Hydrogels Based on Natural Polymers 0128164212, 9780128164211

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Hydrogels Based on Natural Polymers

Hydrogels Based on Natural Polymers Edited by Yu Chen School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2020 Elsevier Inc. 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. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-816421-1 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Edward Payne Editorial Project Manager: Joshua Mearns Production Project Manager: Nirmala Arumugam Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

List of contributors Hamideh Aghahosseini Department of Chemistry, University of Zanjan, Zanjan, Iran Nour Elhouda Ben Ammar Laboratory of Useful Materials Valuation, National Center of Research in Material Science (CNRSM), Soliman, Tunisia Mohamed Barbouche Laboratory of Nanomaterials and Systems for Renewable Energy, Research and Technologies Centre of Energy, Hammam-Lif, Tunisia Yu Chen School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Narayan Ch. Das Rubber Technology Centre, Indian Institute of Technology, Kharagpur, India; School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur, India Poushali Das School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur, India Guoxing Deng Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong Miheng Dong School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Sayan Ganguly Rubber Technology Centre, Indian Institute of Technology, Kharagpur, India Mohamed Mohamady Ghobashy Radiation Research of Polymer chemistry department, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt Vishwajeet Sampatrao Ghorpade Department of Pharmaceutics, School of Pharmaceutical Sciences, Sanjay Ghodawat University, Kolhapur, India Ahmed Hichem Hamzaoui Laboratory of Useful Materials Valuation, National Center of Research in Material Science (CNRSM), Soliman, Tunisia Minjian Huang Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong B. Kaczmarek Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Nicolaus Copernicus University in Torun, Torun, Poland Wing-Fu Lai Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong Yilin Leng School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Rong Li Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, P.R. China

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List of contributors

K. Nadolna Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Nicolaus Copernicus University in Torun, Torun, Poland Hassan Namazi Research Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran; Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Science, Tabriz, Iran A. Owczarek Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Nicolaus Copernicus University in Torun, Torun, Poland Jyotishkumar Parameswaranpillai Center of Innovation in Design and Engineering for Manufacturing, King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand Malihe Pooresmaeil Research Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran Ali Ramazani Department of Chemistry, University of Zanjan, Zanjan, Iran Sanjay Mavinkere Rangappa Department of Mechanical and Process Engineering, The Sirindhorn International Thai-German Graduate School of Engineering (TGGS), King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand Suchart Siengchin Department of Mechanical and Process Engineering, The Sirindhorn International Thai-German Graduate School of Engineering (TGGS), King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand ˇ Nataˇsa Skalko-Basnet Drug Transport and Delivery Research Group, Department of Pharmacy, Faculty of Health Sciences, University of Tromsø The Arctic University of Norway, Tromsø, Norway Xiaoyu Sun School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Shuxian Tang School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China ˇ Zeljka Vani´c Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Sandhya Alice Varghese Department of Mechanical and Process Engineering, The Sirindhorn International Thai-German Graduate School of Engineering (TGGS), King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand Wing-Tak Wong Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong Guozhong Wu Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, P.R. China Runyu Wu Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong Jueying Yang School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Jingjing Yuan School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Ronnie Yuan DSM (China) Limited, Shanghai, China Fei Zhang DSM (China) Limited, Shanghai, China

List of contributors xvii Hongbin Zhang Shanghai Jiao Tong University, Shanghai, China Ying Zhang School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China Lin Zhao School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China

CHAPTER 1

Properties and development of hydrogels Yu Chen* School of Material Science and Engineering, Beijing Institute of Technology, Beijing, China

Hydrogels are a type of polymer network that can absorb and store large amounts of water [1]. The hydrophilic groups or segments of the polymer network can be hydrated under neutral conditions to produce a gel structure [2]. The cross-linked network structure prevents the dissolution of the hydrophilic polymer chains or segments. Hydrogels are solid-like and show nonflowing properties as inverted. They can also be described by their fluidics [3]. In general, a low-concentration aqueous hydrophilic polymer solution with no entangled structures exhibits Newtonian mechanical behavior. Cross-linked hydrogels possess rheological properties with extremely high viscosity ( . 105 Pa s) and high elasticity (shear yield stress .2000 Pa), similar to solids. Due to their water insolubility, hydrogels are the research hotspots of swollen polymer networks, as well as their application studies. Hydrogels have been applied to a variety of fields, such as drought resistance in dry areas [4], masks in cosmetics [5], antipyretic stickers [6], analgesic stickers [7], agricultural films [8], dew prevention agents in construction [9], humidity control [10], water-blocking agents in the petroleum industry [11], dehydration of crude oil and refined oil [12], dust suppressants in mining [13], food preservatives [14], thickeners [15], pharmaceutical carriers in medical applications [16], tissue engineering scaffolds [17], wound dressings [18], etc. It is worth noting that different application fields require different polymer materials. In 1960, Wichterle and Lim synthesized a cross-linked HEMA hydrogel for the first time [19]. Its high hydrophilicity and biocompatibility attracted great interest in the biomaterial field and it has been widely used in contact lenses since then. The most important and influential discovery was the successful embedding of cells in an alginic acid microcapsule by Lim and Sun in 1980 [20]. Later, Yannas and his coworkers prepared hydrogels using natural polymers, collagen and shark cartilage, and explored their applications as burn wound dressings [21]. The natural polymer hydrogels have become the research hotspot for cell embedding. Later, with the rapid development of tissue engineering, hydrogels were used as the matrix for cell growth and carrier of growth factors to repair and re-elevate organs of various tissues [22
24]. With the changes in the application purpose and 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00001-X © 2020 Elsevier Inc. All rights reserved.

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Chapter 1

requirement, the research and development of hydrogel have shifted from traditional hydrogels to intelligent hydrogels. Hydrogels can be classified by different methods. 1. Hydrogels can be classified into natural hydrogels and synthetic hydrogels based on their sources. a. Natural polymers, such as collagen, gelatin, hyaluronic acid, fibrin, sodium alginate, agarose, chitosan, dextran, cyclodextrin, etc. are from natural sources with good biocompatibility and biodegradability [25
30]. The hydrogels prepared with these abundant polymers inherit these excellent properties and are sensitive to the external environment. Therefore they have become the research hotspot of hydrogel and are the focus of this book. In addition, most animals and plants contain a large number of natural polymer gels. The study of the structure and properties of such hydrogels is also an important way to study the physiological mechanisms of tissues for biomimetic applications [31
33]. b. Synthetic hydrogels are cross-linked polymers prepared by the addition reaction or ring-opening polymerization under artificial conditions. Polyacrylic acid and its derivatives [34], polyvinyl alcohol [35], polyethylene glycol and its copolymers [36], and polyvinylpyrrolidone [37], etc. are usually used as the skeletons to prepare synthetic hydrogels. Synthetic hydrogels have the advantages of easy industrial production and chemical modification and precisely controllable properties, yet with poor biocompatibility, bioactivity, and biodegradability, as compared with natural polymer hydrogels. 2. Based on the formation mechanism of three-dimensional network structure, hydrogels can be divided into chemical hydrogels and physical hydrogels. a. Chemical hydrogels are formed by the chemical cross-linking between molecules, and this cross-linking is irreversible. Chemical hydrogels usually have the advantages of stable properties, tunable structures, good mechanical properties, etc. [38
40]. b. Physical hydrogels are mainly the three-dimensional networks formed by noncovalent bonds (secondary bonds), such as electrostatic interaction, hydrogen bonding, chain entanglement, and hydrophobic interaction, between linear molecules that form physical cross-linking joints [34,41,42]. Physical hydrogels usually exhibit reversible sol
gel conversion because very low energies are required to break the physical interactions between the molecules [43]. No chemical reaction is involved in their preparation and the preparation conditions are relatively mild, which is favorable to their applications in the biomedical field [44].

Properties and development of hydrogels 5 3. Based on their degradability, hydrogels can be divided into biodegradable hydrogel and nonbiodegradable hydrogel. a. Most of the natural polymer hydrogels are biodegradable hydrogels [45]. The threedimensional structures of these hydrogels can be destroyed by the actions of microorganisms and enzymes under natural conditions. The bonding between the molecular chains and within the molecular chains is then broken and the strength of the hydrogel is reduced. Eventually, the hydrogel is degraded into small molecules. b. Nonbiodegradable hydrogels are a class of hydrogels that are insensitive to environmental stimuli and can maintain stable structural, physical, and chemical properties for a long time. Most synthetic hydrogels prepared by chemical crosslinking are nonbiodegradable hydrogels [46]. 4. Based on their responsiveness to external stimuli, hydrogels can be divided into environmentally responsive hydrogels (intelligent hydrogels) and environmentally unresponsive hydrogels (regular hydrogels). a. Environmentally responsive hydrogels, also known as intelligent hydrogels or smart hydrogels, can reversibly respond to external stimuli. When a hydrogel is exposed to environmental stimuli, such as temperature, pH, ion strength, electric field, light, stress, magnetic field, etc., its three-dimensional network structure changes, swells or shrinks, or transits between the dense phase and the dilute phase, showing dramatically altered shape, mechanical and optical properties, etc. in response to the stimuli. The hydrogel will automatically return to a lower steady state of internal energy as the external stimuli disappear. Thermo-responsive hydrogels are a type of hydrogel showing temperatureinduced volume shrinking or swelling behaviors [47,48]. In general, their volumes can be varied from several times to hundreds of times by temperature changes. A thermo-responsive hydrogel contains both hydrophilic and hydrophobic functional groups with a certain ratio in its network. At low temperatures, the hydrophilic groups on the polymer chain form hydrogen bonds with water molecules. The polymer
water interaction is stronger than the polymer
polymer interaction. The hydrophilicity dominates the structure, and the polymer chain is in the extended state. As temperature is increased, the hydrogen bonding between the polymer and water is gradually weakened and the hydrophobic interaction between the molecular chains is enhanced. At temperatures close to the lower critical solution temperature (LCST), the hydrophobic interaction between the polymer chains is stronger than the interaction between the polymer and water. The hydrophobicity of the polymer chain dominates the structure. The hydrogel shrinks and a large amount of water in the hydrogel is released, which is called deswelling. The corresponding temperature is called the volume phase transition temperature (VPTT) of the hydrogel. The hydrogel can reabsorb water and swell again as the temperature decreases below the VPTT, which is called reswelling. In general, the

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Chapter 1 volume change of a thermo-responsive hydrogel is most significant at the VPTT. Poly(N-isopropylacrylamide) hydrogels containing both hydrophilic imido groups (
NHCO
) and hydrophobic isopropyl (
CH(CH3)2) are the most commonly used thermo-responsive hydrogels [49]. A pressure-sensitive hydrogel possesses the structural characteristics of a hydrogel and thermo-sensitivity. Its structure changes at the LCST under the hydrostatic pressure [50], causing swelling behaviors mainly due to the compressible caves in its structure. The pH-responsive hydrogels exhibit volume changes as the pH of the external environment is changed. A pH-responsive hydrogel typically contains weakly acidic functional groups, such as
COOH and
SO3H, or weakly alkaline functional groups, such as
NH2 [51,52]. These functional groups are highly hydrophilic in the ionized state and less hydrophilic in the neutral state. Their degrees of ionization change with pH, which changes the charge density. At the critical pH (usually around the pKa), the osmotic pressure between the inside and outside of the hydrogel is dramatically changed, causing the volume phase transition. The swelling, shrinking, and osmotic pressure change with pH and ion strength of pH-responsive hydrogels makes them suitable for targeted drug delivery [53]. In the early 1980s, Tanaka et al. discovered the volume phase transition of a polymer hydrogel under the stimulation of an electric field, which promoted the research and development of electric field-sensitive hydrogels [54]. Similar to pHsensitive hydrogels, electric field-sensitive hydrogels are usually composed of polymer electrolytes. The counter ions of the charged groups on the polymer network migrate in the electric field. The ion concentration gradient across the inside and outside the gel network changes the osmotic pressure, resulting in volume changes or shape changes of the hydrogel [55]. Therefore such hydrogels can respond to the external electric field with shrinking, swelling, or bending behaviors [56]. Magnetic field-sensitive hydrogel is prepared by hybridizing a magnetic material with hydrogel. The magnetic material in the hydrogel responds to the magnetic field changes with changes of paramagnetism and diamagnetism or magnetic field rearrangement. The force generated by such movement and interaction causes macroscopic changes to the hydrogel [57]. A chemical-responsive hydrogel responds to the concentration changes of a specific chemical, such as glucose, antibody, enzyme, and DNA, with shrinking/ swelling volumetric changes or gel
sol transition [58]. For example, the polymer network cross-linked by the antigen
antibody can respond to the free antigen in the medium with volume phase changes because the free antigen competes with the self-antigen (bonded on the polymer network) to bind the antibody linked to the

Properties and development of hydrogels 7 polymer network. The competition changes the cross-linking density of the network, resulting in responsive behaviors [59]. Photo-responsive hydrogels are a type of hydrogel that undergo volumetric changes, phase changes, or color changes under illumination at certain wavelengths [60]. There are mainly three types of photo-responsive mechanisms. For the first photo-responsive mechanisms, the special photoreceptor(s) is composited with a thermo-responsive hydrogel, which can convert photo energy into heat, causing a local temperature rise. When the temperature reaches the phase transition temperature of the thermo-responsive hydrogel, the hydrogel exhibits responsive behaviors [61]. A photothermal responsive hydrogel has been obtained by introducing a photosensitive chromophore, trisodium chlorophyllinate, into a thermo-responsive hydrogel [62]. The second photo-responsive mechanism takes advantage of the photolysis of photo-sensitive molecules into ions [63]. For the third photo-responsive mechanism, chromophores are added into the hydrogel network that undergoes changes in physical and chemical properties under illumination. The space or geometry of the polymer chain with chromophores is then changed, causing swelling or shrinkage of the hydrogel [64]. The photothermal conversion efficiency can be improved by replacing the infrared light and visible light with high-energy laser or doping nanoparticles, such as carbon nanotube [65], graphene [66], etc., into the hydrogel. Such strategies can also promote the response rate or remotely control the stimuli-responses of hydrogels. Multiresponsive hydrogels are hydrogels that respond to two or more external stimuli. There are a variety of multiresponsive hydrogels, mainly including pH/ temperature dual-sensitive hydrogels [67], light/temperature dual-sensitive hydrogels [68], and light/pH dual-sensitive hydrogels [69]. Multiresponsive hydrogels can be prepared with copolymer structures [70], double networks [71], and core
shell structures [72]. The unique intelligent responsive properties of the environmentally responsive hydrogels render them broad application prospects, such as sensors [73], artificial muscles [74], chemical mechanical devices [56], chemical storage [75], molecular separations [76], enzyme immobilization [77], tissue engineering [78], drug delivery [53], dimming materials [79], and other smart materials. The conventional hydrogel drug carriers deliver drugs by the osmotic action of the drug molecules and the decomposition of the hydrogel matrices. The environmentally responsive hydrogels can be used to deliver drugs by a variety of intelligent regulation means. Hydrogels are often used for cell culture due to their similarity to tissue structures. The dynamic responsive characteristics of environmentally responsive hydrogels facilitate the in situ changes in the hydrogel matrix, such as adhesion and mechanical strength, to dynamically regulate the growth environment of cells [48].

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b. Environmentally unresponsive hydrogels Hydrogels with stable structures and physical and chemical properties under different environmental conditions [80], for example, environmental unresponsiveness, are needed for some special applications. For example, agarose is highly hydrophilic and biodegradable, but has almost no charged groups. Its hydrogel is rarely desaturated by or adsorbed by sensitive biomacromolecules, and thus it is often used as a support matrix for immunoelectrophoresis or gelation [81]. 5. Based on their size, hydrogels can be classified into bulk hydrogels and microhydrogels. a. Bulk hydrogels are the large pieces of hydrogels with certain shapes and sizes [82], usually in the forms of low-strength jelly with certain fluidity and viscosity. They are usually used in beauty salons and food processing [83]. Bulk hydrogels with certain strengths and thicknesses can also be used in tissue engineering, food processing, films, coatings on inert biosensors [84], and antibacterial coatings [85]. Despite its excellent properties and wide applications, the processing and shaping of this soft and low-strength aqueous material remain challenging and its applications in some special fields are limited. Therefore efforts have been made to improve the strength of bulk hydrogels by introducing a reinforcing phase into the matrix, which gives the hydrogel new physical and chemical properties [86]. In particular, nanocomposite hydrogels prepared by dispersing inorganic nanoparticles into hydrogel matrices can fuse the rigidity, dimensional stability, and thermal stability of the nanomaterial with the softness and wettability of the hydrogel, producing better physical and mechanical properties and thermal stability [87]. The enhanced performances of the hydrogels can be explained by the permeation theory, nanoparticle/ polymer cross-linked structure, or polymer/nanoparticle intercalation structure [88]. b. Microhydrogels are individual and smaller hydrogels [89], as compared with the bulk hydrogel. Among them, microhydrogels of nanometer scale are the focus and hotspot of research [90]. The quantum effect and surface effect produced by the small sizes and large specific surface areas of the nano-scale microhydrogels render them excellent performances in mechanics, electricity, optics, magnetism, and catalysis applications [91]. 6. Based on their functions in practical applications, hydrogels can be divided into in situ hydrogels, molecularly imprinted hydrogels, nanohydrogels, and porous hydrogels. a. In situ hydrogels A polymer undergoes reversible changes of state or transformation of conformation under physiological conditions with changes in the external environment, such as temperature, pH, ionic strength, etc., which causes the phase transition from the solution to the semisolid gel, forming an in situ hydrogel [92]. Compared with conventional hydrogels, the unique sol
gel transition of in situ hydrogel endows it great advantages in drug delivery, such as easy preparation, easy operation, strong affinity with drugs and mucosal tissues, and long drug

Properties and development of hydrogels 9 retention time [93]. The in situ hydrogel drug-delivery systems have become a research hotspot in pharmacy. The drug embedded in an in situ hydrogel carrier can avoid digestion in the gastrointestinal tract and elimination in the liver during mucosal administration, and thus exhibits low effective dose and high bioavailability [94]. In addition, the physical and chemical properties and the state of the drug in the body are tunable according to environment changes to achieve timely and effective therapeutic effects. The strong affinity of in situ hydrogel to mucosal tissues, long drug retention time, sustained release or controlled release, good tissue compatibility, and easy operation can reduce the frequency of drug administration and improve the quality of life of patients. In addition, the in situ hydrogel carried drugs can be administered by various routes, such as ocular, nasal, transdermal, rectal, and injection [95]. In situ hydrogels can also be used for targeted drug deliveries [96], whole body application [97], macromolecular drugs [98], cell tissues [99], hydrophilic drugs [100], hydrophobic drugs [101], etc. The hydrogels can be temperature-sensitive, ionic strength-sensitive, or pH-sensitive. b. Molecularly imprinted hydrogels The hydrogels prepared by molecular imprinting exhibit high binding abilities to the template molecules, and thus they can recognize, bind, or release specific molecules automatically [102]. Biological macromolecules, such as proteins [103], peptides [104], nucleotides [105], glucose [106], etc., have been used as templates. The combination of molecular imprinting with smart hydrogel can not only improve the binding of a hydrogel network to specific molecules, but also can switch the memory of a hydrogel of a specific molecule by changing the external environment, realizing automatic binding and releasing of the molecule. Meanwhile, the concentration changes of a specific molecule in the medium can also stimulate the hydrogel to swell or shrink, which can be used for controlled release of the embedded substance [107]. Molecularly imprinted smart hydrogels can sense and respond to specific molecules, control the on/off switch of the memory of these molecules based on the external stimulation signals, and thus have great application potential in biosensors [108], controlled drug release [109], and immunoassays [110]. c. Nanohydrogels Nanohydrogels are highly cross-linked nano-scale hydrogels prepared by chemical or physical methods. They can be used to carry therapeutic compounds with excellent ability to prevent premature leakage of the therapeutic agent and sustainably release the agent [111]. Nanohydrogels can be absorbed by cells and thus they are effective carriers for the intracellular delivery of therapeutic agents [112]. Nanohydrogels retain their payload in the extracellular environment and are triggered to release the loaded agent under the stimulation of one or more intracellular triggers once they are internalized by the target cells, giving good therapeutic effects. Nanohydrogels can be administered intravenously to areas that are not easily accessible. These excellent properties of nanohydrogels have rendered them great

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Chapter 1 application potentials for the encapsulation and smart delivery of therapeutic agents [113]. However, evidences for the drug release from nanohydrogels in vivo or intracellularly are still lacking. The in-depth understanding of their action mechanism in cells is the key to their application. In addition, there are only a few reports of their long-term accumulation and degradation, and more studies of the in vivo behaviors of nanohydrogels are needed before any clinical trials. Further studies on their cytotoxicity, immunogenicity, pharmacokinetics, and in vivo therapeutic effects are still needed before application of the theoretical study results. d. Porous hydrogels The application potentials of a hydrogel can be evaluated with a series of parameters, such as swelling rate, swelling ratio, and mechanical strength. The hydrogels synthesized by most methods are nonporous or porous yet with low porosities, and low swelling rates even with high swelling ratios, which greatly limits their applications in drug delivery and tissue engineering. The numerous pores in the porous hydrogel provide a large specific surface area where more hydrophilic groups can quickly contact water, which, along with the capillary action of the porous structure, greatly improves the water absorption rate of the hydrogel [8]. The porous structure of a hydrogel can be tubed to shorten the time to reach the swelling equilibrium by adjusting the processing parameters, such as the dosage of cross-linking agent, pore-forming method, and the dosage of porogen [114]. The environmentally responsive porous hydrogels usually exhibit quicker responses and better sensitivity [115]. However, extremely high porosities can significantly weaken the overall mechanical strength of the hydrogel. Porous hydrogels have been prepared by the freeze-drying method [116], phase separation method [117], template method [118], porogen method [119], and foaming method [120]. The study of hydrogels has a very long history and improving their environmental responses has become an important research direction to promote their application [121]. However, at present, the response time of most hydrogels is still very long, and the size selectivity is low. In addition, the mechanical properties of hydrogel need to be further improved. Natural polymer materials have become a hotspot for the preparation of multifunctional hydrogels due to their good biocompatibility, biodegradability, and modifiability. This book will also discuss progress in the research and application of natural polymer hydrogel materials, as well as the existing challenges and ways to make improvements.

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35.

CHAPTER 2

Natural polymers and the hydrogels prepared from them Sandhya Alice Varghese1, Sanjay Mavinkere Rangappa1,*, Suchart Siengchin1 and Jyotishkumar Parameswaranpillai2 1

Department of Mechanical and Process Engineering, The Sirindhorn International Thai-German Graduate School of Engineering (TGGS), King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand 2Center of Innovation in Design and Engineering for Manufacturing, King Mongkut’s University of Technology North Bangkok, Bangkok, Thailand

2.1 Introduction to natural polymers Polymers are versatile materials composed of repeating structural units forming a macromolecule. The three major classes of polymers based on their origin are natural polymers, semisynthetic polymers, and synthetic polymers. Natural polymers are of natural origins such as from plants, microorganisms, and animals. They include carbohydrates and proteins, which provide structural support to plants and animals. There are six main types of natural polymer: proteins, polysaccharides, polynucleotides, polyisoprenes, polyesters, and lignin. As compared to the other two classes, natural polymers are economical, readily available, potentially biodegradable, and biocompatible due to their origin [1,2]. Many of these polymers are a part of our day-to-day diet and also possess significant scope in drugs, prosthetics, pharmaceuticals, food, and cosmetic industries [1]. Some of these widely used natural polymers based on polysaccharides and proteins are discussed in the following section.

2.1.1 Natural polymers based on polysaccharides 2.1.1.1 Polysaccharides of plant origin 2.1.1.1.1 Cellulose

French chemist Anselme Payen discovered cellulose in 1838 by isolating it from plant matter and also determined its chemical structure [1,2]. Cellulose is the most abundant organic material on Earth and forms an integral part of cell walls in higher plants [1
5]. 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00002-1 © 2020 Elsevier Inc. All rights reserved.

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Cellulose, along with proteins, provides rigidity and dynamic nature to the cell wall [2]. It can be found in many agricultural products including sugarcane, sorghum bagasse, corn stalks, and straws of rye, wheat, oats, and rice and is the key constituent of cotton (95%), flax (80%), jute (B70%), and wood (B50%) [5]. It is insoluble in water and indigestible by the human body [1,6]. There are many limitations to cellulose, such as low solubility in most solvents, low dimensional stability, and poor antibacterial properties [4]. In composition, cellulose is an organic linear polysaccharide composed of D-glucose units having the formula (C6H10O5)n. The absence of side chains in cellulose molecules brings them close to each other to form rigid structures. The structure of cellulose is shown in Fig. 2.1. Interestingly, cellulose was used in writing materials and lingerie during the periods of the Chinese dynasties and Egyptian pharaohs [2]. Today, microcrystalline cellulose finds major applications in the pharmaceutical industry as a diluent/binder in tablets. Modified cellulose like carboxylated methyl cellulose is used in drug formulations, and as a binder for drugs. Cellulose acetate fibers are used for wound dressings and films [7]. 2.1.1.1.2 Starch (amylose and amylopectin)

Starch is the primary carbohydrate material present in green plants and it is found in seeds and underground parts in the form of granules known as starch grains and is the main source of food for humans [8,9]. In its raw form, starch is a tasteless and odorless white powder. Starch contains a large number of glucose units linked together by glycosidic bonds [9]. Generally starch exists in two forms: linear and helical amylose (α-1,4-linked D-glucose monomers) (20%
25% w/w) and branched amylopectin (a highly branched polymer consisting of both α-1,4- and α-1,6-linked D-glucose monomers) (75%
80%).

Figure 2.1 Structure of cellulose.

Natural polymers and the hydrogels prepared from them 19

Figure 2.2 Structure of starch.

In contrast to amylose, which is a semicrystalline biopolymer soluble in hot water, amylopectin is a highly crystalline polymer insoluble in hot water [9]. Although the concentration of these two components varies depending on the source of starch, the microstructure of starch is identical from all sources [2]. The chemical structure of starch is depicted in Fig. 2.2. Reports suggest that starch was used in cosmetic creams, food thickeners, and in paper production as long ago as CE 700 [2]. In industries, starch finds applications in adhesives, paper, mulch films, packaging, and clothing [2,7]. Starch is a potential candidate for use as biodegradable plastic, especially because of its easy availability and low cost [9]. 2.1.1.1.3 Lignin

The word lignin comes from the Latin word lignum, referring to wood. In 1838 lignin was first identified as a constituent of wood by Ansleme Payen [2]. It is the second most

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Chapter 2

abundant natural polymer after cellulose, and is also the most abundant aromatic polymer in nature [2]. It is the main constituent of the cell wall in plants [10]. Lignin is a derivative of cellulose and is a three-dimensional amorphous polymer of methoxylated phenylpropane structures [11]. Although the precise structure of native lignin is not yet known and the structure of particular lignins depends on the source and extraction method, it is known that lignin is mainly made of methoxyl groups, phenolic hydroxyl groups, and a few aldehyde groups [2,11]. Lignin is widely used in the paper pulping manufacture, in adhesives, for fuel, etc. [10]. 2.1.1.1.4 Inulin

Inulin can be extracted from the bulbs of dahlia (Inula helenium; Compositae), roots of dandelion (Taraxacum officinale; Compositae), burdock root (Saussurea lappa; Compositae), or chicory roots (Cichorium intybus; Compositae) [7]. It is a member of the fructan family, and is comprised mainly of β-(2-1) fructofuranosyl units (Fm), and a terminal α-glycopyranose unit (1-2) (GFn). The chemical structure of inulin is shown in Fig. 2.3 [12]. Inulin finds wide application in the food industry.

Figure 2.3 Structure of inulin.

Natural polymers and the hydrogels prepared from them 21 2.1.1.1.5 Guar gum

Guar gum, also called guaran, is powdered endosperm of the seeds of Cyamopsis tetragonolobus Linn. (Leguminosae). Guar gum consists of two sugars, namely galactose and mannose [7]. The D-mannose monomer units are joined to each other by β-(1-4) linkage, to obtain the main chain with D-galactose branches joined by α-(1-6) bonds [13]. Generally, the galactose branches occur on every other mannose unit, the accurate ratio of galactose to mannose is dependent on the growing season [13]. The structure of guar gum is shown in Fig. 2.4. Guar gum thickens spontaneously without application of heat—this property of guar gum finds application in the oil industry as a thickener for hydraulic fracturing to enhance oil recovery [13]. Modified guar gums are used in drug delivery [7]. It is also used as a natural food supplement due to its high nutritional value [13]. 2.1.1.1.6 Alginate

Alginate is a long-chain hydrophilic polysaccharide obtained from seaweed, where it exists within the cell walls enhancing the flexibility and strength [2,7]. Alginate was used in food dated to 600 BCE, however its purified form was extracted only in 1896 by Akrefting [2]. It was used as a stabilizer and emulsifier in the food industry, in drug delivery, tissue engineering, wound dressings, etc. [4,7].

Figure 2.4 Structure of guar gum.

22

Chapter 2

2.1.1.2 Polysaccharides from animal origin Other than polysaccharides of plant origin, there are also polysaccharides of animal origin like chitosan, chitin, xanthan gums, and hyaluronic acid (HA). The polysaccharides derived from animal origin are discussed below. 2.1.1.2.1 Chitin and chitosan

Chitin is the most abundant animal polysaccharide on Earth and contains both amino and acetyl groups in its basic structure. The structure of chitin is shown in Fig. 2.5. It forms the basic element of the exo-skeleton of insects and crustaceans and the outer skin of fungi [7,14]. Structurally, chitin is similar to cellulose except that the hydroxyl groups at C2 are replaced by N methylamide groups [2,14]. Deacetylation of chitin forms chitosan [2], with the structure shown in Fig. 2.5. Chitosan is widely applied in transdermal drug delivery, controlled drug delivery, and gene delivery [2,7]. 2.1.1.2.2 Xanthan gums

Xanthan gum is a high-molecular-weight extracellular polysaccharide produced by the fermentation of glucose or sucrose by the Gram-negative bacterium Xanthomonas campestris [7,15]. Structurally, it contains a cellulose backbone of β-D-glucose and has a trisaccharide side chain containing D-mannose and D-glucuronic acid groups attached alternatively as shown in Fig. 2.6 [7]. It finds application in oral and topical formulations, cosmetics, as a suspending and stabilizing agent in the food industry, and in the preparation of sustained-release matrix tablets [7].

Figure 2.5 Structures of chitin and chitosan.

Natural polymers and the hydrogels prepared from them 23

Figure 2.6 Structure of xanthan gum.

2.1.1.2.3 Hyaluronic acid

HA is a polysaccharide found in the tissue of vertebrates and is an example of a glycoprotein [2]. Chemically, HA is a linear polysaccharide made up of D-glucuronic acid and N-acetyl-D-glucosamine joined to each other by a β-(1-3) linkage. In an HA polymer chain, 250
25,000 such basic disaccharide units may be present, linked by β-(1-4) linkage. The chemical structure of HA is shown in Fig. 2.7 [4]. It is useful for certain biomedical applications as a biological absorber and lubricant [4]; in combination with alginate it is used in surgical applications for wound healing [2]. HA and its derivatives are of use in tissue engineering in the form of gels, sponges, films, fibers, and microparticles. They are also used in drug-delivery applications [16].

2.1.2 Natural polymers based on proteins 2.1.2.1 Collagen and gelatin Collagen is an animal-based structural protein made up of three polypeptide chains folded into a triple helix structure usually produced by fibroblast cells [2,7]. It is usually found in tissues like muscles, skin, and bones, that impart strength and flexibility. There are many

24

Chapter 2

Figure 2.7 Structure of HA, Hyaluronic acid.

types of collagen depending on the source, the most common being type I collagen [2]. Due to its biodegradability, biocompatibility, availability, and versatility, collagen is widely applied in general surgery, orthopedics, cardiovascular, dermatology, otorhinolaryngology, urology, dentistry, ophthalmology, and plastic and reconstructive surgery [2,16]. Modification of collagen by denaturation and/or physical
chemical degradation forms a high-molecular-weight polypeptide called gelatin [7]. They find applications as emulsifiers, foaming agents, colloid stabilizers, biodegradable film-forming materials, etc. [2,7]. Some of the other natural polymers are discussed in later sections of this chapter. As mentioned in previous sections, biopolymers are widely used for many advanced applications because of their biocompatibility and biodegradability. They are highly recommended for biological applications like tissue engineering and regenerative medicines. For such applications, it is desired to obtain these polymers in a form such that they can replicate the extracellular matrix. One such suggested form is hydrogels, which are discussed in the following section.

2.2 Hydrogels Hydrogels are hydrophilic three-dimensional polymeric networks. They cannot dissolve, but are highly swollen by water [17
21]. The hydrophilic groups (like
OH,
CONH,
CONH2, and
SO3H) present in the polymeric backbone enable the absorption of a large amount of water, while the cross-linked network structure hinders their dissolution. These structural cross-links are the result of covalent bonding, hydrogen bonding, and van der

Natural polymers and the hydrogels prepared from them 25 Waals interactions along with physical entanglements, known as crystallites. The development of the first hydrogel by Wichterle and Lı´m in the 1950s was based on the copolymers of 2-hydroxyethyl methacrylate with ethylene dimethacrylate and is considered to be the first hydrogel used in a medical implant (soft contact lenses) [19]. Hydrogels can be derived both from natural polymers (natural or biohydrogels) and also from synthetic polymers (synthetic hydrogels). While synthetic polymer-based hydrogels offer longer service life, higher water absorption, improved gel strength and tunable properties, naturally derived hydrogels are advantageous as far as biocompatibility and biodegradability are concerned. Various natural polymers employed in the preparation of hydrogels include chitosan, alginates, HA, pectins, gellan gum, dextran, xanthan gum, guar gum, psyllium polysaccharide, tamarind polysaccharide, locust bean gum, sterculia gum, gum tragacanth, etc. The unusual properties of hydrogels including high sensitivity to physiological environments, hydrophilic nature, soft tissue-like water content, and adequate flexibility make hydrogels be potential candidates for various biomedical and agricultural applications [17
22].

2.3 Classification of natural hydrogels The classification of natural hydrogels on the basis of origin, cross-linking, charge, physical state, physical configuration, and composition is shown in Fig. 2.8; these are examined in more detail in the following sections.

2.3.1 Classification based on origin The two major classes of natural polymers forming hydrogels are (1) polysaccharides such as HA, alginate, and chitosan and (2) proteins such as collagen, gelatin, and lysozyme [23
26]. These hydrogels are biocompatible, biodegradable, and nontoxic. However, they have poor mechanical properties and batch variation may lead to poor reproducibility [23,27
29]. Each of these classes is discussed in detail in the following sections. 2.3.1.1 Natural polymers forming hydrogels Many biohydrogels can be readily extracted from their natural source. Collagen, fibrin, and HA are components of the natural extracellular matrix. The hydrogels based on these polymers may possess biofunctional features like modulating cell behavior and matrix production and are known as promoting hydrogels. Alginate and agarose are derived from marine algae, while newly established natural hydrogels like nanofibrillar cellulose hydrogels consist of plant-derived fibrillar glucan chains. They are biocompatible, have low cytotoxicity, and are enzymatically biodegradable in the human body. Biohydrogels of nonanimal origin are easily available and avoid probable sources of viral contamination [28,30]. These are now looked at in greater detail.

26

Chapter 2

•Protein-based hydrogels •Polysacchride-based hydrogels

Origin

•Physical hydrogels •Chemical hydrogels

Cross-linking k

Charge

•Neutral •Anionic •Cationic •Amphoteric •Zwitterionic

•Solid •Semi-solid •Liquid

Physical t state

Physical a t configuration

Composition t

•Crystalline •Amorphous •Semi-crystalline

•Homopolymer •Copolymer •Semi-IPN •IPN

Figure 2.8 Classification of hydrogels.

2.3.1.1.1 Hydrogels from proteins

As compared to their advanced industrial applications, polymers based on proteins are among the most under-used feedstocks. They are effective starting materials for the development of several biomaterials, like films and composites. These proteins include silk

Natural polymers and the hydrogels prepared from them 27 fibroin from spider webs, collagen from skin, keratin from wool/hair, bone, and tendons, elastin from elastic tissues, fibrin from blood clots, and resilient from insect tendons. 2.3.1.1.1.1 Preparation methods Generally, the free-radical polymerization method is employed to synthesize protein-based hydrogels, where protein molecules remain in the three-dimensional interpenetrating hydrogel network. Graft polymerization of vinyl monomers on to the protein backbone using initiators in the presence of cross-linkers produces hydrogels with improved properties. Superabsorbent polymer hydrogels based on collagen and cotton seed proteins are synthesized by the graft polymerization technique [31]. Collagen and gelatin hydrogels Collagen is a fibrous protein and forms the major component of the extracellular matrix. The presence of specific peptide sequences in collagen promotes cell adhesion. Due to its low immunogenicity it has been extensively used in biomedical applications. Collagen constitutes 25%
35% of protein content in the body, possesses excellent hydrophilicity, in vivo stability, and pore structure that make it a desirable substrate for stem cell culture. The most utilized form of collagen is collagen type I from various tissues including skin and ligaments, and is prepared through enzymatic and acidified processes. Collagen is generally dissolved in dilute acid and forms a hydrogel once the reaction is neutralized (usually with sodium hydroxide), and gently heated to body temperature. Collagen is mostly used for tissue regeneration applications, wound dressings, cellular transplant vehicles within tissue engineering, and pharmaceutical, photographic, and biomedical products. However, collagen suffers certain drawbacks like poor mechanical properties, handling difficulty, and the possibility of inducing immune reactions and less control over biodegradability [21,32
37]. Gelatin is a derivative of collagen, resulting from physical and chemical degradation of the triple helix structure of collagen. Due to its thermoresponsive nature, gelatin has the ability to undergo sol
gel transition depending upon the environmental temperature. Below ambient temperature (B30 C), there is a solution to gel transition and heating to physiological temperature reverses this state. Usually, enzymatic cross-linking using enzymes such as tyrosinase and transglutaminase (TG) is used for cross-linking gelatin and collagen [21,36]. Chemical cross-linking via aldehydes like glutaraldehyde may affect the biocompatibility of the hydrogel [30]. The porous structure, solubility, transparency, and biocompatibility make gelatin an ideal candidate as a natural scaffold. As in the case of many biopolymers, it suffers drawbacks like poor mechanical properties and needs extensive cross-linking, including physical, chemical, or enzymatic cross-linking, to be functional [21,34,35,37]. Silk proteins/fibroin Silk fibroin is a natural protein produced by the silkworm Bombyx mori and is a class of secretion animal fibers. The primary structure of silk consists of a simple amino acid mixture of glycine and alanine. Fibroins possess properties such as excellent elasticity, biocompatibility, and high mechanical strength. The repetition in their primary structure results in a very consistent and homogeneous secondary structure giving strong yet

28

Chapter 2

flexible materials, with mechanical properties superior to other synthetic as well as natural polymers [21,33]. These silk fibroin gels exhibit shear-thinning behavior and find application as injectable tissue engineering scaffolds for the homogeneous administration of viable cells, wound dressings, as well as articular cartilage repairs [31,38]. Fibrin Fibrin is a biopolymer with high water content formed by the enzymatic polymerization of fibrinogen in the presence of thrombin. Fibrin derived from animals and humans plays a major role in the blood clotting mechanism. The drawbacks of fibrin include its low mechanical strength and rapid degradation behavior that affects its stability. The addition of protease inhibitors, that inhibit the action of enzymes responsible for degradation and chemical modifications like cross-linking, can control the rapid degradation to a degree. Their excellent biocompatibility and biodegradability make them suitable for use in tissue engineering cartilage, bone, and adipose tissues, as a substrate for cell adhesion and guiding cell migration, as a sealant, an adhesive in surgery, and for skin graft fixation [30,32,34]. 2.3.1.1.2 Polysaccharide-based natural hydrogels

Polysaccharides are another class biopolymers used for hydrogel development. Properties such as nontoxicity, high swellability, and high mechanical properties, in addition to their biodegradability, biocompatibility, and biostability make them suitable for many biomedical applications such as cell/drug delivery, gene delivery, regenerative medicine, cell culture, and tissue engineering. Prominent polysaccharides are alginate, chitosan, gelatin, carrageenan, gellan gum, guar gum, pectin, cellulose, agarose, xanthan gum, etc. 2.3.1.1.2.1 Preparation methods Polysaccharide hydrogels can be synthesized either by chemical cross linking methods or physical methods like freeze
thawing. Freeze
thawing produces hydrogels which are more biocompatible and biodegradable than those prepared by chemical methods [31]. Hyaluronic acid hydrogels HA, an anionic polysaccharide, is a linear copolymer of 2-acetamido-2-deoxy-D-glucose and D-glucoronic acid. It was first isolated from the vitreous fluid of cow’s eyes in 1934 by scientists from Columbia University in New York [30]. It forms one of the major components in natural extracellular matrices. HA is highly flexible in its physical structure which permits it to exist as firm hydrogels, viscoelastic liquids, meshes, fibers, sponges, sheets, or nanoparticulate fluids. High-molecular-weight HA is highly viscous and difficult to handle. Low-molecular-weight HA can be prepared by degradation either by acid or base treatment and is preferred for preparing hydrogels. Modification can be done to enhance hydroxyl and carboxylic groups in HA to give amine, hydrazide, thiol, acrylate, and phenol functionality. HA is nonimmunogenic and biodegradable, however the mechanical properties are limited [30]. The applications of HA include cartilage regeneration, wound dressing, as injectable thermosensitive hydrogels, as a sponge base for treatment of osteochondral dysfunction, and promoting chondrogenesis within the affected tissue. HA is

Natural polymers and the hydrogels prepared from them 29 also used within drug-release mechanisms including ophthalmic, nasal, pulmonary, parenteral, and topical methods of drug administration [21,32,33,37]. Alginate hydrogels Alginate is an anionic polysaccharide produced from brown seaweed by the action of two types of bacteria, namely Azotobacter and Pseudomonas. The extraction of alginate from algae involves dehydration by the action of brine. It is the most investigated natural polymer for use in the encapsulation of living cells and is a copolymer of a linear block containing polyanionic blocks (1,4)-linked D-mannuronic β-(M unit) and a-L guluronic (block G) acid. Among the two copolymer blocks, the mannuronic acid segment exhibits linear and flexible confirmation, while the G blocks initiates a steric hindrance and only the G blocks participate in ionic interactions with divalent cations, which is needed in the process of hydrogel synthesis. Alginate with a structure similar to native extracellular matrix can be modified to obtain derivatives with various desirable properties. Chemical cross-linking by use of aldehydes is generally employed for alginates. These hydrogels are especially considered in biomedical applications and cell cultures for wound healing, the encapsulation of therapeutic agents, and tissue engineering applications, because of its biocompatibility, availability, low toxicity, flexible gelation rate, and low cost. However, it suffers drawbacks like very low degradation rate and low mechanical stability. When combined with other biopolymers, alginate gels produce even more effective results [21,30,32,33,37]. Chitosan hydrogels Chitin, a derivative of glucose, is found in nature as a component in the skeletons of invertebrates. Upon deacetylation, chitin forms chitosan, which is comprised of D-glucosamine residues-D-glucosamine and N-acetylglucosamine. Chitosan is a semicrystalline, cationic polysaccharide and is considered to be the most abundant natural biopolymer after cellulose. Chitin deacetylation is carried out by chemical hydrolysis under alkaline conditions or by enzymatic hydrolysis in the presence of specific enzymes, such as chitin deacetylase. Acidification of chitosan solutions, followed by neutralization with basic glycerophosphate produces chitosan-based hydrogels. As unmodified chitosan is insoluble and hydrophobic in water and other solvents, water-soluble chitosan derivatives can be developed by chemical modifications of chitosan with different hydrophilic moieties. These modified hydrogels are mucoadhesive and easily form gel at lower pH. Similar to other biopolymers, chitosan has good biocompatibility, biodegradability, nontoxicity, and unique physicochemical and biological characteristics. Chitosan hydrogels can be developed into chitosan beads which are micro/nano-sized, spherical particles within which drugs or other bioactive substances such as proteins or enzymes can be incorporated for transport. In acidic conditions, these beads tend to swell, thereby causing the substances embedded within them to be released. Hence these beads are ideal candidates for drug-delivery applications including colonic and hepatic drug targeting. The beads also find application in enzyme immobilization, which is used in the areas of biosensors and bioprocessing. More recently, chitosan hydrogels have been used as vehicles for radioisotope drugs in brachytherapy, for controlled, site-specific dosages [21,30,32,36,39].

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Chapter 2

Dextran Dextran, a bacterial homopolysaccharide, was first isolated in 1861, utilizing a bacterial culture of the genus Leuconostoc, which produced dextran from sucrose during bacterial growth [30]. It has a linear 1,6-glycosidic bond, with a degree of branching through 1,3-linkage. Even at concentrations above 20 wt.%, dextran forms low-viscosity solutions in water. Chemical modifications can introduce aldehydes, (meth)acrylate, thiol, phenol, maleimide, and vinyl sulfone groups in dextran. Dextran suffers disadvantages of high cost and nonavailability. Applications of dextran include sustained protein and drug delivery, in tissue-engineered scaffolds, as an antithrombolytic agent, and as a bioadhesive. Dextran-based injectable hydrogels are also developed as a site-specific, trackable, chemotherapeutic device [21,30]. Cellulose Cellulose is a hydrophobic polysaccharide found in plants and natural fibers such as cotton and linen. It is the most abundant biopolymer in nature. Some specific bacteria (e.g., Acetobacter xylinum) can biologically synthesize cellulose. Aqueous cellulose ethers upon cross-linking form stable, irreversible, or reversible cellulose hydrogels. Cellulose can be chemically modified through esterification or etherification of its hydroxyl groups to produce cellulose derivatives called cellulosics. Some common examples of cellulosics are methylcellulose (MC), hydroxypropyl MC (HPMC), ethyl cellulose, hydroxyethyl cellulose (HEC), and sodium carboxy methyl cellulose (NaCMC). Pure cellulose, which is insoluble in water due to strong hydrogen bonding, can be made water soluble by the substitution of hydrogen on the hydroxyl groups with more hydrophobic units such as methyl or hydroxypropyl groups, thereby forming MC or HPMC. MC is used for thermosensitive drug delivery but its low mechanical strength limits its application possibilities [21,36]. Cellulose and its derivatives have been successfully applied to burn wound healing, as well as cardiac, vascular, neural, cartilage, and bone tissue regeneration, and they are considered as attractive candidates for a broad spectrum of applications in different fields, especially in biomaterials engineering and biomedical science [26,36,37].

2.3.2 Classification of natural hydrogels based on the nature of cross-linking Hydrogels do not disintegrate during swelling due to their cross-linked structure. Such cross-linked structures can be synthesized physically or chemically. Chemically cross-linked systems have stable junctions as covalent bonds present between the polymer chains, while only a physical interaction exists between the chains in the case of physical cross-linking, resulting in temporary junctions and hence these physical hydrogels are reversible [40,41]. Configurational changes takes place in chemical hydrogels, resulting in permanent and irreversible systems. Due to the absence of toxic cross-linking agents, physical cross-linking has gained greater importance compared with chemical cross-linking [24].

Natural polymers and the hydrogels prepared from them 31 2.3.2.1 Physical hydrogels Physical cross-linking can be achieved via hydrophobic association, chain aggregation, crystallization, polymer stereocomplexation, chain complexion, and hydrogen bonding [24]. These hydrogels have a great number of uses in pharmaceutical and other biomedical applications due to the absence of toxic cross-linking agents. Nevertheless, it is difficult to control the material properties of the physical hydrogel, such as gelation time, network pore size, chemical functionalization, and degradation time due to lack of a cross-linking agent. This also restricts the improvement in mechanical properties [26]. The various methods reported for physical cross-linking are listed below. 2.3.2.1.1 Crystallization

A strong and highly elastic gel can be obtained by crystallization, which involves a repeated freezing
thawing process. Such a formation was first reported in synthetic hydrogel poly (vinyl alcohol) (PVA) hydrogels. Nowadays, these have been largely implemented in biotechnology fields, especially in proteins and peptides. PVA/chitosan, PVA/starch, and PVA/gelatin hydrogels prepared by freeze
thawing are found to have applications in tissue engineering [25,31]. Dextran hydrogels and microspheres can also be prepared by crystallization. In concentrated aqueous solutions of low-molecular-weight dextran, precipitation was observed which was accelerated by stirring and the presence of salts. The precipitates hence produced though insoluble in water at room temperature readily dissolved in boiling water or dimethyl sulfoxide [42]. 2.3.2.1.2 Stereocomplex formation

This method utilizes the formation of stereocomplexes between the polymers of opposite chirality to form physically cross-linked hydrogels for drug-delivery systems. Such hydrogels offer the advantage of easy formation by dissolving each product in water and mixing of the solution. However, only a limited range of polymer compositions is exploited for such formations. Examples of such systems include physically cross-linked dextran hydrogels by the stereocomplex formation of lactic acid oligomers. However, hydrogel formation required a minimum of 11 lactic acid units in the grafts [30,43]. Protein-loaded hydrogels were produced by suspending the protein in dextran-g-oligolactate solutions preceding the mixing stage. The gels were found to be entirely degradable with the physiological conditions. The degradation time depended on the composition of the hydrogel, that is, the number of lactate grafts, the length and polydispersity of the grafts, and the initial water content [25]. 2.3.2.1.3 Ionic interaction

Ionic interactions, does not require the presence of ionic groups in the polymer and can be cross-linked under normal conditions, that is, at room temperature and physiological pH. A prominent example is alignate which has mannuronic and glucuronic acid remains and can

32

Chapter 2

be cross-linked with calcium ions. Such gels find application as a matrix for the encapsulation of living cells and for protein release. Similarly, cross-linking of chitosan with glycerol-phosphate disodium salt produces hydrogels. Below room temperature chitosan solutions maintain a liquid nature in the presence of this salt, but rapidly become a gel under heating. These gels can deliver active protein induced bone and cartilage formation. Another example is carrageenan, a polysaccharide, composed of 1,4-linked-α-Dgalactose and 1,3-linked-β-D-galactose with an erratic part of sulfate groups, which forms a gel either with potassium ions or under salt-free conditions. Stronger hydrogels can be produced in the presence of metallic ions [25,31,40,44]. Dextran, another natural polymer, also forms a hydrogel in the presence of potassium ions. However, as gel is unstable in water it is less suitable for drug-delivery purposes [44]. 2.3.2.1.4 Hydrogen bonding

A hydrogen bond, which is formed through the association of an electron-deficient hydrogen atom and a functional group of high electron density, can participate in hydrogel formation as in the case of gelatine-based hydrogels. The molar ratio of each polymer, polymer concentration, the type of solvent, and the solution temperature are few among the factors that need to be considered in this type of physical cross-linking [23,25,31,44]. Blends of natural polymers like gelatin
agar, starch
carboxymethyl cellulose, and HA
MC form injectable physically cross-linked hydrogel structures [42]. 2.3.2.1.5 Maturation (heat-induced aggregation)

Maturation forms hydrogels with precisely structured molecular dimensions. A prominent example is the heat-induced gelation of gum arabic. The thermal treatment brings about aggregation of the proteinaceous components present in gum arabic, which leads to an increase in the molecular weight and subsequently forms a hydrogel with improved mechanical properties and water-binding capability [31]. 2.3.2.1.6 Hydrophobized polysaccharides

Polysaccharides including chitosan, dextran, pullulan, and carboxymethyl curdlan can be physically cross-linked by hydrophobic modification. Such cross-linked hydrogels have found applications in drug delivery. Hydrophobized water-soluble glycol chitosan substituted with palmitoyl chains is found to be biocompatible and hemocompatible, and capable of entrapping water-soluble drugs [40,44]. 2.3.2.2 Chemical hydrogels In chemically cross-linked gels, the presence of covalent bonds between different polymer chains prevents their dissolution in solvents and gives them stability unless the covalent cross-links are cleaved. In comparison with physically cross-linked gels, chemical gels have a higher mechanical strength, stability, and longer degradation time [26,40,42,45].

Natural polymers and the hydrogels prepared from them 33 The different techniques employed to synthesize chemically cross-linked gels are as follows: 2.3.2.2.1 Cross-linking by functional groups

The hydrophilic groups like
NH2 and
COOH are used for hydrogel formation. 2.3.2.2.1.1 Cross-linking with aldehydes/dihydrazide/Schiff-base formation When an appropriate bifunctional cross-linker is added to a dilute solution of hydrophilic polymer with adequate functionality, hydrogels can be obtained. A most common cross-linker that is of use is aldehyde and dihydrazide (e.g., glutaraldehyde, adipic acid dihydrazide) [23,25]. To initiate cross-linking, conditions such as low pH, high temperature, and addition of methanol as quencher are usually employed. An amino group present in the polymer can react with the aldehyde resulting in Schiff-base formation for hydrogel preparation. However, at low pH, hydrolysis of the imine bond can lead to degradation of Schiff bases. Cross-linked proteins like gelatine, albumin, and the amine-containing polysaccharides can be prepared by using dialdehyde or formaldehyde as cross-linking agents [25,40]. HA-based hydrogel films were obtained by first derivatization of HA with adipic dihydrazide and then cross-linking with poly(ethylene glycol)-propiondialdehyde. These hydrogels find applications in controlled drug delivery [42,44]. 2.3.2.2.1.2 The Michael addition reaction The Michael addition reaction between a nucleophile (an amine or a thiol group) and an electrophile (vinyl/acrylate/maleimide group) can be used to prepare injectable hydrogels for tissue engineering. Two polymers bearing nucleophilic and electrophilic groups are mixed to form the hydrogel. Natural polymers such as HA, dextran, and chitosan have been conjugated with these groups to prepare hydrogels by Michael reactions. Michael addition reactions take place under physiological conditions; the reactions do not seriously influence cell viability during hydrogel formation. Generally, these hydrogels have moderate gelation times (,0.5 to B60 minutes) and moderate mechanical strength, they form a network structure rapidly and quantitatively under ambient conditions and are unaffected by the presence of water and oxygen, resulting in uniform polymer networks. The incorporated cells in hydrogels remain feasible and survive for days to months. However, use of an excess of thiol functional groups may cause cell death and hence care needs to be taken [30,46]. 2.3.2.2.1.3 By condensation reaction Polysaccharide hydrogels can be synthesized via Passerini and Ugi condensation reactions, a carboxylic acid and an aldehyde or ketone are condensed with an isocyanide to yield an α-(acryloxy)amide in the Passerini condensation followed by Ugi condensation, where an amine is added to this reaction mixture, finally yielding an α-(acylamino) amide. The reaction takes place in water at slightly acidic pH and at room temperature. Hydrogels obtained via the Passerini condensation have ester bonds in their cross-links, which degrade at ambient temperature and pH 9.5 and those

34

Chapter 2

prepared using the Ugi condensation contain amide bonds in their cross-links, which were stable under these conditions [42]. Condensation cross-linking can be used to obtain alignate-based hydrogels with improved mechanical properties when compared to ionically cross-linked gels [31,40,44]. 2.3.2.2.2 Free-radical polymerization

Free-radical polymerization of low-molecular-weight monomers in the presence of a cross-linking agent can be used to produce hydrogels. Free-radical polymerization is frequently used to prepare hydrogels for bioapplications. This is a very efficient and widely used method and results in the rapid formation of the gel, even under mild conditions [31]. The amount of cross-linker used controls the characteristics of hydrogel and the swelling of hydrogel in particular. Prominent natural hydrogels that can be chemically cross-linked by this technique are HA, dextran, and chitosan. This method involves the chemistry of typical free-radical polymerization, the main steps being initiation, propagation, chain transfer, and termination. In the initiation step visible, thermal, ultraviolet, or redox initiators form free radicals. These radicals upon reaction with monomers convert them into active forms which then react with further monomers and so on in the propagation step. Termination is brought about either through chain transfer or through radical combination developing polymeric matrices. This method of hydrogel preparation can take place either in solution or in bulk. For the production of large quantities of hydrogels, solution polymerization is preferred and water is the most commonly used solvent. Bulk polymerization is faster than solution polymerization and is free from use of solvent [23,25]. In addition to radical polymerization of mixtures of vinyl monomers, hydrogels can also be obtained by the radical polymerization of water-soluble polymers derivatized with polymerizable groups [24,40]. For example, polymers like dextran, albumin, (hydroxyethyl) starch, and HA can be derivatized with (meth)acrylic groups and polymerized [42,44]. 2.3.2.2.3 Cross-linking by UV light

UV-induced polymerization, an ultra-fast cross-linking mechanism to produce hydrogels has gained importance in recent years, especially to prepare patterned structures. The type of photoinitiator and the solvent in which it is dissolved should be selected with care as they may leak out from the hydrogel once it is formed. When polymerization is carried out in the presence of a protein, the radicals formed can potentially damage the protein structure. As the heat release during the cross-linking process may cause cellular necrosis, the intensity of the UV light is limited to approximately 5
10 mW/cm2 in order to prevent cell damage [30,44]. 2.3.2.2.4 Cross-linking by high-energy radiation

High-energy radiation, like gamma rays or an electron beam, can be used to polymerize unsaturated substances and has been extensively applied in order to induce cross-linking in

Natural polymers and the hydrogels prepared from them 35 polysaccharides. The irradiation technique does not require any initiator and the extent of swelling of hydrogels, which depends on the degree of cross-linking, can be controlled easily by adjusting the radiation dose. Relatively pure, initiator-free hydrogels are obtained by this approach. In addition, cost-effectiveness, fast and identical formation of free radicals for initiation make radiation-induced synthesis of hydrogels very useful for many biomedical applications [23,25]. 2.3.2.2.5 Cross-linking by enzymatic reaction

Enzymes are highly substrate specific and suppress side reactions during cross-linking. TG is a calcium-dependent enzyme capable of catalyzing covalent cross-linking reactions via the formation of an amide linkage between the carboxamide and primary amines on polymers or polypeptides; TG catalyzed hydrogel formation has been successfully used in a variety of systems like polypeptide hydrogels. The gelation times can be shortened to a few minutes by judicious designing of the peptide sequences. As these hydrogels possess good adhesive properties, they are used as surgical tissue adhesives. Similar approaches have been adopted to cross-link hydrogels based on HA, dextran, cellulose, and alginate using horseradish peroxidase and H2O2 [3,25,40,42,44]. The various crosslinking methods are summarised in Table 2.1.

Table 2.1: Various crosslinking methods for natural polymers. Cross-linking mechanism

Polymers Physical cross-linking

G G G G

G G

Crystallization Stereocomplex formation Ionic interaction Hydrogen bonding Maturation Hydrophobized polysaccharides

Dextran, PVA/chitosan, PVA/starch, PVA/gelatine Dextran Alignate, chitosan, carrageenan Blends of gelatin
agar, starch
carboxymethyl cellulose, and hyaluronic acid
methylcellulose Gum arabic Chitosan, dextran, pullulan, and carboxymethyl curdlan Chemical cross-linking

G

By functional groups Schiff-base reaction (aldehydes) Michael addition reaction Condensation reaction Free-radical polymerization High-energy radiation Enzymatic reaction

Hyaluronic acid-based hydrogel dextran, chitosan alignate

G

G G G G G

PVA, Poly(vinyl alcohol).

Hyaluronic acid, dextran, and chitosan Polysaccharides Polypeptides, hyaluronic acid, dextran, cellulose, and alginate

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Chapter 2

2.3.3 Basis of charge On the basis of the type of charges present on the polymer network, hydrogels are grouped into five types [21,25,27,29,30,41,45]: 1. Nonionic (neutral), for example, dextran, agarose, pullulan; 2. Anionic, for example, carrageenan; 3. Cationic, for example, chitosan; 4. Amphoteric electrolyte, for example, collagen; 5. Zwitterionic (polybetaines), for example, coacervating polyanionic xanthan with polycationic chitosan [17,23,26].

2.3.4 On the basis of physical state The three-dimensional microstructure of hydrogels is responsible for the stabilization of nonextracellular and extracellular matrices. Based on the physical state, hydrogels are mainly classified into three types: (1) solid (2) semisolid, and (3) liquid hydrogels [31]. 2.3.4.1 Solid hydrogels Solid hydrogels are mostly chemically cross-linked, and they are solid in nature at room temperature, but can swell in aqueous media like water, buffer solutions, and biological fluids. They can be used for the preparation of hydrogels for biomedical, environmental, and ecological applications as they can mimic the physical, chemical, electrical, and biological properties of most biological tissues. The mechanical properties are improved by incorporation of nanoparticles into the polymer matrix. Examples include methacrylate gelatin reinforced with multiwalled COOH-functionalized Carbon nanotubes (CNTs), and gelatin-collagen modified with bioactive glass nanoparticles for myocardial tissue engineering [31,47]. 2.3.4.2 Semisolid hydrogels Semisolid hydrogels are characterized by their adhesive interactions with interfacial (van der Waals, hydrogen bonds, and electrostatic) forces and soft-tissue networks. These hydrogels are sometimes called bioadhesive or mucoadhesive hydrogels due to their bioadhesive property. They find applications in prolonged drug delivery and effective dosage applications in the biomedical field [31,48]. Hydrogels synthesized with natural polysaccharide sterculia gum and poly(vinylpyrrolidone) (biological in nature) fall under this category. Recently a starch nanocrystal-based hydrogel was developed for transdermal application [48].

