Functional polysaccharides for biomedical applications [First edition] 9780081025567, 0081025564

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Functional Polysaccharides for Biomedical Applications

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Woodhead Publishing Series in Biomaterials

Functional Polysaccharides for Biomedical Applications

Edited by

Sabyasachi Maiti Sougata Jana

An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2019 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-08-102555-0 For information on all Woodhead publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Sabrina Webber Editorial Project Manager: John Leonard Production Project Manager:   Joy Christel Neumarin Honest Thangiah Cover Designer: Miles Hitchen Typeset by SPi Global, India

Contents

Contributors xi About the editors xv Preface xvii 1

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Introduction to natural polysaccharides Biswajit Maji 1.1 Introduction 1.2 Structure and chemical composition 1.3 Natural occurrence and classifications 1.4 IUPAC nomenclature 1.5 Structure determination (ring size and linkage type) 1.6 Molecular modification 1.7 Conclusion References An overview on the potential biomedical applications of polysaccharides Rajalekshmy, G.P., Lekshmi Devi, L., Jasmin Joseph, Rekha, M.R. 2.1 Introduction 2.2 Biomedical applications 2.3 Gene delivery systems 2.4 Wound-healing materials 2.5 Tissue engineering and regenerative medicine 2.6 Polymer drug conjugates 2.7 Conclusion References Further reading Polysaccharide-based superporous hydrogels for therapeutic purposes Sabyasachi Maiti, Biswanath Sa 3.1 Introduction 3.2 Synthesis of SPHs 3.3 Chitosan (CS)-based SPHs 3.4 Alginate-based SPHs 3.5 Conclusion References

1 1 2 7 9 13 17 26 27 33 33 33 34 48 68 72 77 77 94 95 95 96 100 117 119 125

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Fabrication of polysaccharide-based materials using ionic liquids and scope for biomedical use Abul K. Mallik, Md. Shahruzzaman, Asaduz Zaman, Shanta Biswas, Tanvir Ahmed, Md. Nurus Sakib, Papia Haque, Mohammed Mizanur Rahman 4.1 Introduction 4.2 Selective properties of ionic liquids for dissolving polysaccharides 4.3 Mechanism of dissolving polysaccharides in ILs 4.4 Polysaccharides 4.5 Polysaccharide-based materials prepared by using ILs 4.6 Scope of biomedical applications 4.7 Future scope of polysaccharide-based materials processing with the aid of ILs 4.8 Conclusions References Further reading Polysaccharides as potential materials for the delivery of therapeutic molecules Sougata Jana, Sabyasachi Maiti, Subrata Jana 5.1 Introduction 5.2 Polysaccharides-based systems for delivery of therapeutics 5.3 Conclusion References Polysaccharide-based scaffold for tissue-regeneration Stefano Rimondo, Giuseppe Perale, Filippo Rossi 6.1 Introduction 6.2 Principles of tissue regeneration 6.3 Polysaccharide characteristics and properties 6.4 Homo-polysaccharide 6.5 Hetero-polysaccharide 6.6 Scaffold preparation methods 6.7 Summary References Hyaluronic acid as potential carrier in biomedical and drug delivery applications Vipul D. Prajapati, Pankaj M. Maheriya 7.1 Introduction 7.2 Sources, structure, and properties of HA 7.3 Chemical modification in synthesis of HA 7.4 HA and HADs in biomedical and drug delivery applications 7.5 Patents on HA

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131 133 135 138 144 159 160 161 161 171 173 173 174 183 183 189 189 190 192 193 199 208 209 209 213 213 217 223 233 250

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7.6 Conclusion References Further reading 8

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Green synthesis of polysaccharide-based inorganic nanoparticles and biomedical aspects Amin Shavandi, Pouya Saeedi, M. Azam Ali, Esmat Jalalvandi 8.1 Introduction 8.2 Polysaccharide-mediated synthesize of NP versus conventional techniques 8.3 Preparation of polysaccharide-based metal NPs 8.4 Characterization of NPs 8.5 Biomedical applications of polysaccharide-based AuNPs and AgNPs 8.6 Catalysis of polysaccharide-based AuNPs 8.7 Catalysis properties of polysaccharide-based AgNPs 8.8 Toxicity of polysaccharide-based AuNPs 8.9 Toxicity of polysaccharide-based AgNPs 8.10 Conclusion References Further reading Graphene oxide-based nanocomposites and biomedical applications Tanmay K. Ghorai 9.1 Introduction 9.2 Mechanism of graphene oxide formation 9.3 Nanographene oxide 9.4 Different structures of graphene oxides 9.5 Synthesis 9.6 Biomedical applications of graphene oxide 9.7 Other applications of GO 9.8 Conclusion Acknowledgments References

10 Biomedical applications of green-synthesized metal nanoparticles using polysaccharides Ayan Kumar Barui, Sourav Das, Chitta Ranjan Patra 10.1 Introduction and background 10.2 Biomedical applications of green-synthesized metal nanoparticles 10.3 In vivo toxicity studies of green-synthesized metal nanoparticles 10.4 Challenges and future prospects of green-synthesized metal nanoparticles Acknowledgments References

250 256 264 267 267 268 268 270 273 291 293 293 294 295 295 304 305 305 307 307 309 311 313 321 324 324 324 329 329 332 347 348 349 349

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11 Application of polysaccharides in enzyme immobilization Sadia Sharmeen, Md. Shirajur Rahman, Md. Minhajul Islam, Md. Sazedul Islam, Md. Shahruzzaman, Abul K. Mallik, Papia Haque, Mohammed Mizanur Rahman 11.1 Introduction 11.2 Enzyme and enzyme immobilization 11.3 Polysaccharides 11.4 Types of polysaccharides used for fabrication of immobilization supports 11.5 Application of polysaccharides in enzyme immobilization 11.6 Future perspective—The challenges in polysaccharide-based enzyme immobilization research 11.7 Conclusion References 12 Biosensor platforms for detection of cardiovascular disease risk biomarkers Mintu Pal, Raju Khan 12.1 Introduction 12.2 Biomarkers for early detection of CVD 12.3 Current approaches for detection of biomarkers 12.4 Conclusion and perspective Acknowledgments References 13 Modification of liposomal surface by polysaccharides: Preparation, characterization, and application for drug targeting Jaroslav Turánek, Josef Mašek, Milan Raška, Miroslav Ledvina, Ema Paulovičová, František Hubatka, Jan Kotouček 13.1 Introduction 13.2 Liposomes as drug delivery system 13.3 Bioconjugation reactions of polysaccharides 13.4 Oxime ligation for modification of liposomes by HA and mannan 13.5 Conclusion Acknowledgments References 14 Aptamer-conjugated functionalized nano-biomaterials for diagnostic and targeted drug delivery applications Vishal Das, Channakeshavaiah Chikkaputtaiah, Mintu Pal 14.1 Introduction 14.2 Aptamer: Its structure and function 14.3 Nanoparticles for targeted pharmacotherapy 14.4 Aptamer-based NPs for targeted drug delivery applications 14.5 Aptamer-small molecule conjugated systems

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357 358 366 367 372 387 388 388 397 397 399 407 421 422 422 433 433 436 441 446 460 460 460 469 469 470 472 473 473

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14.6 Aptamer-nanomaterial conjugated systems 14.7 Organic and inorganic nanomaterials 14.8 Aptamer application for targeted cancer therapy 14.9 Properties of aptamers and their biological applications 14.10 Future perspective and conclusions References Further reading

474 474 484 485 486 487 494

Index 495

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Contributors

Tanvir Ahmed Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh M. Azam Ali  Center for Bioengineering and Nanomedicine, Department of Food Science, University of Otago, Dunedin, New Zealand Ayan Kumar Barui  Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, India Shanta Biswas Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Channakeshavaiah Chikkaputtaiah Biotechnology Group, Biological Sciences and Technology Division, CSIR-North East Institute of Science & Technology, Academy of Scientific and Innovative Research, Jorhat, India Sourav Das  Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Vishal Das  Biotechnology Group, Biological Sciences and Technology Division, CSIR-North East Institute of Science & Technology, Academy of Scientific and Innovative Research, Jorhat, India Tanmay K. Ghorai  Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India Papia Haque Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh František Hubatka  Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Md. Sazedul Islam  Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

xiiContributors

Esmat Jalalvandi  School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom Sougata Jana  Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol; Department of Health and Family Welfare, Directorate of Health Services, Kolkata, India Subrata Jana  Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India Jasmin Joseph  Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, India Raju Khan  CSIR-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India Jan Kotouček Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Miroslav Ledvina  Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Prague, Czech Republic L. Lekshmi Devi Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, India Pankaj M. Maheriya Department of Formulation and Development, Ajanta Pharma Limited, Mumbai, India Sabyasachi Maiti Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak, India Biswajit Maji Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India Abul K. Mallik Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Josef Mašek Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Md. Minhajul Islam Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

Contributorsxiii

Md. Nurus Sakib  Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Mintu Pal  Biotechnology Group, Biological Sciences and Technology Division, CSIR-North East Institute of Science & Technology, Academy of Scientific and Innovative Research, Jorhat, India Chitta Ranjan Patra  Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Ema Paulovičová Department of Immunochemistry of Glycoconjugates, Immunology & Cell Culture Laboratory, Institute of Chemistry, Center for Glycomics Slovak Academy of Sciences, Bratislava, Slovakia Giuseppe Perale  Biomaterials Laboratory, Institute for Mechanical Engineering and Materials Technology, University of Applied Sciences and Arts of Southern Switzerland, Manno, Switzerland Vipul D. Prajapati Department of Pharmaceutics, SSR College of Pharmacy, Silvassa, Union Territory of Dadra and Nagar Haveli, India Mohammed Mizanur Rahman  Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh G.P. Rajalekshmy Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, India Milan Raška Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno; Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic M.R. Rekha Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, India Stefano Rimondo  Department of Chemistry, Materials and Chemical Engineering “Giulio Natta,” Politecnico di Milano, Milan, Italy Filippo Rossi Department of Chemistry, Materials and Chemical Engineering “Giulio Natta,” Politecnico di Milano, Milan, Italy Biswanath Sa  Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India

xivContributors

Pouya Saeedi  Department of Human Nutrition, University of Otago, Dunedin, New Zealand Md. Shahruzzaman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Sadia Sharmeen  Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Amin Shavandi BioMatter, BTL, The Interfaculty School of Bioengineers, The Free University of Brussels, Brussels, Belgium Md. Shirajur Rahman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh Jaroslav Turánek  Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Asaduz Zaman Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering and Technology, University of Dhaka, Dhaka, Bangladesh

