationic nature of chitosan gives an opportunity to interact with anionic glycosaminoglycans (GAG) existing in the extracellular matrix of human tissues (Iovene et al. 2021). Along with its cationic characteristics, nontoxicity, biodegradability, biocompatibility, antibacterial, anti-inflammatory, and antimicrobial activity are a few other associated properties of chitosan (Kravanja et al. 2019). Chitosan gels have shown much successful application in the field of regenerative medicines and tissue engineering. They act as carriers for various bioactive/ pharmacological molecules at the same type as crucial scaffold components for successful tissue regeneration. The biodegradability products of chitosan have shown safe interaction in in vivo and in vitro conditions (Pang et al. 2017). Chitosan gels can be added as a scaffold to modify the inner structure, texture, and mechanical property, which are acute for cell growth and proliferation. These gels are reported to have special self-healing features, pH sensitivity, temperature sensitivity, and ionic sensitivity. The application and benefits of chitosan gels offer a broad possibility of tissue regeneration which is witnessed in wound healing, gene therapy, and tissue engineering (Sultankulov et al. 2019; Shi et al. 2006). Similarly, a group of pH/thermoresponsive injectable gels of chitosan has been majorly studied for the regeneration of bone and dental tissue (Tang et al. 2020). This chapter has summarized the gelling concepts of gels made up of chitosan and its derivatives. Further, the application of these gels in regenerative medicine as promising vehicles for drug delivery, cell therapy, and tissue engineering has been discussed in detail.
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Mechanism of Chitosan Gelation
In recent times, different methodologies for the development of chitosan gels have been adapted. These methods can be classified as physical gelation, self-assembly, ionic gelation, polymerization, coacervation, and cryogelation. The methodology for the fabrication of chitosan gels will differ based on mechanism, reaction conditions, and characteristics which are being discussed in the subsequent section.
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Physical Gelation
The physical gelation method of synthesizing chitosan gels is interesting because it does not involve using an emulsifier, crosslinker, or chemical for inducing gelation. The conversion from solution to gel is a highly tunable process that can be achieved by altering the physical parameters such as pH, temperature, and ionic strength. The van der Waals force and electrostatic interactions are the two vital forces that are involved with the physical gelation of chitosan. One of the approaches to form a physical gel with physical gelation is to perform controlled re-acetylation of a fully deacetylated chitosan (Sacco et al. 2018). The formation of chitosan gels through this method occurs in a series of steps. The multifaceted approach and the need for highly specialized equipment have led to a limited number of studies where chitosan gels have been prepared using this method. The re-N-acetylation occurs by using acetic anhydride, which is responsible causes acetylation of chitosan at amino and hydroxyl groups. Here, the proposed gelation mechanism is the molecular aggregation among the acetylated chitosans, which forms a rigid, colorless, and transparent gel. Studies suggest that an increase in the temperature, the concentration of acetic anhydride, and chitosan aid in gelation (Montembault et al. 2005). The formation of gels can also be enhanced in few other possible ways. Firstly, an alteration in the solubility of chitosan can promote polymer-polymer interactions. This can be possible if the repulsive interaction between the chitosan chains can be condensed and achieved by lowering the charge density of chitosan. It has been proposed the application of gaseous ammonia that raises the pH of chitosan solution to 9, which decreases the charge density of the chitosan chains by the deprotonation of amino groups. This phenomenon results in the initiation of the gelation of chitosan molecules (Wang et al. 2017). The chitosan gels, formed from gaseous ammonia to induce physical gelation, have shown regenerative potential in the case of myocardial infarction (Fiamingo et al. 2016). These gels were found to be proper for cardiac tissue engineering and did not display any toxicity. Secondly, the concentration of the chitosan should always be greater than the chain entanglement concentration. The presence of the chitosan molecules above this critical concentration promotes improved networking of the polymeric chitosan chains (Brunel et al. 2008). An extension to the above work was performed by Brunel et al. (2013). They have successfully loaded copper in chitosan gel using the above technique. The chitosan gel thus fabricated was explored for the antimicrobial role (Brunel et al. 2013). In the typical pH range of 4 and 6, chitosan behaves like polyelectrolytes where they can make bonds with oppositely charged small ions like Cu2+. Altogether, chitosan physical gels are an attractive system due to their low toxicity and easy tunability.