Natural polymers and the hydrogels prepared from them 37 2.3.4.3 Liquid hydrogels Liquid hydrogels, as the name indicates, are in liquid phase at room temperature but at a specific temperature have a soft tissue-like elastic phase with good functionality. These hydrogels are injectable and have a variety of applications in the biomedical field. High mannuronic alginate hydrogels used for wound dressing in dermal wound-healing fall under this category. A smart injectable hydrogel prepared from microbial TG and human-like collagen have potential applications as soft materials for skin tissue engineering. Keratinsilica hydrogel can act as a suitable dressing material [31].

2.3.5 Basis of configuration of polymer chain Based on their physical configuration, hydrogels can be classified as: (1) amorphous, (2) semicrystalline, or (3) crystalline [17].

2.3.6 Basis of composition According to the composition of polymers, hydrogels can be classified as (1) homopolymeric hydrogel, (2) copolymeric hydrogel, (3) semiinterpenetrating networks (semi-IPNs), and (4) IPNs [17]. 2.3.6.1 Homopolymeric hydrogel Homopolymers hydrogels form polymer networks derived from a single type of monomer. A cross-linking agent may or may not be used for the network formation. Among natural polymers, cellulose hydrogel is an example of homopolymeric hydrogels synthesized by a one-step polymerization technique in which cellulose was dissolved in a urea/NaOH solution. Epichlorohydrin is added as the cross-linker resulting in transparent hydrogel. 2.3.6.2 Copolymeric hydrogel Copolymeric hydrogels consist of two types of monomers in which at least one has a hydrophilic nature, and the monomers can be arranged in various configurations along the polymer chain network, including random, block, or alternating arrangements. Copolymeric hydrogel based on carboxy MC and carboxymethyl chitosan were utilized for metal ion adsorption. 2.3.6.3 Semiinterpenetrating network If one polymer is linear and penetrates another cross-linked network without any other chemical bonds between them, it is called a semi-IPN [17]. Here, one polymer is crosslinked and the other is a noncross-linked polymer. The constituent linear or branched polymers can be separated from the constituent polymer networks without breaking chemical bonds; these are polymer blends.

38

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2.3.6.4 Interpenetrating network IPNs are conventionally defined as the intimate combination of two polymers, at least one monomer is polymerized/cross-linked in the presence of the other. A typical procedure involves immersing a prepolymerized hydrogel into a solution of monomers and an initiator. The interlocked structure of the cross-linked IPN components improves the stability of the bulk and surface morphology. By the use of IPN formation, relatively dense hydrogel matrices can be produced with stiffer and tougher mechanical properties. Drug delivery is more efficient by IPN hydrogels when compared to conventional hydrogels [23,24,27,41,49
51].

2.4 General characteristics of hydrogels Some of the general characteristics of an ideal hydrogel are high fluid absorption, adequate porosity, desirable photostability, appreciable durability and stability especially during storage, low cost, low content of monomer and other residues, good biodegradability without evolution of toxic matter during degradation, pH neutrality, must be colorless, should not produce any odor, and rewettability as and when required. Although it is impossible for a hydrogel to possess all the characteristics mentioned above, a balance of the properties must be maintained depending on the application [17]. In the following section we discuss the properties and characterization of hydrogels.

2.4.1 Physical and chemical properties 2.4.1.1 Swelling and solubility As already mentioned, hydrogels are three-dimensional network structures swollen in an aqueous medium. The fluid serves as a membrane for free diffusion of some solute molecules, while the polymer in its cross-linked structure acts as a matrix. A hydrogel is estimated to absorb up to 1000 times its dry weight in water. At the initial stage of absorption, the first water molecules entering the network will hydrate the most polar, hydrophilic group which is called “primary bound water.” With time, these polar groups get hydrated, and the water then interacts with the hydrophobic group and forms “secondary bound water.” The primary bound water formed in the first stage together with the secondary bound water is termed total bound water. After the absorption of “total bound water” by the interaction with hydrophilic and hydrophobic groups, an additional amount of water is then absorbed which is called “free water” or “bulk water,” which fills the space between the network chains, and/or the center of larger pores, macropores, or voids. This is due to the osmotic driving force of network chains toward infinite dilution. This process of imbibing is opposed by physical cross-linking in the matrix structure, and forms an elastic

Natural polymers and the hydrogels prepared from them 39 network retraction force. The hydrogel thus reaches an equilibrium swelling level. As the structure swells, the gel will disintegrate and dissolve if the network chains or cross-links are degradable, at a rate depending on its composition [19,52]. The extent of solubility of the hydrogels can be calculated using the equation   Wd 3 100 Gel fractionð%hydrogelÞ 5 Wi where Wi is the initial weight of the dried hydrogel and Wd is the weight of the dried sample after immersing in distilled water (48 hours at room temperature). The swelling ratio (degree of swelling) can be calculated using the equation Swelling 5

Ws 2 Wd Wd

where Ws is the weight of hydrogel in the swollen state (48 hours), while Wd is the initial weight of the dried hydrogel. Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) can also be used to characterize the water in hydrogels. The interchange of water molecules between the free and bound states is obtained from proton NMR, while DSC calculation is based on the assumption that only the free water may be frozen [19,52,53]. It is important to add that the swelling of hydrogel forms the basics of understanding the cross-linking degree, mechanical properties, degradation rate, etc., of the hydrogels.

2.4.2 Mechanical properties The mechanical properties of the hydrogel can be tuned depending on its application. Increasing the cross-linking density can produce a stiff hydrogel, while heating the material can decrease the stiffness. Various parameters such as storage, loss moduli, and tan δ can be evaluated by a dynamic mechanical analysis device or a rheometer [19]. 2.4.2.1 Porosity and permeation Pores may originate in hydrogels during the phase separation process or may present as smaller pores within the polymer matrix structure. The average pore size, the pore size distribution, and the pore interconnections are together termed tortuosity and are difficult to quantify individually [52]. Probe solute permeation is a useful technique to investigate pores and their interconnections in hydrogels. The permeation coefficient, P, is the product of the partition coefficient, K, and the apparent diffusion coefficient, Dapp. The partition coefficient, K, depends on various factors like the size and shape of pores, net charge of the

40

Chapter 2

hydrogel (the ionic, polar, nonpolar groups of the hydrogel), total available “free” water within the hydrogel, temperature, pH and ionic strength, drying method, etc. The “effective” or “apparent” diffusion coefficient of the samples can be calculated as follows Dapp 5 D0 3

pore volume fraction tortuosity

where D0 is the diffusion coefficient in free water. The pores in the hydrogel exhibit different morphologies such as closed, open, or interconnected. Various techniques can be used to study the pore morphology. The above methods when combined with optical microscopy, stereomicroscopy, electron microscopy (SEM and TEM), scanning tunneling microscopy, atomic force microscopy, and electronic microscopy give an overview of pore size, pore volume, etc. Other widely used techniques for understanding the pore morphology include mercury porosimetry, gas pycnometry, gas adsorption, liquid extrusion porosity, capillary flow porosity, X-ray microtomography, etc. [19,53]. 2.4.2.2 Cross-linking As already stated in previous sections, there are two types of cross-links present in a hydrogel: either physical cross-links or chemical cross-links. The degree of cross-linking must be controlled depending on the end application of the hydrogel. As a result, the same hydrogel can be used for different applications by tuning the degree of cross-linking [43].

2.4.3 Rheology The rheological properties are used to characterize the types of structure (i.e., association, entanglement, cross-links) present in the system. It is well known that polymer solutions are viscoelastic in nature and are essentially viscous at low frequencies while at high frequencies, elasticity dominates (G0 . Gv). This technique has been used to characterize the network structure in seroglucan/borax hydrogel, chitosan-based cationic hydrogels and a range of other hydrocolloids [53].

2.5 Stimuli-responsive hydrogels Stimuli-responsive hydrogels or smart hydrogels are able to switch from solution to gel form and vice versa when a particular stimulus is applied. These stimuli-responsive hydrogels, unlike conventional hydrogels, can respond to external stimuli including temperature, pH, electric field, solvent composition, and salt concentration. The stimuli used to activate the hydrogels can be generally classified into two groups: physical stimuli (temperature, electric or magnetic fields, photoresponse, etc.) and chemical stimuli

Natural polymers and the hydrogels prepared from them 41 (pH, ionic factors, chemical agents, etc.). Another recently developed stimulus category is the biochemical stimuli which include responses to antigen, enzyme, ligand, and other biochemical agents [42].

2.5.1 Physical stimuli 2.5.1.1 Temperature-responsive hydrogels Temperature-responsive hydrogels are of medical importance as they switch from gel form to sol form by a change in stimuli. The change in stimuli can be brought about upon the injection of the hydrogel into the body, by this process the temperature of the hydrogels increases from room temperature to body temperature of 37 C. Hydrogels basically fall into two categories: upper critical solution temperature at which the sol
gel phase transition occurs and lower critical solution temperature at which gel
sol transition takes place. Many polysaccharide hydrogels, including those based on cellulose, chitosan, dextran, and proteins like gelatin, can exhibit such responses. Such hydrogels finds applications in drug delivery, tissue regeneration, etc. [38,42,54]. 2.5.1.2 Photoresponsive hydrogels Photoresponsive hydrogels switch their physical and/or chemical properties such as elasticity, viscosity, shape, and degree of swelling by photoirradiation [46]. Such systems offer a high level of control in response to the stimuli. However, they find applications only in ex vivo cases due to inability of ultraviolet radiation to penetrate tissues. The applications of such hydrogels includes photo-controlled enzymatic bioprocessing, targeted drug-delivery systems, and separation/recovery systems [42]. 2.5.1.3 Electrically responsive hydrogels Polyelectrolytes are usually used to develop electrically sensitive hydrogels. These hydrogels undergo shrinkage or swelling in the presence of an applied electric field. Such hydrogels finds application in drug delivery and artificial muscles [38].

2.5.2 Chemical stimuli Physical stimuli-responsive hydrogels require continuous stimuli, as soon as the stimulus is cutoff there is a discontinuous response from the hydrogel. Chemical stimuli-responsive hydrogels are advantageous in this regard. 2.5.2.1 pH-responsive hydrogels Here the ionic pendant groups present in the hydrogels will ionize at suitable pH and develop charge. The accumulation of surface charge will result in electrostatic repulsion that causes the swelling behavior at the appropriate pH. Two classes of hydrogels exist depending on their ionization behavior. Anionic hydrogel networks swell at pH . pKa

42

Chapter 2

(acid dissociation constant) while cationic hydrogels swell at pH , pKa. The biopolymers offer the advantage of degradation within the body over time [38]. 2.5.2.2 Glucose-sensitive hydrogels Glucose-sensitive hydrogels are of particular interest in self-regulated insulin-delivery systems. Unlike other drugs, insulin must be delivered at the correct time and in the correct amount. This calls for a system which can sense glucose and deliver insulin accordingly. For example, lectins are an excellent glucose-sensitive hydrogel. They consist of a glycosylated polymer backbone and physically immobilized concanavalin A [38]. 2.5.2.2.1 Applications of natural hydrogels

Natural hydrogels are widely used in agriculture, drug-delivery systems, water purification, food industry, biomedical applications and regenerative medicine, wound dressings, biosensors, etc. [17,19,24]. As a detailed discussion on each of these applications is beyond the scope of this chapter, therefore we will briefly outline some of the important applications of natural hydrogels in the following sections. 2.5.2.2.1.1 Biomedical applications As mentioned in the above paragraphs, hydrogels can mimic the response of the human body to variation in environmental conditions such as pH, temperature, enzymes, etc. Therefore hydrogels can be used for various biomedical applications. These include in wound dressings, and targeted drug delivery, tissue engineering, and regenerated medicines [24]. Wound dressing Hydrogels can assist the wound-healing process, therefore they are widely used for wound dressing. They find application as debriding agents, moist dressings, components of wound care pastes, and also for emergency burn treatment. The moist environment provided by hydrogels promotes quick healing. Hydrogels also possess the advantage of being nonadhesive to the skin, and hence they can be removed easily. In addition, when biopolymers are used, biocompatibility, biodegradability, and their resemblance to extra cellular matrix (ECM) are added advantages. Many hydrogels are transparent, and a transparent wound dressing helps to examine the healing process. Various biohydrogel-based wound dressings are commercially available. Some of the common biohydrogels used as wound dressings are dextran, cellulose and its derivatives, chitosan, alginate, collagen, gelatin, and silk fibroin [22,55
62]. Drug delivery Hydrogels have gained considerable attention in drug-delivery applications due to their unique properties [63]. The drug delivery can be either a targeted release or controlled release of the drug. Intelligent hydrogels are used for efficient delivery of different drugs varying from low to high molecular weight. The advantage of a hydrogelbased system is that the drug can be effectively trapped inside the hydrogel and can be protected from the external environment, and also the rate of drug delivery can be easily

Natural polymers and the hydrogels prepared from them 43

Hydrogel Drug source

Drug delivery Drug source

Figure 2.9 Drug delivery mechanism of hydrogels. Table 2.2: Hydrogels used for drug-delivery applications. Hydrogels Xylan Chitosan

Cellulose Guar gum Dextran Collagen

Applications in drug delivery Inhibits cell mutation, inherent immunological defense, anticancer and antioxidant properties, resistant against digestion in the human stomach and intestines [45] Promotion of hemostasis, epidermal cell growth, lowering cholesterol, antihypertension, and pain alleviation, antiulcer, antimicrobial properties, facilitates paracellular transport of drugs and promotes intestinal adsorption [65] Controlled drug release (for oral and colon-specific drug delivery) [56], topical gel for the treatment diabetic foot ulcers [66] Controlled drug release (for oral and colon-specific drug delivery) [65] Used for drug delivery in the colon [65] For repair of bone fracture, oral maxillofacial reconstruction, spinal fusion [66]

tuned by using environmental stimuli such as temperature, pH, electrical and magnetic fields, solvent composition, light, etc. [45]. Once the drug is loaded into a hydrogel, it can be released via several mechanisms, namely, diffusion controlled, swelling controlled, chemically controlled, and environmentally responsive release [55,64]. A schematic diagram showing the drug-delivery mechanism of hydrogels is shown in Fig. 2.9. Some of the polymers used for drug-delivery applications and their properties are discussed in Table 2.2. 2.5.2.2.1.2 Tissue engineering Tissue engineering is the replacement of failed organ/tissue partially/wholly with a natural or synthetic substitute. Hydrogels can be used as matrices for tissue engineering. The pores present in hydrogels are large enough to a hold living cells. However, the hydrogels are difficult to handle due to their poor mechanical strength. Natural hydrogels like HA, alginate, collagen, chitosan, fibrin, etc., have been widely used for tissue engineering for the past few decades [52,67,68]. 2.5.2.2.1.3 Agriculture and horticulture Superabsorbent hydrogels are capable of absorbing and preserving a large amount of water. This property of hydrogels find applications in agriculture and horticulture for controlled release of water and nutrients in fertilizers. The

44

Chapter 2

advantages of this technique are that the need for irrigation and plant nutrition can be reduced. Hence it helps in cultivation of plants in desert areas, where water scarcity is an issue. Polysaccharides, such as chitosan, pectin, and carboxymethyl cellulose have been used as superabsorbent hydrogels for fertilizers and water release [24,56]. 2.5.2.2.1.4 Separation technology The inorganic sewage released from various industries like the textile and colored paper industries contains toxic dyes and metal ions in large quantities which pose a dangerous threat to the environment and food chain. Being easily soluble in water, these toxic substances can easily contaminate water resources and cause serious health issues. Hydrogels with their high swelling capability can adsorb these contaminants and hence hydrogel systems are proposed for efficient water purification. The hydrophilic groups
COOH,
OH,
NH2 present in hydrogels can interact with dye molecules and heavy metal ions and become attached to them. Various aquatic pollutants such as metal ions (transition or radioactive), dyes (cationic or anionic), and other ions (nitrogenous or phosphorous) can be easily removed using hydrogels as separators [24,51]. Biopolymers like cellulose or chitin hydrogels are usually used for removing toxic substances. 2.5.2.2.1.5 Hygiene products Another application of superadsorbent hydrogels is in personal hygiene products, especially diapers. Most superadsorbent diapers are now based on acrylamide. However, these disposable diapers are difficult to dispose of as they are produced in huge quantities [19]. Completely biodegradable cellulose-based hydrogels were introduced to overcome this environmental issue. NaCMC and HEC cross-linked with divinyl sulfone behave as a superadsorbent polymer [55]. The main benefit offered by cellulose-based hydrogels over current acrylamide-based super adsorbent polymer (SAP) lies in their biodegradability and environmentally friendly nature. Use of radiation crosslinking of cellulose is recommended [56]. 2.5.2.2.1.6 Cosmetics Another prominent application of hydrogels is in the cosmetic industry. Novel cosmetic products are introduced with hydrogels as active components. Chitosan hydrogel-based materials, such as chitosonic acid and carboxymethyl hexanoyl chitosan, are now accepted by the Personal Care Products Council as cosmetic ingredients. They possess good compatibility with other ingredients that are generally used in cosmetic products [24].

2.6 Conclusion The past few decades have witnessed rapid progress in the field of hydrogels, especially biohydrogels. More recently natural hydrogels have been widely used in agriculture, drugdelivery systems, water purification, the food industry, wound dressings, biosensors, etc. Natural hydrogels are biocompatible, have low toxicity, flexible gelation rate, and low cost. However, despite all their beneficial properties, there are still several challenges to overcome for clinical application of many of these hydrogels.

Natural polymers and the hydrogels prepared from them 45

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1767.

Further reading I. Syed, S. Garg, P. Sarkar, Entrapment of essential oils in hydrogels for biomedical applications, in: K. Pal, I. Banerjee (Eds.), Polymeric Gels: Characterization, Properties and Biomedical Applications, Woodhead Publishing, 2018.

CHAPTER 3

Physical hydrogels based on natural polymers Shuxian Tang, Lin Zhao, Jingjing Yuan, Yu Chen* and Yilin Leng School of Material Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China

3.1 Introduction Hydrogels are three-dimensional networks composed of cross-linked hydrophilic polymer chains [1]. Attributed to the simultaneous presence of three-dimensional cross-linked structures and the hydrophilic functional groups such as OH, CONH , CONH2, and SO3H [2], hydrogels are capable of containing a large amount of water or biological fluids without losing their structural integrity [3]. Due to their high water affinity and content, hydrogels usually appear to be wet, soft, and elastic, rendering them with a resemblance to living tissues, such as extracellular matrix and soft tissues [4]. In addition, the special properties of hydrogels such as sensitivity to temperature, pH, light, magnetism, and other stimuli, have attracted a great deal of attention. In recent years, different types of hydrogels, including self-healing hydrogels, environment-responsive hydrogels, shape-memory hydrogels, self-assembled hydrogels, supramolecular hydrogels, and conductive hydrogels have been widely developed [5]. They possess broadly tunable physical and chemical properties and are widely applied in various fields, such as biomedicine, soft electronics, sensors, and actuators [6,7]. According to the type of cross-linking, hydrogels can be divided into chemically crosslinked hydrogels (also termed chemical hydrogels) and physically cross-linked hydrogels (also termed physical hydrogels) [8]. Chemical hydrogels are formed by introducing covalent cross-links to construct permanent junctions within the matrix. In contrast, physical hydrogels are formed by dynamic and reversible cross-links based on noncovalent interactions, such as crystallization, ionic interactions, hydrophobic interactions, electrostatic interactions, hydrogen bonding, or combinations of these [8 10]. However, physical hydrogels usually have poorer mechanical properties than chemical hydrogels as the physical interactions are weaker than the covalent bonds [11]. Nevertheless, those physical interactions are sufficient to make hydrogels insoluble in aqueous media [12]. 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00003-3 © 2020 Elsevier Inc. All rights reserved.

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The gelation method of many physical hydrogels depends on the intrinsic properties of the polymers and this dependence limits the ability to adjust the attributes of hydrogels. Nonetheless, gelation is easy to carry out without modification of the polymer chains, and is usually easy to reverse when necessary [7]. Due to the dynamic and reversible nature of noncovalent interactions within the physical hydrogels, they may have rapid energy dissipation and reformation of the transient junctions [13]. Their dynamic cross-linking exchange and excellent shear recovery can be attractive for use as injectable and selfhealing hydrogels [14,15]. In contrast, chemical cross-linking can be used to allow for more controllable, precise management of the cross-linking procedure, potentially in a spatially and dynamically defined manner [7]. However, in many chemical hydrogels, the crosslinked junctions are too strong and stable to be broken or reformed, and the assistance of catalysts is often required for the recovery process [16,17]. Chemical hydrogels are difficult to remold once formed, which has limited their responsiveness to external stimuli [11]. For chemical hydrogels, a purification step is usually performed after hydrogel elaboration, aiming to eliminate the residues of chemical cross-linking agents (cross-linker) or other chemical compounds [3]. The main advantage of physical hydrogels is not using the chemical cross-linking agent, which can sometimes be toxic and decrease the biocompatibility of hydrogels [18]. In addition, the preparation conditions of physical hydrogels are relatively milder than those of chemical hydrogels, which usually require high temperature, radiation, and organic solvents [19]. Natural polymers are a class of polymers that have natural origins, such as polysaccharides (e.g., chitosan, alginate, cellulose, and hyaluronic acid), proteins (gelatin, collagen), and DNA. Owing to their abundant availability, renewable resources, eco-friendly properties, low cost, and biocompatibility, a global revival with regard to the utilization and interdisciplinary research into natural polymers has been promoted [20,21]. Because natural polymers are macromolecules with high molecular weight, they can be used to prepare hydrogels without adding any initiators to start the polymerization. For hydrogels that are used in biomedical applications such as drug delivery, tissue engineering, and cell therapies, the properties of hydrogels and the method of interaction with the biological surroundings are extremely important. This may involve factors like biocompatibility, biodegradability, mechanical strength, ionic charge, and surface properties [4]. For example, the hydrogels must be immunocompatible and not cause significant inflammatory response for use in in vivo microenvironments. Removal of small molecules such as unreacted monomers, initiators, and cross-linkers used during hydrogel preparation and other undesired byproducts is essential because they are toxic to host cells both in vivo and in vitro [22,23]. Hydrogels based on natural polymers mimic many features of the extracellular matrix and many have demonstrated better biocompatibility and biodegradability than those based on synthetic polymers [24]. In addition, in order to relieve the increasing environmental effects caused by the fossil fuel industry, there is an urgency to develop and utilize hydrogels based on natural polymers [25].

Physical hydrogels based on natural polymers 53 Therefore, hydrogels prepared via physical cross-linking based on natural polymers can avoid the toxicity of residual chemical cross-linkers, initiators, and monomers and retain the excellent biological properties of natural polymers. The preparation and subsequent process are relatively simpler than those chemically cross-linked hydrogels polymerized by small monomers. The combination of the advantages of both natural polymers and physical hydrogels make them an ideal class of biomaterial, which have been extensively used in the biomedicine field [26 30]. In this chapter, we aim to provide a comprehensive survey of physical hydrogels based on natural polymers, including their formation mechanisms, structures, properties, and applications, and to give an overview of recent related works.

3.2 Types of physical hydrogels based on natural polymers Natural polymers are capable of forming physical hydrogels via different interactions or combinations of interactions. The form of the physical interactions mainly includes ionic interactions, crystallization cross-linking, hydrophobic association, and hydrogen bonding. In the following description, physical hydrogels based on natural polymers are introduced according to their cross-linking interactions, and the corresponding examples are presented to elucidate the formation mechanisms, structures, properties, and applications of those physical hydrogels.

3.2.1 Ionic interaction The preparation of physical hydrogels by ionic interactions can be divided into two classes: the electrostatic interactions between a polyelectrolyte (PE) and an ion with opposite charges, and the electrostatic interactions between two or more PEs with opposite charges. 3.2.1.1 Formation of hydrogels via the electrostatic interaction between natural polyelectrolytes with opposite charges A PE is defined as a macromolecule carrying plenty of ionizable functional groups, converted into a charged long chain with counter ions in polar solvent. PEs can be divided into polycations, polyanions, and polyampholytes according to the type of electrolyte group of the main chains [31 33]. There are many natural PEs among animals, plants, and microbes, such as chitosan from crustacean shells, hyaluronic acid from the extracellular matrix of connective tissues of animals, carrageenan from eucheuma, sodium alginate from kelps, and ε-polylysine from Streptomyces albulus ssp. [34 38]. Polyelectrolyte complex (PEC) is prepared mainly via the electrostatic interaction between the oppositely charged PEs under suitable conditions without any chemical covalent crosslinkers [39 42]. The formation of PEC leads to an obvious phase separation in solution due to the strong and rapid interaction when mixing positively charged polycations and negatively charged polyanions together [43 46]. Based on the above interaction, the PE

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complexation method is suitable for the preparation of physical hydrogels due to its several advantages, including, but not limited to: (1) the combination of desired characteristics of at least two components; (2) the rapid and simple reaction process under mild conditions; (3) no need for the toxic organic solvent or chemical cross-linking agent; and (4) multifaceted tunable factors and properties [47,48]. Natural PEC hydrogel is a three-dimensional network composed of oppositely charged natural PEs principally via an electrostatic interaction, which works as physical cross-linking points to provide mechanical strength, making it a physical gel due to the free chemical cross-linkers [49 51]. Thus, the toxic issues of hydrogels caused by the substantial use and residual of cross-linkers are avoided through PE complexation, rendering PEC hydrogels with good biosafety. Generally speaking, there are three methods ordinarily applied for the preparation of natural PEC hydrogels: blending the natural PE solutions, electrostatic screened complexation by salt, and semidissolution acidification sol gel transition, respectively. 3.2.1.1.1 Blending of natural polyelectrolyte solutions

The most facile and commonly used way to prepare PEC hydrogel is by simply mixing the reaction components in an aqueous media. For instance, Zhang et al. [26] obtained a homogeneous solution of chitosan, heparin, and poly(γ-glutamic acid) by magnetic stirring and ultrasonication, then the PEC hydrogels were formed instantly after adding 1% (v/v) acetic acid into the solution and stirring. The preparation process is shown in Fig. 3.1, chitosan was

Figure 3.1 Preparation of chitosan/heparin/poly(γ-glutamic acid) composite hydrogels [26]. Copyright (2018) Elsevier.

Physical hydrogels based on natural polymers 55 used to promote cell proliferation, heparin was used for pain remission, inhibition of clotting and inflammation, healing enhancement, and poly(γ-glutamic acid) was composited for its good hydrophilicity and biocompatibility. The advantages of the three raw components were gathered to improve the physical properties of the candidate dressing in wound healing. The electrostatic interaction between counter-polyions within the PEC hydrogels is considerably stronger than most secondary binding interactions [52], and many reported works have demonstrated that the electrostatic interaction is sufficient to sustain the network structure of hydrogels without any chemical cross-linkers [53,54]. However, due to the spontaneously occurring interaction, the blending complexation of PEs with strong ionic groups usually tends to lead to macroscopic complexes or precipitates [55,56]. The PEC precipitates formed through Coulomb forces inhibit the further complexation of PEs, which makes it difficult to develop PEC hydrogels with the desired shapes and uniform compositions [44]. In general, there are two methods to overcome this problem. 3.2.1.1.2 Electrostatic screened complexation by salt

Moreira et al. [57] evaluated the electrostatic interaction between the components of chitosan/gelatin PEC hydrogels by zeta potential analysis. The zeta value of hydrogel at 37 C showed a significant decrease in relation to pure chitosan solution, confirming the powerful interaction within it. Based on the charge screening effect, the interaction between the PEs is reduced in the presence of the correct concentration of salt [31], which is conducive to avoiding irregular precipitates when preparing PEC hydrogels. Oliveira et al. [58] adopted three distinct concentration conditions (hypotonic, 0 M; isotonic, 0.15 M; hypertonic 0.5 M) of salt (sodium chloride) in PE solutions before mixing. The zeta potential results showed that solutions with higher salt concentrations had a lower potential, which is in accordance with the charge screening effect. Fig. 3.2 shows the sequentially moldable and self-bondable properties of the synthesized chitosan/alginate PEC hydrogels, indicating a shape-controllable structure via an electrostatic interaction with probably other weak ones, such as hydrogen bond and hydrophobic interaction. Similarly, David et al. [59] selected NaCl to prevent the spontaneous association of two PEs. In the chitosan/hyaluronic acid mixture system, the PEC hydrogels are formed by a desalting process (shown in Fig. 3.3) through which the charges of both polysaccharides were slowly revealed and a long-range intermolecular electrostatic interaction was created. Fig. 3.4 shows a proposed nanostructure of the hydrogel containing folded HA chains, hydrogen bonds, and physical cross-linking points such as H-bonds and PEC solid-like aggregates. The highly entangled network has the ability to dissipate energy through the rupture of the hydrogen bonds, endowing the hydrogels with high stretchability. 3.2.1.1.3 Semidissolution acidification sol gel transition

Chitosan is the most frequently used natural polycation for PEC hydrogels. The semicrystalline structure of chitosan makes it insoluble when in neutral and alkaline

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Figure 3.2 Sequentially moldable and self-bondable chitosan/alginate PEC hydrogels: (A) A piece of hydrogel is retrieved in shape 1 and sequentially molded into shapes 2 and 3. (B) Mold used to achieve shape 3. (C) The samples are easily handled using tweezers after partial dehydration using an absorbent filter and (D) can then be adapted to confined geometries, such as a pipet tip. (E) Simultaneous molding and binding of three different hydrogel fragments into one single piece with shape-morphing features [58]. Copyright (2018) American Chemical Society.

Figure 3.3 The dialysis process [59]. Copyright (2017) Royal Society of Chemistry.

Physical hydrogels based on natural polymers 57

Figure 3.4 The proposed nanostrucure of the chitosan/hyaluronic acid hydrogels prepared by controlled complex coacervation by dialysis in an acidic solution at pH 5 2.5 when allowed to rest (A) and under stretching (B) [59]. Copyright (2017) Royal Society of Chemistry.

solvents [46]. Consequently, an acidifying agent is always needed when preparing the chitosan solution. Soluble insoluble transition occurs around pH 6 6.5, at the pKa value [53], and the amine groups of chitosan are protonated. Thus, the positively charged ammonium ions can form electrostatic interactions with negatively charged groups of polyanions, like the carboxyl groups from alginate and the sulfate groups from carrageenan. In order to avoid the instantaneous aggregation of chitosan with other polyanions, a relatively slow and mild sol gel transition method, called semidissolution acidification, was developed to prepare natural PEC hydrogels. Zhao et al. [60] prepared a slurry-like mixture of insoluble chitosan powder and alginate solution at first, then the mixture was exposed to an acetic acid atmosphere for several hours to perform the sol gel transition. In this process, the chitosan powder was protonated by the infiltrated gaseous acetic acid, thus the ammonium could form ionic bonds with the carboxyl groups from alginate. Chen et al. [30] used the same method to prepare chitosan/ poly(glutamic acid)/alginate PEC hydrogels—the formation mechanism is shown in Fig. 3.5. Lv et al. [27] successfully obtained a homogeneous carboxymethyl chitosan/ alginate hydrogel (Fig. 3.6) using the above method. This method could prepare PEC

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Figure 3.5 Formation mechanism of the chitosan chitosan/poly(glutamic acid)/PEC alginate hydrogels by the semidissolution acidification sol gel transition method [30]. Copyright (2018) Elsevier.

Figure 3.6 Homogeneous carboxymethyl chitosan/alginate PEC hydrogel samples: digital photographs of CMCS/alginate hydrogel film (CA-1) swelled in D.I. water for (a) 0 h, (b) 1 h, (c) 4 h and (d) 24 h, respectively [27]. Copyright (2018) Elsevier.

Physical hydrogels based on natural polymers 59 hydrogels with uniform composition by the gradual sol gel transition method, and no precipitates were observed in the complexation process. 3.2.1.2 Formation of the ionically cross-linked hydrogels based on natural polymers Ionically cross-linked (IC) hydrogels are three-dimensional network systems of polymers cross-linked by ions, which are capable of forming ionic interchain bridges or chelation interactions with functional groups or atoms of the polymers [61,62]. Generally, monovalent, divalent, or trivalent metal ions (such as Ag1, Zn21, Ca21, Cu21, Fe31, and Al31), are selected to interact with functional groups of polymers, such as carboxyl and amino groups, bringing strong affinity to the above metal ions, to form physical crosslinking points [63,64]. Compared with covalent cross-linked hydrogels, chemical crosslinkers are not necessary during the preparation of IC physical hydrogels, thus the toxicity of organic agents is eliminated. In addition, some metal ions used as the physical crosslinker have been demonstrated to have specific properties. For example, calcium ion cross-linked alginate hydrogel films were used for wound healing based on the hemostatic characteristic of the calcium ion [28]. Copper ion cross-linked nanofibrillated cellulose hydrogel reported by Basu et al. [29] showed bacteriostatic activity toward Pseudomonas aeruginosa and Staphylococcus epidermidis. Therefore natural biopolymers with excellent biological properties, such as alginate and chitosan, are very suitable for combining with inorganic metal ions to construct IC physical hydrogels, which can be used as structural or functional biomaterials [18,65]. 3.2.1.2.1 Ionically cross-linked hydrogels based on alginate

Alginate is a natural linear polysaccharide extracted from algae, consisting of (1,4)-linked β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit). This copolymer is comprised of M unit and G unit residues interspersed with MG sequences [34]. Many of the carboxyl, hydroxyl, and ether oxygen atoms on the alginate chains provide copious active sites when cross-linked by ions. Due to the conformation difference between the two residues, the G residues have greater possibility to form coordination with cation ions, such as calcium or strontium ions. An “egg-box” model [66] was proposed to depict the cooperative mechanism of the ion-chain binding. As shown schematically in Fig. 3.7 [67], the divalent cations are like packed eggs, and the twofold screw symmetry chains behave as the “box.” The most commonly used method to prepare IC alginate hydrogels is to combine the ionic cross-linker with an alginate solution. Taking the frequently reported calcium IC alginate hydrogels as an example, while adding the sodium alginate solution into the calcium chloride solution dropwise, the hydrogel would be formed instantly. As shown in Fig. 3.8 [68], Ca21 ions were diffused into the gaps between the alginate chains to initiate crosslinking, thus ionic cross-linking points were formed and gelation occurred, leading to alginate beads. The above mechanism of hydrogel formation is called the external gelation

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Figure 3.7 The “egg-box” model [67]. Copyright (2015) Royal Society of Chemistry.

Figure 3.8 Mechanism of external gelation for bead formation: (a) alginate droplet in contact with calcium solution, (b) inward diffusion of calcium ions, (c) inward gelation of droplet, and (d) completed gelation [68]. Copyright (2016) Elsevier.

Physical hydrogels based on natural polymers 61

Figure 3.9 Hydrogel film appearance after cross-linking at different concentrations (%w/v) and contact times (min) [70]. Copyright (2017) Elsevier.

method [69]. Many researchers have adopted the external gelation method as a facile process to prepare IC alginate hydrogels under mild conditions, and free of chemical crosslinkers. For example, Rezvanian et al. [70] prepared an alginate-pectin hydrogel film by immersing the noncross-linked film in a CaCl2 solution. They found that a high CaCl2 solution concentration (greater than 1% w/v) and a longer immersion time (greater than 2 minutes) would result in a rigid and inflexible hydrogel film with wrinkled edges (Fig. 3.9), which might correspond to the high cross-linking degree of polymers. Some other metal ions can also cross-link with alginate according to the external gelation mechanism [71]. Reddy et al. [72] demonstrated that the trivalent metal ions, such as aluminum ion or ferric iron ion, had a faster and more stable cross-linking ability than divalent ions due to their higher valency. As the poor stability of the IC hydrogels prepared from Ca21, they may be degraded in a physiological environment, which is likely to result in the undesired rapid leakage of loaded materials in a drug-release system [73]. Therefore, Banerjee et al. [74] used the trivalent Al31 ion to form a pH-sensitive alginate/methyl cellulose (MC) blend hydrogel bead. The cation size and supramolecular structure are beneficial to the subsequent coordination of Al31 ions with hydroxyl oxygen atoms of the alginate chains located in the close vicinity of the cation. The study of equilibrium swelling and drug release showed a prolonged and slow release of diclofenac sodium from the Al31 cross-linked hydrogel bead, indicating its potential for controlled oral release application. Similarly, Swamy et al. [75] prepared an Fe31 cross-linked alginate/carboxymethyl cellulose

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(CMC) blend hydrogel bead in ferric chloride solution. Then the hydrogel was studied for in vitro release of metformin, showing a higher swelling degree by two orders of magnitude at gastric conditions as compared to an acidic environment. The intense sensitivity to the pH of the hydrogel makes it a promising carrier for targeted colon drug delivery. In contrast with external gelation, internal gelation means the formation of a gel by slowly releasing or producing ionic cross-linkers within the polymer solution system. For instance, the most common method is to combine water-insoluble calcium carbonate (CaCO3) with D-glucono-δ-lactone (GDL) [76]. The latter is able to acidify the sodium alginate solution and slowly release the calcium ions to initiate gelation. In this process, the calcium ions were uniformly distributed in the sodium alginate matrix, thus leading to homogeneous gels [77]. Kuo and Ma [78] used a CaCO3 GDL system (internal gelation) and calcium sulfate (CaSO4) (external gelation) as the cross-linker for alginate gelation, respectively. The results showed that the IC hydrogels produced by the CaCO3 GDL system were uniform, transparent, and three-dimensionally defined (Fig. 3.10). While the gelation rate of CaSO4 cross-linked hydrogels was too fast to control, the nonuniform gels formed, consisting of lumps of varying density. Other researches also observed the differences between the gels formed through internal and external gelation. Lupo et al. [79] found that the calcium crosslinked alginate hydrogel beads by internal gelation were softer than those prepared by external gelation, due to the more rigid shell of the gel beads formed by external gelation when the calcium ions diffused from the exterior to the interior of the droplets. Through scanning electron microscope observations, Liu et al. [80] concluded that the calcium crosslinked alginate hydrogel beads with an external calcium source had a denser structure and smaller pore size than those with an internal calcium source.

Figure 3.10 The uniform and transparent IC hydrogels produced by CaCO3 GDL system [78]. Copyright (2001) Elsevier.

Physical hydrogels based on natural polymers 63

Figure 3.11 Images of patterned Fe31 cross-linked hydrogel (left) and Ca21 cross-linked hydrogel obtained from it (right) [82]. Copyright (2015) Elsevier.

The attractive homogeneous gel produced by internal gelation is desirable. Brady et al. [81] used a novel glass-GDL system to internally form IC alginate hydrogels for the treatment of intracranial aneurysms. The glass (mole fraction composition of 0.33SiO2
0.18Ga2O3
0.23CaO
0.11P2O5
0.15CaCl2) could deliver a steady release of multivalent ions, controlling the rate of gelation and the strength of the hydrogel. Bruchet and Melman [82] developed a method for preparing homogeneous Ca21 cross-linked IC alginate hydrogels via reductive cation exchange in homogeneous Fe31 cross-linked alginate hydrogels. The reduction was performed photochemically by using ascorbic acid as a sacrificial photoreductant, thus allowing the fabrication of patterned hydrogels by photochemical patterning of Fe31 cross-linked alginate hydrogel, followed by the photochemical reductive exchange of iron cations to calcium. Images of Ca21 cross-linked and Fe31 cross-linked patterned hydrogels are shown in Fig. 3.11. 3.2.1.2.2 Ionically cross-linked hydrogels based on chitosan

Chitosan is the highly deacetylated product of chitin, with many active amino groups ( NH2) and hydroxyls ( OH) on the polymer chains [83]. Because the nitrogen atoms of NH2 and the oxygen atoms of OH have lone pair electrons, they are capable to coordinate with many metal cations [84 86]. The coordination bonds are quite strong as compared to the electrostatic interaction and other secondary bonds [87]. Therefore, metal cations with coordination ability can be used as the cross-linker to form IC chitosan hydrogels. For example, Sun et al. [88] reported a study on an ultrafast gelation via complexation between chitosan and transition metal ions, including Ag1, Cu21, Co21, Ni21, Zn21, Cd21, and Pd21. The Ag1 IC chitosan hydrogels (Ag-CS) were further investigated as a model sample. The results of time-dependent UV/vis absorption showed that the gelation process was completed within 1 minute, due to the fast coordination between the chitosan polymer chains and the Ag1. In addition, these hydrogels can maintain stability at room temperature for up to 6 months. Based on the antibacterial activity of Ag [89], Li et al. [90] prepared an Ag1 IC chitosan hydrogel for wound dressing. The preparation was done by putting the AgNO3-containing acidic chitosan solution under a gaseous ammonia atmosphere. Before gelation, the protonated NH31

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Figure 3.12 A schematic graph of the ion diffusion technique [92]. Copyright (2014) American Chemical Society.

repulsed with Ag1, which increased the solubility of chitosan. In the gelation process, the NH31 was deprotonated by gaseous ammonia, and thus was able to coordinate with Ag1 to form a cross-linked network. The hydrogels showed excellent and broad antibacterial activities against both Gram-negative and Gram-positive bacteria. The mechanical studies showed that the Ag1 IC chitosan hydrogel had the highest ultimate stress of 0.33 MPa. Under acid conditions, the amino groups of chitosan are protonated. The positively charged chains are able to form ionic bonds with small anionic molecules [90]. For example, Shu and Zhu [91] prepared IC chitosan beads using three different anionic cross-linkers: sulfate, citrate, and tripolyphosphate (TPP). In order to avoid the formation of an inhomogeneous system caused by the fast complexation, Sacco et al. [92] exploited an ion diffusion technique to prepare TPP cross-linked chitosan hydrogels, as shown in Fig. 3.12. The TPP slowly diffused into the acidic chitosan solution through the dialysis membrane, and the diffusion process was performed for 24 hours under moderate stirring at room temperature. In addition, the nongelling ion, Na1, was also added to further slowdown the gelation rate by screening the strong electrostatic interactions. Morphological analyses pointed out a uniform hydrogel network with considerable mechanical properties. 3.2.1.2.3 Reversible double-network hydrogel

The conception of a double-network (DN) gel was first proposed by Gong [93]. The DN gel consists of a rigid and brittle polymer as the first network, and another soft and ductile polymer as the second network. The specific structural characteristics render the DN gel

Physical hydrogels based on natural polymers 65 with excellent mechanical properties. The dual covalently cross-linked DN gels have poor fatigue resistance due to the irreversible failure of covalent bonds in the first network. One way to overcome this drawback is by introducing reversible noncovalent bonds instead of sacrificial covalent bonds [44,94]. Based on the reversible nature of ionic bonds and coordination interaction, the IC network is suitable to participate in the DN. For example, Sun et al. [95] mixed two types of cross-linked polymer, covalently cross-linked polyacrylamide (PAAm) and IC alginate, to form a DN hydrogel. As shown in Fig. 3.13, the hydrogel was highly stretchable, with a stretch of 23 at rupture, much larger than that of

Figure 3.13 Images of the stretched hydrogels: (a) A strip of the undeformed gel was glued to two rigid clamps. (b) The gel was stretched to 21 times its initial length in a tensile machine (Instron model 3342). The stretch, λ, is defined by the distance between the two clamps when the gel is deformed, divided by the distance when the gel is undeformed. (c) A notch was cut into the gel, using a razor blade; a small stretch of 1.15 was used to make the notch clearly visible. (d) The gel containing the notch was stretched to 17 times its initial length. The alginate/acrylamide ratio was 1:8. The weight of the covalent crosslinker, MBAA, was fixed at 0.0006 that of acrylamide; the weight of the ionic crosslinker, CaSO4, was fixed at 0.1328 that of alginate [95]. Copyright (2012) Springer Nature.

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alginate gel (1.2) and PAAm gel (6.6). In addition, the DN gels showed pronounced hysteresis in the cycle of loading and unloading, which could be explained by the mechanisms of deformation and energy dissipation in these gels. The alginate network unzipped progressively, while the PAAm network remained intact when the stretch increased. The ionic cross-link points can reform during a time interval after their break. Thus the DN gels exhibited obvious hysteresis and little permanent deformation. Li et al. [96] prepared a dual IC DN hydrogel by the coordination bonds of Fe31 with the carboxyls from both alginate and poly(acrylamide-co-acrylic acid). Due to the dynamic feature of metal-ligand coordination bonds, the Fe31 dually cross-linked hydrogels showed good three-dimensional printing feasibility and pH-triggered healing capability. Similar to the DN, and inspired by the titin configuration in muscles, Ghanian et al. [97] synthesized a dual cross-linked (DC) hydrogel with fatigue resistance, cytocompatibility, and in situ formability. The Ca21-binding derivative of alginate partially substituted by furan was prepared as an injectable or moldable pregel. Then the four-arm poly(ethylene glycol)maleimide was used as an elastic cross-linker to form covalent bonds between furan and maleimide by the well-known Diels Alder reaction. Fig. 3.14 shows the biologically

Figure 3.14 Biologically inspired DC alginate hydrogel and its photographic and schematic illustration: (A) Biologically inspired design of tough hydrogels based on dual cross-linked (DC) alginate. (B) Photographic and schematic illustration of the ionic/click DC alginate hydrogel: ionic cross-linking by calcium ions and Diels-Alder click cross-linking by a four-arm PEG cross-linker [97]. Copyright (2018) American Chemical Society.

Physical hydrogels based on natural polymers 67 inspired DC alginate hydrogel, the low-density covalent cross-links were incorporated within the transient physical network. Compared with DN hydrogels with two individual networks, the DC hydrogel had a homogeneous polymer chain structure and the cross-links were imposed in a single network. As a result, the molecular rearrangement was strictly limited by the permanent covalent cross-links. Thus, the dissociated physical cross-links could return rapidly to their original positions once the load was released, leading to a surprisingly swift self-recovery ability in the successive loading unloading cycle experiment. Unconfined compression studies also showed that the addition of ionic crosslinks into the chemically cross-linked hydrogels could promote their mechanical strength, by the high-energy dissipation of transient ionic cross-links and retarding the rupture of covalent cross-links.

3.2.2 Formation of the hydrogels via the crystallization cross-link Formation of hydrogels via the crystallization cross-link has been reported for chitosan, gelatin, and cellulose. Typically, in thermosensitive sol gel reversible hydrogels prepared by gelation or polysaccharides, renaturation to the triple helical conformation in gelatin and double helical conformation in polysaccharides drives the nucleation and growth of crystallites during gel formation [98]. Helix formation followed by aggregation of the helices results in a junction point (Fig. 3.15). At high temperatures, they are assumed to

Figure 3.15 Gelation mechanism of polysaccharides in water [99]. Copyright (2002) Elsevier.

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Figure 3.16 Structure of a chitosan/poly(vinyl alcohol) (PVA) complexed hydrogel [100]. Copyright (2003) Elsevier.

have a random coil conformation. On reducing the temperature, they start to form double helices and aggregates that act as knots, that is, the physical junctions of the gels [99]. Those junctions act as physical cross-linking points to form a physical gel. Berger et al. [100] reported an interesting approach to prepare physically cross-linked hydrogels of chitosan via complexing of chitosan/poly(vinyl alcohol) (PVA). The structure of chitosan/PVA hydrogels is shown in Fig. 3.16. The crystallite junction zones between PVA polymeric chains are formed with the freeze thaw method. PVA hydrogels can be prepared via a freezing/thawing process [101,102], which is mild in the sense that the use of cross-linking agents and organic solvents can be avoided [103]. PVA is water-soluble and biocompatible. In addition, the mechanical strength of PVA is sufficient. Hydrogels that are composed of PVA and natural polymer combine the advantages of both components, and the freezing/thawing technique has been successfully applied to prepare physically cross-linked PVA hydrogel. Therefore, it is anticipated that the graft copolymer of chitosan/starch and PVA can also form hydrogels in this way. Xiao et al. successfully applied the freezing/thawing technique to prepare physically cross-linked starch-g-PVA hydrogel and chitosan-g-PVA [104,105]. After experimenting [105], soft and elastic chitosan-g-PVA hydrogels were formed. In contrast, chitosan alone remained as a viscous liquid after similar treatment. Under normal circumstances, when we use the method of crystallization cross-link to prepare a natural polymer hydrogel, we mix natural polymer with synthetic polymer such as

Physical hydrogels based on natural polymers 69 PVA, and treated by freezing/thawing. The resulting product has better tensile strength and fracture energy.

3.2.3 Formation of hydrogels via the hydrophobic action between natural polymer groups Hydrophobic association hydrogels (HA-gels) can be formed by the hydrophobic association of amphiphilic polymers obtained by copolymerization of vinyl monomers containing double bonds with hydrophobic monomers or by introducing a small amount of hydrophobic groups by a chemical reaction on a macromolecular backbone. Due to its hydrophilic group and hydrophobic group, the hydrophobic groups will aggregate and associate with each other under the entropy. At a certain concentration, the macromolecule forms a three-dimensional network structure in water. In 1987, Evani and Rose [106] referred to this hydrophobically obtained polymer as a “hydrophobic association polymer.” As a physical cross-linked hydrogel, HA-gel overcomes the shortcomings of traditional chemical cross-linked hydrogels such as low biocompatibility and high toxicity; moreover, the network cross-linking point is a hydrophobic association microregion, and there is an association deassociation equilibrium, which is dynamic and flexible, so that the gel can show the properties of self-healing, self-assembly, easy regulation, and high sensitivity, which could expand its application potential. Compared with synthetic polymer materials, natural polymers have the advantages of being completely biodegradable, nonpolluting, and biocompatible. Due to the many unique structures and properties of HA-gels based on natural polymers, they have important applications in many fields. The following is a brief introduction to the research progress of two kinds of natural polymers, chitosan and cellulose, in HA-gels. 3.2.3.1 Hydrophobic association hydrogels based on chitosan The most common preparation method of chitosan physical gel is to regenerate its aqueous acetic acid solution in an excessive alkaline coagulation bath. In an alkaline environment, the amino group on the chitosan molecular chain deprotonates, which in turn leads to a three-dimensional network structure formed by hydrogen bonding and hydrophobic interaction between the molecular chains [107,108]. Ladet et al. [109] dissolved chitosan in an acetic acid/polyol/water mixed solvent and removed water by heating to obtain a chitosan alcohol gel; the alcohol gel was intermittently placed in a NaOH coagulation bath for regeneration, and the “onion” structure chitosan multilayer hydrogel was first prepared (Fig. 3.17). Chitosan temperature-sensitive hydrogels have attracted much attention due to their good biocompatibility and injectability. CS itself does not have temperature-sensitive properties. Generally, CS temperature-sensitive hydrogels are modified by the structure of CS or

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Figure 3.17 Multi-membrane hydrogels. (a) Schematic diagram of the multi-membrane onion-like structures; (b) multi-membrane biomaterial with‘onion-like’ structure based on chitosan [109]. Copyright (2008) Springer Nature.

blended with substances with temperature sensitivity to increase the hydrophobic interaction between CS. This drives the formation of the gel. In recent years, the research on chitosan temperature-sensitive physical hydrogel has focused on the addition of basic salts to chitosan solution, modification of chitosan, and blending with other polymers. 3.2.3.1.1 Chitosan/glycerophosphate hydrogel

Chitosan is an alkaline polysaccharide that can be dissolved in dilute acid solutions. In the dilute acid solution, the NH2 on the chitosan molecule is protonated to NH31, the hydrogen bond between the molecules is destroyed, and the OH binds to the water molecule, causing the chitosan molecule to swell. However, when the pH of the solution is near neutral, chitosan will precipitate. Glycerophosphate (GP) is a polyhydroxy weak base salt. Under low-temperature conditions, GP binds to the amino group by electrostatic binding, which weakens the electrostatic repulsion on the chitosan molecule and protects the hydration state of the macromolecular substance. Under the combined effect of these two effects, even if the chitosan is in neutral conditions, precipitation will not occur. However, as the temperature increases, the hydrogen bonding between the molecules weakens, the hydrophobic action of the chitosan molecules increases, and the molecules entangle each other, gradually forming a gel. The intermolecular hydrophobic interaction is the main driving force for gel formation [110 113]. When the temperature is low, the CS molecule binds to a large number of GP molecules by ion or hydrogen bonding, as described above. The hydrophilicity of GP makes it possible to form a protective hydration layer around the partially deprotonated CS molecule. It is the existence of this protective layer that makes the interaction force between CS molecules small and unable to form a gel; when the temperature rises, the mobility of the molecule increases, especially small molecules become extremely active, and the small molecule water and sodium GP bound around the CS molecule has a tendency to leave; when the temperature rises to a certain extent, the externally supplied thermal energy is sufficient to

Physical hydrogels based on natural polymers 71 overcome the binding force between CS and the small molecule, then the water-protective layer around the CS molecule is destroyed, and the CS molecules undergo strong dehydration, which allows the CS macromolecules to interact with each other, and the hydrophobic interaction between the molecules is enhanced, and chain entanglement occurs, thereby forming a cross-linked 3D network. Chenite et al. [111] mixed chitosan with beta-GP to prepare an aqueous solution, which can remain liquid for a long time in the physiological pH range (pH 6.8 7.2) and below room temperature, but can form gel rapidly when the temperature rises to 37 C. At the same time, the problem of acidic and nontemperature sensitivity of chitosan solution was solved, and chitosan was pioneered as a temperature-sensitive physical hydrogel. In addition, osteoinductive growth factor and chondrocytes were mixed into a chitosan/glycerol phosphate solution at low temperature, subcutaneously injected into a mouse to form a gel in situ, and the formation of new cartilage was observed, as shown in Fig. 3.18. The good biocompatibility of CS/GP hydrogels and the prospects for application in tissue engineering such as irregular repair have been confirmed. At the same time, some factors have been studied on the factors affecting the temperature and rate of the gel. It is found that the degree of deacetylation (DD) of CS has a great influence on the gel temperature and rate. The larger the DD, the lower the gel temperature and the faster the rate. The molecular weight of CS has little effect on it. In subsequent studies it was pointed out that the gel temperature depends on the pH of the CS/GP mixture, and the lower the pH, the lower the gel temperature. In addition to the above factors, the gel rate has a close relationship with temperature: the higher the temperature, the higher the gel rate. Ruel-Garie´py et al. [114] explored the physical properties of CS/GP temperature-sensitive hydrogels. The test results showed that the gelation rate mainly depends on the DD of CS and the temperature. At 4 C, the CS/GP gel with a degree of deacetylation of 84% can be stored for about 3 months, and the viscosity does not change significantly during this period. Zhou et al. [115] and Wu et al. [112] studied the effects of different types of glycerol phosphate on the CS/GP system. The results showed that α,β-GP had a faster setting rate than β-phosphate. Since α-phosphate is a linear molecular structure, steric hindrance is small, and β-GP is a nonlinear molecule. Zhao et al. [116] analyzed the dissolution of CS in different dilute acid solutions and its effect on CS/GP gelation. The SEM micrographs of CS/GP hydrogels prepared with different acids are shown in Fig. 3.19, which clearly illustrates the dependence of hydrogel morphology on the type of acids. Network-like structures and many ramified configurations were formed in CS/GP hydrogels and the pores were connected to each other. It was found that the CS/GP system prepared by dissolving CS from monovalent acids (formic acid, acetic acid, lactic acid, etc.) condensed within 2 5 minutes, while the high-valent acid (divalent and trivalent) systems could not form a gel. Because high-valence acid dissolves, CS requires more GP to neutralize, when GP concentration is too high, CS will precipitate, so the system cannot form a gel.

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Figure 3.18 An osteoinductive mixture of proteins (BP) delivered from C/GP gels induces bone and cartilage formation in the rodent ectopic model: (A) Von Kossa stain of C/GP gel without BP demonstrated fibrous, unmineralized tissue. (B) Von Kossa stain of C/GP gel with 10 μg BP demonstrated immature, mineralized bone, mineralized cartilage, and non-mineralized cartilage containing chondrocytes. (C) Toluidine blue stain of C/GP gel without BP demonstrated fibrous, noncartilagenous tissue. (D) Toluidine blue stain of C/GP gel with 30 μg BP demonstrated an abundance of chondrocytes encompassed by a territorial cartilagenous matrix. Original magnification was 4 3 for (A), (B), and (C) and 10 3 for (D) [111]. Copyright (2000) Elsevier.

3.2.3.1.2 Hydrophobic association hydrogels derived from chitosan derivatives

Due to the presence of intermolecular and intramolecular hydrogen bonds in chitosan, it is insoluble in common organic solvents and water, which leads to great difficulties for its wider application. In order to improve its solubility, many modifications have been made.

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Figure 3.19 Scanning electron micrographs of CS/GP hydrogels prepared with different acids: (A) formic acid; (B) acetic acid; (C) propionic acid; (D) butyric acid; (E) isobutyric acid; (F) lactic acid; (G) nitric acid; (H) hydrochloric acid; and (I) chloroacetic acid [116]. Copyright (2009) Springer Science Business Media, LLC.

One´sippe et al. [117] carried alkylation modification of CS by a reductive Schiff-base method. Alkylation of chitosan (ACS) with different alkyl substitution degree and alkyl chain was prepared by changing the reaction conditions, and ACS/GP gel was further prepared. ACS introduces hydrophobic alkyl groups into the CS molecular chain, which increase the chain spacing and disrupt the hydrogen bonding between the CS molecules and the molecules. At the same time, it also destroys the regularity of the CS molecular structure, which reduces the existence of the crystal region to some extent. Combining these two effects, ACS has good water solubility. When the temperature is low, the ACS can stably maintain the solution state because of the good solubility of the ACS itself and the similar effect of the GP in the CS/GP system, forming a water-protective layer around the ACS.

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When the temperature rises, the hydrophobicity of the ACS increases and the solubility deteriorates. In addition, the water-protective layer formed by the GP is also destroyed. When the two effects reach a certain level, the ACS chain is strongly dehydrated, and the ACS molecules interact to form a cross-linked three-dimensional structure in which hydrophobic association and hydrogen bonding play a major role. The gel mechanism of the ACS/GP gel system is similar to that of CS/GP, but as the degree of substitution increases, hydrophobic association is the main reason for the gel formation [118]. 3.2.3.2 Hydrophobic association hydrogels based on cellulose 3.2.3.2.1 Hydrophobic association hydrogels derived from cellulose derivatives

Cellulose is a polysaccharide material with many hydroxyl groups in the molecular chain. They easily interact to form a hydrogen bond network structure, forming a crystalline fibril structure, the crystal regions are closely arranged, and it is difficult for the solvent to enter this region, which in turn causes the cellulose itself to be difficult to dissolve in a common organic solvent. Among the cellulose derivatives obtained by esterification or etherification of the hydroxyl group on cellulose molecules, cellulose ether has good biocompatibility and degradability, in addition to excellent thickening, film forming, water retention, etc. Therefore cellulose ether is mainly used in the skeleton material of the sustained-release preparation and the temperature-sensitive gel material. In reversible thermoplastic hydrogels, hydrophobically modified cellulose is in the majority. When the cellulose is replaced by a partial methyl or hydroxypropylmethyl group, the original hydrogen bond structure of the cellulose is destroyed, resulting in its derivative being water-soluble. Temperature-sensitive cellulose ether contains groups such as methyl and hydroxypropyl, giving the polymer surface activity and unique water-dehydration characteristics. The aqueous solution of temperature-sensitive cellulose ether has a thermogel property, that is, a liquid which is clear and transparent at a low temperature, and when the temperature is raised, the solution becomes an opaque gel state, and the thermogel property is reversible, that is, when the temperature is lowered again and the system returns to a clear liquid [119]. The cellulose ether heat-initiated gel can be prepared when the solution is heated. The spatial three-dimensional network structure produced during the gelation process is mainly caused by the hydrophobic interaction between hydrophobic groups such as methoxy groups on the polymer chain. At present, the “cage-like structure” theory is generally accepted as the gel principle of temperature-sensitive cellulose, and MC and hydroxypropyl cellulose have been extensively studied in this respect [120,121]. That is, at a lower temperature, a strong interaction occurs between the water molecules in the solution and the cellulose ether molecules, and the molecular chains are hydrated, and there is almost no other intermolecular action except for some simple molecular chain entanglements. When the

Physical hydrogels based on natural polymers 75 temperature rises, the molecule can absorb the heat and gradually remove the water molecules bound to the cellulose molecular chain, exposing the hydrophobic group. As the temperature rises gradually to the gelation temperature, the intermolecular interaction occurs due to a hydrophobic interaction, causing the solution to become a turbid liquid state and then further transform into a network gel structure. Sekiguchi et al. [122] investigated how the hydrophobic interactions as well as hydrogen bonds contribute to thermally reversible gelation of aqueous solutions for Omethylcellulose. The results indicate that the gelation behavior of a series of regioselectively substituted 2,3-di-O-methylcellulose (2,3MC-n: n 5 1 3) differed from that for randomly substituted O-methylcellulose (R-MC). And the gelation of the 2,3MC-n and R-MC solutions may be caused by cooperation of the hydrophobic interaction among methyl substituents with the intermolecular hydrogen bonds among hydroxyl groups at the C(6) position, which are dependent on the distribution of methyl groups. Yang et al. [123] prepared a temperature-responsive hydrogel composed of aqueous methylcellulose (MC) blended with distinct concentrations of phosphate-buffered saline, as shown in Fig. 3.20. The salts blended usually have a greater affinity for water molecules than polymers, resulting in the removal of water from polymers and thus dehydrating or “salting out” of the polymeric molecules. This can further increase the hydrophobic interaction among MC molecules and lead to a decrease in their gelation temperature. The developed MC hydrogel underwent a sol 2 gel reversible transition upon heating or cooling at B32 C. This temperature-responsive hydrogel was employed to coat the surface of a polystyrene dish and used to cultivate human embryonic stem (HES) cell clumps for the formation of embryoid bodies (EBs) in a liquid suspension culture (LSC-MC/PS). The HES cells within the EBs were shown to express molecular markers specific for representative cells from the

Figure 3.20 The procedure used to prepare the MC-coated polystyrene dish for the HES cell differentiation culture [123]. Copyright (2007) American Chemical Society.