About the editors

Sabyasachi Maiti is an MPharm, PhD from Jadavpur University, Kolkata, India. He is working as Associate Professor at the Department of Pharmacy, Indira Gandhi National Tribal University (Central University), Amarkantak, Madhya Pradesh, India. He has experience of more than 15  years in pharmaceutical education and research. He is an impressive researcher in the field of drug delivery science and technology. His research works focus on chemical modification of natural polysaccharides, characterization, and their application in the design of novel drug delivery carriers. The outcomes of his research have been appreciated by international peers in this field. He has more than 50 publications to his credit. He also finds a place in the panel of peer reviewers of various international journals of repute. He has written 25 book chapters and is experienced in editing reference books for international publishers. Sougata Jana is a BPharm (Gold Medalist) from West Bengal University of Technology, Kolkata, MPharm (Pharmaceutics) from Biju Patnaik University of Technology, Odisha, India. He was an Assistant Professor of Gupta College of Technological Sciences, Asansol, West Bengal, India. Currently, he is working at the Department of Health and Family Welfare, Directorate of Health Services, Kolkata, India. He is engaged in research for 11 years and that of teaching for 10 years. IPA Bengal branch, Kolkata, India conferred upon him “M.N. Dev Memorial Award” for securing the highest marks in the state of West Bengal in 2005. He bagged “Best Poster Presentation Award” at 21st West Bengal State Science and Technology Congress-2014, and “Outstanding Paper Award” at 1st Regional Science and Technology Congress—2016, organized by Department of Science and Technology, Govt. of West Bengal, India. He published 30 research and review papers in different national and international peer-reviewed journals. He edited books in Springer, Elsevier, and Pharmamedix India Publication Pvt. Ltd. He has more than 25 book chapters to his credit in Elsevier, Springer, Wiley VCH, CRC Press, Taylor & Francis group. His research area of interest includes modification of synthetic and natural biopolymers, microparticles, nanoparticles, semisolids, and interpenetrating network system for controlled drug delivery.

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Preface

In the last two decades, there have been significant developments in the field of drug delivery and biomedical applications using natural polysaccharides. The credit goes to their well-documented nontoxicity, biodegradability, and biocompatibility. Further, a number of functional moieties including hydroxyl, amino, and carboxyl groups available in the polysaccharide structure are amenable for different kinds of chemical modification to make them suitable for drug delivery and biomedical applications. This reference book is designed solely for the postgraduate students, research scholars, teachers, and budding scientists in the field of pharmaceutical, biomedical, and materials sciences. This book contains the core knowledge and application-­oriented up-to-date information in the relevant subject matter. This book is composed of 14 chapters and each chapter has been elaborated meticulously in a systematic manner. The subject matter in each chapter has been illustrated with neat diagrams, figures, tables, and references to help the readers for further clarifications. The book starts with an introduction to natural polysaccharides and in the subsequent chapter, their potential biomedical application has been narrated. Next chapters (Chapter 3–6) are focused on the design of superporous hydrogels, ionic liquids-based novel materials, polysaccharide derivatives, polysaccharide-scaffolds for the purpose of drug delivery and biomedical applications. Hyaluronic acid and its derivatives have drawn a special interest in the domain of drug delivery and biomedical application due to its excellent biocompatibility and biodegradability and selective uptake by tumor cells overexpressing CD44 receptors for hyaluronic acid and thus Chapter 7 has been framed. Chapter 8–10 describes the cutting-edge state of the art on polysaccharide-based inorganic/metal nanocomposites for biomedical applications. Chapter  11 covers the application of polysaccharide in enzyme immobilization. Chapter 12 discusses the bio-sensor-based diagnosis of cardiovascular risk biomarkers. This is followed by a chapter that undertakes the modification of the surface of liposome vesicles by polysaccharides and their drug targeting potentials. The last chapter accolades the inclusion of aptamer-­ conjugated nanomaterials for diagnosis and drug-targeting applications. The authors earnestly believe and feel confident that the readers will be benefited from the contents of this book to grasp the knowledge in the realm of ­polysaccharide-based drug delivery and biomedical aspects. The editors would like to express their sincere gratitude to all the contributors for their valuable inputs. We are also thankful to the John Leonard, Editorial Project Manager, ELSEVIER and other supporting staff for their magnanimous cooperation toward successful completion of this book project. Sabyasachi Maiti Sougata Jana 

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Introduction to natural polysaccharides

1

Biswajit Maji Department of Chemistry, Indira Gandhi National Tribal University, Amarkantak, India

1.1 Introduction Photosynthesis is the most important and fundamental chemical reaction of all living processes. “Inorganic carbon” in the form of carbon dioxide is captured and transformed to carbohydrates next to other important biomolecules in living organisms [1]. Polysaccharides are abundant in nature and regarded as one of the most important biological macromolecules, among others such as proteins, nucleic acids, lipids, etc. Polysaccharides are biopolymers whose monomer units are simple monosaccharides [2, 3]. Monosaccharide units are aldose (six-member pyranose structure) or ketose (five-member furanose structure) sugars present in polysaccharides in numbers of >20 to as high as 60,000. In polysaccharides, monomers are joined by glycosidic bonds with a definite stereochemistry. They are plentiful in nature, produced from various sources, notably, plant origin (cellulose, guar gum, and pectin), animal origin (chitin, hyaluronic acid, and heparin), algae origin (agar and alginate), and microbial origin (gellan and curdlan). Polysaccharides may be linear, branched with a few short branches, long and heavy branches with bush-like structure [4, 5]. Polysaccharides play two major roles in living organisms: (1) key structural machinery of plant cell wall and other [6] (2) used as food reservoir/food storage in the form of starch in plants and glycogen in mammalians [7]. Original polysaccharides, or its modified form, have shown a wide range of pharmaceutical applications with a variety of biological properties, such as antioxidant, antiinflammatory agent, anti-HIV agent, antitumor, and anticoagulant agent, etc. [8–13]. Although, it is obvious that not all the natural polysaccharides are bioactive or have showed bioactivities [14] at the highest level owing to their structure and physical properties, including others. Therefore, molecular modification (chemical, physical, and biological) is highly desirable to have the highest level of bioactivities [15–18]. More recently, polysaccharides conjugates can be utilized in different biomedical applications such as drug delivery and imaging, tissue engineering, and other many biomedical processes [19–22]. In this chapter, we present the introductory level of polysaccharides with structure and composition of monosaccharides, biological functions, natural occurrence, classifications, and molecular modifications, particularly by various chemical methods implemented for natural polysaccharides.

Functional Polysaccharides for Biomedical Applications. https://doi.org/10.1016/B978-0-08-102555-0.00001-7 Copyright © 2019 Elsevier Ltd. All rights reserved.

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Functional Polysaccharides for Biomedical Applications

1.2 Structure and chemical composition Because of the immense importance of polymeric carbohydrates (polysaccharides) to the energy production and structural components in cell walls, along with biomedical applications in various sectors, their fundamental structural features are highly advantageous and significant. In addition, the scientific community working in the field of sugar chemistry, primarily applied research, in biomedical applications and industrial biotechnology needs deeper knowledge on molecular structures and the arrangement of repeating units (monosaccharide) and their linkage technique, etc. Especially, the complex structure of the polymeric form of carbohydrates turns them into an interesting structure-activity relationship in supramolecular chemistry (carbohydrate-carbohydrate interaction or carbohydrate-protein interaction). This involves regulating a variety of biochemical processes, such as cell differentiation, proliferation and adhesion, inflammation, and the immune response [23]. In recent trends, polysaccharides alone or polysaccharides conjugates have shown a great importance in pharmaceutical and medicinal fields. In particular, polysaccharide conjugates are widely used in the field of biomedical research and other biomedical applications, such as drug delivery and imaging, tissue engineering, etc. [19–22]. In order to understand the various biomedical and biochemical processes at the molecular level, the short discussion on biosynthesis and degradation of polysaccharides, including the chemical transformation involved in these processes, is necessary to a great extent. Even more, starting from the biosynthesis of monosaccharides and moving to oligosaccharides and polysaccharides is truly significant to all researchers for a better understanding of the fundamental polysaccharide chemistry and their long-standing applications in various fields.

1.2.1 Monosaccharides In nature, the most frequently encountered carbohydrate units as monosaccharides are: hexoses (six-carbon sugar) and pentoses (five-carbon sugar). Photosynthesis produces a three-carbon-based sugar molecule, which is named 3-phosphoglyceraldehyde [24]. Two molecules of 3-phosphoglyceraldehydes turn into six-carbon sugar, glucose 6-phosphote, effectively via reverse glycolytic pathway. In contrast, 3-phosphoglyceraldehydes can be utilized in the Calvin cycle to access five-carbon pentose sugars, such as ribose 5-phosphate, ribulose 5-phosphate, and xylulose 5-phosphate. The fundamental chemical reactions, such as mutation, epimerization, aldose-ketose interconversions, chain-shortening and chain-elongation modification, and oxidation-reduction including transamination steps, can be used in the biochemical manipulation of monosaccharide structure [25, 26]. Reversible transfer of 2-C “active glycoaldehyde” fragment from a ketose donor (d-xylulose-5-phosphate) to an aldose acceptor (d-ribose-5-phosphate) catalyzed by transketolase enzyme (Thiamine pyrophosphate is the active catalyst) generates d-glyceraldehyde 3-P (chain-shortening) and d-seduheptulose 7-P (Chain-lengthening).

Introduction to natural polysaccharides3

1.2.1.1 Anomeric descriptor (α and β) For d-glucose in open-chain configuration, the free hydroxyl group (OH-) of C5carbon intramolecularly attacks the aldehyde group of C1 carbon to form a mixture of two types of isomers: α- and β-anomers with a new stereogenic center at C1-carbon, which is called anomeric carbon. i) α-anomer means the hydroxyl group at the new stereogenic center C1-carbon points in the opposite direction of the CH2OH group at C5-carbon; whereas, the hydroxyl group at the C1-carbon points in the same direction of the CH2OH group at C5-carbon assigned as βanomer (Fig. 1.1).