2.2
Self-Assembly
Gels formed by the self-assembly of chitosan have shown great potential as delivery systems with better encapsulation and precise release of bioactive compounds (Debele et al. 2016). Self-assembly of chitosan occurs by forces like ionic bonding,
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hydrogen bonding, van der Waals interaction, and covalent interaction. Researchers have explored many organic acids, proteins (e.g., bovine serum albumin), peptides, and salts to form self-assembled chitosan structures. As mentioned above, chitosan readily interacts with the acids like ethylenediaminetetraacetic acid, benzoic acid, caffeic acid, and methacrylic acid by creating an amide group in the middle of the amine group of chitosan and the carboxylic group of acids (Wang et al. 2017). Likewise, peptides and larger globular proteins can form self-assembled chitosan nanogel via electrostatic interaction. The significant parameters of consideration during the formation of these gel are the concentration of chitosan, surface charge density, particle size distribution, and turbidity (Rusu et al. 2020). Therefore, these factors are also known as quantitative functional parameters of the formed gel. The fabrication of chitosan gel via this method is more straightforward and convenient. Simultaneously, unlike the physical gelation of chitosan, there is no dedicated step required for the separation of formed gel in this case. A pH-sensitive gel was prepared with the self-assembly of chitosan and ovalbumin (Yu et al. 2006). The core of the nanogel is formed owing to the crosslinking of chitosan with ovalbumin. Even though the gels, fabricated using this method, have come forward as a sound delivery system, their clinical usefulness must be explored thoroughly. The use of self-assembly has been evaluated with luminescent gold (Au) nanocrystals placed in chitosan gel that can be applied in developing sensors and biomedical devices. This method eases the accessibility of control and targeted release of drugs to specific environment. The photoluminescence of the gels can be improved by the use of Au nano crystal of dissimilar emission color and different thiolate ligands which is known to form complex with metal nano crystals (Goswami et al. 2016). The thiolate ligand provides the negative charge of its carboxylic group to interact with the positive amine group of the chitosan.
2.3
Radical Polymerization
A chemical process of transformation of small molecules into larger ones is called polymerization. As a prerequisite for radical polymerization, chitosan is usually modified to gain polymerizable groups. This can be achieved through the process of styrenation and methacryaltion. Styrenation involves combining chitosan with monomer like alpha-methyl-styrene. However, methacryaltion involves the modification of glycol chitosan with glycidyl methacrylate (GMA) to obtain N-methacrylate glycol chitosan (MGC). MGC is a water-soluble chitosan derivative. The polymerization can be done either by increasing temperature or exposing the chitosan solution to UV light (Brunel et al. 2013). Photopolymerization can occur by the exposure of modified chitosan to UV light in the existence of a photoinitiator (Hu et al. 2012). Despite the disadvantage arises from the use of inorganic chemicals as initiators or crosslinker in this process, it has still shown successful application in regenerative medicines and drug delivery. The process of photopolymerization starts with the free radical generated by the photoinitiator molecules when the reaction mixture is irradiated with light. The free
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Fig. 3 Radical copolymerization for the formation of chitosan nanogel. (Reproduced with permission from Duan et al. 2011a; License number: 5052591486565)
radical transforms the double bond of the polymerization functional groups of chitosan into radicals. This is marked as the beginning of the chain reaction, which propagates to form a crosslinked network. MGC can crosslink upon UV exposure and has been successfully explored for cell encapsulation and drug delivery studies (Amsden et al. 2007). Poly (N-isopropyl acrylamide) (PNiPAm) interacts with acrylic acid-functionalized chitosan derivatives via radical polymerization to form pH or thermos-responsive gel. PNiPAm has gained much attention recently as the lowest critical solution temperature (LCST) value of PNiPAm is close to the body temperature (Peniche et al. 1999). The hydrophobic interaction of PNiPAm is dominant at a temperature above LCST. At this temperature, crosslinking occurs using ammonium persulfate as an initiator of the reaction and N, N-methylenebisacrylamide as a crosslinking agent (Fig. 3) (Duan et al. 2011a). The initiator molecule helps forming sulfate anion, which interacts with the hydroxyl group of chitosan to form alkoxy radicals. In the presence of the crosslinking agent, these alkoxy radicals start the copolymerization of PNiPAm onto chitosan. Apart from the temperature and pH sensitivity characteristics, these chitosan nanogels also enhance drug activity and improve the target release. The chitosan gel developed with this method holds many applications in drug delivery and regenerative medicines.