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three embryonic germ layers. These results indicated that the MC-coated dish can be used to produce a large scale of HES cell derivatives through the formation of EBs. 3.2.3.2.2 Hydrophobic association hydrogels derived from cellulose composites

Cellulose and its derivatives can be blended with natural degradable polymers such as chitin, chitosan, starch, alginic acid, and hyaluronic acid to prepare new materials to meet specific applications. MC and chitosan can form injectable hydrogels by ionic and hydrophobic interactions under mild conditions in the presence of various salts. Tang et al. [124] developed a chitosan/methylcellulose/sodium phosphate hydrogel (CS/MC/Na3PO4 hydrogel) whose formation mechanism is similar to the theory of the “cage-like structure.” CS is solubilized in dilute acid by protonation of the free-amino groups, which then undergo ionic/hydrophilic interaction with water molecules, resulting in solubilization (Fig. 3.21A). Increasing the pH results in progressive deprotonation, gradually increasing the hydrophobic characteristic of CS chains until gelation/insolubility occurs. When the MC solution is added at low temperature to CS solution, a hydrogen bond is formed between CS, MC, and water which can maintain/enhance CS chain dissolution (Fig. 3.21B). These interactions and the low temperature reduce macromolecular mobility, preventing CS chain association/precipitation. When temperature is increased, intermolecular hydrogen bonding interactions are reduced and the energized water molecules surrounding the polymers are

Figure 3.21 Proposed interaction mechanisms for chitosan (CS) and methylcellulose (MC): (A) CS solution (low temperature); (B) CS/MC solution (low temperature); (C) CS/MC hydrogel (elevated temperature) [124]. Copyright (2010) Elsevier.

Physical hydrogels based on natural polymers 77 dissipated, thereby allowing hydrophobic intermolecular chain association, resulting in gelation (Fig. 3.21C). Thus, hydrophobic interactions are presumed to be the main driving force in the gelation of CS/MC systems at elevated temperatures. When used as a scaffold for chondrocytes, CS/MC/Na3PO4 hydrogel resulted in good cell viability and proliferation, indicating potential use as a three-dimensional synthetic matrix for tissue engineering.

3.2.4 Formation of the hydrogels via hydrogen bonding Hydrogen bonding is typically expressed as D H


A, in which D represents the atom possessing higher electronegativity, such as an O, F, or N atom [125]. When a hydrogen atom is bonded to the electronegative atom, negative charges exist at the electronegative atom and partial positive charges exist at the hydrogen atom, and electrostatic interactions occur between the two atoms [126]. Intermolecule and intramolecule hydrogen bonding can play the role of a physical cross-linking point during formation of the hydrogels [127,128]. Hydrogen bonding is attracting more and more attention as a physical interaction. Natural polymers have many functional groups that can form hydrogen bonding, so hydrogen bonding is the main type of cross-linking for the formation of hydrogels based on natural polymers via physical interaction. Taking cellulose as an example, there are abundant hydroxyl groups in the chains, so it is easy to form a hydrogen bonding linked network [129]. And like cellulose, there are many OH and COOH or COO groups in alginate [130,131]. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine units [100]. There are many OH and NH2 groups in the chains, which can also easily form hydrogen bonding [132]. 3.2.4.1 The formation of physical hydrogels via hydrogen bonding interactions During the formation of physical hydrogels, hydrogen bonds play different roles. On the one hand, they are the main intermolecule or intramolecule force. Zheng et al. [133] prepared CMC hydrogels through a two-step method. In the first step, they obtained freeshaped Na-CMC pastes. In the second step, they immersed the pastes into citric acid solution for a certain period of time. In the solution, the H1 ions gradually diffused into the Na-CMC pastes and bound CMC polymer chains via hydrogen bonds, and finally formed transparent and free-shaped CMC hydrogels, which exhibited stretchability and had excellent mechanical properties. The CMC hydrogel can achieve 2.5 MPa of compressive strength and exhibit excellent self-healing behavior with over 80% healing efficiency. The preparation process is shown in Fig. 3.22.

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Figure 3.22 The two steps to prepare CMC hydrogels [133]. Copyright (2015) Elsevier.

A kind of novel hydrogel has been prepared through mixing aqueous solutions of carboxymethylchitosan with cellulose ethers including hydroxyethyl-cellulose (HEC) and MC, and these hydrogels were cross-linked by hydrogen bonds. The result showed that CMC/HEC hydrogels had a better interaction than CMC/MC hydrogels because of the stronger hydrogen bonding of CMC/HEC hydrogels [134]. On the other hand, sometimes hydrogen bonds are not the main force for formation of hydrogels, but they are also the most important mechanism to form of the hydrogels. Low et al. [135] prepared a kind of hydrogel consisting of IC alginate, covalent cross-linked PAAm, and hydrogen bonding interactions among water. Apart from an interpenetrating network of covalent and ionic cross-links, the hydrogen bonds formed by hydrogen bond donor groups of AAm monomers with water and functional groups of alginate are responsible for the excellent toughness of hydrogels. In order to explore the effect of hydrogen bonding on mechanical properties, they synthesized hybrid hydrogels with different ratios of AAM and DMMA. The result showed that with PAAm content going down, there was a significant reduction in the properties due to a decrease in the amount of hydrogen bonding. It has been proved that a great number of chitosan hydrogels are formed via Schiff-base bonds, however, when using vanillin(4-hydroxy-3-methoxybenzaldehyde) as cross-linking agent, researchers [136] found that only one aldehyde group of a vanillin molecule formed a Schiff-base bond with a chitosan, and the other hydroxyl group was able to form hydrogen bonding, which is the basis of the reversible hybrid network (Fig. 3.23).

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Figure 3.23 (A) 5% (w/v) chitosan solution; (B) 5% (w/v) chitosan solution stored at room temperature for 24 h; (C) 10 mL 5% (w/v) chitosan solution with 0.5 g vanillin; (D) the schematic illustration of this Schiff-base bond/hydrogen bond hybrid network [136]. Copyright (2018) Elsevier.

3.2.4.2 The effect of hydrogen bonding on the properties of physical hydrogels The presence of hydrogen bonding will affect the mechanical properties, swelling properties, degradation properties, and biological properties of hydrogels. It was found that hydrogen bonds play an important role in self-healing property. Self-healing hydrogels are hydrogels that can fully or partly recover back to normality automatically after breakage [137]. Self-healable hydrogels can be divided into two main types based on their recovery mechanism, that is, physical self-healable hydrogels and chemical self-healable hydrogels. Physical self-healable hydrogels rely on hydrogen bonding for reconstruction after being damaged [138]. Sodium alginate is a type of polysaccharide that has abundant hydroxyl groups [139], and deferoxamine mesylate (DFA) is a commercial medicine consisting of hydroxyl groups and amine groups [140]. Thus, hydrogen bonding can form between these two materials.

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Xu et al. [141] prepared a modified alginate named SA-DFA, which had deferoxamine molecules linked to an alginate backbone through amide bonds. After the transparent hydrogel was prepared, these researchers dissolved the SA-DFA hydrogels with NaSCN, which is a kind of hydrogen bond broken agent. After an hour of stirring, the transparent hydrogels were totally dissolved into the solution, and the hydrogels were measured by time-dependent alternating stain sweep to investigate their self-healing behavior. When the strain was as large as 800%, hydrogen bonds between SA-DFA molecules were broken, and the hydrogels acted as liquid in broken stage 1 (when the strain was raised up to 800% firstly) and broken stage 2 (when the strain was raised up to 800% again). When the strain changed back to 1%, the hydrogen bonding network reconstructed. Thus, the self-healing mechanism of SA-DFA lies in hydrogen bonding’s reversibility. At the same time, hydrogen bonds can work with other forces to make hydrogels reconstruct after being damaged. Zhang et al. prepared a kind of dual cross-linking self-healing hydrogel termed gelatin-ureido-pyrimi-dinone (Upy)-Fe by a combination of multiple hydrogen bonding and ionic coordination. The hydrogels were cross-linked by an ionic coordination of Fe31 and carboxyl group from the gelatin and the quadruple hydrogen bonding interaction from the ureido-pyrimi-dinone (Upy) dimers. The results of rheological measurements and optical tests reflected an excellent self-healing property of the prepared hydrogel, and the multiple hydrogen bonds contributed to its self-healing property. After the hydrogel was cut into halves, the polymer chains were destroyed and the dimers were dissociated. However, when the separate parts were put together, they healed together and reconstructed the hydrogel network due to the strong quadruple hydrogen bonding [142,143].

3.3 Conclusion and perspectives Hydrogels, a kind of soft material holding a lot of water, have a high similarity with the same hydrous, soft, and wet extracellular matrix and soft tissues of living organisms. As an important member of the hydrogels family, physical hydrogels based on natural polymers have attracted a lot of attention due to their special cross-linking mechanism and natural source. Over the past decade, the number of published works on the physical hydrogels is four times that before 2008. Because of the severe environmental pollution caused by the fossil fuel industry, natural polymers such as alginate, chitosan, cellulose, gelatin, and collagen are being used more frequently. It can be seen from the examples listed in this chapter that the demand for soft materials is higher than before in a variety of fields such as biomedicine, soft electronics, sensors, and actuators. On the basis of sufficient biocompatibility, many studies have also tried to introduce other prominent properties such as self-healing property, stimulus-responsiveness, fatigue resistance, antibacterial property, bioactivity, biodegradability, and in vivo stability. In order to meet the above requirements of hydrogels, the synthesis method and the selected raw materials should be considered carefully.

Physical hydrogels based on natural polymers 81 As mentioned earlier in this chapter, during the preparation process of the physical hydrogels based on natural polymers, toxicity of chemical cross-linkers, initiators, and monomers could be avoided, making them promising biocompatible materials. Furthermore, there are many advantages to physical hydrogels and natural polymers could be developed in the future. The reversible nature of physical interactions makes physical hydrogels ideal self-healing materials. It can be predicted that the trend is to prepare facile and efficient self-healing hydrogels by introducing physical interactions. Another potential tendency of physical cross-linking is to form robust and tough double-network hydrogels by cooperating with a covalent cross-linked network. However, compared with conventional chemical hydrogels, there still remain several challenges to be addressed for physical hydrogels. First is how to make the gelation process of physical hydrogels controllable. The formation process of physical hydrogels is easy and mild, but the fast interactions can lead to instant cross-linking, which makes it difficult to control and tune the gelation. In order to get the desired shaped physical hydrogels with uniform compositions, the key is to disperse polymers and other agents uniformly, and slow down the gelation rate. Second is how to improve the physical and chemical stabilities of physical hydrogels based on natural polymers. Because the noncovalent cross-links are weaker than the covalent bonds, and some natural polymers are biodegradable, the hydrogels may not be able to maintain stability when meeting strong acid and basic agents, high-concentration salt solutions, high temperature, or radiation. For instance, when put in a solution of sodium chloride, the cross-linked network of Ca21 IC alginate hydrogels can be destroyed by the substitution of sodium ions. Their poor stability largely limits their applications in some harsh environments. In terms of application, appropriate physical and chemical stabilities are important for hydrogels, such as when used as in vivo implanted materials or drug-delivery materials, which need to withstand the erosion and decomposition of body fluids and the effect of body temperature during the action time. Third is how to handle the poor mechanical properties of physical hydrogels based on natural polymers. Those physical hydrogels are often broken when subjected to large compressive or tensile forces, especially when the water content is high. Physical hydrogels with great mechanical strength are promising materials in tissue engineering and soft electronics. In short, future efforts will mainly focus on improving controllability during preparation, physical and chemical properties, and mechanical properties.

References [1] D. Seliktar, Designing cell-compatible hydrogels for biomedical applications, Science 336 (2012) 1124 1128. Available from: https://doi.org/10.1126/science.1214804. [2] M. Hamidi, A. Azadi, P. Rafiei, Hydrogel nanoparticles in drug delivery, Adv. Drug Deliv. Rev. 60 (2008) 1638 1649. Available from: https://doi.org/10.1016/j.addr.2008.08.002.

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CHAPTER 4

Preparation of hydrogels based on natural polymers via chemical reaction and cross-Linking Vishwajeet Sampatrao Ghorpade* Department of Pharmaceutics, School of Pharmaceutical Sciences, Sanjay Ghodawat University, Kolhapur, India

4.1 Introduction Hydrogels are polymeric biomaterials which show a wide range of applications in the pharmaceutical and biomedical fields. These are three-dimensional network structures arising from the interconnection between hydrophilic polymer chains. They have the ability to absorb and retain a large amount water, without getting dissolved. Their water retention capacity, flexible nature, and permeability are the most important characteristics of hydrogels, which make them suitable for pharmaceutical and biomedical applications [1]. Hydrogels can be prepared from natural or synthetic polymers. Besides a few disadvantages related to pathogenicity and inflammatory responses, hydrogels made from natural polymers are biocompatible, biodegradable, and possess biologically recognizable moieties that support the cellular activities [2]. For this reason, natural polymer-based hydrogels have attracted the attention of scientists dealing with biomaterials. The natural polymers which have been most widely investigated for the preparation of hydrogels mainly include polysaccharides and proteins. The common methods used for the preparation of natural polymer-based hydrogels involve physical, chemical, and radiation cross-linking. The physically cross-linked hydrogels can be prepared without chemical modification of the polymers or use of crosslinking agents which may render hydrogels toxic. However, these hydrogels exhibit poor mechanical properties due to which they dissipate from their site of application within a short period of time. On the other hand, chemical cross-linking results in the formation of covalent bonds between the polymer chains and prevention of dilution and dissipation of


corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00004-5 © 2020 Elsevier Inc. All rights reserved.

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the hydrogel matrix [3]. Despite the high cost of some reagents and rigorous procedures involved, the chemical cross-linking method is popular amongst research scientists dealing with natural polymer-based hydrogels. Although most of the chemical crosslinking agents are toxic, a proper precaution taken during synthesis of hydrogels followed by stringent washing procedures has helped most researchers to obtain biocompatible hydrogels. This chapter focuses on various methods of preparation of natural polymer-based hydrogels using chemical cross-linking and the reactions involved therein.

4.2 Chemical cross-linking methods The chemical reactions which are most commonly involved in the formation of natural polymer-based hydrogels usually require an aqueous environment. Some of the most commonly used chemical reactions for hydrogel preparation include Schiff base reaction, epoxide coupling, addition reaction, click reaction, condensation reaction, and free radical polymerization. In addition, some other methods such as genipin coupling and polycarboxylic acid-based esterification cross-linking have been used to obtain natural polymer-based hydrogels with good mechanical and swelling properties.

4.2.1 Schiff base reaction The natural polymer-based hydrogels with dynamic properties are prepared using a Schiff base reaction. This involves an interaction between two polymers containing amine and aldehyde groups, respectively, leading to the formation of imine bonds (see Fig. 4.1). The imine cross-links formed are pH dependent, and impart self-healing properties to the hydrogels [4,5]. The natural polymers which do not contain aldehyde or amine groups are usually modified to incorporate these groups in the polymer chain.

Figure 4.1 Representation of Schiff base reaction between polymers containing amine and aldehyde groups.

Preparation of hydrogels based on natural polymers 93 Table 4.1: Natural polymer-based hydrogels prepared using Schiff base reaction. Polymers

Modification/functionalization

Interacting components

References

Sodium alginate and gelatin Chitin and sodium alginate Gelatin and hyaluronic acid Starch and polyvinylamine

Oxidation of OH groups of alginate to CHO

CHO of alginate and NH2 of gelatin

Acrylamide functionalization of chitin and oxidation of alginate to alginate aldehyde

NH2 of chitin and CHO of alginate

Nguyen and Lee [9] Ding et al. [10]

Carbohydrazide modification of gelatin (GelCDH) and aldehyde functionalization of hyaluronic acid (HA-mCHO) Oxidation of starch

NH2 of Gel-CDH and CHO of HA-mCHO

Hozumi et al. [11]

CHO of oxidized starch and NH2 of polyvinylamine

Li et al. [12]

Tan et al. prepared injectable in situ forming biodegradable chitosan-hyaluronic acid hydrogels for tissue engineering applications. Initially, chitosan was modified into N-succinyl-chitosan (S-CS) and hyaluronic acid was oxidized using sodium periodate to obtain aldehyde hyaluronic acid (A-HA). Mixing the aqueous solutions of S-CS and A-HA led to the formation of hydrogels [6]. Recently, injectable chitosan-hyaluronic acid hydrogels were prepared for abdominal tissue regeneration, where a similar interaction occurred between N,O-carboxymethyl chitosan and A-HA [7]. In order to promote Schiff base reaction between carboxymethyl chitosan (CMCh) and sodium alginate so as to obtain soft adhesive material for tissue engineering applications, additional amino groups were incorporated into chitosan using ethylenediamine in the presence of N-(3dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride, and sodium alginate was modified by sodium periodate-mediated oxidation [8]. Some other examples of natural polymer-based hydrogels prepared using Schiff base reaction are given in Table 4.1. The advantage of this reaction is the nonrequirement for purification due to the absence of cross-linking agents. There are few cross-linking agents, such as glutaraldehyde, poly(ethylene glycol) (PEG)dialdehyde, and vanillin, which utilize the Schiff base reaction mechanism for the formation of cross-links between natural polymers containing an amine group. These cross-linking agents contain aldehyde groups at the terminal ends of the molecule which react with the amine groups present on the adjacent polymer chains (see Fig. 4.2). Due to its high aqueous solubility and low cost, glutaraldehyde is the most widely used cross-linking agent [13]. It has been extensively used for the preparation of chitosan hydrogels [14]. The aldehyde groups of glutaraldehyde react with the amine groups of chitosan to form cross-linked hydrogels. Similarly, glutaraldehyde has been used for the preparation of gelatin and collagen hydrogels due to presence of NH2 groups belonging to

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Figure 4.2 Schiff base reaction with cross-linking agents like glutaraldehyde (A and B), PEG-dialdehyde (C) and vanillin (D).

Preparation of hydrogels based on natural polymers 95 the amino acids in these polymers [1517]. At acidic pH, glutaraldehyde can also react with the hydroxyl groups present on the polymer chains. Distantina et al. have synthesized carrageenan hydrogels using glutaraldehyde as the cross-linking agent, where glutaraldehyde reacted with OH groups of polymer in an acidic environment to form cross-links [18]. The major drawback of using glutaraldehyde as a cross-linking agent is its biocidal nature. Therefore a lower concentration of glutaraldehyde is often recommended while using it as a cross-linking agent [19]. PEG-dialdehyde cross-links the amino group containing polymers by the same mechanism as exhibited by glutaraldehyde. The Schiff base intermediate formed in the case of PEG-dialdehyde-based cross-linking can be further reduced using NaBH3CN [20]. Hyaluronic acid hydrogels were prepared for the delivery of various therapeutic agents at the wounded sites [21]. The hyaluronic acid was modified into hyaluronic acid-adipic dihydrazide for the incorporation of NH2 groups in the polymer chain. The modified polymer was further cross-linked using PEGdialdehyde. PEG-dialdehyde has also been used for the preparation of collagen/elastin hydrogels intended for tissue engineering purposes. It was found that PEG-dialdehyde improved the mechanical and thermal stability of the hydrogels, without affecting their biocompatibility [22]. Vanillin is a commonly used natural flavoring agent in the food and pharmaceutical industries [23,24]. It has gained recognition as a bio-based cross-linking agent because of the aldehyde and hydroxyl group in its structure [25]. The aldehyde forms an imine bond with the amine group of the polymer, whereas the hydroxyl group participates in hydrogen bonding. Thus vanillin makes use of chemical as well as physical cross-linking for the formation of hydrogels (see Fig. 4.2). Xu and co-workers have reported the formation of self-healing chitosan hydrogels using vanillin as a cross-linking agent. At low concentrations, the self-healing ability of the hydrogels was retained; however, at high concentrations, stiff hydrogel network structures were formed due to an increase in hydrogen bonding [26]. Some aldehydes such as formaldehyde and glyceraldehyde are also known for the crosslinking of the natural polymers containing NH2 groups via Schiff base mechanism [27,28]. It is known that the Schiff base is formed as an intermediate during the cross-linking reaction but the actual mechanism of cross-linking for these agents remains unresolved [29]. Table 4.2 represents the hydrogels based on natural polymers prepared using aldehyde group-containing cross-linking agents.

4.2.2 Epoxide-based cross-linking Epoxides are water-soluble compounds which exhibit high reactivity toward nucleophiles like amines and alcohols [40]. In an aqueous environment, nucleophiles attack the carbon atom of the epoxide ring. Due to the ring strain, epoxides readily undergo reaction at room

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Polymers

Cross-linking agent

Chitosan

Glutaraldehyde

Acetylated chitosan and Glutaraldehyde aminated hyaluronic acid derivatives Chitosan and collagen

Glutaraldehyde

Bacterial cellulose and gelatin

Glutaraldehyde

Hyaluronan and chondroitin sulfate modified with adipic hydrazide Chitosan

PEG-dialdehyde

Chitosan Chitosan Soy protein

Interacting components

References

CHO of glutaraldehyde reacting with NH2 of chitosan CHO of glutaraldehyde reacting with NH2 of acetylated chitosan and aminated hyaluronic acid derivatives CHO of glutaraldehyde reacting with NH2 of chitosan and collagen CHO of glutaraldehyde reacting with NH2 of gelatin and -OH of cellulose CHO of PEG-dialdehyde reacting with NH2 of modified hyaluronan and chondroitin sulfate

Rohindra et al. [30]

CHO of vanillin reacting with NH2 of chitosan Formaldehyde CHO of formaldehyde reacting with NH2 of chitosan D,L-Glyceraldehyde CHO of D,L-glyceraldehyde reacting with NH2 of chitosan D,L-Glyceraldehyde CHO of D,L-glyceraldehyde reacting with NH2 of proteins Vanillin

Crescenzi et al. [31]

Wu et al. [32] Treesuppharat et al. [33] Kirker et al. [34]

Zhang et al. [35], Abraham et al. [36] Sadeghi et al. [37] Oliveira et al. [38] Caillard et al. [39]

temperature without any requirement for catalyst [41]. Depending upon the difference in nucleophilicity, epoxides show more reactivity toward amine-containing polymers than polymers containing hydroxyl groups. Some of the commonly used epoxides for the preparation of natural polymer-based hydrogels include epichlorohydrin, ethyleneglycol diglycidylether (EGDE), polyethyleneglycol diglycidylether (PEGDE), and butanediol diglycidylether (BDDE). The reaction by which these cross-linking agents form cross-links is shown in Figs. 4.3 and 4.4. Epichlorohydrin consists of two reactive functional groups, namely epoxide and chloroalkyl groups. Under alkaline conditions, these groups can react with OH groups of anhydroglucose units of natural polymers such as cellulose, starch, xanthan gum, chitosan, dextran, and guar gum [4247]. In the case of polymers such as chitosan, epichlorohydrin can also react with the NH2 group on one polymer chain by opening an epoxide linkage via Schiff base reaction, and the chloride group can react with the OH group on the other polymer chain. In order to prevent the reaction between epichlorohydrin and NH2 groups of chitosan, chitosan is initially treated with aromatic aldehydes for the formation of Schiff base followed by the addition of epichlorohydrin. The previously formed Schiff base is

Preparation of hydrogels based on natural polymers 97

Figure 4.3 Formation of epoxide cross-links by epichlorohydrin (A), ethyleneglycol diglycidylether (EGDE) (B), polyethyleneglycol diglycidylether (PEGDE) (C), and butanediol diglycidylether (BDDE) (D).

removed later by treatment with alcoholic HCl [48]. Mohamed and Sabaa have synthesized antimicrobial CMChsilver nanoparticle (Ag) hydrogels using epichlorohydrin as a crosslinking agent where, under alkaline conditions, the epoxide group of epichlorohydrin interacts with the NH2 group on one polymer chain and chlorine reacts with the COOH

98

Chapter 4

Figure 4.4 Formation of epoxide cross-links by epichlorohydrin (A), ethyleneglycol diglycidylether (EGDE) (B), polyethyleneglycol diglycidylether (PEGDE) (C), and butanediol diglycidylether (BDDE) (D) under mild alkaline conditions.

Preparation of hydrogels based on natural polymers 99 Table 4.3: Natural polymer-based hydrogels prepared using epoxy cross-linking agents. Polymers

Cross-linking agent

Cellulose

Epichlorohydrin

Hydroxyethylcellulose and soy protein

Epichlorohydrin

Konjac glucomannan and xanthan gum

Epichlorohydrin

Cellulose and chitosan Hyaluronic acid Cellulose (cationic)

Collagen

Ethyleneglycol diglycidylether (EGDE) EGDE Polyethyleneglycol diglycidylether (PEGDE) Butanediol diglycidylether (BDDE)

Interacting components

References

Epoxide ring and chloride group of epichlorohydrin react with OH groups of cellulose Epoxide ring and chloride group of epichlorohydrin react with OH groups of cellulose and -NH groups of soy protein Epoxide ring and chloride group of epichlorohydrin react with OH groups of glucomannan and xanthan gum Epoxide rings of EGDE react with OH groups of cellulose and NH2 groups of chitosan

Navarra et al. [50] Zhao et al. [51] Paradossi et al. [52] Li et al. [53]

Epoxide rings of EGDE react with OH groups of hyaluronic acid Epoxide rings of PEGDE react with OH groups of cellulose

Tavsanli et al. [54] Kono et al. [55]

Epoxide rings of BDDE react with NH2 groups of collagen

Koh et al. [56]

group on the other polymer chain [49]. Table 4.3 shows examples of polysaccharide hydrogels prepared using epichlorohydrin as a cross-linking agent. The bisepoxides can readily react with the NH2 groups on the polymers under mild alkaline conditions [57]. They can react with the OH groups at high pH. The bisepoxides like EGDE, BDDE, and PEGDE have been used to obtain hydrogels with good tensile strength. The chain length of these cross-linking agents increases in the order: EGDE , BDDE , PEGDE. This can be helpful in obtaining hydrogels with low weight (due to short chain in the case of EGDE) or high swellability (due to spacer effect of long chain in the case of PEGDE). The reaction by which the bisepoxides like EGDE, BDDE, and PEGDE cross-link the polymer chains in hydrogels is shown in Fig. 4.3 and the relevant examples are given in Table 4.3.

4.2.3 Addition reaction The Michael addition reaction, also known as the conjugated addition reaction, involves the addition of nucleophiles to the conjugated (unsaturated) compounds such as α,β-unsaturated aldehydes/ketones, vinyl esters, or vinyl sulfones. The nucleophiles mainly include amine or thiol groups on the polymer chain which interact with the electrophilic groups present on

100 Chapter 4

Figure 4.5 Michael addition reaction of polymer containing thiol or amine groups with acrylate and vinyl sulfone-modified polymer.

the adjacent polymer chain such as acrylate, methacrylate, or vinyl sulfone [58]. When the nucleophile is a thiol group, this reaction is also known a ThiolMichael addition reaction. This reaction is primarily used for the preparation of in situ gelling injectable hydrogels. Fig. 4.5 depicts the formation of hydrogels by the Michael addition reaction. This reaction is mostly preferred because it is highly selective and does not lead to the formation of toxic intermediates.

Preparation of hydrogels based on natural polymers 101 Photopolymerizable chitosan derivatives were prepared, where a reaction was carried out betweenNH2 of chitosan and C 5 C of ethylene glycol acrylate methacrylate by Michael addition to produce water-soluble (methacryloyloxy) ethyl carboxyethyl chitosan (MAOECECS). MAOECECS was blended with a photoinitiator such as Darocur 2959 and water. This blend was subjected to ultraviolet (UV) irradiation to form hydrogels, intended for tissue engineering applications [59,60]. However, the in situ gelation of photocrosslinkable (meth)acrylated polymers is limited in vivo due to minimum penetration of UV rays through skin [61]. To overcome this limitation, in situ gelling hydrogels have been prepared by the Michael addition reaction between thiols and acrylates. Chitosan, hyaluronic acid, chondroitin sulfate, and gelatin were modified with thiol groups in order to promote the addition reaction with PEG-diacrylate [6266]. Thiol-modified HA has also been cross-linked with PEG vinylsulfone (PEG-VS) by Michael addition to form hydrogel under physiological conditions [67]. In another study, dextran hydrogels were prepared by an interaction between dextran thiols and PEG tetra-acrylate or dextran vinyl sulfone conjugate [68]. The acrylated or divinyl sulfone derivatives of natural polymers like dextran and hyaluronic acid were synthesized to facilitate Michael addition cross-linking with specific cross-linkers containing thiol groups such as dithiothreitol and four-arm mercapto poly(ethylene glycol) [6972]. Few researchers have used divinyl sulfone as a cross-linking agent for the preparation of cellulose-based hydrogels [73]. Divinyl sulfone is used as a cross-linking agent for the preparation of polysaccharide-based hydrogels [74,75]. It known to react with hydroxyl groups on the adjacent polymer chains by oxa-Michael addition.

4.2.4 Click chemistry Click chemistry has emerged as a bioorthogonal technique for the development of in situ forming hydrogels with pharmaceutical and biomedical importance. It is highly selective and avoids the formation of toxic by-products during synthesis of hydrogels. Some of the click reactions involved in the formation of hydrogels include cycloaddition of azide and alkyne, DielsAlder reaction between furan and maleimide, and thiolene reactions [76]. In 2001, Sharpless and co-workers suggested the copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the preparation of functional hydrogels [77]. As CuAAC reactions can occur under mild conditions and are highly selective, there is vast scope for the incorporation of various functional groups in the hydrogel matrix, thus resulting in the development of responsive biomaterials. In this reaction, the azide and alkyne functionalized polymers interact by cycloaddition reaction in the presence of Cu to form triazole-based cross-links (Fig. 4.6). Hyaluronic acid-based hydrogels were prepared by Crescenzi and co-workers using Cu(I)catalyzed Huisgen 1,3-dipolar cycloaddition, for controlled release of doxorubicin [78]. In order to facilitate the reaction, hyaluronic acid was initially modified by incorporation of

102 Chapter 4

Figure 4.6 Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between polymers containing alkyne and azide functional groups.

azide and alkyne functional groups by amidation of hyaluronic acid, employing an aminoazide bifunctional linker and propargylamine. The other natural polymers which have been cross-linked using a CuAAC reaction to form hydrogels include cellulose, chitosan, chondroitin sulfate, gelatin, and guar gum [7982]. Despite the high efficiency and specificity, traces of copper within the hydrogels prepared by CuAAC reactions can prove to be toxic. Therefore metal-free click chemistry has been developed which mainly includes strain-promoted azide-alkyne cycloaddition reaction (SPAAC), oxanorbornadiene (OB) cycloaddition reaction, DielsAlder reaction, thiolene reaction, and oxime reaction. The ring-strained alkynes which are deficient in electrons can undergo cycloaddition reaction in the presence of azides, without any requirement for a metal catalyst [83]. Truong et al. have prepared in situ gelling chitosan-poly(ethylene glycol) hydrogels via SPAAC using azide functionalized chitosan and PEG-propiolate. Also, some researchers have used strain-promoted cyclooctyne-azide cycloaddition reaction for the preparation of natural polymer-based hydrogels. Injectable hyaluronan hydrogels were developed by Takahashi and co-workers by a reaction between cyclooctyne-modified hyaluronic acid and azide-modified hyaluronic acid [84]. Similarly, cyclooctyne and azide derivatives of dextran have been used for the fabrication of injectable dextran hydrogels [85]. OB cycloaddition involves 1,3-dipolar cycloaddition accompanied by elimination of furan molecules and formation of a triazole linkage [86]. The generation of triazole is based on the retro-DielsAlder reaction. The advantage of this reaction is that it can occur at room temperature. In situ forming chitosan-hyaluronan hydrogels have been prepared by modifying chitosan and hyaluronic acid with OB and 11-azido-3,6,9-triox-127 aundecan-1amine [87] (Fig. 4.7). The DielsAlder reaction is another thermoreversible cycloaddition reaction between a diene and a dienophile [88]. It is a highly selective and efficient type of “click” reaction which is rapid as compared to the metal-free azide-alkyne reactions. In the case of the

Preparation of hydrogels based on natural polymers 103

Figure 4.7 Representation of strain-promoted azide-alkyne cycloaddition reaction (SPAAC) and oxanorbornadiene (OB) cycloaddition reaction.

DielsAlder reaction, polymers are most commonly modified with furan and maleimide functional groups. By controlling the molar ratio of furan to maleimide, the mechanical properties of the hydrogels can be tuned. Nimmo et al. have synthesized hyaluronic acid hydrogels for tissue engineering applications where furan-modified hyaluronic acid was cross-linked using dimaleimide poly(ethylene glycol) [89]. In another work, biodegradable hyaluronic acid hydrogels were prepared by Fan and co-workers using DielsAlder reaction [90]. Furan- and maleimide-functionalized hyaluronic acid underwent cycloaddition, resulting in formation of hydrogel in an aqueous environment. There are few reports where maleimide-based cross-linkers have been used by the authors for cross-linking the furan-modified natural polymers. Garcı´a-Astrain et al. have designed biopolymeric hydrogels by cross-linking furan-functionalized gelatin with Jeffamine®based bismaleimide cross-linker. Similarly, furan-modified hyaluronic acid was crosslinked with bismaleimide enzyme-cleavable peptide to obtain hydrogels for studying breast cancer cell invasion [91]. In recent years, inverse electron demand DielsAlder cycloaddition reaction between tetrazine and alkene has been utilized for the preparation of natural polymer-based hydrogels. The tetrazine- and norbornene-modified alginate was mixed together by Desai and co-workers, to form “click” hydrogel. Trans-cyclooctene functional group has also been used as an alternative to norbornene in some cases [92].

104 Chapter 4

Figure 4.8 Representation of DielsAlder reaction and inverse electron demand DielsAlder reaction.

The strain produced within the structure of trans-cyclooctene helps to promote the cycloaddition reaction with tetrazine (Fig. 4.8). Thiol-ene “click” chemistry deals with the reaction between thiol groups and alkene. The attribute that distinguishes the thiol-ene hydrogels from other click hydrogels is the simplicity of introducing thiol and alkene groups in the polymers as compared to the other functional groups. The thio-lene reactions can be divided into two types: thiolMichael type reactions and radically mediated thiol-ene reactions. In thiolMichael type reactions, thiolate anion is formed due to extraction of proton from thiol by the base. This anion reacts with electrophilic β-carbon of an alkene to form a carbanion intermediate which extracts the proton from the conjugate acid resulting in the final product. The radically mediated thiol-ene reactions require an initiator for the formation of radicals. Once the initiator is selected, radicals can be generated using thermal, oxidation-reduction, or photochemical processes. After initiation, thiyl radical, which is formed from the attack of generated radical on thiol, attacks the C 5 C of alkene. This leads to the formation of thiol-ene addition product along with a new thiyl radical. The alkenes used in the thiolene reactions include vinyl sulfone, maleimide, norbornene, acrylates, and allyl ethers. In both reaction types, the reaction continues until one of the reactants is completely

Preparation of hydrogels based on natural polymers 105

Figure 4.9 Representation of thiol-ene and oxime reaction.

consumed [93]. Arslan et al. have synthesized injectable cyclodextrin (CD) hydrogels for controlled release of poorly water-soluble drugs, using a thiol-ene click reaction between acryloyl-modified β-CD and thiol-bearing PEGylated chitosan [94]. Norbornene functionalized hyaluronic acid was reacted with DTT in the presence of an initiator, namely 2-hydroxy-40 -(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and UV radiation to form hydrogels for biomedical applications [95]. McOscar and co-workers prepared hydrogels from norbornene-functionalized carboxymethylcellulose using 2,20 (ethylenedioxy)diethanethiol (DEG) as the cross-linking agent and UV light as the initiator [96] (Fig. 4.9). The macromolecules modified with aminooxy groups can react with the compounds containing aldehyde or ketone to form an oxime bond. Such oxime “click” reactions are more stable than the thiol-based reactions. These reactions are dependent on the pH and concentration of the catalyst. Very few reports are available on the development of natural polymer-based hydrogels using oxime “click” chemistry. Grover et al. synthesized four armed aminooxy PEG which reacted with the oxidized hyaluronic acid and alginate to form injectable hydrogels [97]. On a similar basis, PEG-containing aminooxy groups at both terminal ends and oxidized hyaluronic acid were used to prepare in situ cross-linking hydrogels containing collagen by oxime reaction [98].

106 Chapter 4

4.2.5 Condensation reaction Condensation reactions which are frequently used for the synthesis of polyamides and polyesters can also be used for the preparation of hydrogels. These reactions occur between hydroxyl or amino groups and carboxylic acid or its derivatives. The commonly used reagent for the preparation of natural polymer-based hydrogel is N, N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC). In order to avoid the possible side reactions and to improve cross-linking efficiency, N-hydroxysuccinimide (NHS) is added to the reaction mixture. EDC and NHS are known as zero-length cross-linkers which activate the COOH groups on the polymer chains so as to interact with hydroxyl or amine groups present on the adjacent polymer chains leading to the formation of ester or amide bonds. Tomihata and Ikada cross-linked hyaluronic acid using EDC to obtain hydrogel films. EDC helped in the formation of ester cross-links between the hyaluronic acid chains [99]. The authors also prepared hyaluronic acid films in the presence of L-lysine methyl ester and found that the stability of the films was improved which was attributed to the formation of amide bonds. Kuijpers et al. used EDC/NHS cross-linking for the preparation of gelatin gels [100]. Here, EDC promoted the formation of amide linkages between the gelatin chains. The carboxymethyl dextran and poly(amino acid) hydrogels have also been synthesized using an EDC/NHS-based condensation reaction [101,102]. Passerini and Ugi condensation reactions have been exploited for the preparation of polysaccharide hydrogels [103,104]. Passerini condensation involves three components, namely carboxylic acid, carbonyl, and isocyanide. These components are condensed together to form α-(acyloxy)-amide. On the other hand, Ugi condensation is a fourcomponent reaction where, initially, amine and carbonyl condense to give imine. The protonated imine, carboxylate, and isocyanide then react with each other to form α-(acylamine)amide [104]. At high pH ( . 9.5), the hydrogels prepared by Ugi condensation are found to be more stable due to the presence of amide cross-links. Alginate hydrogels were prepared by Bu and co-workers using an Ugi reaction between formaldehyde, 1,5-diaminopentane as the bifunctional cross-linker agent, alginate, and cyclohexyl isocyanide [105]. The basic requirement for Ugi condensation to proceed is the presence of an acidic environment.

4.2.6 Free radical polymerization Chemically cross-linked hydrogels can also be obtained by free radical polymerization of the monomer species in the presence of cross-linking agents. It is beneficial over other polymerization methods in terms of high reactivity and the requirement for a mild aqueous environment. At the beginning of radical polymerization, initiator decays to form a radical which attacks the monomer species. The decay of the initiator can be brought about by

Preparation of hydrogels based on natural polymers 107 thermal, chemical, or photochemical triggers. The monomers participating in the radical polymerization often contain molecules with groups bearing C 5 C bonds, for example, vinyl, acryl, methacryl, or allyl groups. Based upon the composition of the hydrogels, free radical polymerization can be classified into homopolymerization (polymerization between the same monomer species in the presence of an initiator and cross-linker) or copolymerization (polymerization between different monomer species) [106]. In order to fabricate natural polymer-based hydrogels using radical polymerization, they must be derivatized with the polymerizable groups. Dextran hydrogels were synthesized by copolymerization technique where dextran was initially acrylated using acrylic acid glycidyl ester [107]. Adding the initiator system composed of N,N,N0 N0 -tetramethylene-diamine and ammonium peroxydisulfate to the aqueous solution of acrylated dextran and N,N,methylenebisacrylamide resulted in the formation of hydrogels. Kim et al. introduced the vinyl groups into dextran using maleic anhydride to obtain the hydrogel precursor, dextranmaleic acid (Dex-MA) [108]. The hydrogel was formed when the aqueous solution of Dex-MA was irradiated with UV rays (360 nm) in the presence of a photoinitiator, 2,20 -dimethoxy-2-phenyl acetophenone. A copolymerization technique was also used for the preparation of hydrogels from the polysaccharide blend [109]. Potassium persulfate (initiator) and acrylamide were added to the aqueous solution of the polysaccharide blend (chitosan, alginate, and starch). Methylenebisacrylamide (cross-linker) was added to this mixture, followed by treatment with microwave or UV rays to promote grafting and formation of hydrogels. The other natural polymers which have been derivatized for the preparation of hydrogels by radical polymerization technique include cellulose and its derivatives [110,111], starch [112] and hyaluronic acid [113].

4.2.7 Enzyme-catalyzed cross-linking In recent years, scientists have developed their interest in enzyme-catalyzed cross-linking methods due to their mildness, avoidance of side reactions due to substrate specificity, and their occurrence at neutral pH and moderate temperature [114]. Due to their mild nature, they are more suitable for the preparation of hydrogels based on natural polymers which may lose their bioactivity under a harsh chemical environment. The enzymes which have been used to prepare natural polymer-based hydrogels include transglutaminase, tyrosinase, lysyl oxidase, and horseradish peroxidase [115]. Transglutaminase is an acyl-transfer enzyme that catalyzes transamidation reaction. It has been used for the preparation of gelatin and chitosan hydrogels [116,117]. In addition, transglutaminase has been used for the synthesis of genetically engineered polypeptide hydrogels intended for cartilage repair [118]. Tyrosinase is known to catalyze the oxidation of phenols into activated quinines. These quinines can undergo a Michael type addition reaction with polymers containing hydroxyl or amine groups to form hydrogels. Similarly to transglutaminase, tyrosinase has also been used for the preparation of gelatin-chitosan hydrogels. Tyrosinase was also used

108 Chapter 4 for the cross-linking of silk proteins with chitosan [119,120]. Lysyl oxidase promotes crosslinking between macromolecules containing amine groups via Schiff base formation. It has been employed for the preparation of peptide-based hydrogels [121]. Amongst the peroxidases, horseradish peroxidase and soy bean peroxidase are commonly used for the preparation of hydrogels. In most cases, these enzymes require hydrogen peroxide as a substrate to support cross-linking through conjugation of phenol and aniline groups. In some studies, natural polymers were conjugated with tyramine, which in the presence of peroxidase formed hydrogels [122,123]. Recently, Chen et al. developed injectable hydrogels of carboxymethylated pullulan and chondroitin sulfate by modifying both the polymers with tyramine and cross-linking them in the presence of horseradish peroxidase and hydrogen peroxide [124]. Natural polymers like chitosan, alginate, and gelatin have also been suitably derivatized to produce injectable biomaterials using peroxidases [125127]. Although the enzyme-catalyzed cross-linking reactions have some considerable advantages over the other cross-linking methods, they have a few drawbacks such as instability of specific enzymes and poor mechanical strength of the hydrogels.

4.2.8 Miscellaneous chemical cross-linking reactions The other reactions used for the synthesis of natural polymer-based hydrogels include genipin coupling and polycarboxylic acid-mediated esterification cross-linking. Genipin is obtained from hydrolysis of geniposide, an iridoid glucoside, by β-glucosidase [128]. It is a water-soluble bifunctional cross-linking agent which exhibits low toxicity as compared to glutaraldehyde [129]. It spontaneously cross-links the free amino groups present in the polysaccharides. There are various mechanisms presumed by the authors by which genipin is supposed to cross-link the amino group containing polymers [130]. In an acidic or neutral environment, the C3 carbon of genipin is attacked by a primary amino group (nucleophile). This leads to an opening of the dihydropyran ring followed by an attack of the secondary amino group on the newly formed aldehyde group [131]. However, under basic conditions, the hydroxyl ions present in the aqueous solution act as nucleophiles and attack the genipin, generating the intermediate aldehyde groups by ring-opening. These intermediate aldehyde groups undergo aldol condensation whereas the terminal aldehyde groups of the polymerized genipin react with the amino groups on the polymer via Schiff reaction resulting in cross-linking [132]. Genipin has been used as a cross-linking agent to prepare hydrogels based on chitosan and its derivatives [133136], gelatin [137140], collagen [141143], and amino acids [144146]. Di- and polycarboxylic acids are also capable of cross-linking the polysaccharide chains. At high temperatures, they form cyclic anhydrides which can esterify the hydroxyl groups present on the neighboring polymer chains and thus covalently link the chains [147].

Preparation of hydrogels based on natural polymers 109 Sodium hypophosphite is used to enhance the rate of reaction by minimizing the hydrogen bonding between the carboxylic acid groups [148]. The dicarboxylic acids such as fumaric acid, malic acid, and succinic acid have been used to cross-link the cellulose derivatives to form hydrogels [149,150]. Citric acid has emerged as a nontoxic and cheap cross-linking agent for the preparation of hydrogels in the last few years. Reports are available on cellulose-based hydrogels where citric acid was used as a cross-linking agent [151156]. Citric acid cross-linked starch films were prepared by Reddy and Yang to improve the tensile strength of the films. Mali et al. synthesized hydrogel films based on carboxymethyl tamarind gum and tamarind gum using citric acid as a cross-linker, for application in drug delivery [157,158]. Despite being a natural polysaccharide, alginic acid has also shown the tendency to crosslink the amino group containing polymer like collagen [159]. The overall cross-linking resulted from intermolecular hydrogen bonding and formation of amide bonds between alginic acid and collagen. Currently, scientists are focusing on chemical cross-linking techniques which can avoid the formation of toxic byproducts and can cross-link the natural polymer chains under mild conditions. Addition reactions, metal-free click reactions, and free radical polymerization can fulfill these requirements to a greater extent. Genipin and polycarboxylic acid could be a better alternative to the toxic cross-linking agents like aldehydes and epoxides which are meant for cross-linking the complementary groups like OH and NH2. However, one should examine the thermal stability of the natural polymers while using polycarboxylic acids to cross-link the polysaccharides. The combination of physical and chemical cross-linking can be useful for synthesizing the biocompatible hydrogels with better mechanical properties. In the near future, innovative chemical cross-linking methods will be developed to obtain smart hydrogels based on natural polymers.

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CHAPTER 5

Preparation of polysaccharide-based hydrogels via radiation technique Rong Li and Guozhong Wu* Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, P.R. China

5.1 Introduction Hydrogels are three-dimensional (3D) networks of hydrophilic polymers, and capable of swelling in water without dissolution [1]. Since the pioneering research on hydrogel in the 1960s [2], hydrogels have been extensively investigated. According to the nature of crosslinking bonds, the preparation methods of hydrogels can be mainly classified into physical and chemical cross-linking methods [3]. In terms of hydrogels prepared using physical methods, the network formation occurs due to various weak interactions, such as the entanglement of the polymer chains, hydrogen bonds, or van der Waals interactions [4]. However, these hydrogels can be dissolved in solvents over time since such structures are not permanent [5]. In order to obtain stable and permanent hydrogels, the polymer chains should be cross-linked by covalent bonds. The chemically cross-linked network cannot be dissolved in solvent without the degradation of polymer [6]. Therefore hydrogels prepared using chemical methods are usually preferable in the majority of the application fields. Hydrogels are currently broadly applied in various fields. In the field of medicine and pharmacy, hydrogels are used mainly in drug delivery [7], tissue engineering [8], and wound dressing [9,10]. Moreover, hydrogels are used in hygienic products such as diapers [11]. In addition, a large number of researches focus on applications in the fields of agriculture [12], wastewater treatment [13,14], etc. Most commercially available hydrogels are synthetic polymers originated from petroleum-based acrylate monomers. Synthetic polymer-based hydrogels exhibit excellent swelling properties and responsive behavior [15]; however, their potential application is restricted in certain fields. For example, their applications in agriculture are seriously hindered by their poor biodegradability [16]. There is increasing interest in renewable resources materials substituting for synthetic polymers. Such materials are not only cheap and available in abundance but also more


corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00005-7 © 2020 Elsevier Inc. All rights reserved.

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120 Chapter 5 environmentally friendly. The most important renewable materials are polysaccharides. Polysaccharides, such as starch, sodium carboxymethyl cellulose (CMC), chitin/chitosan, carrageenan, sodium alginate, hyaluronic acid, have attracted substantial interest in recent years due to their low cost, abundant resources, and eco-friendly properties. Polysaccharides possess intriguing properties for the development of biomaterials, such as biocompatibility, biodegradability, and nontoxicity. Polysaccharide-based hydrogels have been widely investigated and applied in fields including structural transplants, targeted drug delivery, tissue engineering, wound dressing, antibacterial agents, biosensors, adsorbents, etc. [17]. High-energy irradiation has been widely and maturely used for commercial processes to prepare various kinds of functional polymeric materials or sterilization, etc. [18,19]. Commercial radiation sources usually include a γ-ray irradiator and electron beam (EB) accelerator. γ-Ray and EB can efficiently induce the formation of radicals in the chains of polysaccharides; the radicals can then be used to initiate grafting and cross-linking of functional vinyl monomers onto polysaccharide polymer chains [20]. Here we chiefly introduce the progress in the preparation and application of polysaccharide-based hydrogels using the radiation technique in recent years.

5.2 Structures of polysaccharides 5.2.1 Starch Starch, as a typical kind of polysaccharide, consists of glucose units, which are joined by glycosidic bonds. Starch derives from different sources, such as maize, amylomaize, waxy maize, bread-wheat, manioc, rice, potato, cassava, and haricot bean [21]. Starch is one of the most abundant natural polysaccharides. The structure of starch is mainly composed of two major macromolecular chains, that is, amylose and amylopectin [22]. As shown in Fig. 5.1, amylose is an α(1-4)-linked linear glucose with a degree of polymerization as high as 600; amylopectin, α(1-4)-linked glucose with α(1-6) branch points, is the major component of the granule (30%99%), and contains about 5% of branch points [23]. Amylose and amylopectin exhibit different physical and biological properties due to their different structures. Both amylose and amylopectin can form complexes with iodine [23]. For amylose, the complex is pure blue, whereas for amylopectin, the complex is blue-violet. Amylopectin takes up less iodine than amylose. This method can be used to determinate the amyloseamylopectin ratio of starches.

5.2.2 Sodium carboxymethyl cellulose Cellulose is considered to be the most abundant natural polysaccharide. Cellulose derives from a wide variety of sources, such as cotton, wood, hemp, jute, rice hulls, and wheat straw [24]. Fig. 5.2A illustrates the linear molecular chain structure of cellulose, which is

Preparation of polysaccharide-based hydrogels via radiation technique 121 (A)

(B)

Figure 5.1 Schematic diagram of (A) amylose and (B) amylopectin. (A)

(B)

Figure 5.2 Schematic diagram of (A) cellulose and (B) CMC (degree of substitution: 1.0).

composed of hydroglucose units linked by a β(1-4) glycosidic bond [25]. Cellulose is insoluble in water and most organic solvents. Therefore cellulose is usually modified to improve its solubility and expand its application. CMC, as an important water-soluble cellulose derivative, can be prepared via the reaction of hydroxyl group in the “CH2OH” group with chloroacetic acid (CH2ClCOOH). Fig. 5.2B presents a conceptualized representation of the molecular structure of CMC [26].

122 Chapter 5

5.2.3 Chitosan Chitin is the second most abundant natural polysaccharide after cellulose, and is mainly extracted from the exoskeleton of shrimps and crabs [27]. In industry the extraction of chitin includes three steps: (1) acid treatment to dissolve calcium carbonate; (2) alkaline treatment to solubilize proteins; (3) decolorization to remove leftover pigments to obtain a colorless product [27]. Fig. 5.3A shows the structure of chitin, poly(β (1-4)-N-acetyl-Dglucosamine) [27]. However, chitin is insoluble in all the usual solvents. This insolubility seriously limits the development and application of chitin. Chitosan is obtained by deacetylation of chitin using alkaline hydrolysis. When the degree of deacetylation of chitin reaches about 50%, it is called chitosan. Fig. 5.3B illustrates the structure of chitosan, which is soluble in aqueous acidic media, and is the only pseudonatural cationic polymer [28]. Being soluble in aqueous solutions, chitosan is extensively investigated and used in various fields in different forms including solutions, gels, films, fibers, etc.

5.2.4 Sodium alginate Sodium alginate, a linear natural polysaccharide, is extracted from a wide variety of brown algae such as Laminaria hyperborea and Lessonia [29]. As shown in Fig. 5.4, sodium alginate consists of α-L-glucuronic acid and β-D-mannuronic acid linked by a glycosidic bond, and is soluble in water [30]. Sodium alginate has been widely used as a stabilizer, emulsifier, and gelling agent in various fields, such as the food, textile printing, and pharmaceutical industries [29].

(A)

(B)

Figure 5.3 Schematic diagrams of (A) chitin and (B) chitosan.

Preparation of polysaccharide-based hydrogels via radiation technique 123

Figure 5.4 Schematic diagram of sodium alginate.

Figure 5.5 Schematic diagram of hyaluronic acid.

5.2.5 Hyaluronic acid Hyaluronic acid is a linear polysaccharide with repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine, as shown in Fig. 5.5 [31]. Hyaluronic acid is separated from the dermis and epidermis of normal human skin and from normal and hypertrophic scar tissue. The molecular weight of hyaluronic acid ranges from 50 kDa up to 2 million kDa [32]. Hyaluronic acid is nontoxic, nonimmunogenic, and noninflammatory, and is the only nonsulfated glycosaminoglycan in the extracellular matrix of all higher animals. It is one of the most widely used biomedical polymers owing to its high water retention capacity, viscoelasticity, biodegradability, and excellent biocompatibility [33]. One of the largest markets for hyaluronic acid is its use as a subcutaneous or intradermal filler [34].

5.2.6 Carrageenan Carrageenan is the general name for a group of galactans, the commonest and most abundant cell wall constituents derived from red algae. The original source of carrageenan is the red seaweed Chondrus crispus [35]. With the extensive application of carrageenan, the increasing demand for the raw material has resulted in the introduction of the cultivation of species of Eucheuma, referred to as Kappaphycus alvarezii and Eucheuma denticulatum, respectively [36]. As shown in Fig. 5.6, the structures of kappa (κ)-, iota (ɩ)-, and lambda (λ)-carrageenans are anionic, sulfated linear polysaccharides consisting of galactose units in D-galactose and 3,6-anhydro-galactose copolymer, linked by alternating

124 Chapter 5 (A)

(B)

(C)

Figure 5.6 Schematic diagram of (A) kappa (κ)-, (B) iota (ɩ)-, and (C) lambda (λ)-carrageenans.

α(1-3) and β(1-4) bonds. Carrageenan can be classified into six basic types based on their source, sulfate content and solubility, including kappa (κ)-, iota (ɩ)-, lambda (λ)-, mu (μ)-, nu (ν)-, and theta (θ)-carrageenans. Of these, κ-, ɩ-, and λ-carrageenans are of commercial importance owing to their viscoelastic and gelling properties [35]. Carrageenans are widely used as emulsifiers, and gelling, thickening, and stabilizing agents in pharmaceutical and industrial applications.

5.3 Radiation preparation of polysaccharide-based hydrogels Unlike the chemical initiator used for inducing the formation of free radicals, irradiation can directly induce the formation of free radicals in the chains of polysaccharides at room temperature and atmospheric pressure without the addition of an initiator [37]. These radicals can induce the cross-linking of polysaccharide chains to obtain hydrogel; meanwhile, these radicals can also be used to induce grafting of vinyl monomer onto polysaccharide chains with or without cross-linkers to prepare hydrogels. In addition, radiation preparation of polysaccharide-based hydrogels is usually performed in solution,

Preparation of polysaccharide-based hydrogels via radiation technique 125 since their chains are highly mobile in aqueous solutions. In terms of the traditional chemical cross-linking method, the cross-linking agents, such as divinyl sulfone epichlorohydrin and glutaraldehyde, were usually used to react with hydroxyl groups in the polysaccharide chains; however, these cross-linking agents are toxic [38].

5.3.1 Starch-based hydrogel It has been widely demonstrated that γ-ray/EB irradiation mainly leads to the degradation of starch in both aqueous solution and solid state. The molecular weight and degree of polymerization decrease with an increase in the absorbed dose [39]. Radiation cross-linking of starch cannot be directly achieved. Therefore starch-based hydrogels are usually prepared by radiation-induced grafting and cross-linking of acrylate monomers with or without crosslinking agents. Functional vinyl monomers such as acrylic acid and acrylamide are often grafted onto starch to prepare hydrogel. 60Co γ-ray irradiation-induced grafting of acrylic acid onto three types of starch was performed at room temperature by Geresh et al. [40]. The starches were derived from potato, corn, and rice, respectively, and were pregelatinized before mixing with acrylic acid solution. It was found that the starch-based hydrogels are suitable for use as slow-release matrices for drugs. Zhang et al. synthesized the poly(acrylic acid) grafted amylose and amylopectin hydrogels by γ-ray irradiation in the presence of N,N0 -methylene bisacrylamide used as a cross-linker [41]; this is illustrated in Fig. 5.7. The prepared hydrogel shows good water absorption properties [amylose-g-poly(acrylic acid): 530 g/g; amylopectin-g-poly(acrylic acid): 355 g/g]. In addition, in comparison with the Heating Starch granule H2O

Gelatinized starch

OHγ-Radiation

HO • + H • •

O

γ-Radiation

Gelatinized starch

Gelatinized starch

/ HO •

(n+1) H2C CH ( AA(Na) ) γ-Radiation COOH(Na)

/ HO •

COOH(Na) CH2 CH

•

CH2 CH

n

• ( PAA(Na)

)

COOH(Na) •

O

Starch

Gelatinized starch MBA •

PAA(Na)

Cross-linker MBA PAA(Na) chains

Figure 5.7 Mechanism for the formation of starch-g-poly(acrylic acid) hydrogels induced by γ-ray irradiation.

126 Chapter 5

Figure 5.8 Effect of clay on water absorbency of hydrogel (absorbed dose rate: 27.5 Gy/min; absorbed dose: 1.1 kGy; cross-linker content:0.027%; acrylamide/acrylic acid: 4/1).

hydrogels synthesized using the chemical initiator potassium persulfate (K2S2O8), the results indicated that γ-ray irradiation is more time-saving, energy-saving, and efficient than the chemical initiator. Luo et al. synthesized the starch-based hydrogel by grafting of acrylamide and acrylic acid onto starch using a 60Co γ-ray irradiation method [42]. Different synthesis parameters were investigated, including the ratio of acrylamide to acrylic acid, absorbed dose, dose rate, cross-linker (N,N0 -methylene bisacrylamide) dosage, and montmorillonite clay content. The optimum parameters were 4:1 (weight ratio of acrylamide to acrylic acid), 1.1 kGy (absorbed dose), 27.5 Gy/min (dose rate), 0.027% (cross-linker content), and clay content (2 wt.%), respectively. The result showed that the swelling ratio of the hydrogel in water can reach up to B1200 g/g (Fig. 5.8). In addition to the radiation-induced grafting method, the starch derivative, such as carboxymethyl starch, can be directly cross-linked by radiation without the addition of vinyl monomer or cross-linkers due to its better water solubility than starch. This offers an optional route to prepare starch-based hydrogels. Nagasawa et al. has proved that carboxymethyl starch (degrees of substitution: 0.15) in paste-like condition can be crosslinked by radiation using a dynamitron 3 MeV EB accelerator [43]. The prepared hydrogel (carboxymethyl starch: 40 wt.%; absorbed dose: 2 kGy) is environmentally friendly and safe. The results indicated that the swelling ratio of the hydrogel was 500 g/g in distilled water and 26 g/g in 0.9% NaCl.