In another way, if the absolute configurations of the two stereogenic centers of sugar molecules (C1-anomeric center and highest chirality center) are the same (R, R or S, S), they are termed as β-anomer or α anomer if they are alike. A few examples of α and β anomers are depicted as Fig. 1.1.

1.2.1.2  d/l-Notation The d/l notation can be assigned on the basis of the chirality (as shown in Fischer projection) at the bottommost chiral center (highest numbered chiral center). Note: if hydroxyl group at the highest numbered chiral center is positioned toward the right side, it is named as d or l if positioned to the left side (Fig. 1.2).

1.2.2 Oligosaccharide Oligosaccharides are formed by typically 2–5 monosaccharides and as high as 20 monosaccharide units. Each monosaccharide unit is linked with others by a stereoselective α or β-glycosidic bond through a specific carbon center. The biochemical synthesis of natural oligosaccharides or polysaccharides (more than 20 monosaccharides

Fig. 1.1  Anomeric descriptor.

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Functional Polysaccharides for Biomedical Applications

Fig. 1.2  d/l notations in a monosaccharide unit.

joined together in the linear or branched form) depends on an activated sugar bound to a nucleoside diphosphate. The UDP is typically encountered as a nucleoside diphosphate, although sometimes ADP and GDP are also involved. As depicted in Fig. 1.3, an activated sugar UDP-glucose is formed by the nucleophilic displacement reaction of α-d-glucose 1-P with UTP. Next, SN2-type of displacement reaction with a suitable nucleophile [-OH groups of sugar molecule] generates a new oligosaccharide or polysaccharide along with free UDP. Mechanistically, this reaction is SN2-type, so the absolute configuration at anomeric center (C1) would be inversion of configuration, i.e., β, if the starting d-glucose is with α-configuration [26]. Many times the linkages between glucose monomers are shown to have α-linkages (maltose or maltotriose). This is possible if double inversions occur at the reactive centers; first SN2 inversion takes place involving enzyme and followed by second SN2-displacement by an appropriate sugar molecule (Fig. 1.3) [26]. Simple oligosaccharide molecules can be exemplified as maltose, a hydrolysis product of starch that consists of two glucose units linked through α 1→4 glycosidic bond, and lactose, which is found in cow’s milk and has galactose linked with glucose through β 1→4 glycosidic bond (Fig. 1.4).

1.2.3 Polysaccharide Polysaccharide means a polymer of >20 to 60,000 monosaccharide units linked by O-glycosidic bonds in linear or branched fashion. Mostly, the monosaccharides are joined in a linear fashion in the structural polysaccharides, and several linear polymeric chains are closely packed together one after another. The presence of weak secondary interaction, predominantly hydrogen bonding between the hydroxyl groups of the chain layers, makes them more rigid with high tensile strength and high viscous material. Cellulose, a natural polysaccharide developed by both plants and animals, although almost exclusively by plants, is structural polysaccharides of glucose molecules joined by β 1→4 glycosidic bonds and growth observed in a linear approach [27]. The glucose chains (approximately 60–70 glucose chains) lay side by side to make a thicker, stronger, and fibrous bond in nature. Large numbers of hydrogen bonding among the free hydroxyl groups of the glucose chains makes a structure called microfibril, and it provides enormous physical strength (Fig. 1.5). On the other hand, the polysaccharides, which serve mainly as energy storage in plants or animals in the form of starch or glycogen, respectively, are commonly

Introduction to natural polysaccharides5

Fig. 1.3  Oligosaccharide biosynthesis.

Fig. 1.4  Representative examples of oligosaccharide: Maltose and lactose with structure.

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Functional Polysaccharides for Biomedical Applications

Fig. 1.5  Schematic diagram of glucose polysaccharide layers, which are held together by hydrogen bond.

branched or in some cases (starch) a mixture of linear or branched. Obviously, the branched polysaccharides are less packed thus making them easily soluble in water. Very often this type of polysaccharides has a helical shape, rather than a linear shape. Plants produce starch as their main food reserve, and it is composed of a glucopyranoses unit with two different polysaccharides named amylose and amylopectin. Amylose is a linear shaped polysaccharide composed of glucopyranoses linked by α 1→4 glycosidic bond. Whereas, amylopectin is a branched polysaccharide assembled with glucose residues linked by both the 1→4 linearly and α 1→6 branched glycosidic bonds (Fig.  1.6). Amylose structure contains fewer glucose residues (about 1000– 2000) than amylopectin. Amylopectin is a much larger polymer with glucose units as high as 106, and it forms a branched polymer. At every 20 glucopyranoses linearly linked with α 1→4, a branch starts with α 1→6 glycosidic bond, and the branch propagates up to 20 residues linked with α 1→4. The next subsequent second branch starts

Fig. 1.6  Structure of amylose and amylopectin polysaccharides.

Introduction to natural polysaccharides7

with α 1→6 glycosidic bond, and the process continues. Finally, the overall structure of starch looks like a tree structure. In mammalian carbohydrates, storage polymer is a glycogen, which is analogous of amylopectin molecule in structure, although more branched-chains at every 10 residues of glucopyranoses are observed [4, 28–30].

1.3 Natural occurrence and classifications The isolation, synthesis, and structural modification of natural polysaccharides and their applications in the various areas, particularly in the biomedical field, recently have progressed rapidly and can now be considered as the most important biopolymers existing at a higher percentage in nature along with others such as proteins and nucleic acids, etc. Their systematic structural chemistry and classification is highly important; however, it is not an easy task to classify the polysaccharides in one dimension. Because of their great variety in polymolecularity and structure with different functionality [3, 31], the classification of polysaccharides always remained a great challenge to the scientific community. In this section, we attempt to categorize the natural polysaccharides based on origins or sources, shape, charge, chemical and structural features of monosaccharide units, and physiochemical properties. The generic term “glycose” stands for a monosaccharide, and “glycan” stands for a polysaccharide. On the basis of type of monosaccharides, a polysaccharide can be divided in two parts: homopolysaccharides (homoglycans), which consist of only one kind of monosaccharide such as cellulose or glycogen, and heteropolysaccharides (heteroglycans), which may have more than two or more different kinds of monosaccharides linked with a variety of glycosidic bonds. Most importantly, in heteroglycans, the different kinds of monosaccharide units are arranged with uniform and definite repeating structures, rather than a random fashion. Heparin, a heteroglycan [32], consists of α-l-iodopyranosyluronic acid 2-sulfate and 2-deoxy-2-sulfoamino-α-d-glucopyranose 6-sulfate. The linking pattern of these monosaccharides are either α- or β at the anomeric center. Thus on the basis of linkages, a polysaccharide may be classified as homolinkages if all the glycosidic bonds are α- or β-configuration and as heterolinkages if the bonds are mixing of both α- and β-configurations. Polysaccharides can be linear or have a branched structure. In branched types, there is a variety: a few long branches, branch-on-branch structures formed in clusters, or bush-like structures. In a polysaccharide chain, only one reducing end is present. Thus, a polysaccharide chain can have different sequences of monosaccharide units with a uniform repeating pattern [33], different sequences of glycosidic linkages, and different kinds of branching. In respect to mass, linear polysaccharides are the most abundant in nature and are found mostly in higher plants, marine algae, and weeds; however, branched polysaccharides have also been found in nature. The degree of polymerization (DP) or degree of polydispersity, i.e., the number of monosaccharides linked in a polymeric carbohydrate, average molecular weight, and range of molecular weights, can vary from source to source because polysaccharides are polydisperse, which means the biosynthetic route of polysaccharides is not a template-basis [34]. In addition to simple monosaccharide units, polysaccharides may contain other functionality such as ester (acetate, phosphate, glycolate, succinate, sulfate, etc.), ether

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Functional Polysaccharides for Biomedical Applications

(methyl and ethyl ether mostly), acetals including amido groups, in which the acid moiety is acetic, glycolic, and sulfuric acids (amino sugars). Polysaccharides may be attached to other important biopolymer protein (protein-polysaccharides if originated from plants and proteoglycans [35] if originated from animals) and lipids as well. Polysaccharides attached to lipids along with O-antigen are called lipopolysaccharides [36] and are found in outer cell membranes in Gram-negative bacteria. Glycans are attached to amino acids in the form of peptides called peptidoglycans, which are mostly present in all bacterial cell walls as structural components [37]. On the basis of charge, polysaccharides can be classified in three categories: neutral, cationic, and anionic forms [38]. The plant kingdom produces a variety of polysaccharides in nature. The animal kingdom, including microbial (fungal and bacterial) and marine algae or seaweeds, are also equally effective to produce polysaccharides with different physiochemical properties [39]. These natural occurring polysaccharides containing a wide variety of structural components that show tremendous applications in biomedicines and tissue engineering owing to their biocompatibility, nontoxicity, biodegradability, and some specific wound healing and drug delivery properties. In industrial applications, generally, natural polysaccharides can be categorized on the basis of sources or origins (Table 1.1), or on the basis of structure and function (Table 1.2), which is illustrated as follows with some selective examples [5]. Table 1.1  Natural polysaccharides originated from various sources Classification of polysaccharides by source/origin Origin

Selective examples with composition

Plant sources

Dietary fiber and wood Cellulose [β-(1→4) linked d-glucopyranose, linear and homopolysaccharides] Hemicelluloses [Four classes of structurally different cell-wall polysaccharides including xylans, mannans, β-glucans with mixed linkages, and xyloglucans] Pectins [α-(1→4)-d-galacturonic acid and rhamnose backbone, arabinose, galactose, xylose side chains, partially O-methyl/ acetylated] β-Glucans [β-(1→4)-d-glucose and β-(1→3)-d-glucose] Gums [Galactan, xylan, xyloglucan, glucuronic mannan, galacturonic rhamnosan type] Inulin Xylan Glucomannans Arabinans Galactan Herbs Ginseng polysaccharides [(1→4)-Linked homogalacturonan backbone. (1→2 or 3)-Linked rhamnose on position 4 as a part of backbone or ramified regions. (1→5 or 2)-Linked arabinose with branch points at position 3. (1→3 or 4)-Linked terminal galactose Astragalus polysaccharides [α-(1→4)-d-glucan with α-(1→6)-branches ●