2.4
Ionic Gelation
One of the commonly adapted fabrication methods for chitosan gel having high water content is ionic gelation. Herein, the gelation occurs between chitosan and other polymers (e.g., alginate, hydroxyapatite) decorated with functional groups of opposite charges. Several parameters, including temperature, pH, time, molecular
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mass, molar ratio, flow rate, and mixing speed, can help in the precise ruling of the dimensions of the formed gel. The technique of ionic gelation to form chitosan gel has been applied to polymer-drug interaction, the structure and function studies in tissue-specific regeneration, and the absorption/release mechanism of pharmacological compounds. Polyphosphate is often used due to its nontoxicity. Its multivalent nature readily forms ionic gels with chitosan (Pant and Negi 2018). The polyphosphate is dissolved in an acidic solution where polyanionic polyphosphate is added dropwise to obtain cationic charge chitosan (Debnath 2011). The interaction takes place amid the positive charge amino group of chitosan and negatively charged phosphate. The size of the gel can be controlled by regulating various parameters such as concentration of polyphosphate, pH, and degree of deacetylation of chitosan (Huang and Lapitsky 2017). These chitosan gels are recognized for their ability to promote the bioavailability and stability of pharmacological compounds. Chitosan and polyphosphate system is one of the most broadly explored gel systems, studied for drug delivery and tissue regeneration. These gels have been extensively studied along with other polymers and additives like alginate, polysorbate, mannose, BSA, glucosamine, etc. (Wang et al. 2017). The ionic gelation method is also examined to look for the effect of the surface charge of the gel upon absorption at the intestinal segments. Insulin is a commonly used treatment modality in the case of diabetes mellitus. A regular injection of insulin in patients results in a reduction in the quality of life. Since charged particles are quickly taken up by cells, insulin-incorporated chitosan and carboxy-methyl chitosan (CMCS) gels have been explored for this purpose (Fig. 4) (Wang et al. 2016). The weight ratio of CMC-to-chitosan is used to maintain different surface charges.
2.5
Coacervation
Coacervation is a method of phase separation between the two liquid phases. This is one of the most adapted techniques for the microencapsulation of drugs and bioactive agents. The gel size and homogeneity prepared from this method can be modified by altering the molecular weight and amount of the polymer used. The basic principle behind coacervation is the difference in the ionic forces of the polymer that can be adjusted with the knowledge of isoelectric point (pI). The process depends upon the involvement of the number of polymers and is majorly categorized into two sets: simple and complex coacervation.
2.5.1 Simple Coacervation The simple coacervation involves the use of only one polymer. In this process, the induction of phase separation of the polymer occurs by the addition of salts that have less affinity towards the polymer than the water. This can even be achieved with an alteration in the environmental conditions (e.g., pH and temperature) that can promote aggregation of the polymer chains. Simple coacervation is used to prepare
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Fig. 4 The schematic representation of chitosan(CS)/CMCs nanogel loaded with insulin. (Reproduced with permission from Wang et al. 2016; License number: 5053671234643)
crosslinked chitosan gels using epichlorohydrin or glutaraldehyde that are known to react with the amine group present in chitosan (Gonçalves et al. 2005).