5.3.2 Carboxymethyl cellulosebased hydrogel Due to the poor solubility of cellulose in water, CMC, one of the cellulose derivatives, has been extensively investigated and applied due to its good solubility in water. Like carboxymethyl starch, CMC can also be directly cross-linked by γ-ray or EB irradiation.

Preparation of polysaccharide-based hydrogels via radiation technique 127 Wach et al. investigated the effects of EB irradiation on CMC with a degree of substitution (DS) of 2.2 [44]. The results illustrated that irradiation of CMC with a DS of 2.2 in solid state and in low concentration in aqueous solution resulted in the degradation; irradiation of CMC with suitable concentrations in aqueous solution (paste-like mixture) can be effectively cross-linked. The absorbency of CMC hydrogels (CMC: 50 wt.%; absorbed dose: 5 kGy) was more than 800 g/g in water. Moreover, a preliminary biodegradation study confirmed that the cross-linked CMC hydrogel can be digested by a cellulase enzyme. Fei et al. [45], Liu et al. [46], and Fekete et al. [47] also carried out γ-ray irradiation crosslinking of CMC. The results indicated that a low absorbed dose (usually less than 20 kGy) is optimum for preparation of CMC hydrogel, and the swelling ratio of hydrogel in water decreases with an increase in the absorbed dose due to the increasing gel fraction. Meanwhile, the DS and the concentration of CMC in aqueous solution have a significant effect on the cross-linking of CMC. This is similar to the work by Wach et al., in which γ-ray irradiation of CMC (a DS of 2.2 in solid state and a DS of 0.7 in 10% aqueous solution) also resulted in degradation; however, γ-ray irradiation of CMC (a DS of 1.32 in a 5 wt.% aqueous solution) resulted in cross-linking, and the highest gel fraction was obtained at 20 wt.% CMC in aqueous solution. In addition, the gel fraction of CMC with a DS of 2.2 was higher than that with a DS of 1.32 at lower absorbed doses with the same concentration. This indicated that the high DS and concentration in an aqueous solution were favorable for high cross-linking of CMC. It was proved that the cross-linked CMC hydrogel was able to be digested by a cellulase enzyme. Besides the radiation cross-linking of CMC, functional acrylate vinyl monomer can be grafted onto CMC to prepare hydrogels using a radiation method. El-Salmawi et al. synthesized CMC-g-poly(acrylic acid) hydrogels using γ-ray irradiation in the presence of montmorillonite clay and N,N’-methylene bisacrylamide [48]. The hydrogels showed higher swelling ratio in water than in sodium chloride solution. In addition, they exhibited a higher swelling ratio in basic medium rather than in acidic medium. The increasing concentration of montmorillonite in the hydrogels decreased the water retention. CMC-g-polyacrylamide hydrogel was synthesized by Hemvichian et al. using 60Co γ-ray irradiation in the presence of N,N0 -methylene bisacrylamide as a cross-linking agent [49]. The obtained hydrogel exhibited a swelling ratio of 190 g/g in water. In addition, CMC can be cross-linked with different polymers using the radiation method as well. Abd El-Rehim et al. prepared cross-linked CMC/polyacrylamide hydrogel using EB irradiation [50]. CMC and polyacrylamide were mixed with water, put in polyethylene bags, and irradiated using EB (beam energy: 1.45 MeV; EB current: 4 mA; scanner width: 90 cm). The gel fraction increased with the absorbed dose and polyacrylamide concentration. The prepared hydrogel showed good swelling ratio in water (180 g/g) and simulated urine (40 g/g). Raafat et al. [51] prepared cross-linked CMC/polyvinylpyrrolidone hydrogel using 60Co γ-ray irradiation. The gel fraction increased with an increase in both the polyvinylpyrrolidone amount and the absorbed dose. It was found that the swelling ratio

128 Chapter 5 was sensitive to the ionic strength and cationic type in the following trend: Na1 . K1 . Mg21 . Ca21. Moreover, the swelling ratio in sulfate (SO22 4 ) solutions is less 2 than that in chloride (Cl ) solutions.

5.3.3 Chitosan-based hydrogel High-energy irradiation is also suitable for preparation of chitosan-based hydrogel. Yang et al. synthesized chitosan/poly(vinyl alcohol) gels by γ-ray irradiation [52]. While the gels showed high swelling ratio in water, the mechanical properties were poor. However, the mechanical strength of the irradiated hydrogel samples can be greatly improved by freezethawing treatment (Fig. 5.9). Zhao et al. prepared carboxymethyl chitosan-based hydrogels by EB irradiation [53]. Similarly to CMC, carboxymethyl chitosan can be crosslinked by irradiating at high concentration solutions without other additives. Carboxymethyl chitosan-based hydrogels presented good swelling properties and antibacterial activity. Wasikiewicz et al. investigated the preparation of both carboxymethyl chitin and carboxymethyl chitosan by EB and γ-ray irradiation-induced cross-linking [54]. The gel fractions of carboxymethyl chitin and carboxymethyl chitosan hydrogels by EB irradiation were 72% and 50%, respectively, at a concentration of 40 wt.% and at room temperature. In this work, the maximum swelling ratios of carboxymethyl chitin and carboxymethyl chitosan hydrogels in water were 241.2 and 63 g/g, respectively. Biodegradation tests, including enzymatic-controlled microorganism degradation and soil burial, indicated that both hydrogels degraded spontaneously (Fig. 5.10). Functional vinyl monomers were also grafted onto chitosan to prepare hydrogel. Sokker et al. [55] prepared chitosan-g-polyacrylamide hydrogel by 60Co γ-ray irradiation. The swelling ratio in water increased within low acrylamide concentration (2030 wt.%), and then decreased with a further increase in the acrylamide concentration (3040 wt.%). Meanwhile, the swelling ratio in water reduced with the increase in the absorbed dose due to the increasing gel fraction. Shim et al. prepared chitosan-g-poly(acrylic acid) hydrogel by

Figure 5.9 The morphologies of hydrogels made by (A) pure irradiation, (B) irradiation followed by freezethawing, (C) pure freezethawing, and (D) freezethawing followed by irradiation. The composition of the hydrogels is 7 wt.% poly(vinyl alcohol) and 3 wt.% chitosan.

Preparation of polysaccharide-based hydrogels via radiation technique 129

Figure 5.10 (A) Carboxymethyl chitosan and (B) carboxymethyl chitin kept in soil for 10 weeks.

Co γ-ray irradiation [56]. The chitosan-g-poly(acrylic acid) hydrogels showed the highest swelling ratio in water at conditions of 30 vol.% of acrylic acid, 0.1 wt.% of chitosan, and 30 kGy of absorbed dose. Also, the swelling ratio in water increased with an increase in the pH and exhibited the highest value at pH 12. Sahar et al. also prepared chitosan-g-poly (acrylic acid) hydrogels using 60Co γ-ray irradiation [57]. The absorbed dose, the ratio of chitosan/acrylic acid, and the concentration of acrylic acid were investigated to obtain the optimum process conditions. The swelling ratio of chitosan-g-poly(acrylic acid) hydrogel reached 300 g/g in water and the swelling kinetics followed a Fickian type of water diffusion. The Fickian constant value was more than 0.5.

60

5.3.4 Sodium alginatebased hydrogel Considerable works have been devoted to synthesize various sodium alginatebased hydrogels via radiation grafting of vinyl monomers such as methyl acrylate, acrylamide, and acrylonitrile. A nano-porous sodium alginate-g-poly(2-dimethylaminoethyl methacrylate) hydrogel was prepared by γ-ray irradiation at room temperature without chemical cross-linking agent and initiators [58]. The hydrogel displayed the highest swelling ratio (230 g/g) in water at the conditions of 1.5 g/L of sodium alginate, 2.1 mol/L of 2-dimethylaminoethyl methacrylate, and 5 kGy of absorbed dose. The swelling ratio of the hydrogel increased with the pH from 2 to 7, and decreased from 7 to 12. Sodium alginate-g-polyacrylamide was prepared using 60Co γ-ray irradiation-induced grafting of acrylamide onto sodium alginate [59]. Sodium alginate-g-polyacrylamide hydrogels with the ratios (w/w) of sodium alginate/acrylamide (1.5:0.5 and 1.0:1.0) showed the gel fractions of 87% and 66%, and swelling ratios of 3.7 and 5 g/g in water for 40 minutes, respectively; in addition, the hydrogels showed higher swelling ratios in basic solution than in acidic solution (Fig. 5.11). Mohamed et al. synthesized a pH-sensitive sodium alginate-g-poly (acrylic acid) hydrogel using γ-ray irradiation (Fig. 5.12) [60]. The hydrogel reached equilibrium swelling in water for 6 hours. The swelling ratio increased sharply with

130 Chapter 5

Figure 5.11 The gel content (A), swelling degree (B) dependent on the sodium alginate (w/v) content, (C) effect of pH on the equilibrium swelling (g/g) of sodium alginate-g-polyacrylamide, and (D) swellingdeswelling cycle of sodium alginate-g-polyacrylamide.

Figure 5.12 The scheme for preparation of sodium alginate-g-poly(acrylic acid) hydrogel using γ-ray irradiation.

Preparation of polysaccharide-based hydrogels via radiation technique 131 3500 3000 Swelling (%)

2500 2000 1500 1000 500 0 2

4

6

8

pH

Figure 5.13 The swelling ratio for sodium alginate-g-poly(acrylic acid) hydrogel at different solution pH (sodium alginate content: wSA 5 0.1; absorbed dose: 10 kGy).

increasing pH up to 6.0, and then decreased with further increasing pH to 8.0 (Fig. 5.13). Erizal et al. prepared sodium alginate-g-poly(acrylamide-co-acrylic acid) hydrogels via γ-ray irradiation-induced grafting and cross-linking [61]. The effect of absorbed dose (20a40 kGy) and sodium alginate concentration (0.1%a0.7%) on the swelling ratio were investigated. Low sodium alginate concentration (0.1%) made the swelling ratio of the hydrogel reach 800 g/g, and the gel fraction increased with absorbed dose up to B99%. The increasing concentration of sodium chloride significantly decreased the swelling ratio of the hydrogel. This indicated that the hydrogel was very sensitive to the ionic strength of medium. Raafat et al. prepared a hydrogel biocomposite consisting of graphene oxide and sodium alginate-g-poly(acrylic acid) by 60Co γ-ray irradiation-induced grafting and crosslinking [62]. The presence of graphene oxide effectively made the hydrogel bypass the acid simulated stomach medium (pH 1) without significant swelling and maintain it for a longer period of time in simulated intestine medium (pH 7). In addition to radiation-induced grafting and cross-linking of vinyl monomers, the sodium alginate can also be cross-linked with polymers, such as CMC, poly(vinyl alcohol), polyvinylpyrrolidone. Sodium alginate/CMC hydrogels were prepared by γ-ray irradiationinduced cross-linking in the presence of N,N0 -methylene bisacrylamide [63]. The results showed that the gel fraction was proved to increase with increasing absorbed dose up to 20 kGy; the swelling ratio of sodium alginate/CMC hydrogel increased with increasing concentration of sodium alginate, and reduced with increasing absorbed dose; the tensile strength of the hydrogel increased with the CMC content (Fig. 5.14). El-Naggar et al. also prepared sodium alginate/CMC hydrogels using 60Co γ-ray irradiation-induced cross-linking in the presence of N,N0 -methylene bisacrylamide [64]. The gel fraction of the hydrogel decreased with increasing sodium alginate content, while the swelling ratio increased with

Figure 5.14 The effect of different CMC contents in the blend on gel fraction (left), swelling ratio (middle), and break stress (right) at different absorbed doses.

Preparation of polysaccharide-based hydrogels via radiation technique 133 increasing sodium alginate content at a constant absorbed dose of 2.5 kGy. El-Din et al. prepared sodium alginate/poly(vinyl alcohol) hydrogels using EB irradiation [65]. The results showed that the sodium alginate/poly(vinyl alcohol) hydrogels possessed higher thermal stability than pure poly(vinyl alcohol) hydrogel. Meanwhile, the swelling ratios of sodium alginate/poly(vinyl alcohol) hydrogels were higher than pure poly(vinyl alcohol) hydrogel, and increased with the sodium alginate concentration. Moreover, the swelling ratio of sodium alginate/poly(vinyl alcohol) hydrogels was sensitive to the temperature and the pH depending on the composition. Singh et al. prepared sodium alginate/ polyvinylpyrrolidone hydrogels with incorporated silver nanoparticles using γ-ray irradiation [66]. The maximum gel fraction was obtained at conditions of 15 wt.% of polyvinylpyrrolidone and 0.5 wt.% of sodium alginate. The swelling ratios of the hydrogels were about 1881%2361% at 24 hours. The moisture vapor transmission rate of hydrogels with incorporated silver nanoparticles was 278.44 g/(m2 h) at 24 hours.

5.3.5 Hyaluronic acid Hyaluronic acidbased hydrogels can also be prepared by grafting and cross-linking of vinyl monomers, cross-linking of carboxymethyl hyaluronic acid in paste-like condition, and cross-linking with other polymers using γ-ray/EB irradiation. Cross-linking of carboxymethyl hyaluronic acid by γ-ray or EB irradiation is a green method without addition of initiators and cross-linkers. Relleve et al. synthesized carboxymethyl hyaluronic acidbased (DS: 0.5) hydrogel by γ-ray irradiation-induced cross-linking in paste-like condition (namely high concentration) without the addition of any initiator or cross-linker [67]. The results showed that the gel fraction increased with the absorbed dose from 50 to 100 kGy. Fig. 5.15 illustrates photographs of the dry, swollen, and boiled carboxymethyl hyaluronic acid hydrogel. The swelling ratios ranged from 46 to 168 g/g in water, and ranged from 21 to 31 g/g in 0.9 wt.% NaCl solution, respectively. Zhao et al. prepared a novel hydrogel, consisting of hyaluronic acid, chondroitin sulfate, and poly(vinyl alcohol) with various compositions, using the γ-ray irradiation method without additional initiators or cross-linkers [68]. The preparation routine is portrayed in Fig. 5.16. Prior to preparation,

Figure 5.15 (A) Dry gel, (B) swollen gel, and (C) boiled gel of carboxymethyl hyaluronic acid cross-linked at 100 kGy.

134 Chapter 5

Figure 5.16 The schemes of the synthetic procedures for (A) hyaluronic acid, (B) chondroitin sulfate, (C) methacrylated hyaluronic acid, (D) methacrylated hyaluronic acid, (E) poly(acrylic acid), and (F) hyaluronic acid/hyaluronic acid/poly(acrylic acid) hydrogel.

Preparation of polysaccharide-based hydrogels via radiation technique 135 a vinyl group was introduced onto both the hyaluronic acid and chondroitin sulfate. The gel fractions of the hydrogels were 85%88% with the absorbed dose of 15 kGy. These hydrogels reached an equilibrium swelling state within 24 hours. The water contents were higher than 90%, and gradually increased with the concentration of hyaluronic acid and chondroitin sulfate. In addition, the hydrogels containing high hyaluronic acid and chondroitin sulfate content or immersed in higher concentrations of hyaluronidase solution exhibited proportionally higher enzymatic degradation rates. Zhao et al. also prepared hyaluronic acid/chondroitin sulfate/poly(acrylic acid) hydrogel by γ-ray irradiation without other initiators or cross-linkers [69]. The gel fractions of the hydrogels increased with the absorbed dose up to 15 kGy, and decreased with a further increase in the absorbed dose. The gel fractions of the hydrogels were 91%93% at the absorbed dose of 15 kGy. The water contents of the hydrogels were higher than 90%, and reached an equilibrium swelling state within 24 hours. The enzymatic degradation kinetics of the hydrogels also were dependent on both the hyaluronidase concentration in solution and the compositions of the hydrogels. Biocompatible and biodegradable hyaluronic acid/polyvinylpyrrolidone and hyaluronic acid/gelatin hydrogels were prepared using γ-ray irradiation without crosslinkers added [70]. The gel fractions were between 55% and 85%, and increased with increasing concentrations of gelatin or polyvinylpyrrolidone. The gel fraction of the hyaluronic acid/gelatin hydrogel was 30% larger than that of hyaluronic acid/ polyvinylpyrrolidone hydrogel at the absorbed dose ranging from 30 to 50 kGy. The hyaluronic acid/gelatin (1:10) and hyaluronic acid/polyvinylpyrrolidone (1:5) hydrogels exhibited the highest swelling ratio at the absorbed dose of 30 kGy. The hyaluronic acid/ gelatin hydrogel was different from the hyaluronic acid/polyvinylpyrrolidone hydrogel. The hyaluronic acid/gelatin hydrogel was more sensitive to the pH value, and showed a high swelling ratio in aqueous solution at pH 2 and above pH 5, but the swelling ratio was low at pH 4.

5.3.6 Carrageenan Carrageenan-based hydrogels have also been widely investigated due to their viscoelastic and gelling properties. Carrageenan-g-polyacrylamide hydrogel was prepared via γ-ray irradiation-induced grafting of acrylamide onto carrageenan followed by alkaline (sodium hydroxide) hydrolysis treatment [71]. The swelling ratio of the hydrogels decreased with increasing absorbed dose and acrylamide concentration due to the increasing gel fraction. The obtained maximum swelling ratio in water was 704 g/g (Fig. 5.17). The prepared hydrogels were sensitive to the pH and ionic strength of the medium owing to the polyelectrolyte property. The synthesized hydrogels showed higher swelling ratios in basic aqueous solution than in acid aqueous solution, and higher swelling ratios in Li1, Na1, and K1 aqueous solutions than in Ca21 and Fe31 aqueous solutions. Tranquilan-Aranilla et al. prepared carboxymethyl κ-carrageenan hydrogels via γ-ray irradiation-induced cross-linking

136 Chapter 5 750

Swelling (g/g)

700 650 600 550 500 450 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

NaOH concentration (N)

Figure 5.17 Influence of NaOH concentration on the swelling of hydrolyzed carrageenan-g-polyacrylamide hydrogel (carrageenan: 20 g/L; acrylamide: 180 g/L; absorbed dose: 10 kGy; NaOH concentration: 3 mol/L; hydrolysis time: 60 min; temperature: 80 C).

without addition of cross-linkers [72]. Carboxymethyl κ-carrageenans with various degrees of substitution were synthesized by a multistep reaction technique prior to γ-ray irradiation. Like carboxymethyl starch, CMC, carboxymethyl chitosan, and others, carboxymethyl κ-carrageenans in paste-like condition (30 wt.%) can be successfully cross-linked by γ-ray irradiation at 30 kGy in this way. Abad et al. synthesized κ-carrageenan/poly(vinyl pyrrolidone) hydrogel using γ-ray irradiation without addition of cross-linkers [73]. Thermogravimetric analysis, X-ray fluorescence spectrometry, and Fourier transform infrared spectroscopy of the hydrogels indicated the grafting of poly(vinyl pyrrolidone). The degree of grafting was dependent on the concentrations of κ-carrageenan and poly(vinyl pyrrolidone). An optimum degree of grafting was obtained at higher carrageenan concentration and lower poly(vinyl pyrrolidone) concentration.

5.4 Potential applications of polysaccharide-based hydrogels prepared by radiation techniques A considerable number of workers have investigated and discussed the potential applications of polysaccharide-based hydrogels. Polysaccharide-based hydrogels can be used in the fields of biomedical materials, the environment, super water absorbents, and additives used to improving the compressive strength of cement, due to their excellent properties.

Preparation of polysaccharide-based hydrogels via radiation technique 137

5.4.1 Biomedical materials Polysaccharide-based hydrogels can be used as vehicles for controlled release of drugs, wound dressings, and scaffolds for soft-tissue regeneration due to excellent biocompatibility and biodegradability. 5.4.1.1 Drug delivery One of the most important applications of hydrogels in the medical field is drug delivery. Synthesized functional hydrogels can control the release of drugs over an extended period of time, and maintain the constant concentration of drug in the physiological environment and have a continuing effect. Zhao et al. prepared hyaluronic acid/chondroitin sulfate/poly(acrylic acid) hydrogel by the γ-ray irradiation-induced cross-linking method [69]. Cefazoline and theophylline were used as ionic and nonionic model drugs, respectively, to carry out in vitro drug release trials. The two model drugs were incorporated into the hydrogel samples by the swelling-loading method. As illustrated in Fig. 5.18, in comparison with that of theophylline, the release rate of cefazoline was slower. This result indicated that the prepared hydrogels can effectively manipulate the release rate of ionic drug by the interaction of ionic groups within the hydrogels with the ionic groups within the ionic drug molecules. Ta¸sdelen et al. synthesized a hydrogel composed of chitosan/hyaluronic acid/hydroxyapatite using γ-ray irradiation without the addition of cross-linkers [74]. 5-Fluorouracil (5-FU) was incorporated into the hydrogel by the swelling method, and used as a model anticancer drug to determine the drug uptake and release rates of the hydrogels. The uptake amounts of 5-FU in the three hydrogel samples increased with the increasing amounts of both hyaluronic acid and hydroxyapatite due to the fact that more function groups were introduced into the hydrogels (Table 5.1). The release rates of 5-FU for the three hydrogel samples initially exhibited a rapid increase, and gradually reached equilibrium within 24 hours (Fig. 5.19). The 5-FU release rates increased with the increased amount of both hyaluronic acid and hydroxyapatite in hydrogel. This can be attributed to the high gel fraction decreasing the free volume in the hydrogel. The prepared hydrogel might be an appropriate delivery vehicle used for drug release in the human body. Chitosan-g-poly(acrylic acid) hydrogel was prepared using 60Co γ-ray irradiation without the addition of cross-linkers [56]. The gel fraction was higher than 96%, and increased with the concentrations of both acrylic acid and chitosan. The swelling ratio increased with the increase of pH up to pH 12. 5-FU was also used as the drug model. The results showed that more than 90% of the loaded 5-FU released in the first 1 hour at the intestinal pH and the rest of the drug released slowly; meanwhile, the release rate of drug was able to be manipulated via modulating the gel fraction, and the compositions of the hydrogels.

138 Chapter 5

Accumulated release amount (%)

(A) 100

HA/CA/PAAc-73 (theophylline) HA/CA/PAAc-55 (theophylline) HA/CS/PAAc-37 (theophylline)

80 60 40 20 0

0

10

20

30

40 50 60 Time (h)

70

80

90 100

70

80

90 100

Accumulated release amount (%)

(B) HA/CA/PAAc-73 (cefazoline) HA/CA/PAAc-55 (cefazoline) HA/CA/PAAc-37 (cefazoline)

80

60

40

20

0

0

10

20

30

40 50 60 Time (h)

Figure 5.18 In vitro release behaviors of drugs from hyaluronic acid/hyaluronic acid/poly(acrylic acid) hydrogel in PBS at 37 C: (A) release kinetics of theophylline from the hydrogels and (B) release kinetics of cefazoline from the hydrogels. Each point represents the mean standard deviation of three samples. Table 5.1: Variation of 5-FU uptake with hyaluronic acid content in the hydrogel. Hydrogel composition (w/v%) Chitosan 1 1 1

Gelation

5-FU uptake

Hyaluronic acid

Hydroxyapatite

(%)

mg/g dry gel

1 2 3

0.5 1 1.5

84.5 90.8 94.2

8.52 10.88 12.74

Preparation of polysaccharide-based hydrogels via radiation technique 139

Figure 5.19 Release profiles of 5-FU from hydrogel samples (see Table 5.1) in phosphate buffer solution (pH 7.4 and 37 C).

5.4.1.2 Wound dressings Wound dressings are another significant application of polysaccharide-based hydrogels. They are not only able to prevent infection but also promote the healing of wounds. Singh et al. prepared carrageenan/polyvinyl pyrrolidone hydrogels containing nanosilver using γ-ray irradiation without the addition of cross-linkers [75]. The hydrogel composed of polyvinyl pyrrolidone (15 wt.%) and carrageenan (0.25 wt.%) showed high gel fraction at the absorbed dose of 25 kGy. Nanosilver was then loaded into the hydrogel. The fluid-handling capacity of the hydrogel increased from 2.35 6 0.39 g/10 cm2 to 6.63 6 0.63 g/10 cm2 with the time extension from 2 to 24 hours. Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans were not observed in the presence of hydrogels containing 100 ppm nanosilver after 36 hours. This indicated that the hydrogels containing 100 ppm nanosilver exhibited potential microbicidal activity against these wound pathogens. Moreover, the impermeability of the hydrogel matrix was not affected in the presence of nanosilver. The results illustrated that the prepared hydrogel with silver nanoparticles can be used as wound dressings to control infection and facilitate the healing process of wounds induced by burns or other skin injuries. 5.4.1.3 Scaffolds for soft-tissue engineering Polysaccharide-based hydrogel can also be used as scaffolds for soft-tissue engineering. Zhao et al. synthesized a novel hydrogel composed of hyaluronic acid, chondroitin sulfate, and poly(vinyl alcohol) by the γ-ray irradiation-induced cross-linking method [68]. Human keratinocyte cell growth in the hydrogels gradually increased with culture time; after 7 days, the viability of human keratinocyte cells in all hydrogel samples was greater than 80% compared to the control, where the human keratinocyte cells were cultured on a culture plate; moreover, the viability of human keratinocyte cells increased with an

140 Chapter 5

Cell viability (%)

80 1 day

60 3 days 5 days

40

7 days

20

0 HA/CS/PVA-73-G15k

HA/CS/PVA-55-G15k

HA/CS/PVA-37-G15k

Figure 5.20 Viability of HaCaT cells cultured after seeding on hyaluronic acid/chondroitin sulfate/poly(vinyl alcohol) hydrogels. Values represent the mean and standard deviation (n 5 4). Hyaluronic acid (35%)/chondroitin sulfate (35%)/poly(vinyl alcohol) (30%)/15 kGy (left); hyaluronic acid (25%)/chondroitin sulfate (25%)/poly(vinyl alcohol) (50%)/15 kGy (middle); hyaluronic acid (15%)/chondroitin sulfate (15%)/poly(vinyl alcohol) (70%)/15 kGy (right).

increase in the content of hyaluronic acid and chondroitin sulfate in the hydrogel (Fig. 5.20). The prepared hydrogel with a high content of hyaluronic acid and chondroitin sulfate has great potential used as a scaffold for soft-tissue engineering.

5.4.2 Metal ion adsorption Polysaccharide-based hydrogels with functional groups can be used to remove metal ions from aqueous solutions, and might be applied in the environmental field due to their nontoxicity and biodegradability. Hong et al. prepared carboxymethyl chitosan/CMC grafted sodium styrene sulfonate hydrogel by γ-ray irradiation-induced grafting and crosslinking [76]. A schematic diagram is shown in Fig. 5.21. The gel fraction increased with an increase in both absorbed dose and sodium styrene sulfonate concentration. The removal ratio of metal ions was dependent on the swelling ratio of hydrogels. As can be seen in Fig. 5.22, the removal ratio of metal ions increased with an increase in the swelling ratio of hydrogels as the ratios of (CMC/carboxymethyl chitosan) to sodium styrene sulfonate decreased from 6:0 to 4:2; meanwhile, the removal ratio of metal ions did not present an obvious change as the ratio of CMC/carboxymethyl chitosan to sodium styrene sulfonate further decreased from 4:2 to 2:4. This result indicated that the high swelling ratio was an important factor for removal of metal ions from wastewater in addition to the functional groups in the hydrogels.

Figure 5.21 Proposed scheme for the forming of CMC/carboxymethyl chitosan/sodium styrene sulfonate hydrogel.

Figure 5.22 Swelling ratio of prepared hydrogels versus removal ratio of metal ions in aqueous solution (CMC:carboxymethyl chitosan 5 1:1).

142 Chapter 5

5.4.3 Super water absorbent Polysaccharide-based hydrogels with low gel fraction often show high water absorbency, and can be used as the super water absorbent used in the fields of agriculture and hygiene, etc. El-Rehim prepared sodium alginate/polyacrylamide cross-linked hydrogels using EB irradiation without addition of cross-linkers [77]. The swelling ratio of the hydrogel composed of sodium alginate (20 wt.%) and polyacrylamide (80 wt.%) reached 600 g/g in pH 7 water. The small quantities of hydrogel added to sandy soil significantly improved its water retention property. The pot experiment showed that the growth of a bean plant cultivated in a soil with the hydrogel was better than that cultivated in soil with polyacrylamide (Table 5.2 and Fig. 5.23). The difference between polyacrylamide and sodium alginate/polyacrylamide hydrogel might be due to the latter partially undergoing enzymatic degradation in soil to become oligo-alginate, which can be considered as a plant growth promoter. The increase in faba bean plant performance using sodium Table 5.2: Total dry weight of faba bean plant planted in soil containing different types of hydrogels (12 weeks). Hydrogels used for planting Control Polyacrylamide Polyacrylamide/sodium alginate

Total dry weight (g) 21 30 39

Figure 5.23 Faba bean plant planted in soil, 9 weeks after planting: (A) untreated (control), (B) treated with polyacrylamide, and (C) treated with polyacrylamide/sodium alginate.

Preparation of polysaccharide-based hydrogels via radiation technique 143

Figure 5.24 Protocol of development of cross-linked sodium cellulose-g-poly(acrylic acid) hydrogel.

alginate/polyacrylamide hydrogel suggested that it might be able to be used as a soil conditioner in agriculture, providing the plant with water as well as oligo-alginate growth promoter. Fernando et al. prepared cellulose-g-poly(acrylic acid) hydrogels by γ-ray irradiationinduced grafting of neutralized acrylic acid onto two cellulose samples in the presence of N,N0 -methylenebisacrylamide in a nitrogen gas atmosphere [78]. The preparation process is depicted in Fig. 5.24. The swelling ratio in distilled water reached 325 g/g. When the soil contained the hydrogel, watering at 3-day intervals resulted in a good yield from Ipomoea aquatica (Fig. 5.25); meanwhile, cut Chrysanthemum instagram flowers survived 1213 days in biocide- and sucrose-added hydrogel media without petal discoloration or wilting (Fig. 5.26). Synthesized hydrogels with super water absorbency can enhance the water retention property of soil, facilitate the growth of the plants, and be useful in agricultural applications.

5.4.4 Use as an additive to improve the compressive strength of cement Polysaccharide-based hydrogels with good water retention property can also be used as the additives for improvement of the compressive strength of cement. Ghobashy et al. prepared sodium alginate/polyacrylamide cross-linked hydrogels using 60Co γ-ray irradiation method without the addition of cross-linkers [59]. The swelling ratios of hydrogel were higher in basic than acidic aqueous solutions. This implied that the synthesized hydrogel could be

144 Chapter 5 (B) 35 30 25 20 15 10 5 0

30 25 Yield (g)

Date of seedling wilt

(A)

20 15 10 5 0

SAPAHP SAPMwA Coir dust

3 days

Type of materials in survival media

Watering intervals

(D) 120

14 12 10 8 6 4 2 0

Plant height (cm)

Leaf area (cm2)

(C)

5 days 7 days

100 80 60 40 20 0

3 days

5 days 7 days

Watering intervals

3 days

5 days 7 days

Watering intervals

Figure 5.25 (A) Variation of mean date of survival of transplanted Capsicum annuum; (B) variation of mean yield of Ipomoea aquatica; (C) variation of mean leaf area of I. aquatica; and (D) variation of mean plant height of I. aquatica.

Figure 5.26 (A) Dates of Chrysanthemum instagram flowers with full bloom with no petal discoloration or wilting and (B) cut C. instagram flowers after 13 days.

used as an internal curing agent for concrete. As compared with the control, the maximum compressive strength of 39 kN of the cement with only 0.2 wt.% of hydrogel was reached after 7 days (Fig. 5.27). This was attributed to the added hydrogel retaining the water to ensure complete hydration that led to minimum pore size and pore distribution as seen in the obtained SEM images.

Preparation of polysaccharide-based hydrogels via radiation technique 145

Figure 5.27 Compressive strength of cements versus hydrogel content (wt.%).

5.5 Conclusion Various polysaccharide-based hydrogels can be efficiently prepared by the EB and γ-ray irradiation method. This technology can be easily scaled up and commercialized as all its knowledge base is mature. The application of such polysaccharide-based hydrogels in various fields has a bright future due to its natural advantages, biodegradability, biocompatibility, nontoxicity, etc.

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CHAPTER 6

The physical and chemical properties of hydrogels based on natural polymers B. Kaczmarek*, K. Nadolna and A. Owczarek Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics, Nicolaus Copernicus University in Torun, Torun, Poland

6.1 Introduction Nowadays, hydrogels are a type of material which is of great interest to scientists working in a number of different fields. Hydrogels are three-dimensional polymeric networks which are able to bind water due to the hydrogen bond formation. They can be formed from molecules containing hydrophilic groups where the polymeric chains are in a colloidal state in the dispersion medium. Hydrogels have been defined in different ways. The most accurate is that hydrogels are water-swollen materials based on cross-linked polymeric chains. Another definition describes hydrogels as a type of material which exhibits a high ability to swell without changes to its structure, volume, or shape. Hydrogels can be flexible and easy to shape, depending on the application [1]. Such materials can be obtained from natural and/or synthetic polymers, which turn into gel according to various factors, for example, temperature, ionic strength, pH, and UV irradiation. A unique three-dimensional structure is formed due to the high hydrophilicity of polymers [2]. Hydrogels show swelling behavior dependent on environmental conditions, such as pH, temperature, ionic strength, and electromagnetic radiation [3]. Also, the properties of hydrogels including shape, mechanical flexibility, opacity, and porosity can be modified by changing these parameters. The water in hydrogels can be classified as water bound to the polymeric chain, free water, and semibound. When dry hydrogels are introduced into water, first water molecules penetrate the hydrogel and hydrate the hydrophilic groups (primary bound water). When groups are hydrated the polymeric network swells and exposes hydrophobic groups which interact with water molecules resulting in hydrophobically bound water molecules (secondary bound water). After the hydrophilic and hydrophobic sites of the hydrogel 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00006-9 © 2020 Elsevier Inc. All rights reserved.

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152 Chapter 6 interact with water molecules, the network imbibes additional water due to the osmotic forces of the polymeric network chains toward infinite dilution. Such additional swelling is resisted by chemical and physical cross-linking, resulting in the elastic hydrogel properties. The additional water which is absorbed by the hydrogel is called “bulk water” or “free water” and fills the spaces between the polymeric network chains [4]. Semibound water has intermediate properties between free water and those bound to the polymeric chain. Hydrogels can be divided into two main groups: based on synthetic polymers and natural polymers. Both types of hydrogel have advantages and disadvantages. However, considering the medical application of hydrogels, it can be assumed that natural-based materials are biocompatible, biodegradable, and nontoxic for the human body. Such features make them better materials than synthetic compounds. On the other hand, synthetic-based hydrogels have better mechanical parameters which enhances their potential industrial applications. Also, composite materials, which are a mixture of synthetic and natural polymers, are very common. There are different types of hydrogels: 1. Homopolymeric hydrogels—this network refers to a type of monomer, basic structural unit. They can have a cross-linked skeletal structure depending on the polymer and cross-linking type. 2. Copolymeric hydrogels—hydrogel is obtained from two or more monomer species, and one has to be hydrophilic. They can be arranged in various configurations. 3. Multipolymeric hydrogels—two independent cross-linked natural or synthetic polymers which form the network [5]. Hydrogels can also be classified depending on their charge (nonionic, ionic, amphoteric electrolyte, zwitterionic), physical appearance (matrix, film, or microsphere), based on the type of cross-linking (chemical or physical cross-linking methods), and the configuration (amorphous, semicrystalline, and crystalline). Natural polymer-based hydrogels recently replaced synthetic polymer-based materials, because they have several advantages. First, they are biocompatible and after application give excellent cellular and tissue responses. Proteins as well as polysaccharides can be used to design new hydrogels for tissue engineering purposes. Second, hydrogels obtained from natural polymers are biodegradable, and there is no formation of toxic products during the degradation process [6]. Hydrogel properties depend on the type of polymer, including the presence of hydrophilic groups attached to the polymeric chains ( OH, CONH , COOH, CONH2, SO3H). For instance, the presence of alcohol, amides, and carboxylic acids as hydrophilic moieties enhances hydrogel stiffness and the capacity to absorb water. Moreover, the presence of functional groups influences the type of cross-linking process needed for hydrogel formation.

The physical and chemical properties of hydrogels based on natural polymers 153 Hydrogels are a class of soft material with elastic properties. The low mechanical properties of hydrogels restricts their applications. Due to the presence of many functional groups they are easy to cross-link and as a result possess good mechanical strength [7]. Depending on the polymer properties used to obtain hydrogels and the density of the network, various amounts of water can be absorbed in the swollen state. Usually the mass of water is higher than the mass of polymer. Hydrogels are present in a variety of forms, such as slabs, membranes, beads, microgels, nanogels, cryogels, or aerogels. Hydrogels are very attractive materials due to their multifunctional properties which allow for the wide range of applications in different aspects of life, especially in biomedical science. They have the ability to absorb water and dissolved compounds, resulting in their application as napkins or superabsorbents used for water conservation. Hydrogels can be described as “smart materials” because they respond to various external factors including pH, temperature, and osmotic pressure. Such influences of external factors can find application in controlled-release systems in the fields of medicine or agriculture [8]. Hydrogels have found wide application in a variety of fields, such as agriculture, drug delivery, tissue engineering, water purification, contact lenses, and sensors [4].

6.2 Hydrogel preparation Hydrogels can be obtained in different chemical and physical ways. Hydrophilic and hydrophobic monomers are used to regulate hydrogel properties for specific applications. Hydrogels can be produced by reactive cross-linkers, copolymerization, or free-radical polymerization. Such material form can be obtained in a number of ways including ionization, linking polymer in a chemical reaction, or physical interactions. Bulk polymerization is the method used to produce hydrogels from one or more types of monomers. For the formulation a small amount of cross-linker has to be added. Then the initiator, as radiation, ultraviolet, or catalysts, is applied. The initiator used depends on the polymer structure and solvent nature. Such polymerization can be used to produce hydrogels including rods, particles, emulsions, membranes, and films. The other method, free-radical polymerization, can be applied to monomers as vinyl lactams, amides, or acrylates, with appropriate functional groups or which can be functionalized with radical polymerization. It includes the chemistry of typical free-radical polymerization steps, such as propagation, chain transfer, initiation, and termination. The free-radical method is based on monomers modifying into active forms. In solution polymerization the ionic or neutral monomers are mixed with cross-linking agents. The polymerization can be initiated thermally by UV irradiation or by the redox

154 Chapter 6 initiator system. In this method solvent (ethanol, water-ethanol, or benzyl alcohol) serves as a heat sink. The obtained hydrogels need to be washed in water to remove the initiator, unreacted monomers, oligomers, cross-linkers, and other impurities. Hydrogels based on natural polymers can be obtained by one-step polymerization, for example, in situ polymerization where the cross-linkers and nanoparticles can be incorporated. Such composites can find application as delivery systems, sensors, in tissue engineering, etc. [9]. Moreover, multiple-step polymerization can also be used to obtain hydrogels from polymers with reactive groups and their subsequent cross-linkers [5]. Many hydrogels are produced using hydrogen bond formation. The factors influencing the efficiency of this method are temperature, molar ratio of components, polymer structure, and the type of solvent.

6.3 Hydrogels from polysaccharides Polysaccharides are natural polymers constructed by repeating units of different saccharides bound together by glycosidic linkages. They range in structure from linear to highly branched. Polysaccharides can be extracted from different natural sources. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. When all the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present they are called heteropolysaccharides or heteroglycans. Polysaccharides are widely used to obtain hydrogels due to the presence of hydrophilic functional groups which are able to absorb water and can be easily cross-linked by different chemical and physical methods. Hydrogels based on polysaccharides can be considered “injectable hydrogels” thanks to their ability to be squeezed through the needle of a syringe [6]. This review includes a discussion of hydrogel properties obtained from chitosan, alginate, carrageenan, hyaluronic acid, starch, and cellulose.

6.3.1 Chitosan-based hydrogels The main polysaccharide used to produce hydrogels is chitosan. Chitosan is a copolymer of β-(1-4)-linked 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose. It is a polycationic biopolymer which can be obtained by alkaline deacetylation from chitin, which is the main component of the exoskeleton of crustaceans. For any studies, the deacetylation degree and molecular weight of chitosan have to be detected because such factors have an influence on the polysaccharide properties. Chitosan is well known as a biocompatible

The physical and chemical properties of hydrogels based on natural polymers 155 compound which has found biomedical application in implantation, injection, and as a wound-dressing material. Other advantages include its low cost of isolation, its eco-friendliness, and nonhazardous nature. Chitosan contains amine ( NH2) and hydroxyl groups ( OH). Thereby it can be easily cross-linked to modify its properties. Hydrogels can be formed either from chitosan or from chitosan with other polymers. Chitosan is sensitive to pH changes and can be self-crosslinked by increasing the pH or dissolving in a nonsolvent [10]. There are a wide range of cross-linking methods which include the chain modification, use of the chemical reaction of chitosan and cross-linker, hydrophobic association, electrostatic interactions, or hydrogen bonding [11 14]. Also, chitosan-based hydrogels can be obtained by using physical methods such as UV-light irradiation [15]. Covalently cross-linked chitosan-based hydrogels can be divided into three groups—chitosan cross-linked by itself, hybrid polymer networks, and semi- or fullinterpenetrating polymer networks. Polysaccharide cross-linked by itself may involve two units from one or two different polymeric chains. The final structure has a gel network form. The hybrid chitosan network is formed between a polymeric chain of another polymer and chitosan structural unit [16]. In each cross-linking type covalent bonds are the main interactions, moreover secondary interactions, such as ionic, hydrophobic, and hydrogen bonds, can be formed. The modification processes influence the hydrogel properties, thereby appropriate characteristics of obtained materials can be obtained. Covalent cross-linking is based on the reaction between aldehyde and amine groups, where the imine bonds are formed. The use of aldehydes as chitosan cross-linkers is well-documented and studied. Other compounds which can be used as cross-linkers for covalent bond formation are diethyl squarate, oxalic acid, and genipin. However, the cross-linking mechanism is not wellknown, thereby cross-linked chitosan hydrogels by those agents have to be tested by an in vitro method for biocompatibility studies. The next method to form covalent cross-linking interactions is to add functional biopolymers, such as poly(ethylene glycol), cyclodextrin, and poly(vinyl alcohol). Nevertheless after such modification biocompatibility also needs to be studied. Chitosan hydrogels can also be obtained by complexation with collagen, poly(acrylic acid), chondroitin sulfate, and xylan. Chitosan was cross-linked with tris(2-(2-formylphenoxy)ethyl)amine. Hydrogels have been tested as drug-delivery systems and the results showed the pH and temperature-responsive swelling ratio [17]. Chitosan was also tested in the mixture with, for example, hyaluronic acid, carrageenan, and gelatin [18 20]. For instance, chitosan was tested in the mixture with poly(vinyl alcohol) and nano zinc oxide [21], where chitosan is used to improve the material biocompatibility. Polysaccharide was also cross-linked by genipin with poly(vinyl pyrrolidone) to from swellable hydrogels in different pHs and temperatures [22]. Chitosan was also mixed with cellulose and

156 Chapter 6 cross-linked by glutaraldehyde. The proposed hydrogels showed antimicrobial activity against Gram-positive and Gram-negative bacteria [23]. Chitosan can also form a hybrid composite with beta-glycerophosphate as injectable hydrogels able to mimic the extracellular matrix and provide a microenvironment for cell growth [24]. Chitosan-based hydrogels can also be obtained using an ionically cross-linking method. This avoids the purification and verification step. Chitosan is a positively charged polymer which can react with negatively charged components, ions, or molecules. As a result the ionic bridges between polymeric chains are formed. Mostly, the molecular weight of chitosan influences the cross-linking strength. The nature of the ionic cross-linking depends also on the type of cross-linker. The ammonium groups from the chitosan polymeric chain may interact with metallic ions to form coordinate-covalent bonds. Also, the positively charged ammonium groups may interacts with hydroxyl groups. Other hydrophobic or hydrogen interactions may also occur. The cross-linking density is an important factor which can be modified by the external conditions of the hydrogel preparation. The density influences the mechanical properties and swelling rate. The cross-linker agent structure, size and chemical character, temperature, pH, and solvent type may cause the hydrogel properties to change. The main influence on the ionic cross-linking is the pH of the solution. The acid conditions are necessary to form positively charged ammonium groups of chitosan, which then can be neutralized, for instance, in the dialysis process. The pH value during cross-linking must be in the vicinity of the pKa interval of the chitosan and cross-linker [25]. With high pH positively charged chains are neutralized. It can be assumed that the optimal pH for chitosan cross-linking is about 6. Novel materials based on chitosan contain microspheres or microcapsules with an active substances. They are attached to the polymeric network of hydrogels. Such composition improves skin penetration by the compounds, which enhances hydrogel application for skin moisturizing or regeneration. The important factor of such hydrogels is the pH, which must be appropriate to the pH value (4.9 5.6) of skin. Hydrogels were also prepared by in situ hydrogelation of chitosan biopolymer with nitrosalicylaldehyde in the presence of a model drug, varying the cross-linking density [26]. The injectable hydrogels can be formed from the chitosan and alginate mixture, where such hydrogel application results in an increase in the thickness and integrity of epidermal tissue, increased formation of collagen fibers, and enhanced expression of vascular endothelial growth factor as compared to the control group [27].

6.3.2 Alginate-based hydrogels Alginate is a hydrophilic polysaccharide linear binary copolymer composed of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues as monomers, constituting

The physical and chemical properties of hydrogels based on natural polymers 157 M-, G-, and MG-sequential blocks. Commercially available alginates are typically extracted from brown algae (Phaeophyceae), including Laminaria hyperborea, Laminaria digitata, Laminaria japonica, Ascophyllum nodosum, and Macrocystis pyrifera. Alginates can be also obtained by bacterial biosynthesis from Azotobacter and Pseudomonas [28]. Hydrogels based on alginate can be formed under very mild conditions, at room temperature, and in the absence of toxic solvents. They are ideal materials for biomedical applications, however, they need to be modified to resemble the mechanical, chemical, and structural properties. Alginate has wide applications to produce hydrogels due to its biocompatibility and biodegradability, gel-forming properties, and low costs. Alginate has groups easy to protonate, and in the pH above its pKa the polyanionic chains are formed, where the carboxylic groups are negatively charged. In such a form metallic ions including Ca21, Ba21, Cu21, Co21, Ni21, or Sr21 can cross-link the polymeric chain to form gel-like forms. Generally, alginate is hydrophilic and water-soluble, thickening in neutral conditions what can then be related to in situ hydrogel formation. Alginate-based hydrogels can be classified into physical and covalent gels. Various methods can be employed for hydrogel formation, such as ionic interaction, thermal gelation, cell-cross-linking, “click” reaction, and freeradical polymerization. Also, materials obtained from alginate have pH-responsive properties due to the presence of many carboxylic groups in the backbone [29]. Ionic cross-linking is the most common method to obtain hydrogels from an aqueous solution. The alginate structure block of M monomer forms weak junctions with divalent cations and the block of G monomers forms tightly held junctions. Different cations can be used for hydrogel preparation, however, calcium ions are most commonly used for alginate chains. Mainly CaCl2 is used for such purposes, however, the cross-linking is too fast to control. Therefore it can be replaced by CaSO4 or CaCO3, which have lower solubility [29]. The thermoresponsive phase transition can be proposed for alginate-based hydrogel formation simply as the temperature increase above the critical solution temperature. The thermosensitivity of an alginate hydrogel is achieved by incorporating, for example, poly(N-isopropylacrylamide) into its backbone [30]. The cell-cross-linking is a method which includes the specific receptor ligand interactions to cross-link the alginate-based hydrogels. The main strategy of this method is to introduce ligands, such as arginine-glycine-aspartic acid, onto the alginate for cell adhesion [29]. The formed network shows good biocompatibility, however, it has low strength and toughness. Another method, free-radical polymerization, is the process of transforming a linear polymer into a three-dimensional network. This depends on the pH and temperature as well as the addition of initiators.

158 Chapter 6 Alginate can be mixed with chitosan or hyaluronic acid to form novel biocompatible hydrogels [31]. Also, chondroitin sulfate was used as an additive to form hydrogel with alginate polymer, where the main influence on its properties was the molecular weight of the polymers [32]. Alginate-based hydrogels with gum tragacanthin were obtained by a gelation method followed by chitosan polyelectrolyte complexation and were tested as an oral insulin carrier. The proposed polymers are biodegradable, biocompatible, and nontoxic [33]. Alginate can be also mixed with synthetic polymers, for instance, poly(acrylic acid), and cross-linked by gamma irradiation [34]. Polysaccharide was also mixed with peptides, such as silk fibroin [35], collagen [36], and elastin [37]. The obtained materials presented significant biological properties.

6.3.3 Carrageenan-based hydrogels Carrageenan is known to possess multifarious groups including hydroxyl/sulfate groups. These are excellent for various chemical modifications, such as oxidation and carboxymethylation. They can be oversulfated, methylated, acetylated, and phosphorylated. Such modifications prove that carrageenan is a robust polymer and improve its physicochemical properties. Hydrogels based on carrageenan are generally formed through thermoreversible gelation, ionic cross-linking, as well as the photocross-linking of carrageenan backbone by methacrylate moieties [38]. Ionic cross-linking is a method which includes an interaction with K1 and Ca21 ions resulting in brittle hydrogels. Photocross-linking includes the incorporation of moieties such as methacrylate groups followed by UV cross-linking in the presence of photoinitatior (Irgacure 2959) where the hydroxyl groups are replaced by methacrylate moieties. Chemically cross-linked hydrogels had higher water retention capacity resulting in a more flexible network [38]. It can be assumed that higher metacrylation degree results in the lower swelling ratio and lower pore size of interconnected pores. Gradient hydrogels exhibit a gradual change in the viscoelasticity, stiffness, and porosity properties. They can be obtained by various methods, such as electrospinning, microfluidic, and gradient makers. A change in the cell morphology within the gradient hydrogel could be an indicator of the material’s potential to modulate the cell fate [38]. The hydrogels based on carrageenan have good gastroretentive properties. This extended gastroretention can be attained by utilizing the swelling property of the material which absorbs fluid from the surrounding environment, making it flow. It was assumed that the gel strength decreased with an increase in the concentration of the pore-forming agent, as more porous and fragile gel is formed. Hydrogels can be also obtained by the micropatterning method to design patterns in microscale for cell culture substitutes.

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6.3.4 Hyaluronic acid based hydrogels Hyaluronic acid is a nonsulfated polysaccharide that belongs to the glycosaminoglycans group. It is one of the main components of the extracellular matrix of many soft connective tissues. Hyaluronic acid is constructed by repeating units of D-glucuronic acid and N-acetyl-Dglucosamine, linked together via alternating β-1,4- and β-1,3-glycosidic bonds. Hyaluronic acid can be characterized by different molecular weights which influence its properties [39]. Hyaluronic acid is an attractive polymer for biomedical applications because it is biocompatible, biodegradable, nontoxic, bioactive, nonimmunogenic, and nonthrombogenic. It can form gels at high concentration solutions for high-molecular-weight hyaluronic acid which are viscoelastic and do not have longlasting mechanical integrity. It is necessary to modify it in chemical reactions or cross-linking processes. Moreover, additional biological active compounds can be added, for instance, cytokines and drugs. Hyaluronic acid based hydrogels are networks consisting of interconnected chains. Over the past few years researchers have created a wide range of hydrogels based on hyaluronic acid with increasing complexity and diverse functions [40]. Materials based on unmodified hyaluronic acid chains are not useful as biomaterials due to their susceptibility to degradation and inferior mechanical properties. To improve the functions covalent cross-linking has to be carried out. Hyaluronic acid has two functional groups ( COOH and OH) which influence the cross-linker type used for material modification. The carboxylic group can be modified by, for instance, addition of N-hydroxysuccinimide, dicycloheyl carbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The hydroxyl group can be cross-linked by, for example, glutaraldehyde, cyanogen bromide, octeylsuccinic anhydride, and methacrylic anhydride. Compared to native hyaluronic acid, the cross-linked type exhibits more robust mechanical properties and is less susceptible to enzymatic degradation [25,39]. However, the covalent cross-linking method requires toxic reagents which are not suitable for cells and tissues. For tissue engineering purposes the gelation kinetics should be fast enough to allow for cell encapsulation to the cell/gel material.

6.3.5 Starch-based hydrogels Starch is the most abundant storage polysaccharide in plants, as granules in the chloroplast of green leaves and in the amyloplast of seeds, pulses, and tubers. It can be isolated from different sources including corn, potato, tapioca, and wheat. Starch is constructed by a number of monosaccharides or glucose molecules joined together with α-D-(1 4) and/or α-D-(1 6) linkages [4]. Starch includes two main structural components—amylose and amylopectin. The proportions of these components depend on the extraction method and starch source, which affects the crystallinity and molecular order.

160 Chapter 6 Starch changes to gelatin in thermally three-step-assisted hydration-plasticization of the polymeric network. First, swelling is observed by absorbing water molecules. In the second step the gelatinization takes place after the starch dissolves by heating. This results in leaching of the amylose and causes irreversible physical changes. The granulate structure is destroyed. The final and third step is called the retrogradation step, in which the starch hydrogel network is created upon cooling and aging, result in recrystallization and reorganization of the polysaccharide structure [4]. The amylase content and temperature are two important factors influencing the process parameters and obtained hydrogel properties. There are different methods to modify the properties of starch. Starch has hydroxyl groups which can be utilized easily to prepare hydrogels. These modifications can be obtained by chemical reactions by introducing a small amount of ionic or hydrophobic groups into the starch chain. This results in a change to the solution viscosity and association behaviors. Esterified and grafted starches have been proposed to obtain hydrogels. In the etherification process some hydroxyl groups are substituted by ether groups. Also, various vinyl monomers can be grafted onto starch, such as acrylamide and acrylic acid [4]. Starch-based hydrogels have advantages and disadvantages. They are abundant natural biopolymers, biodegradable, with high availability, a renewable resource, economically attractive, easy to prepare, with high swelling capacity. However, they have low surface area and require chemical derivatization to enhance their sorption capacities. Interest in starch as a potential polymer to replace synthetic macromolecules has grown recently. There are two methods to obtain starch-based hydrogels. The first is to graft copolymerization of vinyl monomers on polysaccharides in the presence of a cross-linker. The second is the direct cross-linking of starch. Graft copolymerization begins with the starch reaction with the initiator. Such complexes are dissociated to create carbon radicals on the starch chain. The produced free radicals initiate the graft polymerization of vinyl monomers and cross-linker on the polysaccharide chains [4]. In the other method, the initiator abstracts hydrogen radicals from the hydroxyl groups of the starch to form initiating radicals of the polysaccharide chain. This reaction is influenced by temperature due to the use of a thermal initiator. Starch-based hydrogels have found application in dye removal from industrial wastewaters. Such hydrogels are preferred due to the availability of different adsorbents, their high efficiency, and easy handling. Different factors influence dye sorption, such as dye sorbent interaction, particle size, sorbent surface area, pH, temperature, and contact time. Starchbased hydrogels can be also used to remove heavy metals, which are harmful to humans, plants, and animals. Adsorption is an effective method for removal of heavy metal ions from aqueous solutions. They can also be used as superabsorbent hydrogels to decrease irrigation water consumption, enhance fertilizer retention in the soil, decrease plant death rates, and increase plant growth rates. In agriculture applications, hydrogels can also be

The physical and chemical properties of hydrogels based on natural polymers 161 applied in controlled pesticide formulations that reduce environmental pollution. Hydrogels may deliver pesticides slowly with the purpose of limiting their quantities. Advantages also include decreased leaching, degradation and volatilization, and the reduction of food residues and dermal toxicity.

6.3.6 Cellulose-based hydrogels Cellulose is a natural polymer present in a wide variety of living species including plants, animals, and some bacteria [3]. The most commercial source of cellulose is wood and plant fibers (cotton, jute, flax, etc.). It can also be produced by bacteria, algae, and marine animals. Cellulose exhibits good mechanical properties, and is biodegradable and hydrophilic. Moreover, it is biocompatible and, thereby, it is proposed for biomedical applications. Cellulose is constructed by repeating units of two anhydroglucose units (AGU) linked together through an oxygen covalently bonded to C1 of one glucose ring and C4 of the adjoining ring, β(1-4) glycosidic bond. The hydroxyl groups have high ability to form hydrogen bonds and play a major role in the properties of cellulose-based hydrogels. The intra- and intermolecular hydrogen bonds cause parallel arrangement of cellulose chains, which forms microfibrils. However, cellulose is insoluble in water and most organic solvents [3]. There are different methods to modify the properties of cellulose. Chemical modification includes esterification or etherification causing the obtainment of water-soluble cellulose derivatives. To regulate the solubility and viscosity in water solutions the degree of substitution can be controlled. It is defined as the average number of esterified/etherified hydroxyl groups from AGU [3]. Cellulose derivatives, such as methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), ethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, cellulose sulfate, and cellulose phosphate have been used to obtain reversible or stable hydrogels. Hydrogels can be obtained from cellulose and can form 3D networks. An important feature of hydrogels is their ability to swell and remain insoluble at the same time. Cellulose has excellent biocompatibility and is biodegradable [41]. Hydrogels based on cellulose can be obtained in chemical or physical cross-linking processes. Physical cross-linking is beneficial because it avoids the use of chemical cross-linking agents. It includes physical interactions such as chain entanglements, van der Waals forces, hydrogen bonds, and hydrophobic or electronic associations [3]. Hydrogel preparation can be carried out in a homogeneous medium, where cellulose is solubilized, and heterogeneous, where cellulose fibers are used to modify the mechanical properties of hydrogels. Cellulose can be dissolved in a few solvents, such as lithium chloride, N-methylmorpholine-N-oxide, ionic liquids, alkali

162 Chapter 6 aqueous systems, and alkali/urea aqueous systems. Hydrogels can be also obtained from cellulose nanoparticles, as nanofibrillated cellulose and cellulose nanocrystals. They can be also produced from bacterial cellulose, which has numerous advantages compared to plantderived cellulose, such as surface area, wet tensile strength, purity, crystallinity, and high biocompatibility [3]. Obtaining hydrogel is also possible by chemical cross-linking reactions. Cross-linking agents can be classified into two groups: esterifying agents where the formation of COOR bonds is observed and etherifying ones which result in R-O-R group formation. The most widely used cross-linking agent for polysaccharide chains is epichlorohydrin [42] and poly(vinyl alcohol) [43]. The swelling properties of obtained hydrogels depend on the cellulose/cross-linker ratio. For epichlorohydrin it increases with an increasing amount of cross-linker, which bucks the classical hydrogel trends. Also, hydrogels based on cellulosexanthan can be obtained. An increase in xanthan concentration permits an increase in the swelling of hydrogels [3]. Cellulose-based hydrogels can be obtained by radical polymerization of water-soluble cellulose derivatives with polymerizable groups [44]. Such hydrogels are characterized by high mechanical strength and stiffness, but also flexibility under different loads. Radicals can be formatted in situ of hydrogels by UV or visible light application in the presence of a photoinitiator. In the presence of hydrogel precursors bearing polymerizable groups, such as acrylate or methacrylate moieties, these formed hydrogels. The carboxymethyl cellulose (CMC)-methacrylate hydrogels present the controllable degradation rate, degree of swelling, and mechanical properties. Chemical cross-linking can be carried out with the use of epichlorohydrin, aldehydes and aldehyde-based reagents, urea derivatives, carbodiimides, and multifunctional carboxylic acids [3]. Cellulose can be mixed with chitosan and cross-linked by ethylene glycol diglycidyl ether [45], with sodium alginate, NaOH/urea solvent, and epichlorohydrin as cross-linker [46], with lignin in the presence of epichlorohydrin where the swelling ability is increased with an increasing lignin content [47]. Hydrogels with microfibrillated cellulose (MFC) incorporated in the alginate-based matrix cross-linked by pyrrole monomer by in situ polymerization were tested. The mechanical parameters were improved with a higher amount of TOMFC. Moreover, they were biocompatible and exhibited tunable swelling properties [48]. HPMC and sodium alginate were cross-linked by calcium ions [49]. The most used cellulose derivative as a natural component for hydrogels, due to its water solubility, low-cost, nontoxicity, and environmental friendliness, is CMC. The addition of CMC to cellulose in the NaOH/urea solution with cross-linker as epichlorohydrin contributed to the enhanced size of the pore, whereas cellulose was a strong backbone in the hydrogel to support it in retaining its appearance. Moreover, in different physiological fluids the hydrogels exhibited smart swelling and shrinking, as well as the release behavior of bovine serum albumin that could be controlled by changing CMC content [3].