●

●

●

●

●

●

●

●

●

●

●

Introduction to natural polysaccharides9

Table 1.1  Continued Classification of polysaccharides by source/origin Origin Algae and lichens

Selective examples with composition ●

●

Green algae sulfated polysaccharides [(1→3 or 6)-linked galactose, (1→3 or 4)-linked arabinose, (1→4)-linked glucose and terminal, (1→4)-linked xylose residues. Sulfations occur on O6 of galactose and O3 of arabinose. Sulfate ester content: 9% Brown algae sulfated polysaccharides Fucan: [(1→3)-linked α-fucopyranosyl backbone, mostly sulfated at C4, and branched at C2 with nonsulfated fucofuranosyl and fucopyranosyl units, and 2-sulfated fucopyranosyl units. Sulfate ester content: 30%–34%

Galactan: d-galactopyranose units linked on C3 and C6 and sulfation mostly occur on C4. Sulfate ester content: 21%–24% ●

Bacterial and fungal

●

●

●

●

●

●

Animal origin

●

●

●

●

●

Red algae sulfated polysaccharides Porphyran: Backbone of alternating β-(1→3)-linked d-galactosyl units and α-(1→4)-linked l-galactosyl, (1→6)-sulfate or 3, 6-anhydro-α-l-galactosyl units. Sulfate ester content: 17% Xanthan Dextran Curdlan Pullan Gellan Baker’s yeast glycan Chitin Chitosan Heparin [(1→4)-linked glucuronic/iduronic acid and Nacetylglucosamine disaccharide units with variable 2/3 or 6-O-sulfonation] Chondroitin sulfate [(1→3)-linked glucuronic/iduronic acid and (1→4)-linked N-acetylglucosamine disaccharide units with variable 2/3 or 6-O-sulfonation] Hyaluronic acid

Modified with permission from BeMiller JN. Polysaccharides. Encyclopedia of life sciences. John Wiley & Sons; 2001. p. 1–7.

1.4 IUPAC nomenclature In sugar chemistry, particularly for polysaccharides, a separate and conventional nomenclature is described in detail at the website: http://www.chem.qmw.ac.uk/iupac. In this introductory section, we highlight a simple basis of IUPAC nomenclature for polysaccharides for better understanding, which is discussed below [40]: I. In a polysaccharide, each glycose (monosaccharide) ring size is indicated by an italic f or p for five-membered furanose or six-membered pyranose ring structures, respectively. The term furanose is derived from furan, and pyranose is derived from pyran ring structure,

Table 1.2  Classification of unmodified polysaccharides by structure and functions with examples Classification by structure (chemical composition) Linear molecules

Unbranched Neutral homoglycans 1. Cellulose 2. Laminarans 3. Yeast glucans 4. Cereal β-glucans 5. Amylose 6. Inulins 7. Yeast mannans Neutral diheteroglycans 1. Konjac mannans 2. Agarose component of agar Anionic/acidic homoglycans 1. Lambda-carrageenans 2. Pectins, pectic acids Anionic/acidic diheteroglycans 1. Algins/alginates 2. Kappa-carrageenans 3. Iota-carrageenans Anionic/acidic diheteroglycans 1. Gellan gum Cationic/basic homoglycans 1. Chitosan Linear with short branches/side chain units Branches irregularly spaced 1. Neutral homoglycans (a) Fungal (mushroom)-β-glucans 2. Neutral diheteroglycans (a) Galactomannans (guar gum, locust bean gum, tara gum (b) Flour arabinoxylans (c) Larch arabinogalactan 3. Neutral tetraheteroglycans (a) Xyloglucans Branches regularly spaced 1. Neutral homoglycans (a) Yeast mannan 2. Anionic/acidic triheteroglycan (a) Xanthan gum ●

●

●

●

●

●

●

●

Nonlinear molecules

●

●

Branches in clusters, homoglycans 1. Amylopectins Highly branched/branch-on-branch structures, anionic/acidic 1. Tetraheteroglycans (a) Gum karayas (b) Okra gum 2. Pentaheteroglycans (a) B-type hemicelluloses of cereal brans, etc. (arabinoxylans) (b) Gum arabic (c) Psyllium seed gum

Introduction to natural polysaccharides11

Table 1.2  Continued Classification by functions Cell wall polysaccharidesa

●

●

●

Energy storage polysaccharides

●

●

Higher land plants 1. Cellulose 2. Hemicellulose (a) Arabinoxylans (b) Galactoglucomannans (c) β-Glucans (d) Glucomannans (e) Mannans (f) Xylans (g) Xyloglucans 3. Pectic polysaccharides (a) Arabinans (b) Arabinogalactans (c) Galatans (d) Galacturonans (e) Rhamnogalacturonans Marine algae 1. Algins 2. Cellulose 3. l-fucans 4. Galactans (a) Agars (b) Carrageenans (c) Furcellarans 5. β-Glucans 6. Mannans 7. Xylans Fungi and yeasts 1. Cellulose 2. Chitin 3. β-Glucans Higher land plants 1. Fructans 2. Galactans 3. Galactomannans 4. Glucomannans 5. Starches 6. Xyloglucans Marine algae 1. Fructans 2. α-Glucans 3. β-Glucans 4. Xylans

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Functional Polysaccharides for Biomedical Applications

Table 1.2  Continued Classification by functions ●

●

Freshwater algae 1. α-Glucans 2. β-Glucans Fungi and yeasts 1. α-Glucans 2. β-Glucans

a

Many of those polysaccharides may contain monosaccharide units other than those indicated in the name. For example, xylans often contain uronic acid unit, and l-fucan contains, in addition to main monosaccharide unit l-fucose, dgalactose, d-glucuronic acid, d-mannose, and d-xylose units. Reproduced with permission from BeMiller JN. Polysaccharides. In: Encyclopedia of life sciences. John Wiley & Sons; 2001. p. 1–7.

although both the furanose and pyranose structures have no double bond. The nomenclature for any sugar molecule initiated by anomeric descriptor α- or β- for α/β-anomer and next with the configuration symbols d- and l-configuration with a hyphen is followed by the symbol for a monosaccharide unit (Fig. 1.7). II. A homopolysaccharide (homoglycans) consists of one kind of glycose residue and can be depicted by replacing the ending -ose of the glycose by the suffix -an. For example: xylan for polymers of xylose, mannan for polymers of mannose, and glucan for glucose monomer present both in cellulose and starch. III. A heteropolysaccharides (heteroglycans) consists of two or more kinds of glycose residues: a long chain repeating and similar kind of glycose units (parent or principal chain). It can be cited at the end by replacing -ose with the suffix -an, with other glycose residues written in alphabetical order as glyco- in prefix, if different kinds of monosaccharides present. A heteropolysaccharide, guran composed of d-mannose and d-galactose monosaccharides, where mannose present in a principal chain named as d-mannan at the last and with d-­ galactose present in side chain named as d-galacto- in prefix.

Fig. 1.7  Furanose and pyranose cores in a sugar molecule.

Introduction to natural polysaccharides13

4)- β - D -Manp- (1

4)- β - D -Manp-(1 6

4)- β - D -Manp- (1

1 α - D -Galp

4)- β - D -Manp-(1 6 1 α - D -Galp

D -galacto - D -mannan

IV. For an oligosaccharide, glycose units are linked with a glycosidic bond and the location of linkages is presented in parenthesis between the symbols by an arrow (→/↔) indicating the glycosidic linkage in the direction from the anomeric carbon center to the carbon center of the next monosaccharide unit. Single-headed arrow indicates glycosidic linkage from anomeric carbon center (C1) to the next monosaccharide’s other carbon center except anomeric position. Whereas the double-headed arrow indicates the glycosidic linkage of two subsequent monosaccharide units linked by two anomeric centers. Oligosaccharide without a free hemiacetal group i.e., no reducing end is named as glycosyl glycoside. For example, raffinose is a trisaccharide composed of galactose (Gal), glucose (Glc), and fructose (Fru) monosaccharide units. It can be represented as α-dgalactopyranosyl-(1→6)-α-d-glucopyranosyl-β-d-fructofuranoside, or α-d-Galp-(1→6)- αd-Glcp-(1↔2)-β-d-Fruf (Fig. 1.8). V. Linear oligosaccharide consisting of a free hemiacetal group or with reducing end is illustrated with a nonreducing glycosyl unit on the left side and reducing glycosyl unit on the right side in a linear polymeric chain. As a trisaccharide, cellotriose can be represented as β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-glucopyranose or β-d-Glcp-(1→4)β-d-Glcp-(1→4)-d-Glcp (Fig. 1.9).

1.5 Structure determination (ring size and linkage type) Most polysaccharides except bacterial polysaccharides and some plant-generated polysaccharides are heterogeneous in terms of chemical composition of glycose residues and/or presence of variety of α- or β-linkages at the different positions and other noncarbohydrate

Fig. 1.8  Representation of raffinose polysaccharide.

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Functional Polysaccharides for Biomedical Applications

Fig. 1.9  Representation of cellotriose polysaccharide.

building blocks such as protein, polynucleotide, lipids, etc. In addition, the presence of more substitution patterns and linkages in the sugar molecules make it complicated to solve the structure with ease [41]. Therefore, the absolute structure determination of glycans from extracts of plants or a fermentation culture medium of bacteria, obviously, is not an easy task. Both the chemical methods and instrumental methods are used to determine the most probable statistical structure of polysaccharides. At the primary level, various fractional distillations [42], chemical [43–45], or more specific enzymatic methods [46, 47] are adopted in order to achieve information about monosaccharides, linkages’ types, ring structures, etc. Finally, various spectroscopic techniques [48, 49] are utilized only after subject to appreciable degree of purity of the sample checked by chromatography. In this section, we discuss the techniques, particularly, various chemical/enzymatic methods to determine the primary structure of the polysaccharides, which are as follows: ●

●

Chemical composition of monosaccharides Linkage types

1.5.1 Extraction In order to get a certain degree of purity of the polysaccharides for the next level structure determination, extraction from the sources, whether from plant polysaccharides or from the fermentation of the culture medium in case of bacterial polysaccharides, is very significant. In the laboratory scale, the extraction from plant polysaccharides begins with replacing the noncarbohydrate portions from the sugar residues, and it can be done by adding simple water or, sometimes, alkaline aq. solution to lipids, proteins, or lignins, etc. Subsequently, new polysaccharides are formed, which again can be subjected to precipitation techniques to achieve the highest degree of purity.