2.5.2 Complex Coacervation In a system that is made up of mixed polymers, thermodynamic compatibility and incompatibility are the two possible interactions that can take place among the polymers. At a high concentration and high ionic strength, when polymers carry similar charges, a repulsive force arises between them. This is a scenario of thermodynamic incompatibility, which gives rise to phase separation. On the contrary, low concentration and low ionic strength polymers are thermodynamically compatible due to the presence of a net opposite charge. This state of thermodynamic compatibility is also called complex coacervation. As discussed earlier, the cationic nature of chitosan permits it to form an association with anionic polymers like hyaluronan, alginate, pectins, carrageenans, etc. One of the interesting chitosan coacervation systems is formed with hyaluronic acid (HA). In that case, the gelation is built on electrostatic interaction among the oppositely charged polysaccharides and hydrogen bonding (De La Fuente et al. 2008). Chitosan/HA gels, formed with such type of method,
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Fig. 5 Formation mechanism of chitosan gel using complex coacervation. (Modified from Pal et al. 2013)
have shown excellent potential to deliver drugs, therapeutic protein, and growth factors in pulmonary ailments (Oyarzun-Ampuero et al. 2009). The coacervated system developed using chitosan and alginate is depicted in Fig. 5 (Pal et al. 2013).
2.6
Cryogelation
Cryogels are fabricated at a cryogenic temperature (semi-frozen conditions), usually below the crystallization point of the solvent used for preparing polymeric solutions. The cryogenic treatment follows some basic steps that include freezing the polymer solutions, maintenance of the frozen state for a definite time, followed by defrosting. The frozen polymeric solutions are heterogeneous. This can be ascribed to the fact that the frozen solvent also consists of unfrozen liquid microphase (UFLMP) (Lozinsky et al. 2003). This phase is usually rich in gel-forming agents and thus intensely promotes the formation of the gels. At a lower temperature, the solvent (water) molecules form ice crystals that grow and combine over time. When melted at room temperature, they leave macropores that are scattered in between the polymeric chains. The three-dimensional supra and macroporous crosslinked matrix of cryogels provide structural and chemical stability to the system. A study in this regard has tried fabricating cryogels using chitosan and HA using glutaraldehyde for crosslinking in the cryogenic mixture (Fig. 6) (Kutlusoy et al. 2017). The mixture was allowed to freeze at 12 C for gel formation. The chitosan cryogel, when co-polymerized with HA, has shown improvised cell penetration. Controlling the freezing temperature is an efficient way to modulate the pores that are formed in chitosan cryogels. Thus, altering the freezing temperature can help to improvise the physiochemical properties of the prepared gels (Zhang et al. 2019). Another approach to synthesize chitosan cryogel was made by modifying the chitosan (Evans et al. 2021). Through imine bonding of chitosan’s amine group, an alkyl chain was introduced into the chitosan backbone. It helped in the improvisation of the hydrophobicity of the chitosan gel. This enhanced hydrophobicity in the cryogel
Chitosan-Based Gels for Regenerative Medicine Applications
Fig. 6 Fabrication mechanism of chitosan/HA cryogel. (Reproduced with permission from Kutlusoy et al. 2017; License number: 5058181315646)
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was useful in the adsorption of various anticancer medicines, which are hydrophobic in nature.
3
Application of Chitosan Gel
3.1
Chitosan Nanogels
Nanoparticles as a delivery vehicle in the pharmaceutical industry have gained enormous attention in the current time. With the advanced investigation in the field of nanotechnology, the importance of nano-sized distribution systems is rising rapidly in the field of regenerative medicines. These delivery systems have beneficial therapeutic effects because of their ability to upsurge the concentration of the pharmacological compounds at the damaged/ target site. As a result, the pharmaceutical compound’s systemic contact gets reduced. Further, the varied administration routes of nanoparticles, including parenteral, oral, transdermal, and pulmonary routes, support its wide usage (Islam et al. 2017). Nanogels are 3-dimensional hydrogels formed by the networking of crosslinked polymers (Pathak et al. 2019). Thus, nanogels have a combined property of nanoparticle and hydrogel. Nanogels can retain a large volume of water and can also display high surface energy. The diameter of