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6.4 Agarose-based hydrogels Agarose-based materials can be projected and obtained as hydrogels, injectable hydrogels, self-healing hydrogels, scaffolds, and fibers. Agarose was also combined with other compounds, as well as its derivatives and blends, which have been already proposed. Agarose-based materials found application in tissue engineering, neurogenesis, cartilage formation spermatogenesis, wound healing, and as an artificial pancreas [50]. Agarose (copolymer of 3-linked β-D-galactopyranose and 4-linked 3,6-anhydro-α,Lgalactopyranose residues) is biocompatible polysaccharide which can be extracted from marine red algae. It can be then used to obtain thermal-reversible gel. Agarose can be also isolated from agar and its properties depend on the molecular weight of polymer. Agarose is polysaccharide which has high capacity to absorb water. It has a similar structure to the extracellular matrix, what supports cell proliferation and adhesion. Moreover, agarose has ionizable groups what result in the pH-responsive properties of polymer. The gelation process occurs in three steps, induction, gelation, and pseudoequilibrium, where the hydrogen bonds and other interactions form gel. The gelation process can be investigated by rheological methods and electron microscopy. Cross-linking can then be carried out without the need for a cross-linking agent in addition to the agarose. Hydrogels based on agarose can be obtained by agarose modification through a homogeneous reaction with acrylic monomers, and then by radical copolymerization [51]. The properties of hydrogels were modified by the addition of xanthan and resulted in the prevention of agarose gel aggregate formation [52]. The proposed hydrogels showed softer elasticity and a strong contribution to water molecules. The main factors influencing cross-linked materials are cross-linker type and concentration. Agarose-based hydrogels can be used as a cell immobilization matrix, drug-delivery system, or dental impression material.

6.5 Hydrogels from proteins Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. Proteins can be isolated from natural sources. They cannot be synthesized due to their complicated structure. Proteins are widely used to obtain hydrogels. They have hydrophilic groups present in the polymeric chain which can bind water molecules. Protein-based hydrogels have high ability to absorb water, which is beneficial for application possibilities. In this review hydrogels based on silk, keratin, and collagen are discussed.

164 Chapter 6

6.5.1 Silk-based hydrogels Silk is a natural protein present in the glands of silk-producing arthropods (such as silkworms, scorpions, mites, and bees) and spun into fibers. Silk for commercial use is isolated from silkworm silk, and is mainly produced by Bombyx mori [53]. Silk fibroin has several advantages over other proteins. The isolation process of silk is economically advantageous because silk is an established textile fiber. Silk fibroin can be isolated from wastes produced during silk fiber processing. Also, the purification process is carried out by simple methods, which makes silk available on a large scale. Silk-based hydrogels are eco-friendly and suitable to form biomaterials using different approaches [54]. They are currently receiving a great deal of interest for drug release due to their easy transformation from solution to gel form [55]. Silk-based hydrogels are formed through sol gel transition of aqueous silk solution. However, the sol gel transition time of a silk aqueous solution is long, usually a week or a month, which limits its practical use [56], therefore chemical and/or physical methods need to be applied to stimulate and enhance the gelation kinetics. Hydrogel formation can be carried out by different methods, such as organic solvent introduction, ultrasonification, vortexing, and pH changes. It has been illustrated that gel transformation of silk fibroin is possible by in situ gelation at a temperature 37 C due to self-assembly between the two different silk proteins [57]. Sol gel transition can be accelerated by increasing the temperature, protein concentration, and calcium ion addition. Silk fibroin forms a β-sheet structure which exhibits slow degradation in vitro and in vivo. It has to be cross-linked to improved its degradability and mechanical strength. In recent years silk-based hydrogels have been cross-linked by genipin and glutaraldehyde addition, and ionizing irradiation. Also, enzymatic methods have been applied including tyrosine [58]. Ionizing radiation is a method of hydrogel modification which uses gamma rays, electron beams, or ion beams. It result in radical formation on unsaturated polymer chains and water molecules, inducing intermolecular cross-linking. This proposed method eliminates the need for toxic cross-linking agent addition and is safe for the hydrogel preparation. Hydrogels from silk can have injectable and noninjectable forms. Inorganic particles can be incorporated into a three-dimensional hydrogel structure which improves their bioactivity [59,60]. Rapid cross-linking silk fibroin with surrounding tissue by an in situ method has attracted special attention and enhanced the application possibilities. Hydrogels based on silk fibroin were proposed as a drug-delivery system [55] or for the treatment of burn wounds [57].

The physical and chemical properties of hydrogels based on natural polymers 165

6.5.2 Keratin-based hydrogels Keratin is a fibrous protein which can be classified into two groups: soft keratin and hard keratin. It forms the bulk of cytoplasmic epithelia and epidermal structures. Keratin is abundant in animal hair, nails, wool, horns, and other features [8]. It may present in two conformations, α-helix and β-sheet. Compared to other proteins, keratin-based materials have higher stability and are not degraded by enzymes. Materials are mainly obtained from keratin isolated from wool and human hair [8]. The main source is wool, however, human-derivative keratin is of interest to researchers due to the reduced risk of an immune response. Keratin can be used in the synthesis of scaffolds for long-term cell culture. In hydrogels, keratin allows the formation of porous gel-form materials and creates a suitable environment for cell proliferation. They can be injected into a nerve conduit as a filter to guide nerve regeneration. Keratin-based materials are characterized by high strength and stability in in vivo conditions. They also accelerate cell regeneration and form compatible scaffolds. Keratin hydrogels can be easily rehydrated, which is desirable for invasive injection in prosthetics [61]. The properties of keratin-based hydrogels with collagen-based hydrogels cross-linked by the same cross-linker as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide have been compared [62]. It was assumed that keratin-based materials had a higher storage modulus and loss modulus. Also, the mechanical parameters for keratin without cross-linking were several-fold higher than those for cross-linked collagen. Generally, keratin-based hydrogels have appropriate features for biomedical purposes. Hydrogels obtained from keratin are relatively stable and have good mechanical strength, even without cross-linking modification. Also, the rehydrating methods are fast and easy, and very useful for commercial applications. It is possible to extend the storage of hydrogels by lyophilization. However, further studies of keratin-based hydrogels are needed.

6.5.3 Collagen-based hydrogels Collagen is the main protein in human connective tissues, accounting for 25% 33% of total proteins. It is constructed from repeating peptides (mainly proline, hydroxyproline, and glycine). There are 29 known types of collagen. Collagen I is a fibrous protein which is constructed of three α-chains intertwisting in a right-handed triple helix, stabilized by hydrogen bonds. Type I has been widely used to synthesize biomaterials, due to its abundance and well-understood structure from nano- to microscale. Collagen can be easily extracted from natural sources such as bovine skin or eyeballs, rat tail tendons, and fish scales.

166 Chapter 6 Collagen is widely used to obtain biocompatible, biodegradable, and safe materials including scaffolds, thin films, and hydrogels. The meat industry produces many by-products which contain collagen which can be extracted for further applications. The degree of cross-linking and molecular weight of collagen present in native tissues are significantly across the species. The source of collagen influences its thermal stability, mechanical parameters, solubility, and rheological properties [84]. Such factors play an important role in collagen-based material properties. The main sources of collagen include bovine hide, pig skin, shellfish, fish skin, and rat tendons. Most collagen-based hydrogels are prepared using type I collagen, and are ultimately formed in the presence of a water-based solvent. Along with the collagen content, the temperature of polymerization affects the hydrogel properties. Self-assembly of collagen molecules occurs more rapidly in higher temperatures, resulting in a less ordered structure. The room temperature is not uncommonly used but inhibits the inclusion of cells within hydrogel. Mostly previous studies have used a temperature of polymerization at around 37 C to facilitate cell seeding and viability [63]. Collagen-based hydrogels are sensitive to pH during the fabrication. This influences the structural and mechanical properties of collagen hydrogels. Mostly, hydrogels are formed with pH ranging from 5 to 10 and there is a strong positive correlation between pH and compressive modulus. The pH of collagen hydrogels depends on the type of buffer, the ratio of neutralization agent to acid-solubilized collagen, dilution ratio of collagen in hydrogel, and the soluble collagen concentration. Moreover, the ionic strength affects the collagen-based hydrogels polymerization. It also influences the mechanical and structural properties. Ionic strength is seldom measured directly for collagen hydrogels, rather it is calculated from known or estimated concentrations of all ionic compounds present in the solution [63]. Solubilized collagen must be storage at low pH and low temperature to prevent annealing of dissolved fragments. Hydrogels from collagen can be formed without adding any crosslinker. Fragments of dissolved collagen may aggregate and covalently bond together to reform fibrils in higher temperatures and pH. However, obtained hydrogels have weak mechanical properties, therefore cross-linker addition is necessary to enhance the mechanical strength of collagen-based hydrogels. Chemical cross-linking may also result in the improvement of resistance to degradation. The toxicity and safety of the cross-linking agent have to be considered. This is the reason more attention is now paid to natural cross-linkers [8]. Collagen implanted into the body can be recognized and bound by cells through integrin receptors, degrade collagen via specific enzymes, and synthesize new collagen. It is an important mechanism for tissue reconstruction to decide on the width of collagen application in medicine. However, collagen-based materials are characterized by low mechanical properties and rapid degradation rate, which limit their application [8].

The physical and chemical properties of hydrogels based on natural polymers 167 The kinetics of collagen fibril network assembly is a multistep process which includes fibril formation, fiber nucleation and development, and cross-linking, where the addition of external cross-linking agents affects fibrillogenesis [63]. For polymerization the ideal temperature and pH values have to be considered. Fibrillogenesis can be detected by spectrophotometric methods and allows determination of the degree of polymerization. The mechanical properties of collagen-based hydrogels have to be detected which implies matching both deformation modality and temporal characteristics. The viscoelastic properties can be measured by the deformation modalities including tension, compression, and torsion. However, it is difficult to assume that hydrogels have appropriate parameters for application in tissues, because tissues are in a state of constant change related to the physiological conditions. The settings and conditions of measurement can affect the obtained results. Hydrogels should maintain sample integrity by humidification or immersion in a buffer during measurement. Moreover, the collagen source and fabrication parameters influence the mechanical properties of obtained hydrogels. Recent studies have demonstrated the positive correlation between the collagen concentration and the elastic properties [64 66]. Also, increasing pH increases collagen-based hydrogel modulus [66 68]. The pore size of hydrogels determines the mechanical parameters. The addition of cross-linking agents increases the mechanical parameters of hydrogels as a result of new interactions/bond formation [69 71]. The fiber structure of extracellular matrices is an important quality of hydrogels because it can regulate their cellular morphology, migration, gene expression, and proliferation [70]. Parameters such as fiber diameter, density, and orientation influence material properties. Collagen fibers can be measured by different methods including atomic force microscopy, transmission electron microscopy, or scanning electron microscopy (SEM). Moreover, more advanced techniques can be used, such as cryo-SEM and environmental SEM, two-photon fluorescence, second harmonic generation, as well as confocal reflectance and fluorescence microscopy [72]. Collagen has been grafted to other polymers to form hybrid hydrogels, such as cellulose [73], chitosan [74], alginate [75], hydroxyapatite [44], and silver nanoparticles [76]. Irradiation cross-linking does not require any additives to start the process and the final product contains only polymer. Such a method modifies the hydrogel properties and can also act as a sterilizer. Thereby a one-step process reduces costs and production time. It is a tool in the fabrication of materials for biomedical applications [3].

6.6 Other natural polymers: lignin-based hydrogels Lignin is a phenolic polymer built up by three units: syringyl alcohol (S), guaiacyl alcohol (G), and ρ-hydroxyl alcohol (H). It is the second most abundant biopolymer. Lignin is a

168 Chapter 6 biocompatible, cheap, eco-friendly, and readily accessible material. Moreover, it is a biorenewable material and is available in large amounts as a by-product of forestry. Lignin shows also antimicrobial activities, enabling its biomedical applications. Lignin can be used in different fields due to its dispersing, binding, complexing, and emulsion-stabilizing properties. It can be used as an additive to animal feed, mesoporous materials, catalysts, or phenoplast glues. It can be also used to form organic hydrogels [77]. Hydrogels can be also obtained from lignin, which is a natural polymer, and is biorenewable and produced as a by-product of the forest industry [78]. They can be crosslinked by, for example, poly(methyl vinyl ether-co-maleic acid) [78]. Hydrogels were formed from lignin, with starch and hemicellulose by reactive extrusion [79]. Polymers were cross-linked by citric acid in the presence of a catalyst. The swelling behavior of the obtained hydrogels is highly dependent on the pH of the medium, where it increases at high pH values [79]. Other research includes hydrogel preparation from lignin with poly(ethylene glycol) and poly(methyl vinyl ether-co-maleic acid) through an esterification reaction [80].

6.7 Conclusion Hydrogels are widely studied materials for different industrial applications. They can be obtained from natural or synthetic polymers, which have hydrophilic groups. They have a high ability to swell. Nowadays, many natural polymers from polysaccharide and protein groups have been used for hydrogel preparation. Hydrogels can be obtained from pure polymeric chains by changing the temperature, pH, or solution. However, they are generally obtained in the cross-linking process. The type of polymer and modification procedure influence the hydrogel properties. Therefore it is necessary to detect their properties, such as mechanical parameters, thermal stability, degradation rate, and swelling behavior. It cannot be assumed which polymer is the best for hydrogel preparation, as each has advantages and disadvantages. Research into hydrogel materials is still in progress and are one of the most studied nanomaterial areas.

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CHAPTER 7

The stimuli-responsive properties of hydrogels based on natural polymers Miheng Dong and Yu Chen* School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, China

7.1 Introduction Stimuli-responsive hydrogels change properties according to the external environment. Commonly, hydrogels respond to triggers such as chemical stimulation: pH [1
3], oxidants [4,5], ions [6,7]; physical stimulation: temperature [8,9], electric fields [10], magnetic fields [11], and biostimulation: enzymes [12]. Common natural polymers include alginate, chitosan, starch, dextran, glucan, gelatin, and cellulose [13]. Advanced biocompatibility and biodegradability are common advantages while insufficient mechanical properties are disadvantages of these natural materials [13]. Table 7.1 details the properties of natural materials. Potential applications cover medication, agriculture, environmental protection, and research support (Fig. 7.1). In this chapter, the advancements in stimuli-responsive natural polymer-based hydrogels in recent years are discussed. Topics are categorized by the different types of responses to pH, magnetic, thermal, and multistimulation. In each specific topic, the mechanism of stimuli response and the effects of natural polymers will be introduced.

7.1.1 pH-responsive natural polymer-based hydrogels pH-responsive hydrogel is a hot topic in recent research [18
20]. Ionizable polymers with a pKa value from 3 to 10 are generally considered as proper candidates for pH-responsive hydrogel [21]. The main in vivo applications of this type of hydrogel are to control the release of drug in specific organs, where the environment changes due to pathological situations or intracellular compartments [6,22]. Modification of natural polymers is generally required for pH-responsive applications. The existence of abundant hydroxyl groups on the surface of cellulose leads to strong 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00007-0 © 2020 Elsevier Inc. All rights reserved.

173

174 Chapter 7 Table 7.1: Properties of natural polymers. Type

Properties

Alginate

Biocompatible and biodegradable polymer; suitable for in situ injection; cross-linking is under very mild conditions; water-soluble polymer; mechanical weakness; difficulties in handling, storage in solution, and sterilization [13]. Excellent biocompatibility and good host response; unique biodegradability by lysozyme and other enzymes; high antimicrobial activity; hydrophilic surface provides easy cell adhesion, proliferation, and differentiation; mechanical weakness; very viscous polymer solution; water-soluble polymer only in acetic medium; high purification cost [13]. Water-soluble polymer; inexpensive; in vivo biodegradable; biocompatible; easy to modify with other polymers; difficulties in cross-linking itself; mechanical weakness; needs modification to enhance cell adhesion [13]. Water-soluble polymer; in vivo biodegradable by α-amylase; biocompatible; good proliferation and differentiation behavior; expensive polymer; mechanical weakness; needs modification to enhance cell adhesion [13]. Water-soluble polymer, but yeast-glucan is not soluble in water; biocompatible/ biodegradable polymer; has excellent antibacterial and antiviral activities; fast wound-healing rate [13]. Water-soluble polymer; obtained from various animal by-products; forms thermally revisable and high mechanical hydrogels; widespread in biomedical application; easily forms films and matrix hydrogels; very viscous polymer solution; very fast biodegradation; lower thermal stability at high temperatures [13]. Most abundant renewable source in nature; hydroxyl groups with tuneable properties [14]; prepared from wood fibers; might contain lignin and foreign bodies; biocompatibility, biodegradability, and mechanical properties [15]; Applications in nanocomposite materials, biomedical devices, and adsorbents [16]. Prepared by vinegar bacteria; alternative source for pure cellulose [17].

Chitosan

Starch

Dextran

Glucan

Gelatin

Cellulose

Bacterial cellulose

hydrogen bonding interactions between crystal and hydrogel polymers [23]. However, hydroxyl groups do not respond to pH variations and limit the application of cellulose in pH-responsive materials. To fabricate pH-responsive hydrogels, cellulose is generally functionalized with the tertiary amine, amidine, guanidine, imidazole, and carboxylic acid groups via surface modification while maintaining its original crystalline structure [23
25]. Cellulose-based pH-responsive hydrogels can be generally categorized into two types: (1) pH-responsiveness introduced by the polymers instead of cellulose; (2) groups formed by modified cellulose and other precursors that respond to pH stimulations. As demonstrated in Fig. 7.2, in either type, the major response is introduced by the protonation carboxyl groups forming hydrogen bonding causing a reduction in swelling or the deportation of carboxyl groups increasing repulsion in hydrogel networks which enhance the swelling property. Anime groups have a reverse response to pH variations. For chitosan, carboxymethyl chitosan is an important derivative with carboxylate and amine pendants enabling drug delivery [26].

The stimuli-responsive properties of hydrogels based on natural polymers 175

Figure 7.1 Prospects for applications and developments of natural polymer-based hydrogels [14]. Reprinted with permission from Chang, C., Zhang, L., 2011. Cellulose-based hydrogels: present status and application prospects. Carbohydr. Polym. 84(1), 40
53. Copyright 2011 Elsevier.

Figure 7.2 pH-responsive mechanism of functionalized cellulose nanocrystals (CNCs) with either carboxylic acid (CNC
CO2H) or amine (CNC
NH2) moiety renders the CNCs [27]. Reprinted with permission from Way, A.E., Hsu, L., Shanmuganathan, K., Weder, C., Rowan, S.J., 2012. pH-responsive cellulose nanocrystal gels and nanocomposites. ACS Macro Lett. 1(8), 1001
1006. Copyright 2012 American Chemical Society.

176 Chapter 7

Figure 7.3 Scheme of chitosan-graft-poly(acrylic acid)/cellulose nanofibril hydrogel composite formation [28]. Reprinted with permission from Spagnol, C., Rodrigues, F.H.A., Pereira, A.G.B., Fajardo, A.R., Rubira, A.F., Muniz, E.C., 2012. Superabsorbent hydrogel composite made of cellulose nanofibrils and chitosan-graftpoly(acrylic acid). Carbohydr. Polym.87(3), 2038
2045. Copyright 2012 Elsevier.

7.1.1.1 pH-responsive polymers with properties enhanced by natural polymers In this type, natural polymer is used as a material to enhance mechanical properties and enable tunable anisotropic properties. The pH responsibility is introduced purely by the original polymer networks. Natural polymers displayed the feasibility of altering the network structure for enhanced mechanical properties in pH-responsive hydrogels. Spagnol et al. synthesized chitosan-graft-poly (acrylic acid) copolymer with cellulose nanofibrils. The hydrogel matrix cross-linked by chitosan and acrylic acid (AA) is shown in Fig. 7.3. By adding nanofibrils, the increase the averaged porous dimensions was confirmed by SEM characterization. Compared with the chitosan-graft-poly(acrylic acid) hydrogel, which has a water absorption capacity of 381 gwater/gabsorbent, cellulose improved water uptake to 486 gwater/gabsorbent and shortened the time to reach equilibrium. Compared with the hydrogel without nanofibrils, the cellulose-based hydrogel showed significantly enhanced water absorption capability, especially at pH . 6. At pH 5 8, the optimized hydrogel absorbency reached its maximum and started to decrease with an increase in pH. This decrease was attributed to the excess of negatively charged groups in the media, causing
COO2 to recombine back to
COOH, which resulted in the decrease in repulsion of polymer chains [28]. Apart from conventional isotropic swelling of hydrogels, Milani and co-workers recently published an approach to fabricate pH-triggered anisotropic swelling hydrogels via low shear (0.1 s21) gel formation. As illustrated in Fig. 7.4, they mixed vinyl-functionalized nanogels (NGs) with copolymer worms (W), β-sheet peptides (PP), or nanocrystalline cellulose (NCC). Each group of hydrogels contained a different type of aligned rod-like

The stimuli-responsive properties of hydrogels based on natural polymers 177

Figure 7.4 Preparation of aligned doubly cross-linked NG/RLP gel composites (DX NGx/RLP12x) [29]. Reprinted with permission from Milani, A.H., Fielding, L.A., Greensmith, P., Saunders, B.R., Adlam, D.J., Freemont, A.J., et al., 2017. Anisotropic pH-responsive hydrogels containing soft or hard rod-like particles assembled using low shear. Chem. Mater. 29(7), 3100
3110. Copyright 2017 American Chemical Society.

particles (RLPs). The anisotropic swelling property was measured by comparing the change in length at both the collapsed state (pH 5 5) and the swelling state (pH 5 8). The elongation in parallel with the shear force is smaller than the strain in perpendicular to the shear force. This phenomenon was attributed to the restriction caused by the intra-RLP bonding and was strongest for the RLPs with high τ break values and high RLP percentage in the hydrogel. In this case, doubly cross-linked (DX) NGx/NCC1
x showed weaker anisotropic property than hydrogel with W. However, due to the ultra-high τ break value of NCC, the cellulose-based hydrogel presented strong anisotropic mechanical properties. More interestingly, all three types of hydrogels exhibited strain-induced birefringence, which could be potentially applied as strain sensors [29]. Cellulose used in hydrogels could change the porous structure and tune the polymer’s capability to adsorb heavy metal ions. Wang’s research group reported collagen/cellulose hydrogel beads (CCHBs) prepared by reconstitution from a 1-butyl, 3-methylimidazolium chloride ([C4mim]Cl) solution. With an increase in the collagen mass ratio, the beads showed a rapid increase in the equilibrium adsorption capacity (qe) until the collagen/ cellulose mass ratio reached 2/1. Mass ratios of 2/1 and 3/1 presented relatively stable BET surface areas (54.1 m2/g of CCHB2 and 43.2 m2/g of CCHB3) indicating good porous structure. Compared to the CCHB1 (mass ratio 1/1), the aggregates also decreased (Fig. 7.5). The adding of collagen did not only increase the adsorption of beads, it also showed a steady increase in the adsorption of Cu(II) with the increase in pH to 6. The

178 Chapter 7

Figure 7.5 SEM images of the surface of CHBs (A), CCHB1 (C), CCHB2 (E), CCHB3 (G), and cross-sections of CHBs (B), CCHB1 (D), CCHB2 (F), and CCHB3 (H) [30]. Reprinted with permission from Wang, J., Wei, L., Ma, Y., Li, K., Li, M., Yu, Y., et al., 2013. Collagen/cellulose hydrogel beads reconstituted from ionic liquid solution for Cu(II) adsorption. Carbohydr. Polym. 98(1), 736
743. Copyright 2013 Elsevier.

The stimuli-responsive properties of hydrogels based on natural polymers 179 deprotonation of hydroxyl groups and amino groups significantly enhanced Cu(II) ions pairing to form a complex as shown in the equations below. However, based on previous reports, chitosan-based hydrogel adsorption would have decreased after reaching the adsorption maximum value. Moreover, the desorption efficiency (Ds) of CCHB1 (collagen/ cellulose mass ratio 1/1) showed over 95% desorption after four test cycles, indicating good reusability [30]. Possible chemical reactions for Cu(II) adsorption on the CCHB or CHB bead [30]: 21 21 1 H1 R 2 OH1 2 1 Cu -R 2 OHCu 21 21 R 2 NH1 1 H1 3 1 Cu -R 2 NH2 Cu

7.1.1.2 Modified natural polymer network-introduced pH responses A functionalized pH-responsive polymer network based on cellulose and other natural materials shows great biocompatibility. The control release mechanisms are categorized into two main strategies: conformational and/or solubility change of polymers in response of pH variation; and the pH-sensitive bonds for cleavage that enable the release of anchored molecules, the exposure of targeting ligands, and charge modification of the polymer [22]. Cha and co-workers recently reported pH-responsive hydrogel by gelatinizing carboxylated nanocrystalline cellulose (CNCC) and poly(N-isopropylacrylamide) (PNIPAAm). Scanning electron microscopy (SEM) confirmed that CNCC was integrated as a part of the hydrogel network. With the increase of CNCC amount in the hydrogel, it showed a higher equilibrium swelling ratio (ESR) and faster swelling then the pH-influenced swelling which was invisible in pure PNIPAAm gels. These features resulted from the increase of carboxyl groups introduced by CNCC forming more hydrogen bonds. Hydrogels mixed with 10 mL 5 wt.% CNCC and 15 mL H2O showed 17.8% ESR at pH 5 7 and 24 C. Moreover, with the increase in the CNCC ratio in hydrogels, the yield strain and ultimate strain greatly improved, proving the CNCC could enhance the mechanical properties of hydrogels [31]. Akar et al. reported another biodegradable hydrogel synthesized by sodium carboxymethyl cellulose (NaCMC) using fumaric acid (FA) as a cross-linking agent that showed similar properties. At a static pH value, hydrogels with higher NaCMC ratio generally showed higher ESR. It was found that in low-pH solutions, with the increase of cross-linker agent, the decrease in water absorption capability is more apparent [32]. Pandey et al. reported a bacterial cellulose (BC)/acrylamide (Am) hydrogel system through microwave irradiation (Fig. 7.6). To increase the dissolution of BC in the alkali solution, NaOH/eura mixed solution was used. This drug-controlled release system showed noncytotoxicity and biocompatibility during in vitro cytotoxicity and hemolytic tests. Acute toxicity tests on ICR mice also indicated that the hydrogels were not toxic up to

180 Chapter 7

Figure 7.6 Proposed reaction scheme of BC/Am hydrogels synthesis [33]. Reprinted with permission from Pandey, M., Mohamad, N., Amin, M.C.I.M., 2014. Bacterial cellulose/acrylamide pH-sensitive smart hydrogel: development, characterization, and toxicity studies in ICR mice model. Mol. Pharm. 11(10), 3596
3608. Copyright 2014 American Chemical Society.

2000 mg/kg oral dosage. With no observation of toxic response or histopathological changes compared with the control group (equivalent amount of normal saline), the BC/Am system demonstrated BC-based hydrogels were a candidate for oral drug-delivery vehicles. They synthesized a number of hydrogels with different concentrations of NaOH (% w/v)

The stimuli-responsive properties of hydrogels based on natural polymers 181 and urea (% w/v). All BC/Am hydrogels generally presented the property to swell from pH 2 to 7. After reaching maximum swelling degree (SD%) at pH 5 7, the SD% started to decrease with an increase of pH up to 10. Apart from the pH response, the BC/Am hydrogel also showed a property to swell with an increase in temperature from 4 C to 45 C. During the drug-releasing testing two types of tested hydrogels showed a better drugreleasing feature in pH 7.4 buffer than in pH 1.5 buffer. Interestingly, the hydrogel synthesized with 8% w/v NaOH and 4% w/v urea showed high drug entrapment efficiency (54.03% 6 4.80%) and drug loading (81.56% 6 3.21%). Due to the greater number of carboxylic groups and greater porosity, this type of hydrogel showed a higher cumulative drug release percentage [33]. Bai’s group in 2012 reported microgels prepared via emulsion polymerization for in vitro insulin pH-controlled release. Poly(L-glutamic acid-2-hydroxylethyl methacrylate) (PGH) and hydroxypropyl cellulose-acrylic acid (HPC-AA) were used as precursors for synthesis, as shown in Fig. 7.7. New microgels displayed pH-triggered swelling property due to the transformation of poly(L-glutamic acid) from a hydrophobic protonated form to the hydrophilic deprotonated form near its pKa. Microgels also showed a reduction in maximum particle size with the increase in temperature. During insulin drug-release testing, various types of hydrogels formed with different PGH/HPC-AA ratios were investigated in artificial gastric juice (pH 5 1.2) and artificial intestinal liquid (pH 5 6.8) at 37 C. The result showed insulin release in low pH is much less than in high pH. With the increased amount of HPC-AA in the polymer, hydrogels presented less swelling capability and slower drug release in the high pH solution. Therefore the authors concluded that HPC-AA (15 mg) and PGH (35 mg) was the best formula for insulin intestinal delivery in their research [34].

Figure 7.7 Synthetic route of poly(L-glutamic acid-2-hydroxylethyl methacrylate)/hydroxypropyl celluloseacrylic acid (PGH/HPC-AA) microgels [34]. Reprinted with permission from Bai, Y., Zhang, Z., Zhang, A., Chen, L., He, C., Zhuang, X., et al., 2012. Novel thermo- and pH-responsive hydroxypropyl cellulose- and poly (L-glutamic acid)-based microgels for oral insulin controlled release. Carbohydr. Polym. 89(4), 1207
1214. Copyright 2012 Elsevier.

182 Chapter 7

Figure 7.8 Synthetic route of acrylic acid/bacterial cellulose (AA/BC) hydrogels.

Amin et al. synthesized similar pH-responsive hydrogel via polymerization between AA and BC. As shown in Fig. 7.8, the irradiation process converted the water molecules into reactive species which later created active sites on AA and BC for grafting. The intermolecular 1,5 H-shift had been confirmed in electron-pulse radiolysis investigations of acrylates and could proceed to further grafting. Both steps were enabled under the exposure of accelerated electron-beam irradiation. With a higher dose of irradiation, more reactive species would be produced by radiolysis water, causing a higher degree of cross-linking. Hydrogels with a higher amount of cellulose presented better swelling properties in nearneutral solutions. The addition of BC also led to an increase in the pore size and the highest BC/AA ratio hydrogels could reach 90% release of bovine serum albumin within 8 hours, which was higher than the other groups [35]. Zheng et al. prepared pH-responsive EDTA-Ca-alginate for an oral drug-delivery system. In pH below 4.0, the Ca21 was released from the EDTA-Ca compound forming alginate
Ca binding. The gelation process could form hydrogel microspheres. Encapsulation of Lactobacillus rhamnosus ATCC 53103 by the emulsification/internal gelation technique has proved that microspheres can effectively encapsulate and protect target cells against the acidic environment in the stomach. It could also enable intestinal-targeted rapid release as the microspheres dissolve in a neutral environment [36].

The stimuli-responsive properties of hydrogels based on natural polymers 183

Figure 7.9 Synthesis scheme of the N-carboxyethyl chitosan (CEC) polymer (A), dibenzaldehyde-terminated poly(ethylene glycol) (PEGDA) polymer (B), CEC/PEGDA hydrogel (C), and schematic illustration for preparing the Dox-loaded CEC/PEGDA hydrogel (D) [37]. Reprinted with permission from Qu, J., Zhao, X., Ma, P.X., Guo, B., 2017. pH-responsive self-healing injectable hydrogel based on N-carboxyethyl chitosan for hepatocellular carcinoma therapy. Acta Biomater. 58, 168
180. Copyright 2017 Elsevier.

Qu et al. synthesized pH-responsive and self-healing injectable hydrogels using Ncarboxyethyl chitosan (CEC) via Michael reaction and dibenzaldehyde-terminated poly (ethylene glycol) (PEGDA) (Fig. 7.9). Hydrogels were loaded with doxorubicin (Dox) to test the feasibility for hepatocellular carcinoma therapy. The self-healing property of the hydrogels could prolong the lifetime of loaded drugs, protect normal tissues, and reduce the discomfort of patients. The extracellular pH (pH 5 5.7
7.8) of most cancer tissues is lower than that of the bloodstream (pH 5 7.4). The pKa value of D-glucosamine residue of chitosan is about 6.2
7.0. In the low pH environment, amino groups of chitosan are protonated, weakening the Schiff base bonding
CHO and
NH2. Decomposition of crosslinked CEC/PEGDA leads to the release of Dox [37].

184 Chapter 7

Figure 7.10 Schematic representation of the pH-responsive shape memory materials: (A) PECU/CNC
CO2H, (B) PECU/CNC
C6H4NO2.

Cellulose-grafted polymers also enabled shape memory, which is an important quality of stimuli-responsive materials. Li’s research group reported pH-responsive shape memory hydrogels containing functionalized cellulose nanocrystals (CNCs), as illustrated in Fig. 7.10. Poly(ethylene glycol)
poly(ε-caprolactone)-based polyurethane (PECU) was blended with CNC
CO2H or CNC
C6H4NO2 to enable shape recovery in base or acid, respectively. The pH sensitivity mechanism of PECU/CNC
C6H4NO2 hydrogel was based on the damage of H-bonding interactions in acid. After deformation of the hydrogel, the pyridine on the surface of CNC
C6H4NO2 was deprotonated, causing rebuilding of Hbonding and the shape was memorized accordingly. In the shape recovery stage, the hydrogel was immersed in acid again. With protonation of pyridine, the H-bonding disappeared and the original shape was recovered. The shape-memory property of PECU/ CNC
CO2H was similar except the acid and base were exchanged. Throughout the testing, researchers found that hydrogel with 20 wt.% functionalized CNC showed best shape fixity (Rf . 85%) and shape recovery (Rr . 85%). This phenomenon was attributed to the

The stimuli-responsive properties of hydrogels based on natural polymers 185

Figure 7.11 Properties of poly(vinyl alcohol) (PVA)-borax hydrogels [39]. Reprinted with permission from Lu, B., Lin, F., Jiang, X., Cheng, J., Lu, Q., Song, J., et al., 2017. One-pot assembly of microfibrillated cellulose reinforced PVA
borax hydrogels with self-healing and pH-responsive properties. ACS Sustain. Chem. Eng. 5 (1), 948
956. Copyright 2016 American Chemical Society.

enhanced mechanical property (strength, modulus, and rigidity) caused by the CNC. After six cycles of shape memory, Rf remained over 80%, while the Rr reduced to 65%. Hydrogels kept good repeatability although the orientation of modified CNC changed along stretch direction and influenced the Rr [38]. Self-healing could also be enabled in pH-responsive hydrogels using cellulose-grafted polymerization. Lu et al. reported a one-pot synthesis procedure using milled cellulose pulp to reinforce poly(vinyl alcohol) (PVA)-borax hydrogels with self-healing property (Fig. 7.11). The didiol-borax dynamic equilibrium linkages and cellulose fibers enhanced the self-healing and mechanical properties of the hydrogel system. PVA-borax hydrogels reinforced by 3.0 wt% MFC (PB-MFC-3.0) had a higher initial degradation temperature at 331 C compared to pure PB polymer (280 C). Moreover, the DGT curves showed that peak thermal decomposition with maximum weight loss of PB-MFC-3.0 was 366 C compare to neat PB (312 C). The hydrogen bonding of B(OH)42 and OH groups in PVA/ MFC enabled the network formation. Due to this mechanism, the interchain dynamic didiol
borax complexation showed both a pH-responsive property and self-healing properties. Hydrogels demonstrated reversible sol
gel transfer when added to HCl solution (pH 5 3) and NaOH solution (pH 5 11). The self-healing property was probably attributed to the facilitation of hydrogen bond reformation due to the flexibility and hydrophilic nature of polymer and the reversible didiol
borax complex network. With the reformation of hydrogen bonds across the interface and a reversible didiol
borax complex in the network,

186 Chapter 7 after contact of two halves pieces of hydrogels for 10 minutes, the recovered hydrogels exhibited Gv at around 2016 Pa, which was comparable to the original unbroken hydrogel’s value (B2030 Pa) [39].

7.1.2 Magnetic-responsive natural polymer-based hydrogels With the development of industry and scientific technology, the removal of toxic metals contamination has attracted increasing attention [40]. The conventional physical and chemical removal methods include chemical precipitation, membrane separation, ion exchange, evaporation, and electrolysis [41]. Hydrogel adsorption has become a new hot topic in recent years. However, conventional hydrogels could only be separated from the solution by high-speed centrifugation of filtering [42]. To overcome the limitation, a number of researchers has begun to develop magnetic-responsive hydrogel systems to enable quick magnetic recovery with distinct force [41,43]. Magnetic-responsive hydrogels generally use Fe3O4 and γ-Fe2O3 during synthesis to form gels. With this special property, hydrogels can be easily separated from a mixed system by applying external magnetic fields. A significant possible application is reusable materials for chemical enrichment. The use of natural polymers for gelation backbones could also maintain the biocompatibility of hydrogels. Magnetic and other stimuli-responsive systems have great potential in tissue engineering and cell/drug delivery [44]. In this section, two types of magnetic hydrogels are introduced: (1) natural polymer as coatings for magnets; and (2) natural polymers and magnets grafted into polymer networks. 7.1.2.1 Natural polymers as coatings for magnets In most approaches, cellulose-coated magnets could have higher resistance to an acidic environment and better mechanical performance. The carboxyl groups of modified cellulose or hydroxyl groups of cellulose could change microsphere charges or improve thermal stability, respectively. A widely used combination of coating materials was chitosan and cellulose in which chitosan was used to adsorb heavy metal ions. An exceptional example is also introduced which used cellulose as the core material and Fe3O4 as the coating material for hydrophobic applications. Liu et al. coated cellulose and chitosan onto Fe3O4 core to fabricate hydrogels with less tendency to aggregation and oxidation, as shown in Fig. 7.12. The cellulose and chitosan were first codissolved into ionic liquids (ILs) and then used to coat the magnetite particles. The saturation magnetization (σs) of magnetic hydrogel was 14.7 emu/g compared to naked Fe3O4 (82.5 emu/g). The magnetic hybrid hydrogels exhibited selective affinity to Cu21, Fe21, and Pb21. The equilibrium adsorption capacity compared to dried absorbent weight were 44.7 6 5, 94.1 6 7, and 28.1 6 3 mg/g, respectively. Meanwhile, hydrogels showed little affinity to Mn21, Zn21, and Ni21. Selectivity could be explained as Cu21 and Pb21

The stimuli-responsive properties of hydrogels based on natural polymers 187

Figure 7.12 Scheme of the preparation of magnetic chitosan
cellulose hydrogels and adsorption of heavy metals [45]. Reproduced from Liu, Z., Wang, H., Liu, C., Jiang, Y., Yu, G., Mu, X., et al., 2012. Magnetic cellulose-chitosan hydrogels prepared from ionic liquids as reusable adsorbent for removal of heavy metal ions. Chem. Commun. 48(59), 7350
7352 with permission from The Royal Society of Chemistry.

could form a metal-chelate with chitosan and Fe21 could be hydrolyzed and oxidized to Fe (OH)3, which could be absorbed by hydrogels. The adsorption of this hydrogel to Cu21 is comparable with previously reported research. Blank acidic solution immersion testing also proved that hybrid particles had good stability. Fifty milligram hydrogels were immersed in HCl (pH 5 1) solution for 24 hours with no Fe21 or Fe31 detected in the following inductively coupled plasma-optical emission spectrometry testing [45]. Luo et al. synthesized magnetic chitosan/cellulose microspheres using a different approach. Magnetic γ-Fe2O3 nanoparticles were embedded in chitosan/cellulose matrix drops in NaOH/urea aqueous solution to obtain hybrid hydrogel particles called MCCM. At 298K, all samples presented ultrasmall hysteresis loop and low coercivity, indicating the core was not influenced by the coating process. During heavy metal ion adsorption testing, MCCM showed an order of selectivity: Pb21 . Cd21 . Cu21. The equilibrium adsorption capacity qe (mg/g) of Pb21 was around twice as high as the qe of Cr21 and about three times as high as the qe of Cu21. MCCM also exhibited good regenerability. After 10 cycles, the highest efficiencies of Pb21, Cd21, and Cu21 were still 85%, 89%, and 92%, respectively [46]. As illustrated in Fig. 7.13, Zhu and coworkers reported magnetic hydroxypropyl celluloseg-poly(acrylic acid) porous spheres prepared via oil in water Pickering high internal phase emulsions (HIPEs) integrated precipitation polymerization. Mixed solvent of P-xylene, hexamethylene, and modified Fe3O4 stabilizing particles was used to prepare Pickering HIPEs. The modified Fe3O4 and cosurfactant, the proportion of the mixed solvent, and the volume of the disperse phase were parameters that could impact the porous structure of magnetic spheres. Adding cosurfactant could increase the dispensability of Fe3O4
MNPs
M in an emulsion. With the increase of Fe3O4-MNPs-M, the stronger magnetic property could be achieved with a reducing stability of HIPEs and an increase in pore size. In this experiment, 1.0% of Fe3O4-MNPs-M was used as the stabilizer. After organosilicon coating, the saturation magnetization of hydrogel beads dropped from 60.34 to 33.06 emu/g. Nevertheless, the beads could still be effectively separated from the

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Figure 7.13 Synthetic route of the interconnected magnetic porous spheres for removal of heavy metal [47]. Reprinted with permission from Zhu, Y., Zheng, Y., Zong, L., Wang, F., Wang, A., 2016. Fabrication of magnetic hydroxypropyl cellulose-g-poly(acrylic acid) porous spheres via pickering high internal phase emulsion for removal of Cu21 and Cd21. Carbohydr. Polym. 149, 242
250. Copyright 2016 Elsevier.

solution by applying an external magnetic field. During heavy ion adsorption testing, the adsorption equilibrium was reached within 40 minute and the maximal adsorption capacities were 300.00 and 242.72 mg/g for Cd21 and Cu21, respectively. In addition, the beads maintained a strong adsorption capacity after five consecutive adsorption
desorption cycles, suggesting good reusability [47]. Apart from cellulose as the coating material for magnets, Lin’s group synthesized magneticresponsive hydrophobic microspheres using cellulose as the building block coated with Fe3O4 and then poly(DOPAm-coPFOEA), as illustrated in Fig. 7.14. The cellulose microspheres were first prepared via the sol
gel process. With the presence of electron-rich oxygen atoms on the hydroxyl group of cellulose, Fe3O4 was deposited due to the electrostatic interactions. Then, N-(3,4-dihydroxyphenethyl)acrylamide (DOPAm) containing 1,2-dihydroxybenzene (catechol) groups anchored onto the Fe3O4 nanoparticle surface due to its stronger chelating ability with iron atoms. Finally, the PFOEA on the outmost surface resulted in a low surface energy. Compared with conventional PTFE microspheres, poly (DOPAm-co-PFOEA)/Fe3O4/cellulose microspheres could maintain spherical shape and nonwetting to solid surfaces when contacted with solvents with low surface energy such as toluene and ethanol. A digital camera also proved that liquid marble could be easily driven by an external magnet bar in either the vertical or horizontal direction [48].

The stimuli-responsive properties of hydrogels based on natural polymers 189

Figure 7.14 (A) Scanning electron microscopy (SEM) image of a magnetic superhydrophobic poly(DOPAmcoPFOEA)/Fe3O4/cellulose microsphere. The inset is a magnified view of the surface of a microsphere. (B) Schematic illustration of the hierarchically structured microsphere [48]. Reproduced from Lin, X., Ma, W., Wu, H., Cao, S., Huang, L., Chen, L., et al., 2016. Superhydrophobic magnetic poly(DOPAm-co-PFOEA)/Fe3O4/cellulose microspheres for stable liquid marbles. Chem. Commun. 52(9), 1895
1898 with permission from The Royal Society of Chemistry.

7.1.2.2 Natural polymer and magnets grafted into polymer networks Natural polymer and magnets can also be grafted into polymer networks. Apart from the chitosan
cellulose magnetic adsorbents, this approach could prepare biofriendly hydrogels for enzyme immobilization and hyperthermia ablation therapy. Zhou’s research group prepared adsorbent magnetic beads [magnetic-chitosan/poly(vinyl alcohol)/carboxylated cellulose nanofibrils (m-CS/PVA/CCNFs)] by polymerizing Fe3O4 into gelation networks with carboxylated cellulose and chitosan. As illustrated in Fig. 7.15, magnetite nanoparticles and carboxylated cellulose nanofibrils were first prepared and then gelatinized in the presence of PVA and chitosan. Cellulose used in the process could enhance the thermal stability by its strong hydroxyl bonding with chitosan or PVA. In addition, the
COO2 groups in high pH solution significantly increased the electrostatic interaction between adsorbent and metal ions. This feature was proved by Pb(II) adsorbent testing showing that from pH 3 to 4.5, the m-CS/PVA/CCNFs beads had a much higher removal percentage than m-CTS/PVA. However, when the pH continued to increase, the adsorption rate decreased, which might be caused by the Pb(II) ions formed as complex polynuclear species and protonation of the amino groups in chitosan would lead to aggregation [49]. Duan’s research group prepared magnetic cellulose
TiO2 for selective enrichment of phosphopeptides. Magnetic cellulose microspheres (MCMs) were first prepared by sol
gel transition of cellulose
Fe3O4 colloidal microdroplets under microwave heating. Then MCMs were dispersed into TiO2 precursor solution and in situ synthesized TiO2 nanoparticles into porous structure of MCMs to prepare magnetic cellulose
TiO2 nanocomposite microspheres (MCTiMs). MCTiMs showed a decrease in magnetic

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Figure 7.15 Proposed mechanistic pathway for the preparation of (A) magnetite nanoparticles, (B) CCNFs, and (C) m-CS/PVA/CCNFs hydrogels [49]. Reprinted with permission from Zhou, Y., Fu, S., Zhang, L., Zhan, H., Levit, M.V., 2014. Use of carboxylated cellulose nanofibrils-filled magnetic chitosan hydrogel beads as adsorbents for Pb(II). Carbohydr. Polym. 101, 75
82. Copyright 2013 Elsevier.

hysteresis curve but still enabled efficient separation in external magnetic fields. As shown in Fig. 7.16, the phosphopeptides could be effectively bonded to the nanosphere via Lewis acid-base reaction and could be effectively separated. Matrix-assisted laser desorption/ ionization mass spectra of the tryptic digests of β-casein and human serum samples proved that the enrichment was successful and effective [50]. Wang’s research group prepared hydroxypropyl methylcellulose/Fe3O4 injectable hydrogels for magnetic hyperthermia ablation of tumors. In this approach, Fe3O4 was used to convert electromagnetic energy into heat, while cellulose provides hydroxypropyl methylcellulose (HPMC)/Fe3O4 to limit the leakage of materials into other organs and enable biodegradation. The thermal contractibility under an alternating magnetic field could also minimize invasiveness to normal tissue. This feature was attributed to the hydrophobic interactions between the cage-like hydrogen bonds of water and the methoxy groups on the polymer chains of HPMC. As shown in Fig. 7.17, when the hydrogels were heated, the tail-like structure disappeared, and the tumors disappeared with the degradation of hydrogels. With the increase of Fe3O4 content in gels, the polymer showed a faster response to the magnetic hyperthermia analyzer. During in vitro biosafety testing, normal human smooth muscle cells were used. From 50% to 90% Fe3O4 groups, the cell viability decreased but no statistically significant difference was observed at each developmental

Figure 7.16 Schematic illustration of (A) the mechanism using magnetic cellulose
TiO2 nanocomposite microspheres (MCTiMs) for phosphopeptide enrichment and (B) the typical process of selective enrichment of phosphorylated peptides by using MCTiMs and magnetic separation [50]. Reproduced from Duan, J., He, X., Zhang, L., 2015. Magnetic cellulose-TiO2 nanocomposite microspheres for highly selective enrichment of phosphopeptides. Chem. Commun. 51(2), 338
341 with permission from The Royal Society of Chemistry.

Figure 7.17 Injectable and thermally contractible hydroxypropyl methylcellulose (HPMC)/Fe3O4 for the magnetic hyperthermia ablation of tumors [51]. Reprinted with permission from Wang, F., Yang, Y., Ling, Y., Liu, J., Cai, X., Zhou, X., et al., 2017. Injectable and thermally contractible hydroxypropyl methyl cellulose/Fe3O4 for magnetic hyperthermia ablation of tumors. Biomaterials 128, 84
93. Copyright 2017 Elsevier.

192 Chapter 7 stage of the cell cycle (P.0.05). In vivo therapeutic testing on the MB-231 human breast cancer xenograft model proved that the heating temperature of HPMC/Fe3O4 could exceed 60 C, which is above the threshold for cell death or coagulation necrosis (around 47 C). Coagulation necrosis of the ablated tumor could be observed compared with the control group (mice received HPMC/Fe3O4 without exposure to the alternating magnetic field) [51].

7.1.3 Thermal-responsive natural polymer-based hydrogels Thermoresponsive polymers exhibiting a lower critical solution temperature (LCST) form a gel above the critical temperature and this process is usually reversible. For additionally cross-linked hydrogels, increased swelling is observed at temperatures below the LCST. In contrast, polymers with an upper critical solution temperature (UCST) stay solid below the critical temperature [52]. Natural polymer-based hydrogels with a low LCST at around 38 C have huge potential in biomedical applications. Natural polymers are usually used as materials to enhance the performance of hydrogels and/or to improve the thermoresponsiveness of hydrogels. 7.1.3.1 Natural polymer impacts on thermal-responsive behaviors of hydrogels Cellulose derivatives, like (hydroxypropyl)methyl cellulose (HPMC) and methyl cellulose (MC), are temperature-sensitive materials for hydrogel synthesis. At the LCST, hydrogen bonding between polymer and water became weaker than polymer
polymer and water
water interactions. The transition led to rapid dehydration, forming a hydrophobic structure. Moreover, with the higher ratio of cellulose derivatives used as precursors the hydrophobicity increases, leading to a decrease in the sol
gel transition temperature [53]. Barros et al. synthesized chitosan (CH) and HPMC temperature-responsive hydrogels. With the increasing chitosan proportion in CH-HPMC hydrogel, the LCST values ranged from 85.8 C to 87.5 C, which were higher than for the pure HPMC polymer (85.2 C). This feature could be attributed to the increase in HPMC causing an enhancement of hydrophilicity in the polymer. In addition, the increase in NaCl proportion in solution (from 2 to 20 wt.%) could also decrease the HPMC gelation temperature (from 86.7 C to 80.8 C). This salting-out process was caused by a loss of water in the gel during the synthesis of hydrated ions. Dehydration of gel increases the hydrophobic interchain interactions and decreases the LCST value [53]. Hoo’s group prepared thermo-responsive hydrogels by UV-cross-linking in aqueous solutions at room temperature. Methacrylic anhydride (MA)-modified hydroxypropyl cellulose (HPC) was used as the macromonomer. The synthesis process of HPC-MA is illustrated in Fig. 7.18. Two types of hydrogels were synthesized in the experiments. HPCMA-15% and HPC-MA-20% were named by the HPC-MA precursors concentration (w/v), respectively. Both types of hydrogels presented similar properties in turbidimetry

The stimuli-responsive properties of hydrogels based on natural polymers 193

Figure 7.18 Synthesis scheme of HPC-MA [54]. Reprinted with permission from Hoo, S.P., Sarvi, F., Li, W.H., Chan, P.P.Y., Yue, Z., 2013. Thermoresponsive cellulosic hydrogels with cell-releasing behavior. ACS Appl. Mater. Interfaces 5(12), 5592
5600. Copyright 2013 American Chemical Society.

measurement. The turbidity was first observed at 36 C. Above the LCST of HPC-MA (approximately 37 C
38 C), the transmittance decreased drastically with increasing temperature until a plateau was reached at around 45 C. When the temperature was increased above the LCST, the polymer chains undergo a coil
globule transition leading to a more densely packed network with higher storage modulus. HPC-MA-20% presented a higher storage modulus over HPC-MA-15% due to the higher cross-linking density reached in the previous type of hydrogels. As the temperature increased from 25 C to 45 C, the Gv of HPC-MA-15% and HPC-MA-20% rose from 0.7 to 0.9 kPa and from 1.9 to 2.5 kPa, respectively. After 4 days’ incubation, 80%
90% COS-7 cells seeded onto the surface of HPC-MA-20% hydrogels could detach through cold treatment at 4 C for 30 minutes. The mechanism was the cold treatment decreasing the hydrophobic interaction between the cells and gel surface, leading to the detachment of cells [54]. Liu et al. synthesized injectable hydrogels using xanthan gum (XG) and methylcellulose (MC) as precursors. The transition temperatures of 8%, 10%, and 12% MC samples are 36.6 C, 34.2 C, and 32.8 C, respectively; when 3% XG was mixed with 8%, 10%, and 12% MC, the transition temperatures decreased to 28.5 C, 30.2 C, and 28.0 C, respectively. Fig. 7.19 demonstrates the gelation process. When the gelation temperature was reached, the MC network formed via an intermolecular hydrophobic interaction between XG and MC. XG containing carboxylic groups increased the dehydration of MC and reduced the gelation temperature. It was proved that increasing the XG or MC concentration could reduce the transition temperature. When increasing the temperature from the gelation

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Figure 7.19 Illustration of the gelation mechanism of the xanthan gum/methylcellulose (XG/MC) blend solution [55]. Reprinted with permission from Liu, Z., Yao, P., 2015. Injectable thermo-responsive hydrogel composed of xanthan gum and methylcellulose double networks with shear-thinning property. Carbohydr. Polym. 132:490
498. Copyright 2015 Elsevier.

temperature (37 C) to 45 C, the hydrogel mechanical property was also enhanced. Moreover, at the same temperature, the increase in precursor concentration could effectively reduce the gelation time. By in vitro degradation and swelling testing, it was proved that MC added to the network could lead to a gel erosion process which accelerated the release of loaded drugs. In vitro drug release testing of doxorubicin (DOX) showed good drug encapsulation and gel potential to be used as a long-term drug-delivery material. Moreover, in vivo gelation also proved its biocompatibility and biodegradability [55]. 7.1.3.2 Performance improvement of thermal-responsive hydrogels induced by natural polymers Some thermal-responsive hydrogels based on synthetic polymers (e.g., Nisopropylacrylamide) have limited mechanical properties and biodegradability [56]. To overcome these limitations, natural polymers and their derivatives have been utilized in the gelatin network for desired properties. Functionalized natural polymers could alter the thermal sensitivity of hydrogels as described earlier. Herein, some examples will be introduced on the impacts of natural polymers on other properties of thermal-responsive hydrogels including swelling properties, drug release rate, mechanical properties, and the structure of polymer networks. Sanna’s group reported the synthesis of thermoresponsive poly(N-vinylcaprolactam) (PNVCL). Cellulose nanocrystalline was used in the frontal polymerization technique. From the swelling ratio testing, SR% sharply decreased to 970% with 0.2 CNC added to the hydrogel while the original swelling ratio for neat polymer at 3 C was 1200%. The introduction of CNC strongly enhanced the hydrophobic character and increased the crosslink density. A comparison among hydrogels with different CNC ratios showed no change

The stimuli-responsive properties of hydrogels based on natural polymers 195 in the LCST, which was around 33 C
34 C. Generally, from 3 C to 50 C, an increase in temperature led to a decrease in the swelling ratio in each type of hydrogel [57]. PNIPAAm is a widely used polymer that has thermal-responsive properties. Since its unique thermal behavior was reported in the 1980s, research into its potential applications has increased dramatically [58,59]. PNIPAAm a has an LCST temperature at around 32 C. Entropy increases with the loss of interactions between isopropyl groups of the polymer and water molecules [60]. Due to its thermoresponsive property it has been researched for future applications in bioengineering and nanotechnology [61]. Cellulose added to the PNIPAAm will lead to a reduction of the LCST, an increase in the pore size, and higher mechanical strength. In the report from Wang’s group in 2013, they used a semiinterpenetrating polymer network (SIPN) strategy to synthesize cellulose/PNIPAAm hydrogels with N,Nvmethylenebisacrylamide as the cross-linker and benzoyl peroxide as the initiator (Fig. 7.20). The precursor ratios of cellulose (PNIPAAM 1 PMBAAm) from samples GEL1 to GEL4 were 1:1.4, 1:2.4, 1:5.5, and 1:7.4, respectively. All SPIN hydrogels showed thermoresponsive properties. For GEL1 the swelling ratio decreased from 20.5 to 2.4 when the temperature increased from 15 C (,LCST) to 45 C( . LCST). Similarly, during this temperature range, the swelling ratio for GEL2 decreased from 15.8 to 4.0. However, GEL3 and GEL4 had a less significant change. Different properties among hydrogels could be attributed to the different interior morphologies of hydrogels and the different degree of cross-linking. The higher cellulose ratio in GEL1 led to a stronger hydrophobic network and formed a structure with a larger pore size. Hydrogels with a larger pore size showed a higher release rate of the model drug, dimethylmethylene blue [62].

Figure 7.20 The proposed structure model of the semiinterpenetrating polymer network (SIPN) hydrogel [62]. Reprinted with permission from Wang, J., Zhou, X., Xiao, H., 2013. Structure and properties of cellulose/ poly(N-isopropylacrylamide) hydrogels prepared by SIPN strategy. Carbohydr. Polym. 94(2), 749
754. Copyright 2013 Elsevier.

196 Chapter 7 Wei’s research group reported the improvement of thermo-responsive hydrogels using a TEMPO-oxidized bamboo cellulose nanofibers (TO-CNF)/PNIPAAm matrix. When the temperature was kept below 25 C, the ESR was kept at a high value with the hydrogels being transparent. Along with the increase in temperature, the ESR value dropped sharply and reached a constant value above 40 C. At temperatures below the LCST, the electrostatic repulsion between negative charges of TO-CNF microfibrils could greatly enhance the swelling properties. Hydrogels with a higher percentage of TO-CNF showed much higher ESR than pure PNIPAAm gels. Adding cellulose also increased the pore size and mechanical properties of the hydrogels [63]. Wu’s research group enhanced the mechanical property of poly(N-isopropylacrylamide-cobutyl methacrylate) (PNB) nanogels by adding BC whiskers (Fig. 7.21). Differential scanning calorimetry (DSC) data showed that the thermosensitivity of PNB nanogels was unchanged. Adding BC into nanogel complexes increased the transition temperature from 10 C to 45 C. Compared with pure PNB nanogels, with the increase of BC whiskers to 20 wt.% (N-4), the G0 and Gv generally increased. N-4 had G0 and Gv of 500 and 57 Pa, while pure PNB gels were 160 and 90 Pa. The reason for this could be attributed to the hydroxyl groups on the BC whisker surface increasing the hydrogen bonds between water and BC/PNB nanogels. Biotoxicity testing proved that pure PNB had low cytotoxicity in human umbilical vein endothelial cells (HUVECs). A decrease in oxygen levels could stimulate pronounced expression of hemeoxygenase and cytoplasmic free Ca21 in HUVEC cells. With this property, the increase in BC whiskers and the increase in PNB/BC nanogel concentration, which led to a denser gel network, the relative growth rate could exceed the original percentage [64].

Figure 7.21 The structure of temperature-responsive poly(N-isopropylacrylamide-co-butyl methacrylate) (PNB)/bacterial cellulose (BC) nanogels [64]. Reprinted with permission from Wu, L., Zhou, H., Sun, H.-J., Zhao, Y., Yang, X., Cheng, S.Z.D., et al., 2013. Thermoresponsive bacterial cellulose whisker/poly (NIPAM-co-BMA) nanogel complexes: synthesis, characterization, and biological evaluation. Biomacromolecules 14(4), 1078
1084. Copyright 2013 American Chemical Society.