1.5.2 Fractional analysis In a polysaccharide, the presence of the monosaccharide residues (homoglycan or heteroglycan) can be analyzed by the fractional analysis, which is often carried out in the presence of acid-catalyzed hydrolysis. The monosaccharides, or in some cases in the form of oligopolysaccharides, released upon partial hydrolysis are next evaluated both qualitatively and quantitatively by high-performance liquid chromatography or LC/MS spectroscopic techniques. Sometimes, the linkage-types/anomeric centers (C1) bonded to what extent of other carbon centers (C2/C3/C4 or C6) of next monosaccharide unit also can be determined by the stability of the glycosidic bonds [42]. For example, in

Introduction to natural polysaccharides15

hexopyranoses, the 1→6 linkages are comparatively more stable than other linkages (1→2, 1→3 or 1→4), and α-linkages are less stable than its corresponding β-anomers.

1.5.3 Methylation analysis In methyl analysis, particularly, the positions of glycosidic linkages and nature of the ring sizes are determined. In this chemical analysis, polysaccharides are fully methylated in the presence of excess methylating agents [50] and then hydrolyzed in the presence of acidic conditions. In hydrolysis reaction, only the hydroxyl groups involved in glycosidic bond formation now become free, except all other hydroxy groups remained methylated. Next, the individual monosaccharide units and/or oligosaccharides are characterized further by spectroscopic techniques to get the information about the position and nature of the linkages. To illustrate this method, a simple lactose disaccharide can be considered. Lactose, a disaccharide is composed of two monosaccharide subunits; d-galactose in acetal form linked with 1→4 glycosidic bond of d-glucose, which is in hemiacetal form. On exhaustive methylation reaction (Ag2O, MeI, or Hakomori reagents [50], NaH, MeI in dry DMSO), all the hydroxyl groups in lactose are methylated. Fully methylated lactose is hydrolyzed in acidic condition. On hydrolysis, two acetal linkages hydrolyzed and provided two different products, 2,3,4,6-tetra-O-methylgalactose and 2,3,6-tri-O-methylglucose, which can be characterized by spectroscopic techniques. This provides information about the position of the linkage and nature of the subunits whether in acetal or hemiacetal form (Fig. 1.10).

1.5.4 Periodate oxidation The stoichiometric HIO4-oxidation of vicinal diol (1,2-diol) provided oxidized products aldehyde by cleaving C-C single bond. This technique is employed mostly to determine the ring sizes of monosaccharides subunits present in a polysaccharide. Oxidation of vicinal triol by HIO4 afforded 1 mole of formic acid and 2 moles of aldehydes (Fig.  1.11). To evaluate the furanose or pyranose ring structure and the

Fig. 1.10  Representative example of methylation analysis of lactose sugar.

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Functional Polysaccharides for Biomedical Applications

Fig. 1.11  HIO4-mediated oxidation analyses in structure determination of polysaccharide.

differentiation among the linkages in a sugar molecule with qualitative and quantitative manner, this technique is very significant (Table 1.3) [51].

1.5.5 Enzymatic method Because of the specificity of the enzymatic reaction in a polysaccharide (glycosidic bond hydrolyzes at a specific position), the enzymatic method is supported to determine the sequence of monosaccharide units [46, 47]. In general, two types of Table 1.3  Quantitative periodate oxidation of various linked hexopyranoses units Hexose linkage

Observation per residue

1,2-linkage:

Inner residues Terminal reducing residue

1,3-linkage:

Inner residues Terminal reducing residue

1,4-linkage:

Inner residues Terminal reducing residue

1,6-linkage:

Inner residues Terminal reducing residue

One periodate consumed Three periodate consumed/one formaldehyde (C6) and two formic acid (C4 and C5) formed No reaction Three periodate consumed/ one formaldehyde (C6) and two formic acid (C1 and C5) formed One periodate consumed Three period ate consumed/ one formaldehyde (C6) and two formic acid (C1 and C2) formed Two periodate consumed and one formic acid (C3) formed Four periodate consumed and four formic acids (C1, C2, C3, and C4) formed

Note: For glycans of hexopyranoses, the terminal nonreducing unit consumes 2-moles of periodate with the formation of one mole of formic acid (C3). Reproduced with permission from Tsai CS. Biomacromolecules. In: Biomolecular structure: polysaccharides. John Wiley & Sons, Inc.; 2006. p. 147–182 [chapter 6].

Introduction to natural polysaccharides17

g­ lycosidase enzymes (exoglycosidase and endoglycosidase) are utilized for the partial or complete hydrolysis at a specific position (Table 1.4). Finally, after completion of chemical/enzymatic methods, the individual monosaccharides, or in some cases, oligosaccharide (partial hydrolysis) are further characterized by the various spectroscopic techniques, such as polarimetric method, mass spectrometric analysis (FAB = fast atom bombardment, MALDI = matrix assisted laser desorption/ionization, ESI = electrospray ionization), nuclear magnetic resonance (1H-, 13C-NMR; 1D and 2D) analysis, etc., to deduce the most appropriate primary structure of a polysaccharide [48, 49, 52, 53].

1.6 Molecular modification The natural polysaccharides obtained from different sources have shown a wide range of bioactivities applied to clinical practices or under clinical trials. Since 1943 [54, 55], many research groups have been devoted to finding out biological activities of natural polysaccharides or its molecular level alteration, which includes a variety of chemical, physical, and biological methods. In recent times, the application of these polysaccharides in modern medicinal fields, particularly in the area of tissue engineering, controlled drug delivery and release and wound healing, much more Table 1.4  Selected enzymes mostly used in glycan structure analysis Enzymes

Source

Specificity

Saccharomyces cerevisiae Almond emulsin Green coffee bean Escherichia coli Aspergillus phoenicis Jack bean Helix pomatia Chicken/porcine liver

Glcα1→4 Glcβ1→4 Galα1→3,4,6 Galβ1→4Glc Manα1→2 Manα1→2,3,6 Manβ1→4 GalNAcα1→

Bovine epididymis Archrobacter ureafaciens Clostridium perfringens

Fucα1→6,2,3,4 NeuNAcα2→6,3 NeuNAcα2→3,6

Pig pancreas Bacillus amyloliquefaciens Streptomyces plicatus/griseus

↓ Glcα1→4Glc ↓ (Man)n-GlcNAc-GlcNAc

Exoglycosidases α-d-Glucosidase β-d-Glucosidase α-d-Galactosidase β-d-Galactosidase α-d-Mannosidase β-d-Mannosidase α-N-acetyl-dgalactosaminidase α-l-Fucosidase α-d-Sialidase

Endoglycosidases α-Amylase Endoglycosidase H

Reproduced with permission from Tsai CS. Biomacromolecules. In: Biomolecular structure: polysaccharides. John Wiley & Sons, Inc.; 2006. p. 147–182 [chapter 6].

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Functional Polysaccharides for Biomedical Applications

significantly. In view of the research results, it is evident that the polysaccharides have shown enormous bioactivities in specific manner owing to their own structural features, i.e., the types of glycosidic linkages, solubility [56], degree of polymerization [57], and stereochemistry [58]. The inbuilt properties of natural polysaccharides, such as bonding type (glycosidic bond), anomeric nature (α- or β), and chemical composition of monosaccharides, are not changeable for augmenting the bioactivities. Although, in the laboratory, some polysaccharides can be synthesized to change these natural properties with the enhancement of bioactivities [15–17]. On the other hand, the rest of the area, particularly, the substituent groups attached at various positions, can be easily functionalized by modification, which mainly includes chemical, physical, and biological modification [18]. Perhaps, the chemical modification is the most acceptable and widely implemented method for the modification or functionalization of polysaccharides besides physical and biological modification techniques, in order to increase the highest level of biological activities. In this molecular modification, a variety of well-studied chemical methods is reviewed. Specific reagents and reaction conditions with general mechanism of chemical methods which include etherification (alkylation, hydroxyalkylation, carboxymethylation, sulfoalkylation etc.), esterification (acetylation, sulfation, phosphorylation etc), selenylation, amine alkylation and others, are discussed in this section (Fig. 1.12) [18].

Fig. 1.12  Schematic representations of different types of modification techniques.

Introduction to natural polysaccharides19

1.6.1 Chemical modification 1.6.1.1 Hydroxyl group modification Etherification Natural polysaccharides used as alcohol in chemical modification plays an important role, and the resultant modified polysaccharides can be utilized in various biomedical applications. Mostly, the study of the polysaccharides like cellulose, starch, arabinogalactan, and pullan consisting only hydroxyl groups is extensively investigated. Halogenated alkane (alkyl halide) under basic conditions is mostly investigated for simple alkylative modification [59, 60]. Hydroxyalkylation reaction is generally carried out using oxirane as an electrophile under basic condition [60]. In this etherified modification, the basic condition is employed in most of the cases for the alkylation via ring opening of oxirane from less steric hindrance side. Carboxymethylation reaction is also carried out by mono chloroacetic acid, which yielded anionic materials used for ion exchange applications. This reaction is generally carried out in organic solvent or aqueous medium. Some reports on successful carboxymethylation of cellulose [61, 62] and chitin [63] polysaccharides have shown better solubility in water and enhanced bioactivities. In addition, the etherification modification with sulfoalkylating agents [64] has also been investigated for the same purposes. The cationic ion exchange resins of cellulose can be prepared by etherification modification employing 2-chloroethyl diethylamine [65]. Silylation of hydroxyl groups in a polysaccharide utilizing trimethylsilyl chloride (TMSCl) or hexamethyldisilazane reagents afforded trimethylsilyl ether, which can be used to increase hydrophobicity of the polysaccharides [66]. Most importantly, in the etherification modification, the electrophile, such as epichlorohydrin consisting bis-electrophilic centers, can be used in order to functionalize in second times. Initially, the hydroxyl group of the polysaccharide reacts with epoxide to form ring-opened product (halohydrin) that subsequently removes HCl to form new oxirane product [67]. Further, various nucleophiles (N-, S-, O-, and carbon nucleophiles) react with epoxide-derived new modified polysaccharide, which gave additional functionalization to the polysaccharides for better bioactivities (Fig. 1.13).

Esterification In 1988, Mizumoto et al. first installed sulfate group to a monosaccharide structure and observed that the sulfated modified structure inhibits the T lymphocyte virus [68]. After that, many research groups are engaged to modify sugar molecules with sulfating agents for the improvement of bioactivities. The most commonly used methods for sulfated esterification are chlorosulfonic acid-pyridine (CSA-pyridine) [69] and amino sulfonic acid-pyridine method (ASA-pyridine) [70]. Besides that, the other methods such as SO3-pyridine [71] and oleum-DMF [72] etc. are also employed to sulfate the hydroxyl groups of the polysaccharides. Judging by the recent trends, phosphorylation of sugar molecules and its bioactivities investigation is very rare, most probably owing to the very limited number of

20

Functional Polysaccharides for Biomedical Applications

Fig. 1.13  Various chemical modifications with hydroxyl group in a polysaccharide.

n­ atural phosphated glycose units present in nature [73]. In synthetic point of view, only a few reagent systems, such as simple phosphoric acid or phosphoric anhydride under acid condition in DMSO solvent, POCl3 and salts of phosphoric acids, are considered to introduce the phosphate functionality to a polysaccharide. By introducing the phosphate group, it has been proven that modified phosphorylated polysaccharides showed specific bioactivities owing to the presence of charged phosphate groups [74, 75]. In addition, this modified polysaccharide can improve the properties related with solubility, molecular weight and change, and sometimes the structural parameters. Selenylated modification to polysaccharides is quite important as selenium (Se) plays a significant role in the reduction of hydrogen peroxide catalyzed by glutathione peroxidase and also acting as a radical scavenger in the living system [76]. Although inorganic selenium compounds are toxic, selenylated polysaccharides, an organic selenium compound, can be treated in a safe mode, and both Se and polysaccharide synergistically works together [77, 78]. This modification can be successfully completed by using selenious acid or selenite in the presence of strong acid conditions. Acetyl modified polysaccharides, mainly, the side chain residue or in some cases ring hydroxyl groups to a polysaccharides are acetylated in the presence of acetic anhydride as reagent in water or DMSO solvents. The acetylated polysaccharide showed a greater level of the solubility in water medium, and so it has shown better biological activities [79].

1.6.1.2 Amine group modification Judging by the recent trends, amine functionalization of mainly chitosan and chitin polysaccharides and, after that, resultant modified biopolymer’s usage, mainly in pharmaceutical, biomedical, and biotechnological fields, has been gradually increased [80, 81]. Various methods such as oligomerization, N-alkylation, and followed by

Introduction to natural polysaccharides21

quaternization, reductive amination (condensation reaction with aldehydes and next reduction), N-carboxymethylation, N-acylation including azidation, urea or thiourea formation by reaction with -NCO or -NCS, and many more are well-studied in the literature [19, 82, 83]. Some methods of amine functionalization are presented in Fig. 1.14.

1.6.1.3 Carboxylic group modification Polysaccharides, such as hyaluronic acid (HA), and alginic acids can be modified by a variety of reactions strategy based on free carboxylic acid group or prefunctionalized activated ester group [83–86]. Among them, Ugi condensation reaction (Figs.  1.15 and 1.16), amidation, and esterification (Fig.  1.17) including ring opening reaction with substituted oxiranes, hydrazination, etc. are important. The carboxylic acid is activated by mostly EDC or DCC reagent for esterification; whereas, NHS or HOBT reagent is for amidation modification (Fig. 1.18).

1.6.1.4 Click chemistry In addition, the most significant modification of polysaccharides investigated, so far, is “click chemistry”. This reaction involved Huisgen 1,3-dipolar cycloaddition between azide and terminal alkyne in the presence of Cu-salt [87]. Both the preinstalled azide group, mostly at the C6-position of sugar molecule, and external terminal alkyne,

Fig. 1.14  A variety of techniques adopted for the amine group modifications.ART: Delete the unwanted arrow

22

Functional Polysaccharides for Biomedical Applications

Fig. 1.15  Ugi reaction, hydrazination, and epoxide ring-opening reactions.

Fig. 1.16  Mechanism of Ugi reaction.

or vice-versa, are involved in this reaction. To introduce the azide functionality, in general, the hydroxyl group is tosylated, a good leaving group, and next using sodium azide, a polysaccharide is azidated via SN2 displacement reaction [88]. Finally, azidated polysaccharide reacts with externally added terminal alkyne in presence of Cu-slat as Lewis acid gives the triazole-modified polysaccharide at C6-carbon center. Alternately, the free hydroxyl group can be propargylated, first under basic conditions

Introduction to natural polysaccharides23

Fig. 1.17  Esterification and amidation of polysaccharides consisting of acid functionality.

Fig. 1.18  Mechanism of EDC/DCC or NHS-coupling reaction.

and followed by click reaction with externally added azide; this could provide the same triazole-based polysaccharide. For chitosan, azide functionality introduced by replacing amino functionality at the C2-position by diazotization step and followed by reacted with sodium azide [83]. Similarly, azidated chitosan reacts with terminal alkyne to afford triazole-modified chitosan. Here the mechanism of click chemistry [89] and “click-chemistry”-induced modification of polysaccharides is illustrated in Fig. 1.19. As an example, both the two differently functionalized hyaluronic acid derivatives prepared by an esterification reaction, react in aqueous medium to afford a new cross-linked hyaluronic acid via a “click” reaction, even in the absence of Cu-catalyst (Figure 1.20). The new cross-linked polymer has lost the hydrophilic properties and forms a very rigid hydrogel due to incorporation of triazole-ring substituted cyclooctane ring as a linker between the hyaluronic acids chain [90].

24

Functional Polysaccharides for Biomedical Applications

Fig. 1.19  General reaction mechanism of click-chemistry and a variety of Cu(I)-mediated polysaccharide modifications.

1.6.1.5 Oxidation in modification Dextran and arabinogalactan are frequently encountered polysaccharides for conjugation via oxidation reaction employing external oxidants NaIO4 [91] and peroxidemediated if vicinal diol (C2-C3-diol). For the C6-OH position, DMP-oxidation and TEMPO [92] are utilized. Next, oxidized products of polysaccharides (aldehydes) react with a variety of polyamines or amine-group containing polysaccharide and

Introduction to natural polysaccharides25

Fig. 1.20  “Click”-reaction in hyaluronic acid for making rigid hydrogel.ART: Delete (XXC)

followed by reductive amination generates a self assemble cationic core in aqueous solution. This kind of polysaccharide, which conjugates with cationic core such as dextran-spermine, is useful in gene delivery (Fig. 1.21) [93].

1.6.2 Physical modification The high molecular weight containing polysaccharides have difficulties exerting a certain level of pharmacological effects for better bioactivities as it would be unfavorable for bioactive polysaccharides to penetrate multiple cell membrane barriers exhaustively. Disconnect the chain length of the natural bioactive polymers without alteration of the composition of monosaccharides or decomposition of main chain by applying physical methods. The physical methods, which mainly include ultrasonic disruption [94], microwave exposure [95], or very powerful radiation-induced treatment [96] to polysaccharide long chain with high molecular weight, may be fragmented. The

26

Functional Polysaccharides for Biomedical Applications

Fig. 1.21  Oxidized dextran conjugate with oligoamine derivatives (MS-Q-Speramine).

lowering of chain length obviously increases the solubility, and a variety of pharmacological properties helps to increase the bioactivities of shorter chain length containing modified polysaccharides. Many natural polysaccharides, such as cellulose, starch, and hyaluronic acid including chitosan, can be modified by applying γ-radiation and, consequently, their solubility level increased satisfactorily [97, 98].

1.6.3 Biological modification The enzymatic degradation of polysaccharides is known as biological modification, which can be applied more specifically at the target area to degrade the polysaccharide backbone compared to other physical and chemical methods owing to high specificity of enzyme actions. The enzymatic modification of polysaccharides is only applicable to a certain type of polysaccharides only, and there is a huge scope to develop this particular area and, consequently, to increase the bioactivities [17, 99].

1.7 Conclusion We highlighted a few basics of natural polysaccharide chemistry, such as natural occurrence, classification, structural components, etc. A simple chemistry, starting from monosaccharide to oligosaccharides and finally ending with polysaccharides, is described. The biochemical reactions in monosaccharides are presented with figures for the better understanding. Next, various important chemical and enzymatic methods are described to analyze the primary structure of polysaccharides. Natural polysaccharides obtained from microorganisms, plants, and animals show a broad spectrum of bioactivities, such as antioxidative, immune regulatory, antiinflammatory, anti-HIV, antitumor, and anticancer activities. Sometimes, modified natural polysaccharides also have shown greater bioactivities compared to natural ones. With this

Introduction to natural polysaccharides27

view, ­various ­molecular modifications, which include chemical, physical, and biological modifications of natural polysaccharides, are illustrated in detail. Some of the chemical modifications are discussed with reaction mechanism in detail for deeper knowledge. Overall, fundamental knowledge about polysaccharide chemistry, which includes origin, classification, and structural parameters, is attempted in this chapter.

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An overview on the potential biomedical applications of polysaccharides

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Rajalekshmy, G.P., Lekshmi Devi, L., Jasmin Joseph, Rekha, M.R. Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, India

2.1 Introduction Polysaccharides are renewable materials that are readily available in nature and have found importance in the field of various biomedical applications. The main sources of polysaccharides are plants, animals, and microbial organisms [1]. Depending on the source of each polysaccharide, its properties such as charge, molecular weight, solubility, mechanical strength, etc. vary. This variability itself gives a wide choice for fulfilling various applications in the development of drug delivery systems, tissue engineering scaffolds, wound-healing materials, etc. The most explored polysaccharides include chitosan, alginates, pullulan, cellulose, hyaluronic acid (HA), starch, etc. [2]. Though there are various limitations associated with the clinical usage of these biopolymers, indomitable research explorations have led to promising developments. For example, FDA approved alginate and chitosan wound dressings/hemostats are now available for clinical use [3].