The stimuli-responsive properties of hydrogels based on natural polymers 197

Figure 7.22 The synthesis process and a schematic representing the structural model, drug loading, and drug release of thermo-responsive poly(N-isopropylacrylamide)-nanocrystalline cellulose (PNIPAAmCNC) hybrid hydrogels [65]. Reprinted with permission from Zubik, K., Singhsa, P., Wang, Y., Manuspiya, H., Narain, R., 2017. Thermo-responsive poly(N-isopropylacrylamide)-cellulose nanocrystals hybrid hydrogels for wound dressing. Polymers 9(4), 119. Copyright 2017 MDPI.

Zubik’s research group synthesized poly(N-isopropylacrylamide) cellulose nanocrystal hybrid hydrogels (Fig. 7.22). At either 20 C or 37 C, the increase in CNC content in hydrogels led to higher G0 and Gv. Hydrogels incubated with 1 mg/mL MZ were named NC-50-1, while gels incubated with 5 mg/mL MZ were named NC-50-5. The testing result proved that at pH 5 7.4, 37 C, in vitro drug release of metronidazole (MZ) proved that 80% and 72% of drug was released in 40 minutes in NC-50-5 and NC-50-1, respectively. After 24 hours, the maximum release of NC-50-5 and NC-50-a was 86% and 82%, respectively. Adding cellulose reinforced the mechanical performance properties and increased the stability of hydrogels, making it a possible new material for injectable hydrogels or wound-dressing materials [65].

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Figure 7.23 Schematic illustration of the microfluidic device and preparation route of poly(Nisopropylacrylamide)/ethyl cellulose (PNIPAAm/EC) core
shell microspheres [66]. Reprinted with permission from Yu, Y.-L., Zhang, M.-J., Xie, R., Ju, X.-J., Wang, J.-Y., Pi, S.-W., et al., 2012. Thermoresponsive monodisperse core
shell microspheres with PNIPAM core and biocompatible porous ethyl cellulose shell embedded with PNIPAM gates. J. Colloid Interface Sci. 376(1), 97
106. Copyright 2012 Elsevier.

Instead of embedding cellulose into the network of hydrogels, Ya-Lan’s group invented the nanosphere system containing a thermo-responsive PNIPAAm core and a biocompatible porous ethyl cellulose (EC) shell. The synthesis was realized by first creating monodisperse water-in-(water/oil)-in-water (W1/(W2/O)/W1) double emulsions. After the solvent diffusion and evaporation, the monodisperse solidified into an EC microcapsule with hollows. Immersed into NIPAM solution, the PNIPAAm hydrogel synthesis was triggered by TMEDA solution. After washing the nanosphere with deionized water, the nanosystem was successfully synthesized. The whole process is demonstrated in Fig. 7.23. In pure EC microcapsule testing, the release rate of vitamin B12 (VB12) at above volume phase transition temperature (VPTT) divided by the release rate of VB12 at below VPTT was CF (controlled factor) 5 1.6. The increase was attributed to the enhanced molecular diffusion caused by higher temperature. In contrast, PNIPAAm/EC core
shell microspheres had

The stimuli-responsive properties of hydrogels based on natural polymers 199

Figure 7.24 Schematic illustration of the expected thermo-responsive behavior of the proposed poly(Nisopropylacrylamide)/ethyl cellulose (PNIPAAm/EC) core
shell microsphere [66]. Reprinted with permission from Yu, Y.-L., Zhang, M.-J., Xie, R., Ju, X.-J., Wang, J.-Y., Pi, S.-W., et al., 2012. Thermoresponsive monodisperse core
shell microspheres with PNIPAM core and biocompatible porous ethyl cellulose shell embedded with PNIPAM gates. J. Colloid Interface Sci. 376(1), 97
106. Copyright 2012 Elsevier.

CF 5 11.7. The process is illustrated in Fig. 7.24. When the temperature increased to above VPTT, the PNIPAAm hydrogels shrunk, leaving the EC shell “open” to the release of VB12 [66]. Eyigor et al. synthesized (1,3)-(1,6) β-glucan and PNIPAAm temperature-responsive hydrogels using tetramethylethylenediamine and potassium persulfate as a redox pair (Fig. 7.25). With the increase in the β-glucan portion, the VPTT of hydrogel increased from that of pure PNIPAAm hydrogel at 32.8 C to 35.5 C. The presence of ionic and hydrophilic components contributed to the increase in VPTT. Above the VTPP, the increase of the β-glucan portion could increase the equilibrium swelling degree of hydrogels and a relatively slow 5-aminosalicylic acid (5-ASA) release rate. This was attributed to the increase in the hydrogen bonding between hydrogels and 5-ASA [67].

7.1.4 Salt-responsive hydrogels based on natural polymers Salt-responsive hydrogels exhibit shape transformation in response to salt solutions, salt concentrations, and counterion types [68]. The mechanism of salt response can be attributed to two major causes. The swelling of hydrogels depends on the balance of osmotic pressure, which results from the internal and external ionic concentrations of hydrogel systems. An increase in the external ionic concentration can lead to a reduction of swelling. Moreover, the ions can also form ionic cross-links within the hydrogels and reduce the capability to swell [32]. Akar et al. synthesized biodegradable sodium carboxymethylcellulose (NaCMC)-based hydrogels using FA as a cross-linking agent. Except for the hydrogels used, 0.03 M FA in gelation (CMC3F), which showed maximum swelling at pH 5 7, hydrogels using 0.01 M

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Figure 7.25 Synthesis of β-glucan-PNIPAAm hydrogels [67]. Reprinted with permission from Eyigor, A., Bahadori, F., Yenigun, V.B., Eroglu, M.S., 2018. Beta-glucan based temperature responsive hydrogels for 5-ASA delivery. Carbohydr. Polym. 201, 454
463. Copyright 2018 Elsevier.

FA (CMC1F) and 0.05 M FA (CMC5F) both showed maximum swelling ratio at pH 5 5. Compared with the swelling in deionized water, all three types of hydrogel showed a decrease in the swelling ratio when immersed into NaCl, CaCl2, and AlCl3. NaCMC introduced to the hydrogels significantly increased the time for half-life degradation. From CMC1F to CMC3F the time used for reaching 50% weight loss in enzymatic degradation increased from 5 minutes to 21 hours. The controllable degradation property as well as the sensitivity to pH and salt stimulations could be applied for scaffold fabrication or a controlled-release system [32]. Wu et al. synthesized salt- and pH-responsive hydrogel actuators using NaCMC and AA. As illustrated in Fig. 7.26, the synthesized p(AAm-co-AAc) hydrogels were cut into strips. CMC and AAm monomers were mixed and gelatinized with the strips to form CMC/ PAAm||p(AAm-co-AAc) hydrogel sheets in the mold. When immersed into Fe31 solutions, p(AAm-co-AAc) gel acted as the folding center and drove the shape change of the whole hydrogel. The carboxyl groups in CMC caused heterogeneous shrinkage when in contact with Fe31 ions. When the hydrogels were immersed in acetic acid solution (pH 5 1), the

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Figure 7.26 A schematic of the fabrication process of CMC/PAAm||p(AAm-co-AAc) hydrogel [69]. Reprinted with permission from Wu, S., Yu, F., Dong, H., Cao, X., 2017. A hydrogel actuator with flexible folding deformation and shape programming via using sodium carboxymethyl cellulose and acrylic acid. Carbohydr. Polym. 173, 526
534. Copyright 2017 Elsevier.

destruction of the monocomplex between Fe31 and the carboxyl group on macromolecular chains of the hydrogels led to the reform of the gel’s original shape. Moreover, the CMC/PAAm in the rest of the actuators also enhanced its mechanical properties due to the mono-, bi-, or tridentates formed with Fe31 ions [69]. Similarly, in Zhang’s research group, hydrogels prepared by poly(acrylic acidcoacrylamide) cross-linked with quaternized tunicate cellulose nanocrystals (Q-TCNCs) showed similar properties. With an increase in pH, the swelling properties of hydrogel significantly improved. As shown in Fig. 7.27, Fe31 ions interacted with carboxyl groups to form dual cross-linked hydrogel networks. In strain
stress testing, the hydrogels in contact with higher Fe31 ion concentrations showed higher elastic modulus and toughness [70]. In the previously introduced work by Spagnol et al., cellulose nanofibrils and chitosan-graftpoly(acrylic acid) were copolymerized. The salt sensitivity of hydrogels was reduced by the addition of cellulose nanofibers. The contraction of gels was more obvious when Ca21 or Al31 ions were added. A possible explanation could be that those ions could cause the formation of carboxamide or carboxylate groups including intramolecular and intermolecular complex or neutralize more charges in gels with the same concentration [28]. Salt-responsive hydrogels are sensitive to changed external electrolytes. The addition of natural polymers could improve the mechanical, salt sensitivity, and degradability properties of salt-responsive hydrogels. Compared to pH- and thermo-responsive hydrogels, this is a new area that remains to be further developed for potential applications in biomedical applications [71], electronic logic gates [72], and water desalination [72].

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Figure 7.27 Schematic illustrations for the preparation of dual cross-linked hydrogels. (A) Use of quaternized tunicate cellulose nanocrystals (Q-TCNCs) in situ polymerization of AA and AM monomers to form mono-cross-linked hydrogel (m-Gel) networks. (B) Fe31 interaction with
COO2 groups in the m-Gel to get dual cross-linked hydrogel (d-Gel) networks. (C) After removal of excess and unstable Fe31, reorganization of ionic coordination to obtain the final hydrogel (D-Gel) [70]. Reprinted with permission from Zhang, T., Zuo, T., Hu, D., Chang, C., 2017. Dual physically cross-linked nanocomposite hydrogels reinforced by tunicate cellulose nanocrystals with high toughness and good self-recoverability. ACS Appl. Mater. Interfaces 9(28), 24230
24237. Copyright 2017 American Chemical Society.

7.1.5 Glucose-responsive hydrogels based on natural polymers Glucose-responsive hydrogels typically contain phenylboronic acids (PBA) and exhibit a change in properties with a shift in glucose concentration. The undissociated form (1) and dissociated form (2) reach an equilibrium in the hydrolysis process. With the existence of glucose, the charged borates form a stable complex (3) (Fig. 7.28) [73]. This process impacts the equilibrium between 1 and 2. PBA and its derivatives have a level of cell toxicity. Natural polymers were added to improve their biocompatibility [74]. Yetisen et al. reported the use of Ca alginate to synthesize glucose-sensitive hydrogel optical fibers. 3-(Acrylamido)-phenylboronic acid (3-APBA) molecules were covalently incorporated into the core made of poly(acrylamide-co-poly(ethylene glycol) diacrylate) p (AM-co-PEGDA) and a Ca alginate cladding for continuous glucose monitoring. The bonding of glucose into the core material tuned the intensity of light output (IO). The refractive index could be calculated to reflect the change of glucose concentration (Fig. 7.29). Ca alginate proved to be efficient for use as fiber claddings [75]. Peng et al. improved the biocompatibility of 4-vinyl-phenylboronic acid (VPBA) by mixing cellulose for potential applications in self-regulated insulin release. The cellulose flasks were dissolved in 1-ethyl-3-methylimidazolium acetate and mixed with VPBA.

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Figure 7.28 Equilibria of (alkylamido)phenyl boronic acid [73]. Reprinted with permission from Kataoka, K., Miyazaki, H., Bunya, M., Okano, T., Sakurai, Y., 1998. Totally synthetic polymer gels responding to external glucose concentration: their preparation and application to on
off regulation of insulin release. J. Am. Chem. Soc. 120(48), 12694
12695. Copyright 1998 American Chemical Society.

Figure 7.29 The design of the glucose-sensitive hydrogel optical fibers. (A) Structural composition of the glucose-sensitive p(AM-co-PEGDA-co-3-APBA) fiber core cladded with Ca alginate. (B) Functionalization of hydrogels with 3-(acrylamido)-phenylboronic acid (3-APBA). (1) PEG-crosslinked polyacrylamide hydrogel, (2) 3-APBA in charged tetrahedral state, (3) glucose. (C) The PBA derivative binding of cis diols of glucose molecules and changes in the refractive index of the hydrogel fiber. (D) Light IO as the approach to glucose concentration quantification [75]. Reprinted with permission from Yetisen, A.K., Jiang, N., Fallahi, A., Montelongo, Y., Ruiz-Esparza, G.U., Tamayol A., et al., 2017. Glucose-sensitive hydrogel optical fibers functionalized with phenylboronic acid. Adv. Mater. 29(15), 1606380. Copyright 2017 Wiley-VCH Verlag GmbH & Co. KGaA.

The cellulose/VPBA solution was then exposed to electric beam irradiation for pregelation and the mold was immersed in coagulator for the polymerization of cellulose/VPBA hydrogel membrane (Fig. 7.30). The interaction of glucose and PBA sharply altered the properties of the hydrogels. Fluorescein isothiocyanate-labeled insulin was loaded into the

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Figure 7.30 Preparation procedures for cellulose/4-vinyl-phenylboronic acid (VPBA) composite hydrogel membranes [74]. Reprinted with permission from Peng, H., Ning, X., Wei, G., Wang, S., Dai, G., Ju, A., 2018. The preparations of novel cellulose/phenylboronic acid composite intelligent bio-hydrogel and its glucose, pH-responsive behaviors. Carbohydr. Polym. 195, 349
355. Copyright 2018 Elsevier.

hydrogel. It was observed that at a glucose concentration of 5.0 g/L, the release rate and final concentration at release sharply increased. There was a threshold of the glucose concentration to trigger the release of insulin from the hydrogel [74]. Similarly, Dong et al. prepared injectable glucose hydrogels for controlled drug release. PBA containing monomer (monomer B) were copolymerized with boc-protected monomer (monomer C). Trifluoroacetic acid was used to remove the boc-protecting group. The polymer BC-NH2 was combined with glucose in the reductive amination to give the final product: polymer (3-propionamidophenyl) boronic acid (N-(3-((2
5,6-pentahydroxyhexyl) amino)propyl)propionamide) (polymer BG) for injection (Fig. 7.31). Rhodamine B was loaded as a model drug and it has been illustrated that polymer BG in the solution with higher glucose concentration released a higher amount of model drug [76]. As described in the above examples, natural polymer-based PBA have been prepared for glucose detection, controlled drug-release hydrogels, and injectable hydrogels. They are a potential candidate for the treatment of diabetes. Further research on cytotoxicity remains to be researched.

7.1.6 Light-responsive hydrogels based on natural polymers Light-responsive hydrogels exhibit changes in properties under exposure to light. Compared to the aforementioned stimuli-responses, photo switching allows highly precise alternation of the dimensional and structural change to the material with localized actuation [77]. The stimuli can be triggered at a remote distance without the necessity for contact or proximity.

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Figure 7.31 Synthetic routes to polymer BG, which can self-assemble into an injectable and glucose-responsive hydrogel [76]. Reprinted with permission from Dong, Y., Wang, W., Veiseh, O., Appel, E.A., Xue, K., Webber, M.J., et al., 2016. Injectable and glucose-responsive hydrogels based on boronic acid-glucose complexation. Langmuir 32(34), 8743
8747. Copyright 2016 American Chemical Society.

Figure 7.32 The rapid shape memory process of PAM-GO-gelatin hydrogels [78]. Reprinted with permission from Huang, J., Zhao, L., Wang, T., Sun, W., Tong, Z., 2016. NIR-triggered rapid shape memory PAM-GO-gelatin hydrogels with high mechanical strength. ACS Appl. Mater. Interfaces 8(19), 12384
12392. Copyright 2016 American Chemical Society.

Examples covering the response to near infra-red, UV, and visible light are introduced in this section. Huang et al. prepared NIR-responsive shape memory PAM-GO-gelatin hydrogels (Fig. 7.32). An interpenetrating network was formed by physically cross-linking the gelatin network and chemically cross-linking the polyacrylamide (PAM) network with graphene oxide (GO). The shape memory resulted from a reversible triple helix structure of gelatin

206 Chapter 7 appearing at low-temperature cross-linking molecular chains forming a network. With the sacrificial gelatin network and GO bridging in deformation energy dissipation, high mechanical toughness can be achieved (strength .400 kPa and broken strain . 500%). GO converts NIR to thermoenergy to increase the temperature of the sample and realize the shape memory property. The shape recovery is confined to the area exposed to NIR and the shape change in the adjacent area was not impacted [78]. Light-responsive cotton fibers realized the absorption and release of water under visible light. ter Schiphorst et al. [79] used an atom transfer radical polymerization (ATPR) initiator to functionalize the cotton fibers. Polymers were then grafted from the fiber surface using activators regenerated by electron transfer-atom transfer radical polymerization with NIPAAm, sodium acrylate, and spiropyran (Sp) derivative (Fig. 7.33). AA provides protons for Sp isomerization to hydrophilic merocyanine-H1 for water uptake. Upon illumination, the hydrophobic Sp isomer forms to release the water (Fig. 7.34).

Figure 7.33 Illustration of the functionalization of cotton fibers [79]. Reproduced from ter Schiphorst, J., van den Broek, M., de Koning, T., Murphy, J.N., Schenning, A.P.H.J., Esteves, A.C.C., 2016. Dual light and temperature responsive cotton fabric functionalized with a surface-grafted spiropyran
NIPAAm-hydrogel. J. Mater. Chem. A 4(22), 8676
8681 with permission from The Royal Society of Chemistry.

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Figure 7.34 Schematic representation of the polymer-grafted light-responsive cotton fibers [79]. Reproduced from ter Schiphorst, J., van den Broek, M., de Koning, T., Murphy, J.N., Schenning, A.P.H.J., Esteves, A.C.C., 2016. Dual light and temperature responsive cotton fabric functionalized with a surface-grafted spiropyran
NIPAAm-hydrogel. J. Mater. Chem. A 4(22), 8676
8681 with permission from The Royal Society of Chemistry.

Reversible photoisomerization of hydrogels based on xylan-type hemicellulose hydrogels prepared by Cao et al. for UV light-enhanced drug release. As shown in Fig. 7.35, the xylan-type hemicellulose was synthesized to hemicellulose methacrylate with glycidyl methacrylate (GMA). 4-[(40 -Hydroxy)phenylazo]benzoic acid and 4-[(4-acryloyloxyphenyl) azo]benzoic acid (AOPAB) was prepared from 4-aminobenzoic acid. Hemicellulose methacrylate and AOPAB were mixed in DMSO and azobis(isobutyronitrile) was added as an initiator for polymerization. Under UV exposure, the thermodynamically stable transconformation of azobenzene structure would change to cis-conformation. This process was reversible under visible light or dark. It was found that UV irradiation changed the hydrophilic hydrogels to a more hydrophobic structure, leading to a higher cumulative release of the loaded hydrophilic drug (vitamin B12) (Fig. 7.36) [80]. Light-responsive hydrogels based on natural polymers respond to stimulation by light
heat transfer, charge change, or photoisomerization. Further in-depth investigation remains to be done to test the in vivo biocompatibility and the application of these properties in medical treatments and precisely controlled actuations, etc.

Figure 7.35 Scheme for preparation of xylan-type hemicellulose-based hydrogel [80]. Reprinted with permission from Cao, X., Peng, X., Zhong, L., Sun, R., 2014. Multiresponsive hydrogels based on xylan-type hemicelluloses and photoisomerized azobenzene copolymer as drug delivery carrier. J. Agric. Food Chem. 62(41), 10000
10007. Copyright 2014 American Chemical Society.

Figure 7.36 The illustration of drug load and the release process of xylan-type hemicellulose-graftphotoisomeric azobenzene hydrogels [80]. Reprinted with permission from Cao, X., Peng, X., Zhong, L., Sun, R., 2014. Multiresponsive hydrogels based on xylan-type hemicelluloses and photoisomerized azobenzene copolymer as drug delivery carrier. J. Agric. Food Chem. 62(41), 10000
10007. Copyright 2014 American Chemical Society.

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7.1.7 Multistimulation-responsive hydrogels based on natural polymers In practice, mono-responsive hydrogels have limited applications. To overcome this problem, multiresponsive hydrogels are becoming an emerging research point which could enlarge the application of stimuli-responsive hydrogels. In this section, hydrogels that respond to multiple stimulations are introduced. As shown in Fig. 7.37, Appel’s research group synthesized hydrogels with ultrahigh-water content. Naphthyl-functionalized cellulose (HEC-Np) and PVA-MV were cross-linked with the existence of cucurbit[8]uril (CB[8]). With an increase in temperature, this type of hydrogel presented a decrease in bulk mechanical properties. This phenomenon was attributed to the concomitant decrease in the association constant of the dynamic ternary complex cross-links. The noncovalently cross-linked structure also displayed recovery of the hydrogel network under high-magnitude deformation in step-rate time-sweep

Figure 7.37 Schematic representation of a supramolecular hydrogel prepared through the addition of cucurbit [8]uril to a mixture of multivalent first- and second-guest-functionalized polymers in water [81]. Reprinted with permission from Appel, E.A., Loh, X.J., Jones, S.T., Biedermann, F., Dreiss, C.A., Scherman, O.A., 201.2 Ultrahigh-water-content supramolecular hydrogels exhibiting multistimuli responsiveness. J. Am. Chem. Soc. 134(28), 11767
11773. Copyright 2012 American Chemical Society.

210 Chapter 7 measurements. Hydrogels also showed sensitivity to external stimuli, including seconding guests and redox conditions. The addition of 2,6-dihydroxynaphthalene or an aromatic solvent such as toluene dissociated the polymer network and led to the loss of bulk mechanical properties. The addition of 2,6-dihydroxynaphthalene did not change the original light-orange color, while the toluene caused a loss in color as the previous competing guest formed a colored charge transfer similar to the original polymer. Hexane as a simple linear hydrocarbon was not a suitable second guest for the CB[8] ternary complex, and no hydrogel alternation was observed. The special feature of the hydrogel network was the one-electron reduction of MV. The addition of sodium dithionite formed the 2:1 MV•1:CB[8] complex and changed the original mechanical properties [81]. Cha et al. prepared N-isopropyl acrylamide-based thermal/pH-responsive hydrogels using CNCC. At 24 C below LCST, an increase in CNCC in hydrogels increased the amount of hydrogen bonds, leading to an increase in the ESR. Compared with pure PNIPAAm hydrogels, the higher amount of CNCC in hydrogels led to a more sensitive pH response. In hydrogels with constant CNCC ratio, the increase in pH led to an obvious enhancement of ESR [31]. Sanjoy et al. synthesized polythiophene-g-poly(dimethylaminoethyl methacrylate)-doped methylcellulose (MC) hydrogel with photoluminescence (PL) property like a AND logic gate. As illustrated in Fig. 7.38, with an increase in temperature, the PD within MC selfassembled a fiber-like structure in gels. The two inputs are pH and temperature, respectively, and PL intensity is measured as the output. PD as a pH-responsive fluorescent graft copolymer has a light-responsive property. In PL measurement, a 417-nm laser was used. With the increase of temperature from the solution state to the gel state, hydrogels with low MC:PD weight ratio showed the largest enhancement caused by temperature variation. However, a high MC concentration could reduce the polarity of the local environment around PT chains and led to higher PL-intensity, the reduced sensitivity to temperature made the hydrogels with low MC concentration (MC:PD wt 5:1) considerable for the logic gate design. At a temperature of 45 C, hydrogels at pH 5 9.2 showed a PL intensity 4.5 times as high as the hydrogels at pH 5 4 and pH 5 7. Given appropriate threshold value, hydrogels showed AND logic gate function with the temperature and pH as two inputs and the PL intensity as an output [82]. Photosensitizer (PS) is widely utilized in current clinically practiced photodynamic therapy (PDT) to kill cancer cells. In a conventional medical treatment, the nonspecific activation of PS could cause damage to body parts exposed to sunlight, so patients were advised to take precautions for an extended period of time after the treatment. To increase the selectivity response of PS, Wooram’s research group conjugated PS (Pheophorbide-a, PPb-a) to a temperature-responsive polymer backbone of biocompatible hydroxypropyl cellulose to develop thermoswitchable polymeric photosensitizer (T-PPS) (Fig. 7.39). At temperatures below the LCST, the cellulose-based T-PPS polymers retained their hydrophilic

The stimuli-responsive properties of hydrogels based on natural polymers 211

Figure 7.38 Schematic presentation of doping of PD within MC and their self-assembly producing a fiber-like structure in the gel with an increase in temperature [82]. Reproduced from Samanta, S., Das, S., Layek, R.K., Chatterjee, D.P., Nandi, A.K., 2012. Polythiophene-g-poly(dimethylaminoethyl methacrylate) doped methyl cellulose hydrogel behaving like a polymeric AND logic gate. Soft Matter 8(22), 6066
6072 with permission from The Royal Society of Chemistry.

random-coil conformation and PS molecules were in an aqueous environment which resulted in a quenching of the photoactivity. When temperatures increased to above the LCST, the backbone cellulose polymer dehydrated leading to a nonpolar microenvironment between the T-PPS molecules which led to a monomeric state and significant light responsiveness of PS. The monomeric state PS had significantly higher photoactivity. Tests proved that at 45 C (above the LCST), there was a strong enhancement in fluorescent emission (F.I.) of the T-PPS aqueous solution. The F.I. of singlet oxygen generation (SOG) at 45 C also increased sixfold compared to testing at 37 C. Throughout in vitro photocytotoxicity of T-PPS, the results showed that at 45 C with the laser on, cell viability of PANC-2 showed the lowest cell viability, proving the potential application in PDT [83]. As demonstrated in Figs. 7.40 and 7.41, Shi et al. utilized the pH-responsive property of
COOH to fabricate in vitro controlled-release hydrogels. Using biopolymer BC and sodium alginate (SA) as precursors, they successfully synthesized hydrogel with swelling property in an alkaline environment. BC slurry and SA solution were mixed first, followed

Figure 7.39 Schematic of temperature-responsive polymeric PS. (A) Chemical synthetic route of T-PPS (PPb-a conjugated HPC); (B) temperature-dependent change in structure and photoactivity of TPPS for thermal cancer therapy [83]. Reprinted with permission from Park, W., Park, S.-J., Cho, S., Shin, H., Jung, Y.-S., Lee, B., et al., 2016. Intermolecular structural change for thermoswitchable polymeric photosensitizer. J. Am. Chem. Soc. 138(34), 10734
10737. Copyright 2016 American Chemical Society.

Figure 7.40 Formation process for the preparation of the drug-loaded nf-BC/SA hybrid hydrogels with a semiIPN structure [84]. Reproduced from Shi, X., Zheng, Y., Wang, G., Lin, Q., Fan, J., 2014. pH- and electroresponse characteristics of bacterial cellulose nanofiber/sodium alginate hybrid hydrogels for dual controlled drug delivery. RSC Adv. 4(87), 47056
47065 with permission from The Royal Society of Chemistry.

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Figure 7.41 Releasing process for the drug-loaded nf-BC/SA hybrid hydrogels under different pH values [84]. Reproduced from Shi, X., Zheng, Y., Wang, G., Lin, Q., Fan, J., 2014. pH- and electro-response characteristics of bacterial cellulose nanofiber/sodium alginate hybrid hydrogels for dual controlled drug delivery. RSC Adv. 4 (87), 47056
47065 with permission from The Royal Society of Chemistry.

by the cross-linking process with CaCl2. Throughout the comparison among a series of hydrogels with different BC/SA weight ratios, the higher BC weight ratio in synthesis led to better swelling property, while causing a delay in the swelling process. nf-BC/SA33 hydrogels [BC/alginate 5 67/33 (wt.%)] presented 11.6 times greater swelling than the dry weight in PBS solution (pH 5 7.4). With the increase in pH, nf-BC/SA33 hydrogels showed a swelling ratio from 8
13 times greater than dry weight in 150 minutes. In drug testing, the nf-BC/SA33 hydrogels in pH 5 11.8 released ibuprofen twice as much as that in pH 5 7.0. At the end of testing, both hydrogels in pH 5 7.0 and pH 5 11.8 released 90% of encapsulated drugs while the acidic group only showed a 60% release. The conversion from alginate acid to sodium salt caused a break in the hydrogen bonding, the expansion of hydrogel, and release of drugs. In the electro-responsive test shown in Fig. 7.42, the external electrical field applied to nf-BC/SA33 matrix enhanced the escape of H1. An increase in the ionizable
COO2 groups strengthened the swelling behaviors of hydrogels, leading to an improved drug-release property [84]. Mahdavinia et al. synthesized a magnetic/pH stimuli-responsive system based on carboxymethyl chitosan and к-carrageenan. The mixture of precursors was dropped into an Fe21/Fe31 solution. After the formation of gels via ionic cross-linking, the beads were immersed into KCl/CaCl2 solutions, electrostatic interaction of K1 with sulfate groups (on к-carrageenan) and Ca21 with carboxylate groups (on carboxymethyl chitosan) enhanced the apparent strength of magnetic beads (Fig. 7.43). Fe3O4 particles in a cross-linked structure enable easy removal from the system by external magnetic fields. Compared to acidic conditions (pH 5 1.2), in neutral pH conditions (pH 5 7.4) hydrogel beads showed

Figure 7.42 Releasing process for the drug-loaded nf-BC/SA33 hybrid hydrogels under an electric field [84]. Reproduced from Shi, X., Zheng, Y., Wang, G., Lin, Q., Fan, J., 2014. pH- and electro-response characteristics of bacterial cellulose nanofiber/sodium alginate hybrid hydrogels for dual controlled drug delivery. RSC Adv. 4 (87), 47056
47065 with permission from The Royal Society of Chemistry.

Figure 7.43 A simple scheme for the synthesis of magnetic CMChitoCar beads [26]. Reprinted with permission from Mahdavinia, G.R., Etemadi, H., Soleymani, F., 2015. Magnetic/pH-responsive beads based on carboxymethyl chitosan and kappa-carrageenan and controlled drug release. Carbohydr. Polym. 128, 112
121. Copyright 2015 Elsevier.

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Figure 7.44 Diagram of drug-delivery behaviors and cell proliferation of xylan-based P(NIPAm-g-AA) hydrogel copolymer networks [85]. Reprinted with permission from Gao, C., Ren, J., Zhao, C., Kong, W., Dai, Q., Chen, Q., et al., 2016. Xylan-based temperature/pH sensitive hydrogels for drug controlled release. Carbohydr. Polym. 151, 189
197. Copyright 2016 Elsevier.

better swelling properties due to the electrostatic repulsion between anionic pendants (sulfate and carboxylate) on the backbone structures. Gao et al. prepared temperature/pH-sensitive hydrogels based on xylan for controlled drug release. Xylan with N-isopropylacrylamide (NIPAm) and AA were cross-linked using N,N0 methylenebis-acrylamide as a cross-linker and 2,2-dimethoxy-2-phenylacetophenone as a photoinitiator via ultraviolet (UV) irradiation. With an increase of temperature (25 C
37 C), the ESR generally decreased. Compared to low-pH conditions (pH 5 1.5), the cumulative release rate of drug in intestinal fluids (pH 5 7.4) is much higher due to the electrostatic repulsions by ionizing
COOH. Xylan has been proved effective in protecting capsulated drugs and has good biocompatibility (NIH3T3) (Fig. 7.44) [85]. Hu et al. synthesized redox/pH stimuli-responsive degradable Salecan-g-SS-poly(IA-coHEMA) hydrogel for DOX controlled release. Salecan is a water-soluble extracellular β-glucan with excellent physicochemical and biological properties. Itaconic acid and 2hydroxyethylmethacrylate (HEMA) were grafted onto Salecan in aqueous media with a disulfide cross-linker N,N0 -bis(acryloyl)cystamine for hydrogels (Fig. 7.45). Doxorubicin hydrochloride (DOX) was loaded as an amphiphilic anticancer model drug. In low pH conditions (pH 5 3.2), protonation of carboxyl groups reduced the electrostatic interactions, triggering the release of DOX. An in vitro cytotoxicity test proved the safety of potential drug-delivery applications. Dithiothreitol was used to cleave the disulfide bond. The results showed the degradation of hydrogel was significantly slower than the drug release rate,

216 Chapter 7

Figure 7.45 Scheme showing the preparation of Salecan-g-SS-PIH hydrogels [86]. Reprinted with permission from Hu, X., Wang, Y., Zhang, L., Xu, M., Dong, W., Zhang, J., 2017. Redox/pH dual stimuli-responsive degradable Salecan-g-SS-poly(IA-co-HEMA) hydrogel for release of doxorubicin. Carbohydr. Polym. 155, 242
251. Copyright 2016 Elsevier.

indicating the Salecan-gSS-PIH hydrogel could be used as a redox-degradable controlled drug-delivery system [86]. Multiresponsive polyethylene-polyamine/gelatin hydrogel was prepared by Zhang et al. polyethylene polyamine (PPA) and gelatin solution were mixed at 60 C and cooled down to 25 C for gelation (Fig. 7.46). During the cooling process, amine groups of PPA molecules formed hydrogen bonds with carboxyl groups of gelatin, creating the major hydrophobic domains as cross-linkers. The hydrogel showed an irreversible pH response after adding acid and forming an opaque suspension. This phenomenon was caused by protonation of the

The stimuli-responsive properties of hydrogels based on natural polymers 217

Figure 7.46 Illustration of the preparation, hydrogen-bond, and hydrophobic-domain categories in the polyethylene polyamine (PPA)/gelatin hydrogel. PPA and gelatin aqueous solution were mixed at 60 C, and only a few hydrogen bonds existed between gelatin chains and PPA (blue circles), gelatin chains (red circles) and PPA (yellow circles) in the solution. Upon cooling to 25 C, due to the increasing formation of hydrogen bonds in this process, the abundant hydrophobic domains consisting of hydrogen bonds between gelatin chains and PPA (blue squares) and gelatin chains (red squares) appeared [87]. Reproduced from Zhang, Z., Liu, Y., Chen, X., Shao, Z., 2016. Multiresponsive polyethylene-polyamine/gelatin hydrogel induced by non-covalent interactions. RSC Adv. 6(54), 48661
48665 with permission from The Royal Society of Chemistry.

amine groups on PPA branches which disassociated the hydrogen bonds between PPA and gelatin. The hydrophobic force aggregated the PPA. Slight aggregation remained when NaOH was added. The inadequate cross-linking points prevented the reformation of the hydrogel network. By adding GO, the photothermal effect could be utilized to enable reversible phase change of hydrogel via NIR. P50/G6 hydrogel also showed self-healing properties [87].

7.2 Conclusion This chapter has covered the pH, magnetic fields, temperature, electric fields, competitive agents, redox, light, glucose and salt stimuli-responsive hydrogels. Natural polymers were proved to be efficient in a variety of stimuli-sensitive applications. Their functionalities cover the backbones of the hydrogel structures, the cladding of fibers, functional groups for stimuli responsiveness, and materials to improve mechanical/biocompatible properties. Stimuli-responsive hydrogels with single or multiresponsive features have huge potential applications in controlled drug release, tissue engineering, sustainable products, etc. As a

218 Chapter 7 biodegradable, natural, and highly stiff material, natural polymer-based hydrogels as a portion of this type of “smart” material will have even greater and broader potential to be used in real-life products. Stimuli-responsive natural polymer-based hydrogels will likely to be a hot research topic in the next few years.

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CHAPTER 8

Self-healing properties of hydrogels based on natural polymers Guoxing Deng, Wing-Tak Wong, Minjian Huang, Runyu Wu and Wing-Fu Lai* Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong

8.1 Introduction Hydrogels are three-dimensional networks formed by physical or chemical cross-linking of polymers. Because of the presence of hydrophilic groups, they can absorb a large amount of fluids. By modulating the compositions of raw materials and by using different crosslinking methods, the structure and properties of hydrogels can be tuned, enabling the properties of hydrogels to be optimized for tissue engineering, organ remodeling, and drug delivery. Over the years, hydrogels with different properties (such as low surface friction, high efficiency of encapsulating drug molecules, or high stimuli-responsiveness) have been designed and prepared in literature [1
11]. Despite these advances, the use of hydrogels still has limitations. This is partly because of the poor mechanical strength and low stability of hydrogels, giving hydrogels poor wear and tear resistance. Moreover, long-term wear and tear in physiological conditions may lead to damage and defects in the structure of the hydrogel material. This greatly increases the difficulty of replacing traditional ceramic and metal implant materials with hydrogels for hard tissue repair. The development of selfhealing hydrogels, which can repair themselves and recover their usual functions after injury as living organisms, is therefore necessary. The self-healing function of hydrogels is attributed to reversible physical or chemical interactions, which respond to damage under certain conditions and lead to rapid autonomous network reconfiguration [4,7,12
14]. Together with the high biocompatibility and biodegradability of natural polymers, self-healing hydrogels generated from natural polymers have high potential in biomedical applications. In this chapter, we will discuss different modes of interactions applicable to the formation of self-healing hydrogels, followed by a review of current strategies for designing the hydrogel microstructure for 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00008-2 © 2020 Elsevier Inc. All rights reserved.

223

224 Chapter 8 self-healing purposes and for characterizing the performance of a self-healing hydrogel generated from natural polymers.

8.2 Overview of hydrogel design Biological tissues have the capacity to repair their own minor damage or to regenerate after injury. Inspired by this, many self-healing materials based on natural polymers have been fabricated. Incorporation of self-healing properties into material development has not only extended the service life of materials but has also reduced the use of limited natural resources. Natural polymer
based materials with self-healing properties possess a maximized life span and enable self-repair after use or implantation. Benefiting from this, the development and application of self-healing implantable devices have been developed rapidly. The generation of self-healing materials can be mediated by diverse bonds or interactions. When a crack is present in a material, the cleavage of covalent or noncovalent bonds at the crack can be initiated. The material then reforms the broken bonds to exhibit a self-healing capacity. Over the past few decades, the preparation of self-healing materials has been achieved by incorporating a number of microcapsules and/or microvascular networks into the material. In addition, the use of healing agents (such as catalysts and cross-linking agents) has been involved in the self-healing process. These agents are often released into the damaged region, followed by the occurrence of in situ polymerization and subsequent repair of the damage. Despite the ability to heal damage, these self-healing materials have drawbacks in many applications, including high complexity of the material structure and poor healing properties [15]. Self-healing hydrogels fabricated from other methods (e.g., reversible chemical cross-linking) have been proposed. Details of these methods for the design of a self-healing hydrogel are discussed in subsequent sections. Regardless of the method used to generate self-healing hydrogel, two common mechanisms generally occur during the self-healing process. One is the process of long-chain interdiffusion at the cleavage regions. The other is the reformation of reversible bonds between the polymer chains, followed by recovery of the damaged surface. These two mechanisms are independent of the type of interactions between the fractured moieties. In general, when the reaction temperature is higher, the diffusion rate will also be increased. The rate of long-chain interdiffusion is largely affected by the length of free chains and also the reaction temperature. The longer the polymeric free chain length is, the stronger and faster the interdiffusion of long chains between the damaged surfaces [16
18]. This may explain why the interdiffusion process usually occurs in polymeric hydrogels rapidly, because those free chains are highly fluidic in an aqueous environment. On the other hand, the self-healing process of semicrystalline or glassy-state materials is constrained owing to the comparatively poor mobility of the polymer chains, confining the reformation of chemical bonds responsible for self-healing to the surface, and thereby reducing the healing efficiency as a whole.

Self-healing properties of hydrogels based on natural polymers 225

8.3 Self-healing mediated by noncovalent interactions Self-healing materials are materials that can respond to external stimuli and reconstruct broken bonds. Some of these may be able to repeatedly undergo several healing processes without stress because the chemical bonds on the damaged surface of the material can be regenerated spontaneously and autonomously. Taking this into consideration, the choice of appropriate chemical bonds is particularly important during the design of a self-healing material. Some reversible noncovalent linkages (including electrostatic interaction, hydrogen bonding, metal
ligand coordinated complexation, hydrogen bonding, peptide and protein self-assembly, and host
guest interactions) can be incorporated into the design of a material. Moreover, the material can incorporate with reversible covalent interactions (such as imine bonds, disulfide bonds, acylhydrazone linkages, and CaC bonds formed in a Diels
Alder reaction) [12
14,19
22]. In addition, the material in a water-filled environment may experience hydrophobic associations between molecules. The hydrophobic association process can be driven by the entropy drive in the process (in particular the more disorderly tendency of the solvent); however, the enthalpy term of the process may play a role in determining whether the process can be triggered by itself [23]. Self-healing hydrogels can heal the damage, cracks, and cuts, but the process does not always take place. A number of studies have shown that the self-healing process is limited to hydrogel that has only just broken [17,24,25]. In other words, after the material is damaged, its self-healing capacity will be reduced over time. This is known as “time dependence,” which is caused by the hydrophobic rearrangement of the damaged hydrogel surface. Due to the directionality of the movement of hydrophobic parts at the contact surface between air and hydrogel, the contact angle of water droplets on the hydrogel can also be relatively high. After damage, the hydrophilic parts of the hydrogel are exposed to air. This initiates rapid migration of hydrophobic molecules to the damaged surface to minimize surface energy. The net result is that a hydrophobic polymeric layer is generated on the damaged surface to inhibit the diffusion of polymeric chains. In addition to the aforementioned, the process can be affected by the ratio of hydrophilic to hydrophobic moieties and by the rate of polymeric long-chain migration. These two processes are closely related to surface recombination [26]. Finally, the process of self-healing can be affected by the rearrangement of reversible interactions. The longer the separation time, the lower the amount of active groups on the damaged surface. This reduces the efficiency of self-repair, resulting in reduced self-healing ability [27]. Apart from hydrophobic associations, other mechanisms such as electrostatic interactions and hydrogen bonding may contribute to the process of self-repair. In the following parts of this section, different modes of interactions applicable to initiate self-healing are discussed.

226 Chapter 8

8.3.1 Electrostatic interactions Electrostatic interactions comprise the attractive or repulsive interactions between charged molecules. In most cases, electrostatic interactions are generally combined with other interactions for material repair. Although materials with the self-healing process based solely on electrostatic interactions are rare, such materials do exist. For instance, the electrostatic interactions between the poly(acrylic acid) (pAA) polymer backbone and free iron ions have been adopted by Wei and coworkers for the design of a self-healing hydrogel [28]. The results of tensile tests have revealed that a proper increase in the Fe31 concentration can improve the self-healing efficiency which, however, decreases as the cross-link density increases. In fact, the use of electrostatic interactions in the fabrication of self-healing hydrogels has been very commonly adopted in the case of peptides. Through self-assembly, short peptides form a variety of ordered nanostructures in aqueous solutions. This process is mainly dependent on amino acid sequences that allow for secondary interactions between amino acid residues. Negatively charged amino acids form stable βstrand or β-sheet structures and active communication patterns with peptides can all lead to self-assembly into hydrogel scaffolds [29]. In addition to electrostatic interactions of charged amino acids (e.g., Asp, Lys), hydrogen bonding (Ser) and π
π stacking (Phe) may initiate the self-assembly process. Furthermore, electrostatic interactions between polypeptides and ions can be adopted to generate hydrogels. For example, Lei et al. [30] have made a self-healing hydrogel with good mechanical stability. Gels are produced via ionic interactions and protein
protein interactions between bovine serum albumin and Ca21 ions (Fig. 8.1). Hydrogels heal spontaneously at physiological pH and at room temperature. Similar to the original sample, after the healing process, the hydrogel fractures in the massive area, but not on the contact surface. Peptide-based materials can be widely used in drug transportation and cell culture because of their self-healing and remarkable mechanical properties. Instead of applying electrostatic interactions alone, many more hydrogels are designed in a way that electrostatic interactions are combined with other cross-links. This can be exemplified by the work reported by Suo and colleagues [31], who have developed a hybrid hydrogel with a three-dimensional network structure by combining ionic crosslinking of alginate with covalent cross-linkings of polyacrylamide. No change in the integrity of the covalent cross-links was observed as the gel elongated. In the material, ionic cross-linking is a time-dependent process. This means that the mechanical stability of the gel does not deteriorate, even if the bond breaks. Intriguingly, regardless of whether the damage to the gel occurs within the permissible length range, the mechanical properties of the gel can be fully recovered. More recently, the use of static interactions

Self-healing properties of hydrogels based on natural polymers 227

Figure 8.1 A schematic diagram showing (A) the gelation process, and (B) the self-healing process in the hydrogel produced. Reproduced from J. Chen, Q. Dong, X. Ma, T.H. Fan, Y. Lei, Repetitive biomimetic selfhealing of Ca(2 1 )-induced nanocomposite protein hydrogels, Sci. Rep. 6 (2016) 30804 [30] with permission from Springer Nature.

228 Chapter 8 between zwitterions has been exploited for fabrication of a hydrogel [26,32]. The selfhealing efficiency of the gel does not change with the passage of time. In addition, polyelectrolyte multilayer assemblies can be easily recovered from mechanical deformation due to the presence of water [33,34].

8.3.2 Hydrophobic associations Hydrophobic associations are a type of interaction in which a hydrophilic group interacts physically with another hydrophobic group. The emergence of the micellar copolymerization technology enables rapid development of hydrophobically associating polymers [35]. An increasing number of scientists have studied hydrophobic polymeric materials. Previously, self-healing hydrophobic association hydrogels were generated by a combination of hydrophobic molecules [36,37]. These gels are physically cross-linked hydrogels formed by hydrophobic interactions. During the gelation process, hydrophobic molecules self-assemble into micelles and act as physical cross-linking points. These micelles can be reformed again, showing the reversibility and self-healing properties of the hydrogel system. The possibility of developing self-healing hydrogels from natural polymers using hydrophobic associations as the mediator has been demonstrated by Li et al. [38], who first modified chitosan with ferrocene, followed by dissolution of the product in an acidic medium for subsequent hydrogel fabrication. When hydrogel is subjected to an external force, the hydrophobically associating ferrocenyl groups act as dynamic crosslinking sites, allowing the stress to disperse and prevent the hydrogel from breaking. The self-healing properties of the gel are mediated by the dynamic nature of the hydrophobic associations. To demonstrate that the hydrophobic portion of the polymer is involved in cross-linking, the mechanical properties of the hydrogels were studied by increasing the number of hydrophobic groups. The higher the number of hydrophobic groups contained in the gel, the stronger the tensile strength exhibited [37]. The self-healing mechanism of a hydrophobic association hydrogel has been studied using a gel prepared by micellar copolymerization of acrylamide and octyl phenol polyethoxy ether acrylate in an aqueous solution containing sodium dodecyl sulfate (SDS) at 50 C [37]. Because the gel contains hydrophobic SDS micelles, when it breaks, the SDS micelles dissociate at the site of damage and cover the exposed surface. After the damaged sites are joined, the SDS molecules form cylindrical micelles at the contact interface. The hydrophobic group of the polyacrylamide chain heals the injured site by associating with the cylindrical micelles [37]. More recently, Okay and colleagues utilized the complexation of acrylamide and n-alkyl acrylate [e.g., stearyl methacrylate (C18) or behenyl acrylate (C22)] to prepare the hydrophobic association gel under ambient conditions [39]. To accelerate the growth of the micelles, NaCl has been introduced into the hydrogel system to improve the solubility of hydrophobic groups [40].

Self-healing properties of hydrogels based on natural polymers 229 Self-healing for recovery of the damaged hydrogel was largely mediated by hydrophobic rearrangement. During self-healing, the hydrophobic groups in the gel migrate to the air
gel two-phase contact interface. Because of this, regardless of the long separation time, the micelles can still be modified by the hydrophobic groups [41]. After extraction of SDS with water, the physical cross-links formed by hydrophobic associations become irreversible, and a loss of the self-healing ability results. This demonstrates the importance of the SDS solution for effective self-healing.

8.3.3 Metal
ligand coordination complexation Formation of metal
ligand coordination complexes is made possible if polymer chains can effectively form coordination bonds with metal ions. Because of the dynamic properties of metal
ligand complexes, the generated material can be self-healable. The technical viability of generating a self-healing hydrogel based on metal
ligand coordination complexation has been partly evidenced by Kikuchi et al. [42], who developed a selfhealing gel via gelation of a metallo-supramolecule [which consists of terpyridineterminated low-molecular-weight three-armed poly(ethylene glycol) (PEG)] with cobalt ions by aerobic oxidation. In this system, the metal ionic ligand complex exhibits reversible sol
gel transformation. Generation of self-healing hydrogels based on metal
ligand coordination complexation can also be inspired by nature, for example, by aquatic mussels, which rely on dopamine in their bodies to provide strong adhesion to various surfaces. Such strong adhesion is mediated by strong reversible complexation between the phenolic hydroxyl groups of dopamine and Fe31 ions. This intrinsic property has enabled the development of biomimetic self-healing hydrogels inspired from mussels [43
46]. A good example is the gel reported by Holten-Andersen and coworkers [45], who introduced bis- and/or triscatechol Fe(III) cross-links into a synthetic, dihydroxyphenylalanine (DOPA)-modified PEG to fabricate self-healing and elastic modulus polymer networks. At low pH (less than 5), only moncatechol
Fe31 complexes are present. These complexes are in a fluid state. When the pH of the surrounding medium is increased, bis- and tris-catechol
Fe31 complexes are formed. This results in the formation of a red elastomer gel at pH 5 12 and a purple gel at pH 5 8. The tri-catechol
Fe31 cross-linked gel can restore the original storage modulus even after fracture, which is shown by the result of the step-strain experiment. Mixing polyallylamine with various metals ions, such as Ga31, In31, and Al31 can also produce hydrogels with similar self-healing properties [43,44]. The mechanical performance of the Fe-catechol gel can be further improved by photopolymerization of the catechol methacrylate
Fe31 complexes with acrylamide [46]. The resulting gel displays a selfhealing efficiency of 80%, and can completely heal itself under the condition that there is no fracture at the healing site after the tensile test.

230 Chapter 8

8.3.4 Hydrogen bonding The third type of interaction that can be adopted for generation of self-healing hydrogels is hydrogen bonding; however, water is an excellent hydrogen bond donor and hence can form hydrogen bonds with polymer chains. This may affect the interaction between polymer chains inside the gel. This may also explain the phenomenon that self-healing gels generated solely based on hydrogen bonding between natural polymers are rare. Despite this, the possibility of using hydrogen bonding for fabrication of self-healing hydrogels has still been demonstrated by a few examples based on synthetic polymers. One example is the physically cross-linked polyvinyl alcohols (PVA) hydrogel that are prepared by freeze
thawing (Fig. 8.2) [17,47]. Since polymer chains can diffuse toward each other after contact on the cutting surface, the formation of hydrogen bonds between PVA chains is the main mechanism for self-healing [17]. The maximum healing efficiency of the hydrogel was estimated to be around 70% [17]. The process of self-healing in the hydrogel can be further facilitated by increasing the concentration of PVA, by shortening the time interval between surface separation and reattachment, and by reducing crystallinity to enhance interchain diffusion [17]. Another example is the reversible hydrogen-bonding ureido-pyrimidone polymer network generated using 3(4)-isocyanatomethyl-1-methylcyclohexyl-isocyanate in the regioselective coupling reaction of multihydroxy functionalized polymers with isocytosines [48]. With

Figure 8.2 Photographs of PVA hydrogel: (A) hydrogels with different colors (the right gel was colored with rhodamine B); (B) two halves of the two hydrogels; (C) a two-colored gel; (D) the bending test of the hydrogel that has undergone self-healing; (E) the tensile test of the hydrogel after the selfhealing process. Reproduced from H.J. Zhang, H.S. Xia, Y. Zhao, Poly(vinyl alcohol) hydrogel can autonomously self-heal, ACS Macro Lett. 1 (2012) 1233
1236 [17] with permission from the American Chemical Society.

Self-healing properties of hydrogels based on natural polymers 231 ureido-pyrimidone dimerization, the material displays good mechanical strength. More recently, a supramolecular hydrogel has been generated from functionalization of poly[2(dimethylamino)ethyl methacrylate] with light-activatable 2-ureido-4-pyrimidone units [49,50]. The hydrogel is responsive to temperature, light, and pH, and exhibits self-healing properties. Because of its stimuli-responsiveness, the gel can undergo reversible collapse with a change in temperature, and reversible swelling with a change in pH. This work has illustrated the feasibility of incorporating stimuli-responsiveness into a self-healing hydrogel to enable the properties of the gel to be tuned independently using different factors.

8.3.5 Host
guest interactions In host
guest chemistry, the binding affinity, specificity, and molecular recognition are mediated by complementary shapes and specific noncovalent interactions. The interaction between the host and guest molecules can be applied in the engineering of self-healing materials. Cyclodextrins (CDs), cucurbiturils, crown-ethers, and calixarenes are the most common synthetic molecules to be used as macrocyclic hosts. By using host
guest interactions, several self-healing hydrogels have been produced. For instance, previously a few host systems (namely pAA-6αCD and pAA-6βCD) from pAA and 6-aminoα-cyclodextrin (6αCD) and 6-amino-β-cyclodextrin (6βCD) have been generated for fabrication of a self-healing hydrogel [51]. Meanwhile, by modifying pAA with ferrocenyl groups, pAA-Fc has been produced as the guest. After adjusting the pH to 9, the host and guest molecules were mixed to form a gel. The gel can undergo sol
gel transition in response to redox changes (Fig. 8.3). Dissolved in the NaOCl solution, the pAA-6βCD/ pAA-Fc system becomes aggregated by undergoing a reduction reaction. Due to the lower affinity of βCD for the ferrocenyl group (Fc1) compared to the neutral Fc, the pAA-6βCD/ pAA-Fc hydrogel can switch to the sol state. Step-strain measurements (in which 90% of the original storage modulus have been found to be recovered in 20 seconds at γ 5 0.1% strain after the polymer pull-apart at γ 5 200%) underpinned the self-healing ability of the gel. The healing efficiency of the gel has been shown to be as high as 84%, as measured by the macroscopic healing test. More recently, N-adamantane has been grafted with polyacrylamide. The product has been used as a guest polymer for βCD [25]. The healing efficiency of such a gel has been shown to be as high as 99%, which may reduce to 84% after 25 hours separation time between cutting and regeneration. Finally, taking advantage of host
guest interactions, the fabrication of the cucurbit[8]uril (CB[8]) gel has been reported [52,53]. With the “barrel” shape, CB[8] could be incorporated with ureido-carbonyl oxygen molecules via the portal domain. It enables two guest molecules to react, and hence features a bigger pore structure compared to its homologues (CB[5
7]). By modifying PVA with methyl-viologen (MV) and hydroxyethylcellulose (HEC-Np) with naphthalene (Np), PVA-MV and HEC-Np have been generated as

232 Chapter 8

Figure 8.3 (A) Two-cut pAA-6βCD/pAA-Fc hydrogel pieces were shown to rejoin after standing for 24 h, with the crack being healed to form one gel. (B) Redox-responsive self-healing undertaken by the pAA-6βCD/pAA-Fc hydrogel as demonstrated by experiments using oxidizing/reducing agents. During the experiment, the pAA-6βCD/pAA-Fc hydrogel was cut in half, and an aqueous solution of NaClO was applied to the cut surface. Healing failed to occur even after 24 h. Readhesion was found 24 h after spreading an aqueous solution of a reducing agent onto the oxidized cut surface. Reproduced from M. Nakahata, Y. Takashima, H. Yamaguchi, A. Harada, Redox-responsive self-healing materials formed from host-guest polymers, Nat. Commun. 2 (2011) 511 [51] with permission from Springer Nature.

Self-healing properties of hydrogels based on natural polymers 233 the first electron-deficient and second electron-rich guests of a ternary complex network. After mixing with CB[8] at the ratio of 1:1:1, the ternary network has exhibited remarkable binding affinity. The introduction of cellulose nanocrystals to the ternary network could result in a tougher material with the low storage modulus being as high as 104 Pa [53]. Fragments of the self-healing gel can remodel in months after physical damage. These examples illustrate the viability of using host
guest interactions to mediate the fabrication of self-healing hydrogels.

8.4 Self-healing mediated by covalent interactions Most self-healing materials undergo noncovalent interactions to achieve self-healing, but covalent bonds may also be used to form self-healing materials. Rowan et al. [54] previously introduced the concept of dynamic covalent chemistry, which enables reversible bond formation and breakage under equilibrium. Chitosan-based hydrogels with reversible Schiff-base bonds have been developed by Zhang and coworkers [55]. Benzaldehydemodified PEG and the chitosan backbone react in less than 60 seconds through interactions of aldehyde groups and amine groups. Moreover, low toxicity and high stability of aromatic Schiff bases are the two outstanding merits for practical applications of the generated hydrogels. Repeated step-strain rheological measurements have confirmed self-healing of the gels by exhibiting a fast reduction of G0 above the critical strain of 100% and a rapid recovery of modulus at smaller strains. A manmade hole whose diameter is 0.9 cm in a hydrogel disc can recover and disappear within in 2 hours. Generation of a pH-responsive material, as the outcome of catechol-modified four-arm PEG (cPEG) cross-linked with 1,3-benzenediboronic acid (BDBA), has been reported by He and coworkers (Fig. 8.4) [56]. Mixing the components at pH 5 9 [optimal pH of the complexation must be located between the pKa of the catechol (9.3) and BDBA (8.7)] leads to the formation of hydrogels. UV
vis and 11B and 1H NMR spectroscopy have been adopted to investigate the stability of boronic esters. After a large strain (1000%) fracture, the hydrogels recovered their G0 almost completely when the strain has returned to 5%. The block fusion experiment has proved the self-healing of the gels. The same phenomenon was later observed with the use of DOPA-modified poly(Nisopropylacrylamide) cross-linked with boric acid [57]. In fact, these covalently crosslinked self-healing hydrogels are only a few examples that have been reported in the literature. In recent years, covalently cross-linked self-healing hydrogels have been developed extensively, and the related chemistry is becoming more and more sophisticated. These hydrogels can in general be generated using three chemical approaches: (1) reversible covalent bonds, (2) self-healing polyurethanes (PURs) based on carbohydrates and heterocyclic compounds, and (3) stable free radical-mediated shuffling reactions. Each of these types of hydrogels is discussed in detail.

234 Chapter 8

Figure 8.4 (A) A schematic diagram showing the pH-responsive hydrogel based on BDBA and cPEG in an aqueous solution at 20 C. (B) Self-healing of the gel generated from cPEG and BDBA at pH 9.0. The gel was fabricated as a cube (a), and then cut into two pieces (b). After that, they are fused together (c), and rejoined 30 s after fusion (d). Reproduced from L. He, D.E. Fullenkamp, J.G. Rivera, P.B. Messersmith, pH responsive self-healing hydrogels formed by boronate-catechol complexation, Chem. Commun. 47 (2011) 7497
7499 [56] with permission from Royal Society of Chemistry.

Two significant factors to design self-healing materials are reversibility of the chemical bonds and the chain mobility. As high-strength bonds, reversible covalent bonds are extremely suitable in developing self-healing materials with strong mechanical properties. One type of covalent mechanism that can be applied to the generation of self-healing materials is reversible cycloaddition reactions. As a matter of fact, both cycloaddition and Diels
Alder reactions have been exploited extensively owing to their suitability for the generation of various polymers. The formation of an adduct (exo- or endotype) by the Diels
Alder reaction between the furan (diene) and maleimide (dienophile) entities can be used to construct a cross-linked polymer network [58,59]. Retro-Diels
Alder reactions caused by elevated temperatures and/or cracks lead to disconnection of the diene and dienophile, followed by reconnection of the covalent bond at the lower temperature to repair the damage [60]. The incorporation of Diels
Alder pairs into epoxies, polyamides, and polyesters results in the formation of self-healing networks with diverse attributes [60].