2.2 Biomedical applications The biomedical applications of polysaccharides span a very wide range from drug delivery to tissue engineering/3D bioprinting. However, the intensely focused areas include [4, 5]. (i) nano or microparticles for delivery of therapeutic molecules such as proteins, drugs, or nucleic acids; (ii) polymer-drug conjugates; (iii) advanced biomaterials for wound dressings; and (iv) scaffolds for tissue engineering and development of bioinks for 3D bioprinting.

Numerous book chapters and review articles are available on drug delivery systems. Here the main focus will be on other topics listed.

Functional Polysaccharides for Biomedical Applications. https://doi.org/10.1016/B978-0-08-102555-0.00002-9 Copyright © 2019 Elsevier Ltd. All rights reserved.

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2.3 Gene delivery systems During the past decades, intensive research works in the fields of chemical engineering, basic life science, and modern medicine have been directed towards the advanced level of gene therapy and regenerative medicine [6]. Gene therapy is defined as the procedure used to introduce foreign genomic materials into host cells and thereby modify a patient’s cells to elicit a therapeutic benefit [7, 8]. Compared with direct administration of protein-based drugs, gene therapy offers enhanced bioavailability, high specificity, less systemic toxicity, good in vivo stability, long-term efficacy by reduced hepatic and renal clearance rate, and reduced cost of manufacturing [9, 10]. The key to successful gene therapy lies mainly in the efficacy of gene transfer. Two major approaches used for gene delivery are; (i) Viral vector, which is an efficient natural transfecting agent (ii) Nonviral vector, in which a polymer or lipid or liposome acts as a carrier to deliver therapeutic gene material and its associated regulatory elements into the nucleus [11–13].

Although viral vectors provide efficient transduction and high levels of gene expression, there still exist limitations of toxicity, immunogenicity, target-cell specificity, and the costs of manufacturing [11]. The above-mentioned critical safety issues for clinical use of viral vectors have opened up the possibility of new targets for therapeutic intervention leading to the development of nonviral vectors [13–15]. In contrast to viral vectors, nonviral vectors offer benefits such as non-immunogenicity, ease of formulation and scale-up, and potential for repetitive administration and are relatively inexpensive [12]. Additional advantages of the nonviral delivery system include its ability to carry large DNA molecules and to modify the chemical structure of vectors accordingly so as to accelerate transfection efficiency as well as transgene expression. In the nonviral gene delivery system, biodegradable polysaccharides play an important role as excipients. They are of natural (animal, plant, algal) origin formed via glycosidic linkages of monosaccharides and its use as a vector for gene delivery applications is attractive because of its peculiarities such as biodegradability, biocompatibility, availability, non-toxicity, and because it is economical [16]. Since polysaccharides offer a large number of available reactive functional groups in their chemical structure including hydroxyl, amino, and carboxylic acid groups, their chemical modification favors specific targeting [17]. Polysaccharide materials can be divided into cationic (chitosan), anionic (alginate, heparin, pectin, HA) and neutral (pullulan, dextran) based on their intrinsic charge [17, 18]. Fig. 2.1 shows the chemical structure of different polysaccharides.

2.3.1 Chitosan Cationic polymers form condensed complexes with negatively charged nucleic acids (polyplexes) via electrostatic interactions [19] to form particles with a diameter in the order of 100 nm. These complexes have the ability to protect the nucleic acid from undesirable degradation during the transfection process and also facilitate cell uptake and intracellular delivery [13]. The mechanism of nonviral gene delivery by cationic

Biomedical applications of polysaccharide

35 O

OH O

O HO

NH2

HO

NH

NH2

HO O

O

OH

O

O HO

O

O

OH

HO

n

O

n

Hyaluronan

Chitosan COO– O

OH

O O

O O

HO

COO–

HO

O O

O O OC OH

OH HO

COO

COO–

OH HO O O –

OH

–

OH O n

Alginate H HO HO

O HO

O O

OH

HHO H H OH H O

O

HO OH

HO

H H OH H O HO

HO

HO

O HHO

O OH

O

HO

HO OH

HHO

H H OH H O HO O HO HH

OH O

HO

OH

H H OH H O

O

HO

HO HO

OH O

OH HHO

H H OH H O HO

n

Dextran

Pullulan

OH HHO

H H OH O

Fig. 2.1  Polysaccharides used for developing gene delivery vectors.

systems includes the following steps: (i) adherence of the complexes to the cell surface via nonspecific interaction, (ii) entry of the complex through the mechanism of endocytosis into endocytosis vesicles, (iii) release of DNA particles from the endosome and entrance into the cytosol in a partially de-condensed form, and (iv) translocation of the DNA particle to the nucleus via passive diffusion or nuclear membrane crossing, resulting in transgenic expression [20]. Among cationic nonviral vectors, chitosan and its derivatives are considered to be one of the most attractive candidates in gene delivery and transfection because of their biocompatibility and biodegradability, and antitumor properties along with low immunogenicity and low toxicity. Chitosan is a natural, linear, semicrystalline polysaccharide consisting of two subunits N-acetyl-D-glucosamine [(1 → 4)-2-acetamido-2-deoxy-β-D-glucan] and D-glucosamine [(1 → 4)-2-amino-2-deoxy-β-D-glucan] which are joined together by

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glycosidic linkage [21, 22]. It is prepared by partial deacetylation of chitin, the second most abundant natural polymer in the world, which is normally found in crabs, cuttlefish, lobsters, shrimps [12], and the cell walls of fungi. In fungi Mucoraceae, chitosan occurs naturally [23, 24]. Chitosan is insoluble in water, organic solvents, and aqueous bases but is soluble in acids with a pKa smaller than 6.2, due to the presence of an amino group having free protons to donate (the pKa of amino groups in chitosan is from 6.2 to 7.0) [12, 24]. It is soluble in acids such as acetic acid, nitric acid, hydrochloric acid, perchloric acid, and phosphoric acid [25–28]. Natural origin, abundance, and reactivity of chitosan offer a unique opportunity for the development of biomedical and pharmaceutical applications. Its antimicrobial and anti-inflammatory nature makes chitosan an excellent candidate for wound healing. Radhakumary et al. reported a new formulation consisting of thiolated chitosan with poly (N-isopropyl acrylamide) loaded with ciprofloxacin, which showed antibacterial properties to E. coli bacteria, supporting its potential as a wound dressing [29]. The antimicrobial effects of chitosan against a broad spectrum of bacteria were reported by Jeon et al. [30]. Nanoparticles of chitosan express its potential applications in drug as well as gene delivery [20, 31, 32]. The ability of chitosan to adhere to the mucosal surface improves the efficiency of drug delivery by oral administration [33, 34]. Due to its positive charge, chitosan can interact with negatively charged nucleic acid leading to the formation of polyplexes, which protects condensed nucleic acid from enzymatic degradation. This property makes chitosan a valuable candidate for gene carrier in gene therapy [35–38]. Various factors such as molecular weight (Mw) and degree of deacetylation (DDA) of chitosan, stoichiometry or the N/P ratio of the chitosan/DNA polyplex, pH of the transfection medium, serum concentration, cell types, etc. may influence the stability and transfection efficiency of chitosan/DNA polyplexes [39].

2.3.1.1 Parameters influencing stability and transfection efficiency of polyplexes Molecular weight In order to obtain high levels of transfection, the molecular weight of chitosan plays an important role which influences chitosan/DNA complex size as well as stability, cellular uptake, and the release of DNA from the polymer/DNA complex after endocytosis [40, 41]. According to Wagner et al., the particle size of a polyplex less than 200 nm is favorable for cellular uptake through clathrin mediated endocytosis [42]. MacLaughlin et al. reported that the size of the complex decreases with a decrease in molecular weight [41]. Huang et al. studied the influence exerted by the molecular weight of chitosan on the size and stability of the polyplex. They observed that the average particle size decreased from 181 to 155 nm when the molecular weight of chitosan decreased from 213 to 48 kDa, while a further decrease of the molecular weight reversed the trend sharply, that is, the polyplex prepared from 17 and 10 kDa chitosan showed the average size as 269 and 289 nm, respectively [43]. Since the transfection efficiency of polyplexes depends strongly on the size of particles, which determines their cellular uptake, chitosan of appropriate molecular weight should be selected [44].

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According to Huang et al., the capacity to retain the condensed DNA was influenced by the chitosan carrier having an adequately high molecular weight [43]. The stability of the polymer/DNA complex becomes high with high molecular weight chitosan (100–400 kDa), which is beneficial for the protection of DNA in the cellular endosomal/lysosomal compartments, but the release of pDNA becomes delayed, thereby delaying the expression [41]. Polyplexes with a rather low molecular weight chitosan are not stable enough to protect DNA effectively due to early dissociation, which results in reduced or no transgene expression. Also, chitosan with a very high molecular weight have low solubility at physiological pH. Hence, it is important to select chitosan with an intermediate molecular weight which can form polyplexes providing both intracellular DNA protection as well as efficient intracellular DNA release to get good transfection efficiency.

N/P ratio The N/P ratio is defined as the charge ratio of amines of chitosan (N) to phosphates of DNA (P). The stability and transfection efficiency of chitosan/DNA polyplexes are strongly influenced by stoichiometry or the N/P ratio of the polyplex [43]. KopingHoggard et al. prepared polyplexes using ultrapure chitosan (UPC), in which 83% of the monomers had primary amines, and stability studies were carried out with sodium chloride (3.5 M), Sodium dodecyl Sulfate (0.5 M), and heparin. None of these conditions released the pDNA from the chitosan, indicating the formation of excessively stable polyplexes. The stability of chitosan polyplex was also checked using DNase enzyme (8U) by incubating UPC polyplexes at a charge ratio of 3:1(+/−) for 1 h and the result showed that the pDNA was protected from DNase degradation. The pDNA was released only after incubation with a chitosan-degrading enzyme chitosanase (120 mU; 5 h). From the gene expression studies at varying N/P ratios, the highest gene expression was obtained at a charge ratio of 2.4:1 (+/−), giving a modest CAT expression of 6.8 ± 1.2 pg/mg proteins [35]. A high N/P ratio indicates high stability of the polyplex in the extracellular environment and the enzymatic degradation of chitosan helps the release of pDNA in endosomal/lysosomal vesicles, thereby increasing the transfection rate. Formation of neutral or negatively charged polyplexes with a very low N/P ratio will show the least (or an absence of) transfection due to the decreased tendency to bind with the cell membrane and also tend to form aggregates [45]. It has been reported that maximum transfection efficiency can be obtained at N/P ratios in the range of 2–5 [46].