Self-healing properties of hydrogels based on natural polymers 235 The most important part of the rational design of self-repairable heterogeneous networks is to enhance fragmentary mobility. In an earlier study, dihydroxyl coumarin derivatives were used to form a linear PUR chain, which, upon being cross-linked with the dimer of coumarin derivatives, can form optical stimuli-responsive PUR under 350 nm UV radiation [61]. The coexistence of coumarin dimer and PEG
PUR segments can lead to the availability of two glass transition temperatures (Tg) when cross-linking has occurred. Only one Tg can be observed under the condition of good miscibility between soft and hard segments when lower-molecular-weight PEG is employed. The dimerization of anthracene groups after photo-cross-linking can significantly increase the Tg from
46 C to 75 C. When the material is damaged, regeneration of building blocks induced by 254 nm photoradiation can be exercised, followed by reorganization of macromolecular segments and then cross-linking at 366 nm radiation [62]. The network can be rearranged and regenerated at room temperature. Another type of covalent mechanism is exchange reactions. Under milder conditions, dynamic exchange reactions between reversible covalent bonds can be used to generate self-healing materials. Exchange reactions, in general, have a lower activation temperature compared to temperature-sensitive reversible Diels
Alder reactions. In an earlier study, star polymers grafted with poly(n-butyl acrylate) were incorporated into poly(ethylene glycol diacrylate) core to undergo S
S bond exchange interactions for fabrication of heterogeneous self-healing networks [63]. Accompanied by an increase in the self-healing capacity, the mechanical strength of the network is reduced. To find the balance between the self-healing capacity and mechanical strength, rigid aromatic domains have been introduced into epoxy matrices [64]. The incorporation of aromatic segments into the epoxy backbone resulted in an increase in the Tg from 246 C for a fully aliphatic network to
3.6 C. The combination of aromatic segments and epoxy matrices, however, has led to a longer self-healing time. The introduction of the inorganic phase may substantially improve the material both in the mechanical strength and self-healing ability when comparing the inorganic
organic hybrid with the epoxy network enhanced by aromatic segments. At the moment, by introducing the heterogeneous phase, the mechanical strength of the material can be enhanced without compromising self-healing abilities. Cross-linked silicone-based materials can also be self-healable via exchange reactions. Their structures can be modeled by using silicone-based polymers synthesized from anionic ring opening copolymerization of octamethylcyclotetrasiloxane (D4) and bis (heptamethylcyclotetrasiloxanyl)-ethane (bis-D4) [65]. Modified with bulky groups, urea also has been transformed into different types of self-healable materials [66]. The dissociation energy of the C
N bond in urea can be reduced, thereby resulting in the dynamic equilibrium between urea and amine (
NH2)/isocyanate (
NCO) reactive groups. In fact, in self-healing hydrogels that are developed based on covalent interactions, the availability of sufficient time for reformation of covalent bonds is pivotal to network remodeling. Owing to the dissociation of covalent bonds, mechanical damage of polymers

236 Chapter 8 often produces free radicals. To reestablish the network, free radicals in the damaged surfaces can play a role during the regeneration of covalent linkages. Self-healing does not proceed when oxidation processes are weakened by unstable free radicals. A dynamic reaction involving the formation of stable free radicals provides an alternative. This can be exemplified by the case of trithiocarbonate (TTC) [67]. In TTC, hemolysis of C
S bonds can be stimulated by UV irradiation at 330 nm, generating stable free radicals which can exchange with adjacent TTC groups so that the process of self-healing can be gained. Thiuram disulfide moieties have also been used to repair covalently cross-linked polymers [68]. Visible light irradiation can initiate homopolymerization of the disulfide, leading to the formation of dithiocarbamate-based free radicals. It has been reported that these free radicals can be stable for more than 2 weeks [69], thereby giving sufficient time for the network to rearrange before reconnection. If a hydrogel is designed in a way that free radicals are adopted as part of the self-healing process, it would be good if the free radicals were stable in air. Such free radicals have been reported to be obtained via cleavage of diaryldibenzofuranone groups. This cleavage event can generate arylbenzofuranone oxygeninsensitive free radicals [70]. The use of oxygen-insensitive free radicals can significantly enhance the application of self-healing hydrogels in practical situations. Finally, cleavage of chemical bonds usually occurs at the weakest position of a polymer chain. Here it is worth noting that in polymers, forces are often unevenly distributed because of the cross-linked structure and polymer entanglements. Thus, all chemical bonds (irrespective of being covalent or noncovalent in nature) may break, depending on the magnitude of the force [71]. This fact can be also exploited for the design of a self-healing hydrogel. For instance, cleavages of cross-linked PUR chains will lead to the generation of diverse free radical intermediates. If those intermediates remain stable until meeting the pendulous chain ends in an extremely short distance, self-healing of the material can be attained. Hence, when it comes to the development of self-healable PUR networks, fourand five-membered heterocyclic compounds [such as oxolanes (OXO) and oxetanes (OXE)] with low activation energy to provide stable free radicals have been extensively adopted [72,73]. After modification of the C6 position of chitosan with OXE, the product has been cross-linked with tri-functional hexamethylene diisocyanate in a PEG solution to form a heterogeneous cross-linked PUR network (OXE
CHI
PUR). When mechanical damage is imposed, self-healing occurs upon exposure to UV irradiation, which can allow the pyranose ring in chitosan to undergo chair-to-boat conformation changes.

8.5 Design of the hydrogel microstructure When the microstructure of a self-healing hydrogel is designed, we may make use of microcontainers that can spontaneously release a healing agent to the damage area. The healing agent is expected to be able to fill the damaged area and restore surface

Self-healing properties of hydrogels based on natural polymers 237 integrity. Additives (e.g., catalysts or healing agents), which are in the matrix, must be rapidly dissolved in the healing agent. Moreover, they must be chemically stable to the matrix, and can tolerate a variety of potential applications or temperatures. Finally, to enhance the efficient use of the catalyst, the microcontainers have to be evenly distributed throughout the matrix. In this section, we will discuss different types of microcontainers (ranging from microcapsules and microvascular networks to nanoparticles) that can be exploited when self-healing hydrogels are designed based on natural polymers in the future.

8.5.1 Microcapsules The use of microcapsules for the design of a self-healing hydrogel has been reported by White and coworkers [74], who successfully introduced the autonomous healing concept, and presented a polymer composite incorporating a catalyst and a healing agent encapsulated within microcapsules (Fig. 8.5). The working principle of using

Figure 8.5 A schematic diagram illustrating the autonomous healing concept. According to the concept, when damage occurs, (A) a crack will be formed. (B) This leads to the release of the healing agent and catalyst, (C) resulting in polymerization to heal the damage. Reproduced from S.R. White, N.R. Sottos, P.H. Geubelle, J.S. Moore, M.R. Kessler, S.R. Sriram, et al., Autonomic healing of polymer composites, Nature 409 (2001) 794
797 [74] with permission from Springer Nature.

238 Chapter 8 microcapsules to mediate self-healing is that, if crack formation occurs, the microcapsules break and release the healing agent and catalyst. Here it is worth noting that, although there are diverse cross-linking mechanisms, there are several limitations to the use of microcapsule technologies. For example, in passive systems, the healing rate often fails to match the damage rate [75]. In addition, the number of healing times depends predominately on the number of capsules present in the material. The higher the number of capsules presents, the stronger the self-healing ability will be. After the mixture is consumed, subsequent injury to the same place will be unable to heal itself again. Even where self-healing can occur, a larger amount of healing agents is generally needed to be released to fill the damaged area completely or even partially. Finally, material properties and processability (extrusion, hot pressing, lamination, injection, powder coating, or resin transfer molding) can also be affected by the addition of microcapsules and catalysts [76]. Practically, to optimize the filling of the damaged site with the healing agent, the capsule size and weight fraction must be properly adjusted according to the predetermined size of the crack [77]. This means that it is necessary to decide in advance which type of damage can be healed by the formulation [78]. Based on the proposed application of the material, the mechanical properties, dimensions, and wall thickness of the capsule shells have to be properly considered. Special treatments (e.g., the use of inert gas) are also needed to prevent the catalyst from reacting with oxygen so that the self-healing ability of the hydrogel can be maintained. To achieve all these criteria, different strategies have been proposed for microcapsule modification. For example, to ensure that the outer wall of the microcontainer can resist mechanical stress during the manufacturing process while it is weak enough to ensure cracking during the self-healing process, various production parameters (e.g., types of emulsifiers, agitation speeds, pH, core/shell mass ratios, and reaction temperatures) have been exploited for use in controlling the mechanical properties [79]. In practice, in situ microencapsulation is generally carried out in an aqueous environment [80]. The core healing agent is hydrophobic, leading to the formation of an emulsion. The diameter of the capsule depends largely on the speed of agitation. The shell monomers are hydrophilic and soluble in the aqueous phase. The polymerization process can be triggered by using catalysts or heat. The final product of this process is a spherical polymer shell [81]. Common examples of capsule materials include melamineformaldehyde, poly(urea-formaldehyde), and melamine urea-formaldehyde. Another commonly used technique for encapsulation is by interfacial polymerization. During this process, hydrophobic prepolymers or monomers are dissolved in an encapsulant, sometimes with the addition of a cosolvent. Then, the mixture is emulsified, with aqueous monomers being added to the bath. The encapsulant
water interface of the droplet can then form the shell wall. One can control the shell wall thickness by changing the shell wall precursor or reaction time [81,82].

Self-healing properties of hydrogels based on natural polymers 239

8.5.2 Microvascular networks When damage occurs, microvascular self-healing materials possessing networks of capillaries (or hollow channels) can be healed using the healing agents released from the networks. Trask and Bond [83] applied the concept of vascular systems to generate highperformance carbon fiber/epoxy composites, after replacing carbon fibers with hollow glass fibers. Bleay and coworkers [84] also adopted hollow glass fibers (outer diameter 5 15 μm; inner diameter 5 5 μm), which are filled with vacuum-assisted capillary action, for the design of a self-healing hydrogel. The healing efficiency has been found to be closely related to fiber characteristic parameters (e.g., the impact energy level, the amount and type of tubing materials, spatial distribution, and thickness of the composite plate). More recently, Toohey et al. [85] generated a material system containing microvascular networks that enable over 60% healing efficiency. These have demonstrated the practical feasibility of using the concept of microvascular networks when designing the microstructure of a self-healing material system. Compared to using microcapsules, there are some advantages to using the microvascular system. First, it can heal cracks of larger dimensions because it can store more healing agents than microcapsules. In addition, it is possible to refill the vascular healing network through an external source or an undamaged vessel from the vascular system. Finally, by increasing the dimensions of the capillary network, the number of healing times can be enhanced [86]. However, although the vascular system enables healing to occur over a larger area, once the microtubules in the damaged area are filled and blocked, the supply of the healing agent to the area (and also to other parts of the vascular system) will be blocked. If the capillary force of the small tube is too high to be completely balanced with the flow characteristics of the healing fluid, the release of the healing agent from the microchannel can cause problems. In particular, for vascular systems with very small diameters, the behavior of microfluids is largely affected by the viscosity of the healing agents inside [87]. During the self-healing process, the viscosity of the healing agents may impede thorough mixing of the agents. Inadequate mixing can reduce the mechanical properties of the healing surface. Finally, because incorporation of the microvascular networks into a material involves multiple processes, production is very expensive and complex. These factors may impede the wide application of microvascular networks during the design of the microstructure of a self-healing material.

8.5.3 Additives and nanoparticles Self-healing of a thermosetting material can be obtained upon the addition of thermoplastic additives, which can flow into the crack to reconnect the matrix material. In an earlier study, Zako and Takano [88] added unmodified solid epoxy particles (50 μm average

240 Chapter 8 particle size) into the glass fiber/epoxy matrix to repair microcracks. The amine in the composite leads to curing of the epoxy resin. Heating at 120 C causes the embedded particles to melt and flow into the crack area. This method enables an increase in initial damage to be restrained by having the crack partially filled. When damage occurs on the same fracture surface, the healing process may possibly be repeated multiple times. Despite this, it is worth mentioning that leakage of unmodified epoxy particles can be a potential problem in self-healing material systems which rely on phase-separated additives and nanoparticles. Such leakage may create gas bubbles in the composites, thereby impairing the performance of the composite. To increase healing rates and to eliminate the effects of damage, healing agents must flow as quickly as possible [78]. In recent years, applications of phase-separated additives and nanoparticles have been continuously exploited in the development of self-healing systems. For instance, by dispersing phase-separated poly(caprolactone) (PCL) in an epoxy matrix, Luo et al. [89] successfully generated a heat-curable epoxy resin. After heating, PCL melts and fills the damaged area for self-healing via thermal expansion. Through nanoparticle reinforcements and the use of an amorphous polymer above the Tg, Picken et al. [90] also generated a polymer system with good healing and mechanical properties. In their work, dispersed particle phases were adopted to fill cracks and defects. Gupta et al. [91] deposited a silicon oxide brittle layer on a polymethylmethacrylate (PMMA) film containing embedded CdSe/ZnS nanoparticles. The interaction of enthalpy and entropy between the PMMA matrix and nanoparticles resulted in migration and clustering of nanoparticles toward the cracks in the SiO2 layer.

8.6 Characterization of self-healing hydrogels Characterization techniques adopted for studying hydrogels fabricated from synthetic polymers can be applied to study hydrogels generated from natural polymers. Among the different techniques, oscillating rheology, which is a method usually used to characterize viscoelastic materials, is widely used to characterize self-healing materials by measuring changes in the storage modulus and loss modulus. This technology can be adopted to study reversible interactions within hydrogels and to explore the relationship between the timescale and deformation frequency during the self-healing process. This is achieved by observing the stress
time function. Periodic sinusoidal shear deformation of a material occurs during measurement. The main parameters are oscillation amplitude (γ0) and oscillation frequency (ω). During measurement, trace samples can be used for preliminary experiments before formal measurements are carried out. The oscillatory shear test can reflect the change of the internal modulus of a gel, and can explore the reversible interaction at the microscale. The linear viscoelastic range of a material refers to the constant strain-independent response below the threshold when the strain increases at a constant angular frequency. When the strain increases further, the cross-linking or interacting cracking in the material can lead to structural damage, which is demonstrated as a sharp decrease in the modulus.

Self-healing properties of hydrogels based on natural polymers 241 Repeated increases and decreases of the strain can be used to indicate whether the recovery process is reversible. In this case, the self-healing gel is expected to restore the original state when the strain decreases. If this is the case, the structural damage can be understood as reversible. In the linear viscoelastic range, a small constant strain is first applied, and then is rapidly increased to a large value, which leads to destruction of the network structure. Subsequently, the strain is restored to the original value. After a few minutes, the degree of recovery (as well as the modulus) of the self-healing material is characterized. Here it is worth mentioning that self-healing properties can also be characterized at the macroscopic level. This can be achieved by comparing the mechanical properties of the healed material with that of the original material [16]. The kinetics of the healing process can be studied by examining the relationship between the healing time and healing efficiency (hence the relationship between the healing rate and hydrophobic rearrangement efficiency).

8.7 Conclusion Ordinary polymers do not exhibit self-healing properties because of their nonfree backbone as well as the lack of bond recombination abilities. Over the past decade, diverse mechanisms have been adopted to provide self-healing materials with reversible bonds. By manipulating the nature of bonds and intermolecular interactions, formation of reversible bonds has been made possible. This introduces molecular-level heterogeneities along the diversity of polymer backbones, thereby facilitating the formation of new materials with highly tunable properties and self-healing capacity. As presented in this chapter, reversible primary and secondary interactions can be employed to develop self-healing hydrogels. Some of these hydrogels are from synthetic materials; while some are from natural polymers. To further improve the self-healing property and versatility of self-healing hydrogels from natural polymers, deeper and more effective methods for characterization of self-healing hydrogels are required. Complete healing in bulk materials should be achieved by adjusting the interactions in the materials. Most of the existing self-healing hydrogels can only allow for self-healing within a short period of time after injury. Therefore, hydrogels with long-term self-healing ability after injury will be one of the research directions that are worth exploration in the future. Because self-healing hydrogels are difficult to endow with strong mechanical properties, improvement of the mechanical properties of self-healing hydrogels is also a direction that warrants investigation.

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233.

CHAPTER 9

The biological properties of hydrogels based on natural polymers Ali Ramazani* and Hamideh Aghahosseini Department of Chemistry, University of Zanjan, Zanjan, Iran

9.1 The importance of hydrogels based on natural polymers with biological properties Recently, advances in polymer science and biotechnology have facilitated the production of biomaterials with excellent biological properties. In biohydrogels water is present in a scaffold which is generally an appropriate natural polymer or a combination of natural polymers and artificial polymers [1,2]. The polymeric matrix gives particular characteristics to the biohydrogel, leading to applications in various fields [3
6]. The structure and biological properties of biohydrogels are extremely important factors in selecting their final applications. Biohydrogels can be classified on the basis of their preparation methods, ionic charges, physical structures, and the nature of their cross-links that lead to their different biological properties. Meanwhile, their mechanical and swelling properties are also important biological factors, which determine their final applications. Hydrogels are mainly designed and synthesized for use in the biological field because of their high water retention capacity, which makes them excellent structures to favorably shelter proteins and cells without altering their characteristics and properties. Currently, hydrogels with biological properties are a main focus in biomedical researches. Many advanced hydrogels based on natural polymers with biological properties have been developed, each possessing unique structures, namely high water swellability, high oxygen permeability, improved biocompatibility, structural diversity, and ease of loading and releasing drugs.



corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00009-4 © 2020 Elsevier Inc. All rights reserved.

247

248 Chapter 9

9.2 Antioxidant properties of hydrogels based on natural polymers Oxidative stress can produce radical by-products, which create significant toxicity and graft loss in cellular transplantation. Antioxidants are compounds that inhibit oxidation reactions that can produce free radicals, which may damage the cells of organisms. They have attracted a great deal of attention in biomedical applications [7]. The use of bare antioxidants in the pharmaceutical, biomedical, and food industries has faced various challenges, including volatilization, instability, and oxidation under ambient oxygen [8,9], hence the functionalization of biopolymers and inorganic materials with antioxidants addresses the aforementioned challenges. Polyphenols are an important class of antioxidants, which can undergo oxidation/reduction reactions [10]. The role of fruits and vegetables in disease prevention can be attributed, in part, to their constituent polyphenols. It has been shown that many polyphenolic constituents derived from plants have antioxidant properties [11]. Tannic acid is a natural polyphenol which is found in many plant species. It has many attractive biological properties such as antimutagenic, antimicrobial [12], antiviral, anticarcinogenic [13], antiinflammatory, and antioxidant effects [14]. Such important biological properties have attracted particular attention from biochemical communities to develop some poly(tannic acid)-based hydrogels as biohydrogels. In this regard, a superporous natural polymeric network of poly(tannic acid) hydrogel was synthesized employing trimethylolpropane triglycidyl ether as an epoxy cross-linker [15]. The antioxidant properties of prepared poly(tannic acid) hydrogel were evaluated by the total phenol contents and the effects of scavenging 2,20 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) radical cation (ABTS1∙) of degraded poly(tannic acid) hydrogels at pH 5.4, 7.4, and 9 and calculated in terms of gallic acid equivalent and trolox equivalent antioxidant capacity, respectively. The results indicated that in acidic media (pH 5.4), the further hydrolysis of tannic acid units of poly(tannic acid) hydrogel to gallic acid units, which have more effective antioxidant molecules than tannic acid [12], represented more antioxidant activity than in pH 7.4 (Fig. 9.1). Meanwhile, the highest antioxidant capacity of degraded poly(tannic acid) hydrogel was observed at pH 9. These results are reasonable because the poly(tannic acid) degraded much more at high pH values than in the other pH solutions (Fig. 9.1). The highly antioxidant nature of poly(tannic acid) hydrogels in addition their antibacterial, antiapoptotic, and antinecrotic properties has highlighted them as a good candidate in the treatment of chronic wounds.

The biological properties of hydrogels based on natural polymers 249

Figure 9.1 Gravimetric degradation of poly(tannic acid) hydrogel at pH 5.4, 7.4, and 9 at 37.5 C [15].

Semiinterpenetrating polymer networks or full-interpenetrating polymer networks based on tannic acid as a natural polymer and poly(acrylamide) as a synthetic polymer were prepared [16]. These hydrogel films were synthesized by incorporation of tannic acid during poly (acrylamide) hydrogel film preparation with and without cross-linking of tannic acid simultaneously. The synthesis of such poly(acrylamide)/tannic acid hydrogels with different amounts of tannic acid was done by concurrent use of redox polymerization and epoxy cross-linking. The antioxidant properties of tannic acid containing semiinterpenetrating and full-interpenetrating polymer networks hydrogels were determined employing FolinCiocalteau and ABTS assay methods as shown in Table 9.1. The results indicated that the antioxidant effect of full-interpenetrating polymer network hydrogel was lower than semiinterpenetrating polymer network hydrogel, possibly due to the cross-linked nature of tannic acid in full-interpenetrating polymer networks, where some of the cross-linker is also released with tannic acid. The poly(acrylamide) hydrogel showed no antioxidant properties. The other antioxidant hydrogels were synthesized by the construction of an interpenetrating chitosan network and its functionalization with gallic acid [17]. The hydroxyl groups on the aromatic ring of gallic acid could play important roles in the defense mechanism against free radicals and reactive oxygen species by breaking the free radical chain reaction [18,19]. Such antioxidant hydrogels can be prepared by the surface modification of poly(2hydroxyethyl methacrylate)-based hydrogels with an interpenetrating polymer network made from methacrylamide chitosan via radical polymerization reaction resulting in the

250 Chapter 9 Table 9.1: The comparison of antioxidant capability of poly(acrylamide)/tannic acid semiinterpenetrating polymer networks (s-IPN) and poly(acrylamide)/tannic acid full-interpenetrating polymer networks (f-IPN) with gallic acid (GA) and free tannic acid (TA) to determine their total phenol content, and trolox equivalent antioxidant capacity (TEAC) values [16]. GA b

Total phenol content (μg/mL) TEAC value (mM trolox equivalent/g)

TA


140 6 5

6TAa s-IPN 171 6 12

4TA sIPN 122 6 5

2TA s-IPN 89 6 20

TA s-IPN TA f-IPN 53 6 11

149 6 17

2.48 6 0.4 21.64 6 0.4 597 6 0.4 522 6 0.2 449 6 0.1 376 6 0.1 564 6 0.5

a

Acrylamide hydrogels with different TA ratio (1:0.025, 1: 0.05, 1:0.1, and 1:0.15, w/w) are represented by TA, 2TA, 4TA, and 6TA. b 170 μg/mL linear TA and TA released solution of hydrogels expresses as μg/mL gallic acid equivalency.

interpenetrating chitosan network hydrogels, which were more functionalized with gallic acid through an amide coupling reaction. It was indicated that the longer chitosan backbone in the gallic acid-modified hydrogels exhibited more antioxidant activity than the shorter chitosan backbone. Indeed the longer chitosan backbone could be attached to a higher amount of gallic acid. Taking advantage of the antioxidant properties of the trans-ferulic acid, a cellulose hydrogel containing this residue has been prepared [20]. The linking of trans-ferulic acid groups on the cellulose hydrogel rather than on its precursor was effected to avoid the inhibitory action of this antioxidant residue at the radical polymerization process. The cellulose hydrogel containing ferulic moieties was employed in inhibiting the lipid peroxidation in rat-liver microsomal membranes [21], induced in vitro by different sources of free radicals including 2,20 -azobis (2-amidinopropane) (AAPH) and tert-butyl hydroperoxide (tert-BOOH). The investigated hydrogel represented a stronger antioxidant activity in protecting the membranes from tert-BOOH- than from AAPH-induced lipid peroxidation. The results strongly indicated that this antioxidant hydrogel neutralized free radicals, which could be successfully applied as a prodrug of trans-ferulic acid in the pharmaceutical field. Catalytically antioxidant hydrogel was engineered based on the autocatalytic, self-renewing cerium oxide nanoparticles which were embedded in alginate hydrogel [22]. In order to enhance stability of the cerium oxide nanoparticles in solution, they were coated with dextran. Dextran compared with other stabilization coatings provides easy fabrication and increased cytocompatibility [23]. The potential of cerium oxide nanoparticle
alginate nanocomposite hydrogel was evaluated in the protection of encapsulated beta cells. It was demonstrated that cerium oxide

The biological properties of hydrogels based on natural polymers 251 nanoparticle
alginate hydrogel provides significant cytoprotection to encapsulated cells from superoxide exposure. Moreover, this cerium oxide nanoparticle-based hydrogel represented negligible cytotoxicity at nanoparticle concentrations 10-fold higher than free cerium oxide nanoparticles. This cerium oxide nanoparticle-based hydrogel platform has significant potential for applications and pathologies which necessitate the localization of potent antioxidants to a specific site. The capacity of cerium oxide nanoparticles to perform enzyme-mimetic electron transfer reactions with hydrogen peroxide (H2O2), 3,30 ,5,50 -tetramethylbenzidine (TMB), or superoxide was verified (Fig. 9.2A). Cerium oxide nanoparticle solutions (1 mM) upon the catalase-like behavior in the reduction of H2O2, display a red spectral shift (Fig. 9.2B), which could arise from the shifting of cerium atoms from the 14 to 13 oxidative states [24]. The reversible activity of dextran-coated cerium oxide

Figure 9.2 Cerium oxide nanoparticles (CONPs) in solution exhibit strong catalytic activity. (A) Hypothesized reactions of CONPs with superoxide and hydrogen peroxide. (B) Reactivity of CONP (1 mM) with H2O2 (1 mM) in solution via spectroscopic assessment of color shift. Return to the initial state is tracked over 14 days. (C) Effect of CONP concentration (0
13 mM) on TMB oxidation, measured via absorbance shift over time. (D) Neutralization of superoxide, generated via the XA/XO system, as measured through the oxidation of cytochrome C without (“XA + XO”, ) and with CONPs (1 mM, “XA + XO + CONP”, ). Additional controls of XA with no XO added (XA, ) and CONP only (“CONP”, £) are also shown [22].

•

•

252 Chapter 9 nanoparticles was evaluated by the incubation of TMB with cerium oxide nanoparticles, resulting in rapid TMB oxidation (Fig. 9.2C). The capacity of cerium oxide nanoparticle solutions to counteract the free radical superoxide (O2) was investigated, whereby superoxide was generated via a xanthine/xanthine oxidase (XA/XO) system. Fig. 9.2D demonstrates that the presence of 1 mM cerium oxide nanoparticles abrogated superoxide levels completely. Alginate/gelatine-blended hydrogels such as green polymer-based hydrogels loaded with guava leaf extract were synthesized employing the simple physical cross-linking in situ gelation method [25]. Guava leaf extract-loaded hydrogels illustrated high antioxidant activity, near 70% 1,1-diphenyl-2-picrylhydrazyl radical scavenging after 30 minutes immersion in PBS solution. An intrinsically antioxidant citric acid-based thermo-responsive hydrogel has been developed [26]. Such a hydrogel was fabricated from citric acid, poly(ethylene glycol) (PEG), and poly-N-isopropylacrylamide, which were copolymerized by sequential polycondensation and radical polymerization. This hydrogel could act as a nanonet for the delivery of nano- to macrotherapeutics where oxidative stress in tissue is a concern. This antioxidant injectable hydrogel showed free radical scavenging activity and inhibited lipid peroxidation. Moreover, the developed hydrogel platform was biocompatible and bioresorbable when evaluated in vivo. Another antioxidant hydrogel which was derived from natural biopolymers was prepared from agar/chitosan thin films [27]. The antioxidant properties of this natural polymerbased hydrogel were evaluated via total phenol content and ABTS1 scavenging assay methods. It was found that this agar/chitosan hydrogel film is a promising candidate as a wound dressing material due to its biodegradable, biocompatible, nontoxic nature.

9.3 Antibacterial properties of hydrogels based on natural polymers Antibacterial materials with effective inhibition of bacterial infections are classified as important biomaterials. Hydrogels with antibacterial properties are a main focus of biomedical research.

9.3.1 Hydrogels based on natural polymers with inherent antibacterial properties Hydrogel with inherent antibacterial activity are hydrogels that contain antibacterial components [28].

The biological properties of hydrogels based on natural polymers 253 Hydrogels with inherent antibacterial activity were developed as effective antibacterial materials with little or even no side effects compared with traditional hydrogels. The main forms of these hydrogels based on natural polymers are discussed below. 9.3.1.1 Hydrogels based on peptides with inherent antibacterial properties Peptides with antibacterial peptides are a diverse group of molecules produced by plant and animal cells [29], which are recognized as a panacea for the treatment of antibiotic-resistant bacterial infections [30,31]. Antibacterial peptides have strong antibacterial activities against many microorganisms [32]. The self-assembling of two antibacterial peptides (KIGAKI)3
NH2 employing a central tetrapeptide linker created an antibacterial hydrogel [33]. The use of bioactive peptide [antibacterial (KIGAKI)3
NH2 peptide] in the design of hydrogel structures rationalized the synthesis of materials with inherent biological properties. The antibacterial activity of this biohydrogel was confirmed by an antibacterial assay against Escherichia coli. Another antibacterial hydrogel based on a long-chain amino acid-containing dipeptide has also been developed [34]. A three-dimensional nanofibrillar structure was suggested for this biohydrogel according to the microscopic imaging studies. This biocompatible injectable hydrogel exhibited remarkable antibacterial activity against Gram-negative bacteria E. coli and Pseudomonas aeruginosa, which are responsible for many common diseases. A biohydrogel of cell-adhesive polypeptides and PEG with inherent antibacterial activity was developed with improved biocompatibility as a potential scaffold for cutaneous wound healing [35]. The concentration of PEG cross-linker for the developed hydrogel was optimized to achieve gelation and support antibacterial activity. A lower PEG concentration resulted in no gelation, while a higher concentration led to loss of the antibacterial activity of the biohydrogel, which was a result of a few amine groups killing the bacteria (Fig. 9.3). 9.3.1.2 Hydrogels based on amphoteric ions with inherent antibacterial properties Amphoteric ion hydrogel via electrostatic interactions with anionic bacterial membranes could facilitate the physical destruction of bacterial membranes and cell death [36]. Quaternary ammonium compound is one of the most well-known antibacterial materials. A cellulose-based hydrogel containing quaternary ammonium groups (Fig. 9.4A) was prepared by simple chemical cross-linking and showed strong antibacterial activity against Saccharomyces cerevisiae [37]. Such quaternized cellulose hydrogel networks, in addition to their antimicrobial activity, could provide good swelling properties with the electrostatic repulsion of their quaternary ammonium groups.

254 Chapter 9

Figure 9.3 Schematic diagram of the interaction between the positively charged polypeptides/poly(ethylene glycol) (PEG) hydrogel and the negatively charged bacterial membranes. (Bacterial cytoplasmic membrane disruption may be caused by the rapid interaction of the amphiphilic structure of antimicrobial peptides that mimic negatively charged lipid bilayers [35].)

Among several low-molecular-weight hydrogels, peptide-based gelators with a high biocompatibility have attracted a great deal of attention [39,40]. Antibacterial hydrogels based on N-fluorenyl-9-methoxycarbonyl amino acid/peptide-functionalized cationic amphiphiles were prepared [38]. The spectroscopic evidence has indicated that the selfassembled gelation process could result from the π
π interaction and intermolecular hydrogen bonding that are oriented through an antiparallel β-sheet arrangement of the peptide backbone (Fig. 9.4B). This Fmoc-based cationic hydrogel exhibited high antibacterial activity against both Gram-positive and Gram-negative bacteria.

9.3.2 Nanocomposite hydrogels based on natural polymers with antibacterial properties Metal and metal-oxide nanoparticles are commonly reported to be incorporated into biohydrogels to obtain nanocomposite biohydrogels. Silver has been used as an antibacterial agent for thousands of years ago. Nanocomposite hydrogels containing Ag nanoparticles including natural polymers or modified natural polymers have been widely reported. A plausible mechanism for the antibacterial activity of Ag nanoparticles which can be generalized to some other metal and metallic oxide nanoparticles is presented in Fig. 9.5 [41,42]. Accordingly, the interaction between silver ions (Ag1) and the thiol group in proteins on the bacterial cell membrane affect the bacterial cell’s viability by inhibiting the replication of DNA (Fig. 9.5).

Figure 9.4 Amphoteric ion hydrogels based on natural polymers. (A) Cellulose-based hydrogel containing quaternary ammonium groups with antibacterial activity [37]. (B) Hydrogels based on N-fluorenyl-9-methoxycarbonyl amino acid/peptide-functionalized cationic amphiphiles with antibacterial activity [38].

256 Chapter 9

Figure 9.5 Antibacterial mechanisms of metal and metallic oxide nanoparticles [41,42].

The polysaccharides, as an important class of natural polymer, are incorporated in the biohydrogel matrix. Alginate, as a linear natural polysaccharide, can form hydrogels via ionic interactions with Ca21. The Ag nanoparticles can embedded into sodium alginate microbeads through an electrochemical process, which produces nanocomposite hydrogels with high antibacterial activity against Staphylococcus aureus [43]. Furthermore, sodium alginate hydrogel was modified to enhance the mechanical strength of the resulting Ag nanoparticle
sodium alginate nanocomposite hydrogel. The calcium- or N,Nmethylenebisacrylamide-cross-linked sodium alginate fibers which were loaded with Ag nanoparticles showed sustained release of Ag and long-term antibacterial activity against Gram-negative bacterium E. coli [44,45]. They could be employed in wound dressings or utilized for other healing purposes [46,47]. Chitosan as a natural polysaccharide with inherent antibacterial ability has been used frequently for the preparation of hydrogel-based natural polymers. Chitosan has displayed low toxicity toward mammalian cells [48]. Taking the antibacterial and metal-binding advantages of chitosan and chitin, antibacterial nanocomposite hydrogels based on these natural polymers have been developed [49
52]. In this regard, chitosan/2-glycerophosphate/nanosilver hydrogels with different chitosan molecular

The biological properties of hydrogels based on natural polymers 257

Figure 9.6 Effects of the nanosilver concentration and molecular weight of chitosan on the inhibition zones (mm) for the degree of deacetylation: 88 chitosan/5% 2-glycerophosphate/Ag hydrogels. Values are mean 6 SD (n 5 10) [49].

weights (113, 146, 160, and 204 kDa) and containing different concentrations of nanosilver (0, 6, and 12 ppm) have been prepared and their antimicrobial activity was evaluated against S. aureus and E. coli (Fig. 9.6) [49]. It was indicated that all of the diameters of the hydrogel inhibition zones for P. aeruginosa and S. aureus increased as the concentration of nanosilver increased. Similar results were observed for the β-chitin/nanosilver scaffolds plotted against S. aureus and E. coli [52]. In order to reduce the cytotoxicity of nanosilver and obtain similar antibacterial activity, the hydrogel could be prepared with a lower molecular weight chitosan and a lower nanosilver concentration. Gold nanoparticles, in addition to their antimicrobial properties, could play an important role in some other biological applications, such as cell imaging, photothermal therapy, and sensing [53]. Moreover, gold nanoparticles are recognized as biocompatible materials. Au/ gelatin nanocomposite hydrogel was developed employing genipin as an excellent natural cross-linker [54]. The encapsulated Au could be released and attached to bacterial membranes and then they kill bacteria by leakage of bacterial contents or by penetration of the outer membrane and peptidoglycan layers. Biohydrogels can also be loaded with metallic oxide nanoparticles and have good antibacterial properties. Photocatalysis is the main antibacterial mechanism of metallic oxide nanoparticles [55]. Sunlight ultraviolet irradiation can produce large amounts of hydroxyl and oxygen free radicals at the surface of metallic oxide nanoparticles. These free radicals can oxidize organic matter in microorganisms, thereby the metallic oxide nanoparticles kill microorganisms in a relatively short time. Zinc oxide is the most popular antibacterial agent among the various metallic oxides [56
58]. Zinc oxide nanoparticles with antibacterial activity and noncytotoxicity at the

258 Chapter 9 appropriate concentrations are used in the formulation of many natural polymer-based hydrogels. Accordingly, chitosan hydrogel/ZnO nanocomposite bandages have been developed for wound healing and collagen deposition [59], such nanocomposite hydrogels have high antibacterial activity against both Gram-positive and Gram-negative bacteria as well as high temperature-resistant and high pressure-resistant bacterial spores [60]. Sodium alginate-based hydrogels were also loaded with zinc oxide nanoparticles and had enhanced swelling, blood clotting, and antibacterial activities against various strains of bacteria [61]. Low concentrations of zinc oxide nanoparticles were nontoxic to human dermal fibroblasts. Another nanocomposite hydrogel was prepared via in situ formation of CuO nanoparticles within swollen carboxymethyl cellulose hydrogel. The resulting carboxymethyl cellulose/ CuO nanocomposite hydrogels exhibited excellent antibacterial effects against both Grampositive and Gram-negative bacteria [62]. Moreover, chitosan
pectin composite hydrogel was loaded with TiO2 nanoparticles and exhibited good antibacterial activity and excellent biocompatibility [63].

9.3.3 Antibacterial agent-containing hydrogels based on natural polymers Antibiotics are the most common and effective antibacterial agents [64], however drug resistance of bacteria is a great challenge in the development and applications of antibiotics. Hence, minimizing conventional antibiotic dosages is more desirable than exploring new antibiotics [65]. Hydrogels based on natural polymers with high water content and biocompatibility could act as a form of local administration matrix for the selective release of loaded drugs at desirable sites [66,67]. 9.3.3.1 Ciprofloxacin-loaded hydrogels based on natural polymers Ciprofloxacin is a fluoroquinolone-based antibacterial agent, with high antibacterial activity against both Gram-positive and Gram-negative bacteria. The antibacterial mechanism of ciprofloxacin is such that the antibiotic resistance develops slowly [68]. Using the advantages of this antibiotic, some ciprofloxacin-based hydrogels based on natural polymers have been developed [67,69]. For instance, an antibacterial hydrogel was developed by selfassembling ciprofloxacin with a tripeptide (D-leucine-phenylalanine-phenylalanine) [67]. This ciprofloxacin
peptide self-assembled hydrogel with high drug-loading efficiency and prolonged release did not show any cytotoxicity in hemolysis assays of red blood cells or in cultures of fibroblast cells. Furthermore, this hydrogel showed high antimicrobial activity against S. aureus, E. coli, and Klebsiella pneumoniae. 9.3.3.2 Gentamicin-loaded hydrogels based on natural polymers Gentamicin is a traditional antibiotic with some application challenges such as systemic toxicity and low plasma concentration.

The biological properties of hydrogels based on natural polymers 259 Local administration of functional gentamicin hydrogels could provide an efficient antibacterial solution, for example, an injectable gellan gum-based hydrogel for embedding gentamicin-loaded poly(lactide-co-glycolide) nanoparticles. This gentamicin-based biohydrogel had antibacterial activity against Staphylococcus saprophyticus without affecting the bone-forming cells [70]. A class of thermosensitive chitosan
glycerophosphate hydrogels was developed via incorporation with nanohydroxyapatite/gentamicin. This antibacterial biohydrogel was introduced into polymethylmethacrylate bone cement and increased the mineralization capacity and antibacterial activity of the resulting cement [71]. 9.3.3.3 Vancomycin-loaded hydrogels based on natural polymers Vancomycin is an antibiotic, and is known as a last line of defense against an infection [72]. Biohydrogels acting as a drug-delivery system could protect and enhance the effectiveness of vancomycin. Some natural polymer-based hydrogels have been employed successfully as vancomycin carriers. For instance, an injectable gellan gum-based poly(lactide-co-glycolide) nanoparticleloaded system [73], injectable Pluronic
α-cyclodextrin supramolecular hydrogels [74], biohydrogels consisting of thiolated chitosan cross-linked with maleic acidgrafted dextran [75], photo-cross-linked methacrylated dextran, and poly(L-glutamic acid)-graft-hydroxyethyl methacrylate hydrogels [76] were developed as vancomycinloaded hydrogels with excellent antibacterial properties and desirable release capabilities. 9.3.3.4 Biological extract-loaded hydrogels based on natural polymers Some biological extracts with antibacterial properties have been employed in the preparation of antibacterial hydrogels. Sodium alginate has morphology, fiber size, porosity, degradation, and swelling ratio characteristics that have resulted in it being introduced as an ideal material for wound dressings [77,78]. Hence, sodium alginate hydrogels encapsulated with essential oils with antibacterial properties, such as lavender, thyme oil, peppermint, tea tree, rosemary, cinnamon eucalyptus, and lemongrass, have been developed as disposable wound dressings [79]. Among animal-based extracts, honey was the most easily acquired extract and has been used for its antimicrobial activity in the management of various wounds [80]. In this regard, a functional wound dressing was developed by carboxymethyl cellulose hydrogel-incorporated propolis honey, prepared by gamma radiation [81].

260 Chapter 9

9.4 Antiviral properties of hydrogels based on natural polymers Viral infections are known as some of the most common diseases affecting people worldwide. New viruses emerge all the time and only a small number of antiviral agents have been discovered to combat viral diseases [82]. Therefore the development of biohydrogels with antiviral properties is of particular importance. Semiinterpenetrating polymer network microspheres of acrylamide grafted on dextran and chitosan were prepared in size ranges of 265
388 μm by an emulsion cross-linking method using glutaraldehyde cross-linker [83]. The resulting hydrogel was employed in the controlled release of the antiviral drug acyclovir. It was found that the acyclovir-encapsulated microspheres prepared by a water-in-oil emulsion method could successfully extend the release time of this antiviral drug [83]. Because of the very short half-life of acyclovir (2
3 hours), special importance has been given to develop sustained-release systems for this drug [84]. Employing dextran/chitosan hydrogel formulations, sustained acyclovir release of up to at least 12 hours was obtained. It was found that the cross-linking density and polymer concentration could influence the controlled release. Moreover it was found that the inclusion of antiviral drug-loaded nanotubes into peptidebased biohydrogels may offer potential antiviral hydrogels [85]. Incorporation of Ag [86] and other metal nanoparticles and nonmetal nanoparticles including TiO2, ZnO, CeO2, CdSe, FeOx, and ZnS [87
90], in hydrogels based on natural polymers may provide effective antiviral biohydrogels [28]. This field of research, including biohydrogels with antiviral properties, requires further development.

9.5 Antifungal properties of hydrogels based on natural polymers Materials with antifungal properties have emerged as an important class of materials with biological properties. An antifungal biohydrogel was developed by adsorption of amphotericin B into a dextran-based hydrogel [91]. Amphotericin B is a potent antifungal agent, which has a broad spectrum of antifungal activity [92]. The resulting biohydrogel can kill fungi within 2 hours of contact. This biocompatible hydrogel can be reused for at least 53 days without losing its effectiveness against Candida albicans. Another amphotericin B-based biohydrogel was developed by amphotericin B-conjugated polypeptide hydrogels to improve the antifungal activity [93]. Such biocompatible and biodegradable hydrogels significantly exhibit antifungal activity against C. albicans. It was found that the developed biohydrogel is a suitable carrier for poorly water-soluble drugs and for enhancement of the therapeutic efficacy of antifungal drugs. Econazole is an imidazole antifungal derivative, which is used primarily for the treatment of both systemic and topical fungal infections and exhibited distinguished antifungal

The biological properties of hydrogels based on natural polymers 261 properties [94]. The poor solubility and lipophilicity or lipophilic nature of econazole have limited its bioavailability. A novel citric acid dendritic hydrogel was developed for the delivery of econazole nitrate [95]. The dendritic architecture and size of citric acid dendrimer hydrogels could improve the solubilization and permeation properties as well as the in vitro release and antifungal activity of econazole. Terbinafine hydrochloride is an allylamine with antifungal properties [96]. It was shown that chitosan hydrogel containing 1% terbinafine hydrochloride exhibited good antifungal activity against Candida krusei and C. albicans. This could be attributed to the inherent antifungal activity of chitosan, which was used as the polymer [97]. Therefore, the antifungal activity of chitosan may have increased the antifungal activity of the active drug. Despite the superior biological properties, biomedical applications of chitosan-based hydrogels are limited because of the toxicity of different organic cross-linkers. Hence, a new strategy has been proposed to produce supramolecular chitosan hydrogels using lowmolecular-weight compounds which are able to form covalent linkages and H-bonds to give a dual cross-linking [98]. 2-Formylphenylboronic acid, was employed to prepare such a three-dimensional chitosan nanostructure which adds the advantage of imine stabilization

Figure 9.7 The schematic representation of an iminoboronate
chitosan cluster [98].

262 Chapter 9 via iminoboronate formation and potential antifungal activity due to the presence of boric acid residue (Fig. 9.7) [98]. The resulting iminoboronate
chitosan-based hydrogels, which were obtained by dual chemophysical cross-linking, proved to have strong antifungal activity against Candida planktonic yeasts and biofilms. An antifungal heterocyclic hydrogel employing citric acid, indole-3-acetic acid, and PEG was prepared via condensation polymerization [99]. The hydrogel sample was tested for antifungal activity against Aspergillus fumigates, Rhizopus oryzae, and C. albicans at different concentrations. The biopolymeric hydrogels could provide an important restriction to fungal infections. The equimolar composition of the hydrogel presented increased antifungal activity compared with other compositions. The cytotoxicity results of hydrogels indicated the nontoxic and biocompatible nature of this antifungal heterocyclic hydrogel. This kind of biopolymeric hydrogel could be a suitable candidate for wound-healing applications.

9.6 Antiinflammatory properties of hydrogels based on natural polymers Inflammation is part of the nonspecific immune response that occurs in reaction to harmful stimuli, such as pathogens, damaged cells, or irritants [100]. Antiinflammatory drugs can reduce inflammation or swelling, while their continued use can lead to significant side effects, including gastrointestinal and kidney problems [101]. Hence, antiinflammatory drugs are well placed to benefit from local delivery strategies. Accordingly, the study of either polymer- or supramolecular-based biohydrogels as localized drug depots has attracted increasing attention due to their advantages in drug administration and low systemic toxicity [102,103]. Hydrogel design with self-assembling prodrugs providing superior characteristics such as inherent biocompatibility and biodegradability, as well as the simplicity of the system design, has attracted a great deal of recent attention for medical applications [104,105]. In this regard, a hydrogel-forming self-assembling prodrug was prepared through the conjugation of a hydrophobic nonsteroidal antiinflammatory drug [(R/S)-ketoprofen] into a short peptide with overall hydrophilicity [106]. The prepared amphiphilic peptide
drug conjugates represented excellent selectivity for the inhibition of cyclooxygenase-2 and inducing apoptosis in fibroblast-like synoviocytes with no associated chondrotoxicity. The particular chosen peptide sequence (valyl
glutamic acid
valyl
glutamic acid), which has alternating hydrophobic
hydrophilic side-chain functionalities, when conjugated to a larger hydrophobic moiety could form hydrogel structures [107,108]. Copaiba oil with antiinflammatory properties are employed as a popular medicine in the Amazonian forest region [109]. Copaiba oil nanoemulsions were incorporated in hydroxyethylcellulose and chitosan as natural polymer-based hydrogels [110].

The biological properties of hydrogels based on natural polymers 263 Hydroxyethylcellulose-based hydrogels incorporated with both positively charged and negatively charged copaiba oil nanoemulsions represented good stability and skin permeability and did not interfere with copaiba oil nanoemulsion droplet size and polydispersity index. An appropriate antiinflammatory effect was observed employing both types of copaiba oil nanoemulsions. A biocompatible collagen type 1/chitosan hydrogel was prepared with dexamethasone as an inflammation-controlling molecule combined in biohydrogel [111]. This biohydrogel secretes interleukin-10 as an antiinflammatory cytokine that has been related to its regeneration capacity. Inflammation-targeting hydrogel microfibers were generated from ascorbyl palmitate [112], which is a highly bioavailable, fat-soluble derivative of vitamin C. The prepared biohydrogel microfibers were loaded with the antiinflammatory corticosteroid dexamethasone and employed as a drug-delivery system to the inflamed colon.

Summary In this chapter the biological properties of hydrogels based on natural polymers including antioxidant, antibacterial, antiviral, antifungal, and antiinflammatory properties were extensively discussed and exemplified. The biological properties of hydrogels based on natural polymers were investigated as three general types, including hydrogels with inherent biological properties, nanocomposite biohydrogels, and drug-containing biohydrogels. Hydrogels with inherent biological properties were developed as effective biological agents with little or even no side effects compared to the others. Nanocomposite biohydrogels generally represented good thermal and mechanical properties and the drug-containing biohydrogels offer additional advantages such as targeted and controlled drug-release potentialities, and controlled degradation rate. Generally, the various types of biohydrogels with biological properties currently being developed promise a bright future for biomedical treatment.

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CHAPTER 10

The application of natural polymer based hydrogels in tissue engineering Jueying Yang, Xiaoyu Sun, Ying Zhang and Yu Chen* School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, P.R. China

10.1 Introduction Recent decades have witnessed the popularity of using engineering science and bioscience to guide tissue remolding. Tissue engineering [1] is normally illustrated as exploiting the application of the principles and methods of life sciences toward the structure function relationship in normal and pathological mammalian tissues in a variety of ways to restore, maintain, or enhance tissues and organs [2]. Tissue engineering, whether in the medical field or in the field of materials, has been applied in bone [3 5], cartilage [6 8], nerve [9 11], vascular [12 14], skin [15,16], and the organization of the gastrointestinal and urogenital systems [17]. The common application of tissue engineering can be roughly divided into four processes [1] as shown in Fig. 10.1. (1) Extract and separate specific cells from living tissues of the patient; (2) cells grow in three-dimensional (3D) scaffolds and expand to form new tissues or structures [18]; (3) cells and scaffold structures are implanted into the site of the body and lead to the formation of new tissue in the scaffold [19]; (4) the scaffold gradually degrades as the tissue develops, and the damaged organ or tissue is rebuilt. Scaffolds used in tissue engineering are one of the vital factors in tissue engineering [20]. For clinical use, some indispensable properties are required (Fig. 10.2), such as higher porosity [8], capability of maintaining structural strength and mechanical stability [21], quality of cell compatibility [20], and biodegradability [22]. In addition, for scaffolds serving as templates for cellular interactions and formation of extracellular matrix (ECM), in the process of degradation, the balance among the speed of material degradation, absorption by the human body, and the mechanical properties of materials must be taken into consideration [18,20]. Scaffolds of biomaterials play a mechanical supporting role in 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00010-0 © 2020 Elsevier Inc. All rights reserved.

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Figure 10.1 An example of a tissue engineering concept that involves seeding cells within porous biomaterial scaffolds [18].

Figure 10.2 Architectural and mechanical challenges associated with creating three-dimensional (3D) scaffolds [24].

The application of natural polymer based hydrogels in tissue engineering 275 cell growth and differentiation of stem cells into specific cells, and play the role of cell active factors in controlling the release of vectors [23]. In the early stages, synthetic materials were commonly used for scaffolds. The commonly used materials were short-chain saturated aliphatic polyesters, mainly polyglycolic acid (PGA) [25], poly(lactic-co-glycolic acid) (PLGA) [26,27], polylactic acid (PLA) [28,29], polycaprolactone (PCL) [30], polyurethane (PU) [31], polyvinyl alcohol (PVA) [32 34], etc., most of which have been approved by the United States Food and Drug Administration for internal use [27]. However, its strong hydrophobicity, inability to load growth factors, the reproduction of living cells, and rapid degradation rate cannot be neglected. It was also found that the fast rate of degradation had a negative effect on cell viability and cell migration because of the rapid accumulation of acidic by-products, which leads to local acidification and affects cell activity and migration [35]. Recently, natural materials have been explored and widely used as they do not interact with biological systems in the host [36]. Commonly, natural polymers include proteins like collagen [37], silk [38,39], polysaccharides like chitosan and its derivatives [12,15,40,41], hyaluronic acid [42,43], alginate [21,36], cellulose [44 46], etc. These materials are originated either from animals or plants and have existed in natural world for several decades. For example, polysaccharides assume the function of correspondent and storage in membranes and intracellular communication; proteins assume the function of structural materials and catalysts [12]; collagen is an important part of the ECM (Fig. 10.3) if used as

Figure 10.3 Content of the extracellular matrix (ECM) [20].

276 Chapter 10 scaffold in tissue engineering, and can not only imitate the environment of the ECM to a great extent, but also avoid immune rejection. Hydrogel is a water gel that swells but does not dissolve with a 3D polymer network, and its water content of over 90%, close to natural tissue, which is widely used in tissue engineering as an alternative to the ECM material used for cell culture [47]. Hydrogel fabricated by natural materials is a hydrophilic polymer network made by physical crosslinking or chemical cross-linking of individual polymer chains. It is also a multicomponent system composed of polymer 3D cross-linking network structure and medium [48]. In addition, it is suitable for cell seeding and encapsulation due to its high fluid content. Its biocompatibility and excellent diffusion properties also make it highly recommended for tissue implantation and other biomedical applications [49]. Due to the fact that its mechanical properties and structural properties are similar to those of tissues and ECM [50], hydrogel’s position as a scaffold material will become increasingly important in tissue engineering. Great efforts have been concentrated on the development of smart hydrogels (Fig. 10.4), which are able to swap from a liquid to gel state depending on external stimuli or initiators, as well as nanogels and microgels [43].

Figure 10.4 Types of hydrogels used for cardiac repair, administration routes that can be used to deliver each hydrogel into the heart, and how hydrogels reach their final structure in the cardiac tissue [49].

The application of natural polymer based hydrogels in tissue engineering 277 Generally, natural polymer hydrogels used in tissue engineering have outstanding hydrophilic quality, biodegradability, and biocompatibility [51]. Taking alginate as an example, each monomer of alginate contains a large number of hydroxyl groups, which greatly improves the hydrophilicity of sodium alginate [43]. Almost all natural polymers like alginate have a large number of hydroxyl groups. Excellent hydrophilic properties also make them well adapted to the ECM environment. The degradation of scaffolds is another significant feature for these materials used inside the human body. The degradation rates of most synthetic polymers like PGA, PLGA, and PLA are related to hydrolysis based on the composition, purity, and processing conditions, which are usually altered by controlling the molecular weight of the polymers. Natural polymers like collagen and silks degrade via the action of proteases, which suggests that their degradation can be controlled by adding enzymes or using enzymes produced by organisms themselves [38]. The excellent advantage that natural biomaterials offer over other materials is that they have intrinsic peptide sequences easily identifiable by cell-surface receptors [12,52]. For example, cell compatibility can be improved by modifying the surface of cellulose fiber or cellulose groups [52]. However, as defects found in natural materials, scaffolds with cellulose, alginate, or chitosan commonly have low mechanical strength. Importantly, materials like lignocellulose have also proven to be immunogenic in some cases for they are exogenous to the human body [49]. To meet the requirements of scaffolds, there are a few reports about the application of pure natural polymers in tissue engineering, using the surface modification of raw materials [46], the modification of functional groups [53], and the combination of two or more materials [21]. Each material has its own strengths and weaknesses, for example, cellulose has high mechanical strength but does not have a 3D structure [45]. If two or more materials are used together, the advantages of materials can be fully exploited and the disadvantages of materials can be minimized. Fig. 10.5 is an illustration of the use of multiple materials to enhance the mechanical properties and promote cell proliferation of hydrogels simultaneously. Also, various ways to fabricate scaffolds have been explored, such as injection [53], freeze-thawing [48], 3D printing [54], and photo-cross-linking [55]. In the rapid development of natural polymer based hydrogels, their synthesis and applications should not be neglected in the process of tissue engineering.

10.2 The properties of hydrogel in tissue engineering Not all biomaterials are suitable to serve as scaffolds in tissue engineering. Natural polymers usually have better proteolytic degradation but poor mechanical strength, especially hydrogels with high porosity. In addition, cell adhesion, internal connectivity, and growth factor binding are the basic factors enabling the success of organ transplantation and tissue regeneration for natural polymers.

278 Chapter 10

Figure 10.5 The process for preparation of silk/calcium silicate/sodium alginate (SCS) composite scaffolds [21].

10.2.1 Cell adhesion property In natural ECM, water-soluble and nonwater-soluble proteins, growth factors, and polysaccharide components create a complex web of supramolecular structures. They provide a physical form for cell growth, and can meet the requirements of specific biochemical properties of the microenvironment [56]. Collagen is widely found in ECM, providing a growth environment with mechanical strength and promoting cell adhesion and growth. The pure collagen hydrogels are poor in mechanical and thermal stability, limiting their widespread use in the field of biomedical materials [57,58]. It was found that pure collagen type I incorporated into gels gives no significant improvements in prolonging human cell adherence [59]. However, if cooperated with alginate gels, collagen acts as a delivery vehicle for cells to a tissue site, and it often acts as an auxiliary material to improve cell compatibility [60]. Another major component in the ECM is polysaccharide, which form a gel-like matrix structure to maintain the stability of the internal environment under atmospheric pressure [61]. Because of repeated units containing a lot of cellulose hydroxyl groups, cellulose

The application of natural polymer based hydrogels in tissue engineering 279

Figure 10.6 Schematic representation of the self-assembling procedure of polydimethylsiloxane-modified chitosan (PMSC) covalent amphiphilic polymer networks (CAPNs). First, the solvation effect from water caused morphological isomerization of the hydrophilic and hydrophobic segments of PMSC. This was followed by thermally induced water elimination; the salt ion effect rising between the abundant COO and NH31 groups contributed to the formation of a dense and hierarchical structure [66].

hydrogels can be obtained by modification of a series of special cellulose derivatives [62,63]. Cellulose and its derivatives are widely used in the preparation of tissue repair materials, porous scaffolds, recycled fibers, and other materials [64,65]. For example, bacterial nanocellulose (BNC) is chemically pure and can be produced in various shapes and sizes, but it exhibits low adherence for cell attachment. Kuzmenko et al. [60] used an effective bioconjugation method to modify the surface of BNC scaffolds with fibronectin and collagen type I, which increased the number of cells attached to the surfaces of human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In addition to cellulose, modified chitosan [66] and alginate [67] can also provide adhesion points for cells, which makes the growth and expansion to form tissues possible. Chitosan has been found to attract extracellular matrix (ECM) molecules (e.g., collagen IV, laminin, and fibronectin) that have been proved to promote cell adhesion, migration, and differentiation [68]. As shown in Fig. 10.6, Huang et al. [66] prepared a kind of amphiphilic hydrogel with polydimethylsiloxane-modified chitosan (PMSC). Based on microphase separation, the self-assembling hierarchical structure increased the stiffness of the PMSC hybrid through the formation of self-assembled aggregates, which provided anchor sites for cell adhesion for long-term proliferation without any adhesive reagent.

10.2.2 The mechanical property Due to its good biocompatibility, stability, porous structure, and low friction coefficient, hydrogel is an ideal scaffold [69,70]. However, the main reason limiting its large-scale application is its low mechanical property, especially those formed by natural polymers [71]. When applied in tissue engineering, the network structure of hydrogels will be

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Figure 10.7 Schematic diagram of chitosan-based hydrogel implant manufacturing process [73].

affected by the differentiation and proliferation of cells, which further deteriorate the mechanical properties of hydrogels. Keeping the natural source of hydrogels guarantees their biocompatibility, and promoting their mechanical properties is a current research hotspot [72]. The concentration and molecular weight of polymers forming a 3D structure is the base mechanical property of hydrogel. Hydrogel is constructed by intertwining mesh chains, so that the higher the concentration and molecular weight are, the stronger the net will be. Nawrotek et al. [73] changed the concentration of chitosan to study its effect based on an electrodeposition phenomenon, aiming at obtaining chitosan-based hydrogel implants intended for peripheral nervous tissue regeneration (Fig. 10.7). They found that when the content of chitosan was increased from 0.4 to 0.6 g, the Young’s modulus increased from 0.52 6 0.06 to 0.92 6 0.13 MPa. Compositing hydrogel with a reinforcer is the most common method to increase the mechanical strength, especially in the field of bone tissue engineering. Hydroxyapatite (HA) is one of the most popular additives as it is a component of bone with a molecular formula Ca10(PO4)6(OH)2. Maiolo et al. [74] found that by adding 3% HA, the tensile strength and compressive strength of PVA hydrogel was increased by 81% and 531%, respectively. For natural polymers, the same law is applied. Chitosan silica hybrid (CSH) hydrogel enhanced by HA (HA-CSH) possesses precisely controllable, high mechanical property and interconnected porous structures, obtained by combining the sol gel method and a 3D plotting technique. Compared with CSH, the compressive strength of the HA-CSH scaffold was increased from 10.99 6 1.29 to 13.55 6 2.52 MPa, supporting the adhesion and proliferation of cells and meeting the mechanical requirement of human trabecular bone. The increase in the mechanical property may originate from the nano-sized HA formed in situ and dispersed uniformly in the hybrid network, which reduced water absorption and increased the mechanical strength of the hybrid scaffold under humidity condition. Apart

The application of natural polymer based hydrogels in tissue engineering 281

Figure 10.8 Schematic representation of in situ synthesized polyacrylamide/sodium alginate/silica glass/ cellulose nanocrystals (PASG/CN) hybrid hydrogel [75].

from HA, the enforcements could be either polymers like cellulose nanocrystals [75] (Fig. 10.8) or inorganic molecules like calcium silicate [21] or calcium phosphate [76]. Another method to improve the mechanical properties is to build a double-network (DN) structure. DN gels consist of two interpenetrating but strong asymmetric networks, where the first network is rigid and brittle, serving as a sacrificial bond to effectively dissipate energy, and the second network is soft and flexible, maintaining the integrity of hydrogel [77,78]. Li et al. [79] fabricated a supramolecular polymer DN hydrogel based on hydrogen bonding cross-linked poly(N-acryloyl glycinamide) and Fe31 coordination cross-linked sodium carboxymethyl cellulose, which not only increased the mechanical property but also satisfied the requirements of recoverability and fatigue resistance (Fig. 10.9). Adopting both strategies like adding reinforcement materials and using DN structures at the same time is an excellent choice. Zhu et al. [80] built a DN hydrogel system consisting of γ-glutamic acid, lysine, and alginate, and meanwhile incorporated bacterial cellulose into the DN structures (Fig. 10.10). Furthermore, they introduced HA particles with two different sizes to fabricate a bilayer hydrogel scaffold based on the bionics principle for osteochondral regeneration, respectively: micro-HA in the top layer for promoting cartilage matrix deposition and HA nanocrystals in the bottom layer for enhancing compression

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Figure 10.9 Photos illustrating the deformations and mechanical performances of the cross-linked poly (N-acryloyl glycinamide)/carboxymethyl cellulose-Fe double-network (PNAGA/CMC-Fe DN) hydrogels: (A) bending; (B) knotting; (C) twisting; (D) compression and recovery of the DN hydrogel; and (E) compression failure of the CMC-Fe hydrogel [79].

modulus and osteogenesis. The scaffolds were evaluated by the osteochondral defect model of rabbits, and the results showed such as-synthesized scaffolds had a good osteochondral repair effect.