Degree of deacetylation (DDA) Like molecular weight, DDA of chitosan has a great influence on the stability as well as gene transfection efficiency of the chitosan/DNA polyplex. DDA denotes the relative percentage of d-glucosamine residues (i.e., deacetylated primary amines) in a chitosan molecule, which considerably affects a number of its properties such as the charge density, solubility, and ordered structure [47, 48]. Kiang et al. studied the effect of DDA of chitosan on the efficiency of gene transfection in chitosan/DNA nanoparticles. They suggest that deacetylation above 80% releases DNA very slowly and that of chitosan having DDA below 80% may improve the release since it ­reduces the charge

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density and increases the steric hindrance in complexing with DNA, which in turn accelerate the degradation rate. The in vivo gene expression studies on mice showed a dramatic increase in luciferase expression when DDA was decreased from 90% to 70%, that is, 62% and 70% deacetylation led to luciferase transgene expression two orders of magnitude higher than chitosan with 90% deacetylation [49]. KopingHoggard et al. reported that DDA of chitosan must exceed 65% to form stable chitosan/DNA polyplexes that can effectively transfect target cells [35]. Lavertu et  al. performed a systemic study on the combined effect of molecular weight (in the range 10–150 kDa) and DDA (in the range 70%–100%) and suggested that maximum transfection efficiency and, hence, maximum expression levels could be obtained either by simultaneously lowering the molecular weight and increasing DDA or by decreasing DDA and increasing molecular weight. They also reported that at a fixed DDA, chitosan having a lower molecular weight requires a higher N/P ratio to condense DNA completely. Similarly, at the same molecular weight, complete condensation of DNA occurs at a lower DDA having a higher N/P ratio [50]. From the above discussion, it is clear that transfection efficiency is a function of chitosan DDA, molecular weight, and N/P ratio, and hence there should be a proper correlation between the three parameters for getting the desired level of gene expression.

The pH of the transfection medium The transfection stability and efficiency of chitosan polyplexes was largely dependent not only on molecular weight and DDA but also on the pH of the culture medium because the change in pH of the transfection medium would influence the protonation of chitosan, affecting DNA binding and the charge density of the polyplexes. H.Q. Mao et al. reported that about 90% of the amino groups are protonated at pH 5.5–5.7 [51], which facilitates the DNA binding potential of chitosan to form small and stable polyplexes. The degree of protonation reduces with increasing pH, and hence aggregation of the complexes occurs with a subsequent positive charge neutralization [52]. Lavertu et  al. observed that the transfection efficiency at pH 6.5 was higher than at pH 7.1 and was comparable to commercially available vectors such as LipofectamineTM and Fugene 6 [50]. Mao et al. reported that the zeta potential of a polyplex decreased from slightly positive to nearly neutral when pH increased from 5.7 to 7.2 [51]. Sato et al. found that the transfection efficiency of chitosan/DNA polyplexes in A549 cells is higher at pH 6.9 than at pH 7.6 [40]. Another study by Nimesh et al. also demonstrated that the particle size of chitosan/ pDNA complexes increased with the increase in pH from 6.5 to 7.4, whereas the zeta potential decreased from 11.4±4.1 at pH 6.5 to −4.9±3.5 at pH 7.4. They also demonstrated that in HEK 293 cells, the transfection efficiency at pH 6.5 was greater than that at pH 7.1 and 7.4 [53]. The transfection of primary chondrocytes in different pH of the transfection medium was investigated by Zhao et al. at pH 6.8, 7.0, 7.2, 7.4, and 7.6 and the highest expression efficiency was obtained at pH 6.8 and 7.0. However, a decreasing tendency in transfection efficiency was observed with more acidic pH (below ~5.5) due to the strong electrostatic interaction between the positively charged chitosan and negatively charged DNA which thereby reduces the release rate of DNA [54].

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Serum concentration Another major parameter which influences the transfection efficiency of chitosan/DNA complexes is the serum concentration. A large number of studies have been conducted by many researchers in order to check the effect of serum on the transfection efficiency and it has been shown that the presence of a low percentage of serum in culture media enhances the chitosan-mediated gene expression [46, 55]. Nimesh et al. carried out the cell uptake of complexes by HEK 293 cells at pH 6.5, 7.1, and 7.4, both in the presence and absence of serum, and observed that the uptake of complexes was higher in the presence of serum at all the three investigated pH. The possible reason may because of the formation of small hydrodynamic diameter complexes between positively charged chitosan with the negatively charged serum proteins which can then be efficiently uptaken [53]. Sato et al. demonstrated that the gene expression level of chitosan/pGL3 polyplexes in 20% serum-containing media was two to three times higher compared to serum free medium and the effect may be due to increased cell function. However, an increase in the serum percentage to 50% showed a reduction in transfection efficiency because of the cell damage induced by the high content of serum [40]. Erbacher et al. observed higher transfection efficiency of chitosan/DNA polyplexes in HeLa cells in the presence of 10% serum than in the absence of serum [56].

Cell type A large number of studies on the transfection of different cell types with chitosan/DNA complexes have been carried out by many research groups. For chitosan-­mediated transfection, the HEK293 cell line is used most often since the best results on DNA uptake were achieved for these cells [57–59]. Corsi et al. reported that the transfection potential of polyplexes was higher in HEK293 than in human mesenchymal stem cells (MSCs) or human osteosarcoma cells (MG63) [60]. The transfection efficiency of ­chitosan-DNA nanoparticles was checked in HEK293, HeLa, IB-3-1 (bronchial epithelial cells), and 9HTEo (human epithelial cells) by Mao et  al. who observed higher gene expression levels in HEK293 cells and IB-3-1 cells compared with those in 9HTEo and HeLa cells [51]. In general, the binding affinity of chitosan for DNA, the stability and the transfection efficiency of the chitosan/DNA complexes, etc. are dependent on several parameters as discussed and the variation in any of these parameters can influence the size, charge, cell uptake, and release of the chitosan/DNA complex and thereby overall transfection efficacy. Hence, there must be a correlation between all these formulation parameters while designing a gene therapy system.

2.3.1.2 Chemical modifications of chitosan The potential of chitosan in gene delivery can be improved by making structural modifications with diverse chemical moieties. The reactive sites of chitosan include primary and secondary hydroxyl groups and a primary amine group. Many reports show that the surface modification in such sites enhances its overall gene transfection efficiency. For example, low aqueous solubility of chitosan at physiological conditions cause inadequate transfection efficiency, which can be improved by quaternization

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of primary amine [61]. Another reason for reduced transfection efficiency of chitosan across intracellular delivery is its poor buffering capacity, which causes inefficient release of DNA in chitosan/DNA complexes from endosomes into the cytoplasm [62]. One of the best solutions for this problem is the modification of chitosan with pH-­ sensitive groups such as imidazole, low molecular weight poly ethylene imine, methacrylic acid, etc. having buffering capacity to overcome the intracellular barrier [63]. Ghosn et al. observed a 100-fold increase in the transfection efficiency of chitosan by the incorporation of imidazole acetic acid (IAA). Water-solubility was also improved by the presence of IAA in the chitosan backbone [64]. Modification of chitosan can be divided into two: non-covalent and covalent. Covalent modification includes hydrophilic, hydrophobic, pH-sensitive, thermosensitive, and cell-specific ligand groups. One of the subtypes of hydrophobic modification is alkylated chitosan. The following section focuses on the different type of chemical modifications carried out for improving the efficiency of chitosan in gene delivery applications.

Non-covalent modification Non-covalent modification of chitosan, which consists of ionic gelation or electrostatic interaction with polyanions, is used to increase the water solubility and, thereby, transfection efficiency of chitosan/DNA polyplexes. Ionic gelation is a physical cross-linking process in which ionic interaction occurs between the positively charged primary amino groups of chitosan and the negatively charged groups of polyanion. Sodium tripolyphosphate (TPP) is one of the most extensively used ion cross-linking agents due to its multivalent properties [65, 66]. Csaba N et al. used TPP as a polyanionic cross-linker for developing nanoparticles of PEG-grafted chitosan (CS-g-PEG) and observed a very good gene expression level [59]. Gaspar et al. investigated the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using TPP as a polyanionic cross-linker. Analysis showed influence on the encapsulation efficiency and release of pDNA and consequently enhanced transfection efficiency [67]. Natural polyanions like HA, poly (g-glutamic) acid, etc. have also been used as complexing agents with chitosan. Low toxicity, high transfection efficiency, high biocompatibility, and good biodegradability make HA a good complexing agent with chitosan [68]. A fourfold improvement in transfection efficiency was obtained on both HEK293 and retinal pigment epithelial cells by the incorporation of HA with chitosan [69]. A combination of HA with ultralow molecular weight chitosan (5 kDa) forms stable polyplexes with high transfection efficiency in 293Tcells. Shu-Fen Peng et al. observed that the cellular uptake of chitosan/DNA complexes was significantly enhanced via the incorporation of poly (g-glutamic acid) [70].

Covalent modification

Hydrophilic modification  Hydrophilic modification of chitosan facilitates the enhanced water solubility at physiological pH, higher intracellular DNA release, reduced opsonization, and decreased sensitivity of polyplex to pH changes [71]. Quaternization of chitosan and incorporation of PEG moieties to the chitosan backbone are the two major strategies to improve the hydrophilicity.

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PEGylation of chitosan increases the stability of complex interactions (as well as minimizing nonspecific ones) between the complex and serum proteins which thereby increase the systemic circulation time [39]. Jiang et al. demonstrated that PEG-grafted chitosan effectively shielded polyplex charge, and due to the reduced surface charge and the steric hindrance of PEG chains, chitosan-g-PEG/DNA complexes were able to maintain increased serum stability and improved transgene expression in rat liver after bile duct and portal vein infusions [72]. Quaternization of chitosan increases the binding efficiency of nucleic acids to the polymer due to its increased positive charge density. Complete alkylation of the amino group is one of the best methods for quaternization of chitosan. Thanou et al. synthesized trimethylated chitosan (TMO) from oligomeric chitosan (