10.2.3 High porosity and internal connectivity The common process of tissue engineering is to isolate specific cells from the patient’s living tissues, to grow and amplify the cells to form structures in 3D porous scaffolds under accurately controlled culture conditions, and then to implant the cells/scaffolds into the required parts of the body, guiding the formation of new tissues in the scaffolds. The tissue is gradually degraded and disappears, enabling damaged organs or tissues to be reconstructed. Therefore its 3D structure and internal porosity are important factors to evaluate whether it is suitable for tissue engineering scaffolds.

The application of natural polymer based hydrogels in tissue engineering 283 (A) (1) 1 M Ca2+

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Osteogenic layer (1) Ca solution (2) RO water

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Figure 10.10 Schematic of hydrogels formation and structures. (A) Schematic illustration of the preparation of bacterial cellulose double-network (BC-DN) hydrogels. (B) Schematic depiction of the preparation of bilayer hydrogel scaffolds. (C) Schematic illustration of the structure of the bilayer hydrogel. (D) SEM images of bilayer hydrogel scaffolds [80].

In the design of scaffolds for tissue engineering applications with the construction of hydrogel microstructure promoting the regeneration of bone and other hard tissues, especially in the application of biomaterial scaffolds, porosity and pore size are the most important factors. Therefore it is very important to prepare a hydrogel mesh with the appropriate size and number to facilitate effective cell activity and material transport, and to realize its application in tissue engineering. It is generally believed that the pores must be connected to each other and be of sufficient size. These allow cell growth, supply nutrients, clean out metabolic by-products rapidly, promote the formation of blood vessels, with the new organization being formed and remodeled, in order to facilitate the integration of implants into host tissue. The formation of micrangium in biological scaffolds has become a critical indicator of tissue repair and improvement, and is a prerequisite for the healing of bone defects [81]. The importance of the porosity and pore size of biological scaffolds is greater, especially in hard tissues such as bones without functional vessels [82,83].

284 Chapter 10 At present, hydrogels with 3D vesicular structure and biodegradable properties have been developed for liver, bladder, nerve, skin, bone, cartilage, ligament, and tissue engineering, as well as oral regeneration [84]. Naturally derived polymer hydrogels are often used in tissue engineering because their composition and structure are similar to those of natural ECM. Porous scaffolds made from the blends of collagen and chitosan can be used as artificial skin. Porous scaffolds can also be prepared by freeze-drying of gelatin and chitosan cross-linked with glutaraldehyde, which can be used in skin and cartilage tissue engineering. For another example, after mixing different proportions of oxidized cellulose and collagen, different oxidized cellulose-modified collagen hydrogels (Fig. 10.12) were obtained. After cryodesiccation, the inner structure of the modified collagen hydrogels was observed by scanning electron microscopy. After cryodesiccation the pure collagen hydrogel had the larger pore size and inhomogeneous distribution; the oxidized cellulose-modified collagen hydrogel had s smaller pore size and more uniform distribution [85]. As we can see, the 3D structure of hydrophilic polymers provides a 3D space for cell adhesion and proliferation, therefore hydrogel scaffolds are widely used in the development of cell delivery and tissue engineering products.

10.2.4 Proteolytic degradation During the tissue engineering process after culture in vitro and amplification, the donor cells are implanted into the scaffolds with biodegradable 3D structures. Then complexes of the cells and material are implanted into the body or defect sites, and the implanted cells continue to proliferate and secrete ECM. With the degradation of scaffold materials, new tissues and organs with the same morphology and function as the repaired tissues or organs are formed, repairing defects and reconstructing functions. Only if materials own biodegradability, they can be replaced by new-developed tissues in the living body, thus achieving the goal of tissue engineering. Or this material may affect the function of regenerated tissue. Good scaffold materials should be able to degrade gradually in vivo as new tissues and organs are formed. If the material does not degrade very well, it will affect the structure, quality, and function of the new tissues and organs. Moreover, the degradation rate of scaffold materials should be in line with the rate of tissue and organ regeneration. Finally, the degradation products from scaffold materials respond to the microenvironment and the organism should be nontoxic. Although the degradation products of some materials are nontoxic to the body, they may cause excessive acid or hypoxia in the local environment, which is not conducive to the proliferation and differentiation of cells.

The application of natural polymer based hydrogels in tissue engineering 285 For example, alginate is a polysaccharide extracted from seaweed. Calcium alginate hydrogel can be formed by ionic cross-linking when calcium exists. Calcium alginate has been successfully used in tissue and cell culture in vitro because of its suitable morphology and nontoxicity. It produces mannuronic acid and glucuronic acid monomers by enzymatic degradation in vivo. These characteristics are favorable for chondrocyte transplantation. Therefore the biodegradability of alginate gel is very important for tissue engineering.

10.2.5 Growth factor binding The binding of growth factors with scaffolds stimulates or inhibits cellular affinity, proliferation, differentiation, migration, adhesion, and gene expression [86]. Cellular affinity has an important influence on cell adsorption, migration, phenotype maintenance, and intracellular signaling of cells in vitro, or cell growth and healing at the scaffold tissue interface of implantation in vitro [87,88]. Compared to synthetic polymers, natural polymers provide bioactivity for the intended tissue phenotype of a specific layer or microregion of the scaffold [54]. In an in vitro environment, it lacks the protection of serum, a complex medium supplement which contains potent stimulators of cell growth, including amino acids, growth factors, vitamins, proteins, hormones, lipids, and minerals [89]. Therefore hydrogel scaffolds can stabilize or release growth factors in several ways. On the one hand, natural polymer hydrogels can mimic the ECM environment. Hydrogels, especially those made completely from native ECM molecules, impart bioactivity through already present adhesion domains and protease cleavable sites [90]. Liang and Andreadis [91] presented a method to incorporate transforming growth factor-β1 (TGF-β1), involved in promoting the differentiation of mesenchymal stem cells (MSCs) to the myogenic lineage, onto fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. It was found that thanks to the fibrin-based constructs similar to ECM, TGF-β1 increases vascular contractility by sustaining the activity of the downstream signaling pathway. On the other hand, hydrogels provide the conditions for survival and controlled delivery of growth factors. Aloysious and Nair [92] fabricated a 3D biodegradable scaffold comprised of natural polymers dextran and gelatin (DEXGEL) for differentiation of adipose stem cells to islet-like clusters (ILCSs). The macroporous nature of the scaffold provides a spatial environment for maintaining the morphology, thereby sustaining the survival and function of ILCSs. The overall findings signify that adipose stem cells cultured on a DEXGEL scaffold in the presence of appropriate growth factors could be a preferred method to generate viable ILCSs.

286 Chapter 10 To some extent, the ability of growth factors to exist and enable binding with scaffolds should not be underestimated. Future research must be done to translate studies of native growth factor expression and knowledge from 2D physicochemical gradient work to new applications in 3D constructs. Time controlled release of growth factors must then be accomplished with a combination of new materials, novel strategies for the stimulation of growth factor release, and suitable 3D structural designs [93].

10.3 The application of cellulose-based hydrogel in tissue engineering As mentioned earlier, synthetic polymers, such as PGA, PLA, PLGA, PCL, PU, and PVA, have higher elasticity but may either be too difficult or too easy to degenerate, and the products after their degradation may not be absorbed and utilized by the body. As a result, their use in the human body is limited. Natural polymers come from a wide range of sources and have already been applied in many life sciences fields. Natural polymers like collagen [94], silk, chitosan, hyaluronic acid, and alginate, owing to their efficient mass transfer, low antigenicity, well-performed cell binding properties, and higher porosity with high internal surface area and volume ratio [95,96], are suitable scaffold materials. However the mechanical properties are limited in these materials, restricting their potential range of application. As with the development of materials for tissue engineering, in order to improve the biocompatibility of natural polymers to make them more popular as scaffold materials, it is a good choice to modify the surface of natural polymers, the combination of these with other materials then changes it to a hydrogel [97]. Hydrogels based on natural polymers are now widely applied as scaffolds to culture stem cells or cells in for instance the urinary system and nervous system, and also applied for cardiac repair and vascular regeneration. They are also used to simulate ECM conditions to form organs including bone [98,99] and cartilage [100]. It is still undeniable that the modification, blending, copolymerization, or even some sources of natural polymers, when they are in direct contact with fresh blood for a long time, can cause coagulation and thrombosis or foreign body reactions, which greatly limits their research and application in the biomedical field [101]. Because of the delicate composition of the human body, how to balance the biocompatibility property and other application requirements is still the main problem that researchers are struggling with to apply in vivo.

10.3.1 Application in stem cells Stem cells can be defined as units of biological organization that are responsible for the development and regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. The sources of stem cells can be embryo stem cells or adult stem cells. Embryo stem cells derived from extrafetal tissues are

The application of natural polymer based hydrogels in tissue engineering 287 stored for potential use, which is expensive and uncommon. In comparison, adult stem cells located in practically all organs and tissues of the adult organism are superior and almost all tissue engineering starts with the culture of adult stem cells [102]. In various types of stem cells, adipose-derived stem cells (ASCs) are multipotent somatic stem cells and have a great potential to differentiate and secrete growth factors [103]. Because adipose stem cells can differentiate into bone [94], cartilage [104], nerve cells [105], and other tissues, their culture is often the first step in tissue engineering. In tissue engineering in vitro, this differentiation can be induced by growing the cells on scaffolds with an appropriate composition, architecture, and physicochemical and mechanical properties [102]. Domingues et al. [43] reported an injectable hyaluronic acid/cellulose nanocrystal bionanocomposite hydrogel (HA-CNCs) for culturing ASCs. HA-CNCs nanocomposite hydrogels exhibited preferential cell-supportive properties in culture conditions in vitro due to the higher structural integrity and potential interaction of microenvironmental cues with CNC’s sulfate groups. Natural hydrogels like cellulose can also be engineered to develop adipose tissue-derived stem cells by cell sheet engineering (Fig. 10.11). Scaffolds do not allow sufficient cell

Figure 10.11 Temperature-responsive culture dishes. (A) During cell culture, cells deposit extracellular matrix (ECM) molecules and form cell-to-cell junctions. (B) With typical proteolytic harvest by trypsinization, both ECM and cell-to-cell junction proteins are degraded for cell recovery. (C) In contrast, cells harvested from temperature-responsive dishes are recovered as intact sheets along with their deposited ECM, by simple temperature reduction [108].

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IFS of ECM proteins in scaffold

Figure 10.12 This schematic depicts the preparation of fibroblast-derived matrix (FDM) [109].

migration, thus high-density cell seeding is applied in static cultures to establish adequate ECM interactions [106]. The hydrogel of methylcellulose (MC) was prepared by pouring aqueous MC blended with distinct salts on tissue culture polystyrene (TCPS) dishes at room temperature and subsequently gelled at 37 C for cell culture [107]. In order to overcome the high viscosity of the MC formulation, a modified MC collagen hydrogel method was developed to work with ASCs of adipose tissue and for the creation of multidimensional cell sheets. MC-aqueous solution was first prepared and then coated with TCPS dishes. The addition (evenly spread) of 200 μL of 2 mg/mL bovine collagen type I (pH adjusted to 7.5) over the MC-coated surface at 37 C, significantly improved ASC cell adhesion and proliferation on the hydrogel system [45]. Apart from ASCs, MSCs are another candidate, especially in bone and cartilage tissue engineering. Park et al. [109] used human bone marrow derived MSCs to test the cell viability, proliferation, and chondrogenic and osteogenic differentiation in integrating alginate hydrogel and fibroblast-derived matrix and finally formed bone tissues, as shown in Fig. 10.12. Bonifacio et al. [110] showed noncytotoxic effects while there was consistent expression of collagen II and high synthesis of proteoglycans, thus indicating the formation of cartilage matrix from human bone marrow derived MSCs. Numerous experiments have shown that the culture and differentiation of stem cells is a fundamental step in tissue engineering.

10.3.2 Tissue engineering scaffolds in vascular regeneration Vascular trauma and defects, as well as cardiovascular disease, have always serious risks to human life/health. The exploration and research of vascular scaffold materials have been developed, coping with vascular injury and vascular graft surgery. Exploring tissue engineered vascular scaffold materials to support the growth of new blood vessel cells and 3D space is the key to the construction of tissue-engineered blood vessels. Natural polymer

The application of natural polymer based hydrogels in tissue engineering 289 materials, such as cellulose, hydrogel, chitin, alginate, and many biopolymer materials, are widely used in the construction of vascular stents because of their excellent biocompatibility and certain mechanical properties. As natural polymer is rich in biosignal molecules, it is conducive to cell adhesion and proliferation, and can provide mechanical support for normal tissues. In recent years, it has been widely used in the preparation of artificial blood vessel stents. Sun et al. [111] combined human collagen and hyaluronic acid in different proportions, and prepared vascular scaffolds by vacuum freeze-drying method. The scaffolds were found to have good cell compatibility, histocompatibility, and degradation properties. Tseng et al. [112] showed that the porous scaffold (C2G5 scaffold) formed by cross-linking chitosan and gelatin at a concentration of 1:1.25 by freeze-drying technique, can provide a 3D growth space for the culture of fibroblasts. The cells are evenly distributed in the C2G5 scaffold material, which can reach the interior of the 3D scaffold, while on other scaffold materials it is limited to the growth of the scaffold surface. Chan et al. [113] found that porous 3D collagen scaffolds can support the formation of capillaries in vitro, and can promote the vascularization of tissues after implantation in vivo (Fig. 10.13). The amount of collagen scaffolds implanted with human adipose derived MSCs was significantly greater than that of nonplanted collagen scaffolds, confirming the feasibility of collagen as a scaffold for studying angiogenesis and 3D cell culture. Because of their ECM-like properties, hydrogels have also been developed to mimic vascular function. Chen [114] applied hydrogel to the surface of a textile-based tubular structure to prepare a textile-based gelatin hydrogel small-caliber artificial blood vessel. The prepared artificial blood vessel wall was more uniform and smoother, the hydrogel and the textile material were well combined, and the elasticity and compliance were good.

Figure 10.13 Capillary formation from human endothelial cells cultured in collagen scaffolds for 3 days. (A) Human dermal microvascular endothelial cells formed CD311 capillaries with clear luminal structures at 3 days in culture. (B) A higher magnification of the inset box in (A) with capillary-like structures identified by arrows. Scale bar 5 100 μm [113].

290 Chapter 10 Jackson et al. [115] prepared an electroresponsive hydrogel by combining Pluronic and methacrylic acid salts. The electroresponsive hydrogel can be delivered to a specific location of the target vessel by an integrated delivery device that creates a long-term occlusion in the vessel. The hydrogel can reduce clogging of blood vessels after implantation into a blood vessel without any significant side effects on the human body. Bacterial cellulose is a kind of natural cellulose synthesized by microorganisms. In recent years, bacterial cellulose has been studied rapidly in vascular tissue engineering. Esguerra et al. [116] implanted bacterial cellulose into the skin folds on the backs of hamsters to assess their biocompatibility and vascularization. The results showed that the compatibility of bacterial cellulose was good, and vascularization occurred around it. This study is based on an in vivo study basis for bacterial cellulose as a vascular tissue engineering material [117]. Because bacterial cellulose has unique physical, mechanical, and biological properties, Wippermann et al. [117] transplanted hollow tubular bacterial cellulose into pigs to replace the carotid artery. After 3 months of implantation, the chronic inflammation, foreign body rejection, cell growth, and lumen patency of bacterial cellulose were evaluated by histological analysis and electron microscopy. The patency rate of tubular bacterial cellulose was 87.5% (one of eight cases were blocked). Finely and smoothly moved to the inner surface of the graft lumen, endothelialization occurs, and a three-layer structure similar to normal blood vessels is formed in the tubular bacterial cellulose wall (inner layer: endothelial cells and basement membrane; middle layer: collagen and smooth muscle; outer layer: fibroblasts). At the same time, there was no obvious inflammatory reaction or foreign body rejection (Fig. 10.14). Due to the good biocompatibility, biodegradability, antibacterial activity, and multifunctional activity of chitosan, studies on the construction of tissue engineered blood vessels in vitro by chitosan and its derivatives have also been reported. Xiao [118] prepared a composite natural

Figure 10.14 Graft histology (H&E staining) at the experimental end point after 3 months: (A) An untreated segment of the carotid artery; (B) the BC graft after 3 months representing a “vessel-like” wall structure (arrow) showing a mild infiltration of lymphocytes; (C) cellulose fragments (arrow) are still detectable, which was not out of the ordinary as cellulose is nondegradable in the human organism [117].

The application of natural polymer based hydrogels in tissue engineering 291 bioscaffold gelatin/chitosan membrane from hydrogel, and evaluated the biological properties, histocompatibility, and cell compatibility of the membrane material, and prepared a tissue engineering artificial blood vessel patch. Xu et al. [119] mixed the in vitro expanded smooth muscle cells with bioprotein glue, inoculated on the outer surface of the chitosan tube, and then inoculated the vascular endothelial cells on the inner surface of the chitosan tube. The results showed that smooth muscle cells survived in the bioprotein gel and formed a 3D structure. Vascular endothelial cells form a continuous monolayer of cells on the inner wall of the chitosan tube, covering the inner wall of the chitosan stent lumen. Badhe et al. [120] fabricated a composite chitosan gelatin macroporous hydrogel-based scaffold with bilayered tubular architecture by solvent casting-co-particulate leaching (Fig. 10.15). This scaffold had a desirable tensile strength and porosity of 82%, indicating that it is an excellent tubular archetype for blood vessel tissue engineering.

Chitosan-gelatin hydrogel with Eudragit® L100 microspheres

Hydrogel poured on 2 mm plastic tube spinning at 20 rpm Drying of scaffold Eudragit® L100 particle leaching in NaOH

Curing in glycerol solution

Freeze-drying

Chitosan-gelatin hydrogel nonporous layer at 20 rpm

Macroporous, bi layered, flexible blood vessel scaffold

Figure 10.15 Preparation process of the macroporous bilayered tubular chitosan gelatin scaffolds [120].

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10.3.3 Tissue engineering scaffolds in myocardial engineering With modern medical standards and the improvement of people’s health awareness, diagnosis and treatment of coronary heart disease have become more standardized. Revascularization by reperfusion, antiplatelet, and anticoagulant therapies can greatly reduce deaths from coronary heart disease, and improve the prognosis of patients with coronary heart disease. However, it is not possible to regenerate cardiac muscle cells; myocardial infarction region will be replaced by fibrous scar tissue. Regional ventricular wall becomes thinner, causing infarction, ventricular dilatation, and eventually lead to heart failure or arrhythmia [121]. At present, a large number of researches have shown that using injectable hydrogels of stem cell transplantation, biological active substance, and drug controlled release treatments can solve this problem to a degree. When environmental factors like temperature, ultraviolet irradiation, pH, solvent exchange, ionic concentration, or injection shear force change, hydrogel can expand or contract, quickly inducing a liquid phase and solid phase change, called phase transformation. Due to rapid phase transformation, hydrogels can be formed in situ during the injection of solution and fill the defect of tissue [122]. Bioactive substances, mixed with water before injecting into the human myocardial infarction area, can achieve controlled release of bioactive substances in the myocardium to help stem cells to repair damaged heart muscle, resulting in an optimal biological activity (Fig. 10.16). Environmental changes are difficult to make in vivo, or this kind of change may affect the activity of enzymes, so it is not easy to achieve the gel sol process. The concentration changes of certain ionic make it possible to form

Figure 10.16 Schematic illustration of injectable hydrogel for tissue regeneration approaches [125].

The application of natural polymer based hydrogels in tissue engineering 293 hydrogel, which provides convenience to construct alginate hydrogels. Leor et al. [123] prepared a calcium cross-linked alginate solution that undergoes liquid to gel phase transition after deposition in infarcted myocardium. Examination of hearts harvested after injection showed that the alginate crossed the infarcted leaky vessels and was deposited as hydrogel in the infarcted tissue. In addition, Fang et al. [124] found that hydrogel with a 0.8% alginate and 20% gelatin formulation resulted in the highest cell viability during the injection process, and hydrogel composed of 1.1% alginate and 20% gelatin maintained the highest cell survival rate after 2 months in culture. The hydrogel-mediated cell delivery system provides a 3D environment similar to in vivo conditions and allows the maintenance of normal cellular function [126]. Fig. 10.17 represents epicardial delivery of in situ forming hydrogel. Studies have shown that by regulating the cell microenvironment of myocardial infarction, improving the infarction area local blood circulation and inducing cell homing strategies can effectively solve the problem of low survival rate of stem cell transplants. Growth factor has a strong regulatory effect on stem cell transplantation. When testing whether sustained delivery of basic fibroblast growth factor (bFGF) enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction, Cho et al. [127] found that combining bFGF delivery and CBMNC transplantation significantly enhanced neovascularization in the ischemic myocardium, as compared with either therapy alone. The enhanced neovascularization was likely due to increased vascular endothelial growth factor (VEGF) and bFGF expression.

Figure 10.17 Epicardial delivery of hydrogelable solution-carrying cells and biomolecular signals, which on administration forms three-dimensional (3D) hydrogel over the infarct site due to cross-linked networks [126].

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10.3.4 Tissue engineering scaffolds in brain tissue Traumatic brain injury (TBI) is an international multiple myeloma disease, which has one of the highest rates of mortality and disability. TBI survivors have residual obstacles in movement, and cognitive and social communication [128]. The brain’s ability to repair itself is limited, which is dependent on the brain-derived progenitor cell response to injury, showing limited ability to produce new cells. These defects are also not suitable for regeneration of nervous tissue in the central, therefore part of the strategy is to improve the treatment of the brain injury microenvironment, making it suitable for nervous tissue repair. We can use exogenous cells transplanted to replace failing or dying cells, making host cells integration of function and repairing neurobehavioral defects. Stem cell transplantation has a significant influence on the cell survival rate and nerve regeneration. The present experimental stereotaxic transplantation of cells around focal cerebral tissue after brain injury induces a low cell survival rate of only 1.4% 1.9% [10,129]. Cell transplantation in skin, bone, cartilage, blood vessels, peripheral nerve, and spinal cord repair in tissue engineering gives inspiration to improve its efficiency. Seed cells in tissue engineering strategies aim at providing adhesion of brackets, adding the correct amount of nutritional factors, so as to create a microenvironment more suitable for cell survival. To determine whether regenerating axons can be guided in a rostrocaudal direction, Guenther et al. [130] implanted 2 mm long alginate-based anisotropic capillary hydrogels seeded with bone marrow stromal cells (BMSCs) expressing brain-derived neurotrophic factor (BDNF) or green fluorescent protein as control into a C5 hemisection lesion of the rat spinal cord (Fig. 10.18). Four weeks postlesion, numerous BMSCs survived inside the scaffold channels, accompanied by macrophages, Schwann cells, and blood vessels. Quantification of axons growing into channels demonstrated three to four times more axons in hydrogels seeded with BMSCs expressing BDNF (BMSC BDNF) compared to control cells.

Figure 10.18 The fabrication of 2 mm long alginate-based anisotropic capillary hydrogels seeded with bone marrow stromal cells (BMSCs) [130].

The application of natural polymer based hydrogels in tissue engineering 295 Often, there is no self-repairing ability after nerve injury. Therefore the application of nerve tissue engineering scaffolds in nerve repair and promoting nerve regeneration has become a research hotspot. The ideal artificial nerve is a kind of nerve conduit with a special 3D structure, which can accept the regenerative axons to grow into and guide axons mechanically [9]. Ma et al. [131] isolated and dispersed neural stem cells and neural progenitor cells from rat embryonic cortex or subcortical neuroepithelium in type 1 collagen. The collagen structure of nerve cells was placed in a medium containing fibroblast growth factor to observe the changes to cells. This is the first time that functional synapses and neural networks originating from central nervous stem cells and progenitor cells have been presented in a 3D matrix. Neural progenitor cells turn to neurocyte in three-dimensional collagen culture medium. Their ability to proliferate and differentiate suggests their potential for promoting nerve regeneration in vitro (Fig. 10.19). The use of different natural polymers as scaffolds for nerve cell culture has been extensively studied [9,10,68].

Figure 10.19 Collagen-entrapped cortical or subcortical progenitor cells differentiate into neurons, astrocytes, and oligodendrocytes [131].

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10.3.5 Tissue engineering scaffolds in urinary system Tissue engineering provides a new method for the repair and reconstruction of the urethra, which has become the most promising physiological repair technique at present. Tissue engineering scaffolds do not only provide structural support for specific cells, but also enable a template action in guiding tissue regeneration and controlling tissue structure. Finding ideal scaffold materials for urethral reconstruction is the focus of current research. The defect filling stent is one of the most important applications. A defect filler support is a scaffold that provides filling, adhesion, and biological properties. Natural polymer hydrogels have good pore size and porosity. The 3D reticular structure is conducive to cell adhesion, growth, and infiltration, promoting cell migration to the material, increasing the process of vascularization, and is thus conducive to tissue regeneration. At the same time, its cell compatibility and biodegradability provide an excellent environment for the regeneration of urethral and bladder tissues. In addition, its mechanical strength also meets the needs of urethral and bladder tissue reconstruction to maintain the desired volume and structural integration during treatment. The cellulose-based hydrogel scaffold has good biocompatibility and epithelial cells grow well on the scaffolds. At the same time, compared with the pure cellulose-based hydrogel scaffold, the composite cell scaffold can promote the regeneration of smooth muscle and blood vessels. In addition, the composite cell scaffold does not produce obvious inflammation and fibrosis in the urethral tissue of the repair segment. The addition of hydrogels in the preparation process provides pore geometry for cellulose. The scaffold modified with the cellulose-based hydrogel consists of a surface compact layer and a loose porous layer at the base. This pore geometry plays an important role in promoting cell growth, infiltration, and vascularization. Before revascularization of the urethral tissue in the repair section, the regeneration of tissue depends mainly on nutrients, oxygen supply, and waste output. Therefore pore structure promotes the regeneration of tissues by promoting the transport of nutrients, oxygen, and waste [132]. In addition, glutaraldehyde-cross-linked collagen hydrogel scaffolds have been successfully used as fillers in the treatment of urinary incontinence [133], and continuous injections are required to maintain their function. Polysaccharide/hyaluronic acid copolymers are used as fillers for the treatment of bladder and ureter reflux, which requires that these copolymers not only be injectable, but also biodegradable [134].

10.3.6 Application in bone tissue Bone tissue defects caused by trauma, infection, or congenital disease are a major problem in repair and reconstructive surgery. Existing treatments include autogenous and allogenic

The application of natural polymer based hydrogels in tissue engineering 297 bone transplantation. However, these have some shortcomings such as increasing trauma, limited blood supply, and immunogenicity, which make it difficult to fully meet the clinical needs. Natural polymer hydrogel not only can provide 3D space for cells to survive, but is beneficial for cells to obtain sufficient nutrients, so that cells can grow on prefabricated 3D scaffolds to achieve the purpose of repairing bone defects. Therefore it has broad application prospects in bone tissue engineering. At present, alginate hydrogel is often used as bone and cartilage tissue repair materials. For example, Cohen et al. [135] studied alginate porous scaffolds prepared by lyophilization and cross-linked by Ca2 . Because of the poor adhesion to the cells, 90% of the cells were aggregated and the cells became spherical after being cultured in vitro for 24 hours. However, after being cultured for 4 days, the cells secreted fibronectin, which indicated that the cells could express their function normally, although the cells did not adhere to the alginate gel. In contrast, bone cells have good compatibility with alginate hydrogel. In addition, Mooney et al. [136] studied the covalent attachment of RGD (Arg-Gly-Asp) to alginate gels to improve their adhesion to skeletal muscle cells and promote the proliferation of skeletal muscle cells. Lee et al. [137] used polyglucuronide hydrogel as scaffold material for bone tissue engineering. After acidolysis and oxidation of alginate, they obtained poly glucose aldehyde. The hydrogels were then chemically cross-linked with adipic dihydrazide as the cross-linking agent. Rat cranial parietal osteoblasts and hydrogel were then injected into the back of the mouse. After 9 weeks, the mineralized bone tissue was well preserved. Hyaluronic acid is a polysaccharide with high molecular weight, which can form a solution of high viscosity. It is widely distributed in the ECM of almost all animals. At the same time, it is nonantigenic, and does not cause inflammation or an allogeneic reaction. It can be degraded into glucosamine in vivo, which is absorbed by human body. Also, it has excellent biocompatibility and bioactivity. In the area of physiological function, hyaluronic acid also regulates osmotic pressure, macromolecular transport, and cellular functions, forms physical barriers, promotes bone cell motility, proliferation, and coagulation, and plays an important role in bone formation [138]. Therefore hyaluronic acid has potential applications as a bone repair material. Kim et al. [139] prepared hyaluronic acid acrylate hydrogel and carried out bone regeneration research with loaded bone morphogenetic protein and MSCs from human bone marrow. Their results showed that hydrogel could be used as a carrier of cell and growth factor for bone repair. Yoshikawa et al. [140] inoculated bone marrow cells with 1 3 106/mL cell density into the pore of the matrix apatite scaffold, soaked in hyaluronic acid solution or culture medium, and then implanted the scaffold material into the subcutaneous tissue in the back of a rat. Four weeks later, histological observation revealed that new bone formation was found in most scaffolds, but not in the samples in the medium environment. Therefore it can be considered that hyaluronic acid provides a 3D space for cells to survive and can effectively promote the formation of new bone.

298 Chapter 10 Fibrin is also a natural ECM component, which has a good function in mediating cell signaling and interaction. Fibrin monomer can be polymerized into 3D network structure fibrin gel by thrombin. The polymerized fibrin gel can promote cell adhesion, proliferation, and secretion of matrix by releasing transforming factor and platelet-derived growth factor, and has good biocompatibility. In addition, fibrin gels have strong plasticity, which can delay the process of fibrin polymerization by reducing the thrombin concentration, and provide sufficient time for gel formation. Fibrin gel is an ideal ECM material, which is derived from the blood and avoids the immunogenicity problem. Nihouannen et al. [141] implanted the composite scaffold of fibrin gel and calcium phosphate particles into the femoral defects of New Zealand White rabbits. After 24 weeks, bone tissue was formed ectopically on the surface of the scaffold, and the new bone was mineralized well. Trabecular bone was also formed between calcium phosphate granules, and its characteristics were like those of cancellous bone. Chung et al. [142] synthesized a nanomicrosphere heparin/fibrin hydrogel complex with highly effective sustained-release bone morphogenetic protein (BMP-2) and implanted it into a rat skull defect model. By X-ray, histological alkaline phosphatase activity, immunohistochemical, and mineral content analysis, it was found that BMP-2 content in the new bone tissue formed by this material was significantly higher than that of the control group. The calcium phosphate ratio of the new bone tissue was close to the normal value, and a large area of mineralized bone was formed.

10.3.7 Application in cartilaginous tissue Different from bone that is continuously remodeled during the lifespan of men, the cartilage can barely heal itself, with some repairs occurring as mesenchymal chondroprogenitor cells invade the lesion and form cartilage. Therefore compared with bone tissue engineering in which the scaffolds should easily integrate with the adjacent bone and allow host blood vessels to colonize, the scaffolds used for cartilage tissue engineering help the growth and reconstruction of chondrocytes, which means that the degradation rate of scaffolds used in cartilage tissue engineering should be matched with that of production of ECM by chondrocytes. In addition, both scaffolds used in bone and cartilage tissue engineering should not only provide proper mechanical strength, but allow the transport of particles, such as diffusion of nutrients and cell waste products between cells and the ECM [5]. Cartilage is an avascular tissue consisting of only one type of cell, chondrocytes, which is embedded in a matrix composed of collagen and proteoglycan [143]. An important application of cartilage tissue engineering is to heal defects in bone joints [144,145]. Since the basic structure of the cartilage scaffold must be 3D, the structure of hydrogel satisfies this requirement perfectly. In addition, hydrogels after modification have absolute advantages in withstanding considerable pressure and mimicking the environment of cartilage tissue that articular cartilage needs.

The application of natural polymer based hydrogels in tissue engineering 299 For example, injectable self-curable silane hydroxypropyl methylcellulose (Si-HPMC) has been designed [146]. This injectable and self-setting hydrogel fabricated by HPMC grafted with silanol groups, allows the maintenance and recovery of a chondrocyte phenotype. Early in 2007, Vinatier et al. [147] illustrated that Si-HPMC hydrogel can be a suitable scaffold for the proliferation of human nasal chondrocyte (HNC)-based cartilage engineering by methyltetrazolium salt assay and cell counting experiments. The ability of HNC/Si-HPMC constructs to form a cartilaginous tissue in vivo was also investigated. Two years later, with further research, Vinatier et al. [148] developed an injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects. This study is the first to prove the concept of associating nasal chondrocytes with a Si-HPMC injectable hydrogel for the treatment of articular cartilage defects in vivo by the experimental use of autologous rabbit nasal chondrocytes (RNC) associated with an injectable self-setting cellulose-based hydrogel (Si-HPMC). Implants were histologically characterized for the presence of sulfated glycosaminoglycans (GAG) and type II collagen. Transcript analysis indicated that dedifferentiated RNC recovered expression of the main chondrocytic markers after in vitro 3D culture within Si-HPMC. Two studies in in vitro or in vivo environments demonstrated that Si-HPMC hydrogel is a convenient approach for cartilage tissue engineering. With the aim of minimally invasive surgery, transplanting Si-HPMC hydrogel containing autologous cells in an articular cartilage defect through percutaneous injection could pave the way for new therapeutic strategies for the treatment of cartilage defects. There has been a recent study about enriching a cellulose hydrogel with a biologically active marine exopolysaccharide for cell-based cartilage engineering [149]. Biological sulfated polysaccharides, such as sulfated GAGs, are major components of articular cartilage ECM [150]. In order to produce GAGs, Alteromonas infernus, an exopolysaccharide, has been shown to produce a branched high-molecular-weight polysaccharide, GY785 (B106 g/mol), which was associated with a Si-HPMC scaffold to develop a hydrogel for cartilage tissue engineering. They found that the Si-HPMC hydrogel with GY785 enriched the development of scaffolds for cartilage regeneration by growth factors tested by surface plasmon resonance and evaluating the biocompatibility of Si-HPMC/GY785 toward rabbit articular chondrocytes and its ability to maintain and recover a chondrocytic phenotype in vitro by methyl tetrazolium salt (MTS) assay, cell counting, and (real-time quantitative-polymerase chain reaction (qRT-PCR), which demonstrated that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering.

10.4 Outlook It has been decades since the concept of tissue engineering was first put forward and, in recent years, it has been developing rapidly. Tissue substitutes, for artificial blood vessels,

300 Chapter 10 skin, bone, and cardiac repair, have been widely studied not only in the research field but also in the clinic. For tissue engineering, scaffolds are an indispensable part, which has also changed from synthetic materials to natural materials with good biocompatibility. Hydrogels prepared from natural polymers have similar properties to the ECM environment, and have the advantages of promoting cell adhesion, proliferation, and differentiation. Due to their biocompatibility, biodegradability, and porosity, they are commonly applied in stem cell culture neurogenesis, cardiac repair, and reconstruction of bone and cartilage. Although much researches have been done on tissue engineering scaffolds, few have been put into practice, even after overcoming the disadvantage of insufficient mechanical properties. Since the human body is delicate, how to match the degradation rate of scaffolds with the formation rate of tissues is one of the most difficult problems restricting their use at present. In addition, how to ensure blood and tissue compatibility to overcome immune rejection also requires a large number of clinical experiments. With the advent of 3D printing technology and artificial intelligence, the manufacture of hydrogel scaffolds will be more convenient and intelligent, and could overcome the existing problems of materials and benefit more patients with organ damage.

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CHAPTER 11

Hydrogels as intrinsic antimicrobials 2, ˇ ˇ * Zeljka Vani´c1 and Nataˇsa Skalko-Basnet 1

Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia 2Drug Transport and Delivery Research Group, Department of Pharmacy, Faculty of Health Sciences, University of Tromsø The Arctic University of Norway, Tromsø, Norway

11.1 Introduction In spite of numerous technological advances in the medical field, the 21st century has seen the return of many infectious diseases that were earlier considered treatable; these infections are now presenting a clear health threat due to the increased antimicrobial resistance and reduced choice of available antibiotics to combat the resistant infections successfully. Antimicrobial resistance has emerged as one of the greatest challenges in current drug therapy [1]. Recent population-leveled modeling analysis in the European Union and European Economic Area indicates that the burden of infections caused by antibioticresistant bacteria has increased since 2007 and is the highest among infants and elderly, the most vulnerable patient populations [2]. Microorganisms are continuously developing mechanisms of resistance against antimicrobials; yet the process for discovery and development of novel antimicrobials is lengthy and expensive, often lasting over 10 years and having a very limited successful yield. New antimicrobials must overcome several types of resistance mechanisms that already exist against established antimicrobials and, simultaneously, avoid the development of novel resistance mechanisms. To add to the already serious situation is the rather limited understanding of the resistance mechanism. It is worth mentioning that antibiotics found in nature often have large, complex structures and multiple interaction sites, making them less prone to resistance development [3]. Academia, research institutions, and the pharmaceutical industry have addressed the increasing concern related to limited options in antibiotic use by different approaches and means. One of the approaches relies on reusing the old antibiotics packed in novel dosage forms and delivery systems that act on improving their efficacy [4,5]. Rather extensive efforts have been put into the search for novel antimicrobials of natural, semisynthetic, and 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00012-4 © 2020 Elsevier Inc. All rights reserved.

309

310 Chapter 11 synthetic origins. However, as mentioned earlier, the processes are lengthy and often with very limited success. Unfortunately, some industrial partners have opted to give up on antibiotic research and development, which is the worst option [6]. Systemic exposure to antibiotics leads to faster development of antimicrobial resistance and one of the means to reduce the resistance development would be to treat localized infections locally rather than systemically. We have in past decades focused on localized therapy as a means to assure improved therapy efficacy of various disease sites, including infections [7,8]. We have been interested in skin infections [9 16] and the treatment of vaginal infections [17 24]. In recent years, and responding to the increasing problem of antimicrobial resistance to antibiotics, we have looked for a material of natural origin with intrinsic antimicrobial properties as the building blocks for our delivery system. The synergy between an antimicrobial, either of natural or synthetic origin, and a building material with intrinsic antimicrobial properties, is expected to lower the doses required to achieve efficient antimicrobial treatment. Applied topically, avoiding systemic absorption, it limits the chances of resistance development. One of the most promising materials with intrinsic antimicrobial properties is chitosan. Chitosan can be used as a building block of various nanosystems as well as a vehicle. We have focused on chitosan as a building block in hydrogels utilizing its antimicrobial activity and contributing to the antimicrobial potential of the final formulation. Chitosan forms hydrogels which are mucoadhesive and have great potential in application on wounded skin and vaginal site [24,25]. Hydrogels have long been used as basic building blocks in tissue engineering and regenerative medicine [26]. However, in spite of tremendous developments of polymer chemistry and innovation pressure on the pharmaceutical industry, only a relatively few “smart materials” have been translated into clinical use [27]. There are different reasons behind this lack of success; however, most of the polymers used as hydrogels served only as vehicles and not as active ingredients. Since this book is dedicated to hydrogels as a dosage form, we have focused this chapter mostly on hydrogels made of material with intrinsic antimicrobial properties, rather than discussing the characteristics of hydrogels. Since chitosan is the most studied polymer with intrinsic antimicrobial properties, we have discussed the mechanism of its antimicrobial action in more detail. A simplified version of the concept discussed in this chapter is presented in Fig. 11.1.

11.2 Intrinsic antimicrobial properties of chitosan Chitosan, [α(1-4) 2-amino 2-deoxyβ-D-glucan], is a cationic polysaccharide produced from the deacetylation of chitin, a natural element abundantly sourced from the shells of

Hydrogels as intrinsic antimicrobials 311

Figure 11.1 Proposed concept of efficient topical therapy of infections.

crustaceans. Over the last 200 years since its discovery, the study and application of chitosan has taken on many different forms [28]. Its biocompatibility, biodegradability, low toxicity, and mucoadhesive and antimicrobial properties are the reasons for its use in a wide range of skin products and biomedical applications. Chitosan is considered to be one of the most successfully developed biodegradable polymers. Chitosan can be dissolved in various organic and inorganic acids. Its reactive amino groups are a good target for possible modifications with different ligands, functional groups, and moieties [29]. Furthermore, the wound healing property of chitosan, which is dependent on the degree of deacetylation (DD) and molecular weight, has been utilized as patches, scaffolds, bandages, and bioadhesive gels where chitosan enhances the function of inflammatory cells, therefore accelerating the wound healing [28,30]. This chapter focuses on chitosan as a polymer with pronounced antimicrobial effects. The effects are contributed to its ability to destabilize the outer membrane of Gram-negative bacteria [31] and permeate the microbial plasma membrane [32]. The molecular weight, degree of acetylation, and

312 Chapter 11 ionic strength and pH of the dissolving medium will affect its antimicrobial properties. By tailoring its formulation (gel, film, etc.) it is possible to optimize its antimicrobial potential [28]. Although the exact mechanisms of the antimicrobial actions of chitosan remain to be discovered, it has been proposed that the interaction between positively charged chitosan molecules and negatively charged microbial cell membranes results in the disruption of the microbial membrane, and consequent leakage of proteinaceous and other intracellular constituents [33]. It was suggested that at a lower chitosan concentration (,0.2 mg/mL), the cationic groups of chitosan bind to the negatively charged bacterial surface to cause agglutination, while at higher concentrations, the larger number of cationic groups forms a net positive charge to the bacterial surfaces forming a suspension [31]. It is well established that the polycationic structure of chitosan contributes to its antimicrobial activity. A higher positive charge density is expected to lead to stronger activity, suggesting that a positive charge is associated with the DD or degree of substitution [33]. Regarding its molecular weight, rather contradictory results were published on the correlation between the chitosan molecular weightage and bactericidal activity. However, there is a consensus that the hydrophilicity of chitosan is crucial for its antimicrobial potential. Due to limited space, we are not able to deeper into various chitosan derivatives and possible tailoring of its properties by chemical modifications in this chapter. The mechanism of antibacterial properties of chitosan remains to be discussed and a deeper insight as well as the proposed mechanisms need to be confirmed. At pH below its pKa (6.3) the electrostatic interactions between its polycationic structure and predominantly anionic parts of the surface of Gram-negative lipopolysaccharides and cell surface proteins are considered major contributors. The number of amino groups linked to C-2 on chitosan backbones is important in electrostatic interaction. The native chitosan with higher DD should exhibit stronger inhibitory activity than chitosan with lower DD. However, at the environmental pH above its pKa, hydrophobic and chelating effects dominate its electrostatic interactions [33]. Low-molecular-weight (LMW) chitosan hydrogel has shown promising anti-Candida activity in an in vivo catheter mouse model [34]. Chitosan was also proposed for possible prevention or treatment of fungal biofilms on central venous catheters [35]. Chitosan at very low concentrations (0.0313%) could kill more than 50% of Candida albicans cells in the early and intermediate phases of biofilm formation, whereas higher concentrations were required to kill cells in mature biofilms [36]. Particularly attractive is the proven ability of chitosan to disrupt bacterial biofilms in bacterial vaginosis. Bacterial vaginosis is one of the most recurrent infections of the genital tract and most therapies fail to completely disrupt the persistent bacterial biofilms. Negatively charged polysaccharide matrix covers biofilm bacteria and prevents/restricts the

Hydrogels as intrinsic antimicrobials 313 penetration of applied antimicrobials. Chitosan gels were able to disrupt Pseudomonas aeruginosa biofilms in a pH-independent manner. Considering that the pKa of chitosan is 6.3, more than 99% of its amino groups will be protonated at pH 4, and only about 60% at pH 6; however, at both pHs the activity was similar and possibly independent of the cationic charge density [37]. The chitosan concentration required to achieve this effect was rather low (0.13%). Its relevance has been also confirmed in clinical settings. Akincibay et al. [38] reported the clinical effectiveness of chitosan in the treatment of chronic periodontitis contributing the observed therapeutic efficacy to the antimicrobial action of chitosan. Similarly, Boynuegri et al. [39] reported that chitosan gel either alone or in combination with demineralized bone matrix/collagenous membrane has beneficial effects on periodontal regeneration. Chitosan has been used intensively to form nanocomposites with other known antimicrobials, such as silver, for example, [40]; however, as the focus is on hydrogels with intrinsic antimicrobial potential, we have not discussed nanocomposites further.

11.3 Antimicrobial hydrogels for wound therapy and treatment of skin infections Hydrogels as wound dressings can play a major role in the wound healing process due to their physical properties allowing absorption and retention of wound exudate, thus promoting fibroblast proliferation and keratinocyte migration; both events are necessary for complete epithelialization of the wound and to reduce scarring [41]. However, this hydrated, water-rich, environment can also facilitate microbial infections. Therefore hydrogels capable of imparting antimicrobial action in addition to their primary role as a wound dressing are of high importance [42,43]. In that context, hydrogels constituted of polymers with intrinsic antimicrobial properties, such as chitosan, are of great relevance, especially considering the treatment of acutely infected wounds, or as dual-acting formulations when incorporating either antimicrobial drugs or nanoparticles, for the treatment of chronic, deep wounds [28]. The tight mesh network within the hydrogel allows incorporation of antimicrobial substances or nanoparticles loaded with antimicrobials; the active ingredients are then gradually released to the infected wound as hydrogel absorbs the exudate and swells [42]. An additional advantage of hydrogel as a wound dressing lies in its cooling effect, which is contributed by high water content, assisting in pain relief [44].

11.3.1 Chitosan-based hydrogels Chitosan-based hydrogels are considered superior as wound dressings because they induce faster wound healing, protect from secondary infection, and minimize scarring. Moreover, chemical modifications of chitosan leading to derivatives such as N,N,N-trimethyl chitosan,

314 Chapter 11 N-succinyl chitosan, N-carboxymethyl chitosan, and thiolated chitosan or combinations of chitosan/chitosan modifiers with other polymers or nanoparticles improve the hydrogel properties related to wound healing [45]. For instance, carboxymethyl chitosan is a watersoluble polymer and its antibacterial activity is found to be superior to that of unmodified chitosan [46]. Hydrogels composed of O-carboxymethyl chitosan embedding lincomycin exhibited good pore structure and swelling characteristics. Compared with plain O-carboxymethyl chitosan gel, the in vitro antibacterial activities of lincomycin O-carboxymethyl chitosan toward Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli significantly improved with the increase in lincomycin concentration within the hydrogel. Interestingly, an increase in the content of O-carboxymethyl chitosan and cross-linking agent in the gel formulation resulted in a decrease in the antimicrobial activities against both tested strains [47]. These results are not so surprising considering that it has been earlier confirmed that, at higher chitosan concentrations ( . 0.2 mg/mL), the large number of positive charges imparted a net positive charge to the bacterial surfaces to keep bacteria in suspension. However, at lower concentrations, the polycationic chitosan is bounded to the negatively charged bacterial surface causing agglutination [31,48]. Targeting the treatment of infected wounds, Hurler et al. [25] developed mupirocin-loaded liposomes incorporated into chitosan hydrogel composed of 2.5% high-molecular-weight (HMW) chitosan. The formulation was found to be nontoxic toward keratinocytes in vitro and exhibited antibiofilm activity against S. aureus biofilms, although the effect was more pronounced toward planktonic bacteria and prevention of biofilm formation than against the mature biofilms. The in vivo wound healing study performed in a mice burn model over 28 days demonstrated that the mupirocin-loaded liposomal hydrogel was safe for use and exhibited the same healing effect as the registered topical formulation of mupirocin. The histological evaluations revealed complete reepithelialization of all wounds, whereas the healing time for the liposomal formulation was shorter in comparison with the marketed formulation. However, due to the limits of the used in vivo method, the authors suggested additional in vivo evaluations to prove the potential of the novel liposomal formulation clinically. Many of the chitosan-based hydrogels investigated for the treatment of infected wounds contained incorporated metallic nanoparticles such as silver, gold, or zinc. Among these metals, silver is the most commonly used because of its good antibacterial properties and relatively low toxicity [49]. The presence of silver nanoparticles inside the matrix of chitosan hydrogel contributes to the hydrogel hardness and therapeutic efficiency. Using the chitosan powder with a DD of 80% 95% and alkali-urea solutions, Xie et al. [50] synthesized the hydrogels with integrated silver nanoparticles, which exhibited pronounced hardness and antibacterial

Hydrogels as intrinsic antimicrobials 315 activities. The inhibition rates of the blank chitosan gel against S. aureus and E. coli were 27% and 12%, respectively, while incorporation of silver nanoparticles resulted in superior antibacterial efficiency that was confirmed in vivo (rat model). The wound healing effect was significantly increased and accelerated in comparison with the plain chitosan gel. Zhou et al. [51] prepared series of gelatin/carboxymethyl chitosan hydrogels comprising silver nanoparticles by radiation-induced reduction and cross-linking at ambient temperature. The hydrogels had an interconnected porous structure and could absorb 62 108 times their dry weight in deionized water. Silver nanoparticles contributed to the antibacterial efficacy of the hydrogels, assuring the controlled release and erosion of the hydrogel. The composite hydrogel incorporating 2 and 5 mM nanosilver inhibited more than 25% and 50% of E. coli, respectively, while the maximal activity was obtained with 10 mM nanosilver. However, the cytotoxicity studies were not conducted to prove the biocompatibilities and lack of toxicity of the developed formulations. Chitosan hydrogels prepared from HMW chitosan at different concentrations of the polymer (3.5%, 1.75%, and 0.875%, w/w) and incorporating silver or gold nanoparticles, were evaluated for their rheological, thermal, antibacterial, and biocompatibility properties. Chitosan gold nanoparticle hydrogels demonstrated better thermal stability than chitosan silver nanoparticle hydrogels, while the viscosities of both types of hydrogel were affected by the concentrations of the embedded nanoparticles in the hydrogel. In vitro antibacterial studies performed against reference Streptococcus mutans, S. aureus, and E. coli strains, as well as four clinical isolates of S. mutans, demonstrated similar efficiency of the hydrogels containing either silver or gold nanoparticles against the tested reference strains. Generally, the activities of the formulations against clinical isolates of S. mutans were significantly lower than toward reference S. mutans strains. Testing of the plain 3.5% chitosan hydrogels prepared with different concentrations of acetic acid showed the highest efficiency for the hydrogel prepared with the highest concentration of acetic acid (6 vol.%), which was similar to efficiency of the hydrogels incorporating nanoparticles prepared with 4 vol.% acetic acid. In vivo wound healing evaluation in a rat model performed with the hydrogels containing the highest concentrations of either silver or gold nanoparticles, demonstrated normal healing at 28 days. Interestingly, the rats treated with chitosan gold nanoparticle hydrogel exhibited scarring at the wound site. The histopathological evaluation of the same group of rats showed a moderate infiltration of leukocytes as compared to the light infiltration in groups of the animals treated with chitosan silver nanoparticle or plain chitosan hydrogels. The findings suggest more delayed wound healing probably as a result of the toxic effects produced by the contact of gold nanoparticles with the skin cells [52]. Sundheesh Kumar et al. [53] incorporated zinc oxide nanoparticles into chitosan hydrogel prepared from LMW chitosan for the possible treatment of infected wounds and diabetic foot ulcers. This flexible and microporous composite bandage displayed controlled

316 Chapter 11 degradation, excellent platelet activation ability, enhanced blood clotting, and in vitro antibacterial activities against S. aureus and E. coli. However, the plain chitosan gel (without ZnO nanoparticles) has not revealed antibacterial activities, possibly due to the neutral pH of the chitosan as a vehicle. The observed antibacterial effect was therefore attributed to only ZnO nanoparticles, which are expected to produce reactive oxygen species. These species, together with zinc ions, attacked the negatively charged bacterial cell wall and caused cell wall leakage. The higher activity attained against E. coli than S. aureus was ascribed to the thick layer of peptidoglycans in the cell wall of S. aureus. The cytotoxicity evaluation on human dermal fibroblast cells demonstrated that plain chitosan hydrogel did not exhibit any toxicity during 24, 48, and 72 hours of incubation. However, the presence of ZnO nanoparticles in the chitosan hydrogel affected the cell viability, which was between 30% and 60% after 24 hours, depending on the concentration of ZnO incorporated in the chitosan hydrogel. Interestingly, after a longer period of incubation, the remaining viable cells began to multiply and the cell viability increased, suggesting a proliferative effect. In vivo wound healing assessment in a rat wound model proved the enhanced healing effect with faster reepithelialization and collagen deposition. Moreover, even plain chitosan hydrogel demonstrated a good healing effect after 1 and 2 weeks. The superiority of the formulation was confirmed by in vivo antibacterial evaluation against P. aeruginosa, Staphylococcus intermedius, and Staphylococcus hyicus, the bacteria isolated from the rat wounds, thus proving the potential of the nanocomposite chitosan hydrogel for the treatment of infected wounds and diabetic foot ulcers. Wahid et al. [54] proposed a similar approach for the treatment of infected wounds. It was based on the in situ incorporation of ZnO nanorods (160 190 nm) into the cross-linked carboxymethyl chitosan hydrogel. The nanocomposite-based hydrogel exhibited excellent antibacterial activity against E. coli and S. aureus bacteria, with S. aureus being more responsive to the formulation. However, the hydrogel without ZnO nanorods showed a poor antibacterial effect, probably due to the cross-linking of carboxymethyl chitosan contributing to a decrease in the positive charge on the formed hydrogel, or due to the poor dispersion of hydrogel in phosphate buffered saline solution. Biodegradability and biocompatibility studies have not been performed to evaluate possible physiological acceptability of the formulation. The properties of the chitosan hydrogels as wound dressings can also be improved by preparing their combination with other polymers of either natural or synthetic origin, to exert the advantages of each component and enhance the therapeutic effect of the final wound dressing. For example, alginate exhibits low cell adhesiveness due to its poor protein adsorption to the hydrophilic surfaces; however, its mixture with chitosan enables improved cell interaction, adhesion, and proliferation [45]. Accordingly, wound dressing based on the alginate/chitosan hydrogel promoted the cell proliferation in vitro and accelerated the wound closing rate [55]. In a relevant study, a hydrogel composed of a mixture of chitosan,

Hydrogels as intrinsic antimicrobials 317 alginate, silk, and dextrin, containing recombinant human epidermal growth factor was prepared and evaluated for in vivo wound healing effect. Although antimicrobial studies were not included, the results demonstrated that this multicomponent natural origin hydrogel could promote the healing process and recovery of deep diabetic wounds in rats [56]. To develop a wound dressing with intrinsic antimicrobial properties, Straccia et al. [57] designed an alginate hydrogel coated with water-soluble chitosan hydrochloride. In vitro antibacterial studies against E. coli confirmed a pronounced bactericidal effect of the chitosan-coated gel, while the noncoated, that is, alginate gel, failed to show any zone of inhibition. A biodegradable hydrogel dressing with high antibacterial efficiency prepared by the reaction between aldehyde and amino groups of oxidized alginate and carboxymethyl chitosan and incorporating tetracycline microspheres has been recently developed. The hydrogel was evaluated for swelling, degradation, compressive modulus, rheological properties, and the drug release profile. Increasing the ratio of microspheres incorporated within the hydrogel contributed to the shorter gelation time, lower swelling rations, and higher strength of the formulation. The composite hydrogel containing 30 mg/mL microspheres showed optimal mechanical properties for wound healing, allowing sustained tetracycline release. The plain alginate/carboxymethyl chitosan hydrogel (without tetracycline) exhibited very small inhibition zones for both S. aureus and E. coli. However, the hydrogels containing either free tetracycline or tetracycline-loaded microspheres demonstrated stronger antimicrobial activities for both tested strains. The inhibition zones for hydrogel containing tetracycline-in-microspheres against E. coli and S. aureus were slightly reduced as compared to those determined for tetracycline-in-hydrogel, probably a result of the slower drug release from the microsphere-based hydrogel [58]. Noppakundilograt et al. [59] synthesized chitosan-grafted poly[(acrylic acid)-co-(2hydroxyethyl methacrylate)] to prepare nanocomposite hydrogels incorporating mica. The higher mica loading produced a rougher surface of the nanocomposite hydrogel, and the water absorbency decreased with increasing levels of mica loading. The in vitro antimicrobial testing against S. aureus demonstrated that the presence of mica inside the hydrogel had no influence on antibacterial efficacy. Regardless of the mica loading levels in the hydrogel the minimum inhibitory concentration (MIC) was constant at 12.5 mg/mL and was significantly higher that the MIC value of the chitosan-grafted poly(acrylic acid) gel (1.56 mg/mL). Go´mez Chabala et al. [60] recently presented an interesting approach in the design of a novel wound dressing. It proposes the development of porous chitosan/alginate matrices embedding Aloe vera gel and silver nanoparticles. A. vera gel is well known for its antiinflammatory, antitumor, immodulatory, and antibacterial properties. A porous structure

318 Chapter 11 with interconnected pores inside the chitosan/alginate matrices showed great capacity for absorbing A. vera gel, which could be gradually released from the formulation following its application. Moreover, the presence of silver nanoparticles, chitosan, and A. vera gel enabled improved antibacterial activities against S. aureus and P. aeruginosa; these effects were comparable to those achieved with classical antibiotics such as tetracycline and gentamicin. Chronic wounds, commonly associated with ischemia, venous stasis diseases, and diabetes mellitus, require specifically designed treatment protocols. They are difficult to cure due to a lack of the growth factors required for the naturally occurring healing process. Moreover, chronic wounds are usually infected with bacteria that may form biofilm. Therefore their management requires simultaneous administration of antimicrobial drugs and tissue grow factors such as platelet-reach plasma (PRP) [61,62]. Targeting the treatment of chronic wounds infected with S. aureus, Nimal et al. [63] developed an injectable nanocomposite hydrogel by incorporation of tigecycline-loaded chitosan nanoparticles (93 nm) and activated PRP powder into chitosan hydrogel prepared from LMW chitosan. The formulation demonstrated shear thinning property, thermal stability, injectability, biocompatibility, and enabled sustained release of tigecycline, which is considered beneficial to reduce the inflammation phase at the wound site. Additionally, nanoparticle-loaded hydrogel could induce the proliferation of fibroblasts at the wound site and speed up the proliferative phase of wound healing in vitro, while cell migration studies confirmed that a PRP-containing gel system was more effective in the migration of fibroblasts compared to the gel system without PRP. In vitro, ex vivo (porcine skin), and in vivo (Drosophila melanogaster infection model) antibacterial studies revealed that the chitosan PRP gel system containing either free or nanoparticle-loaded tigecycline inhibited bacterial growth to a greater extent, while the plain gel system (without tigecycline) failed to show any antibacterial activity.

11.4 Hydrogels destined for administration to the vaginal site Hydrogels are one of the most common dosage forms/delivery systems for the topical vaginal administration of various drugs and active substances, including antimicrobials. They are easily spread over the vaginal surface, allowing good distribution, and retention of the active ingredients inside the vaginal cavity, while their high water content assures a moist microenvironment and a lubrication effect that helps improve the symptoms associated with vaginal dryness [24,64]. Hydrogels can be prepared from different polymers of natural, semisynthetic, and synthetic origin. However, among the various natural origin hydrogels with relevant intrinsic antimicrobial activities, carrageenan, chitosan, and gels from plant extracts are especially attractive.

Hydrogels as intrinsic antimicrobials 319 Carrageenan is a natural, sulfated, anionic polysaccharide obtained from edible red seaweeds, which is typically used in pharmaceutical and food industries as a gelling, thickening, and stabilizing agent [65]. Due to pronounced intrinsic antimicrobial properties, carrageenan-based gels have attracted considerable attention as microbicides in prophylaxis of sexually transmitted infections caused by herpes simplex virus (HSV), human papilloma virus (HPV), and human immunodeficiency virus (HIV). Its antimicrobial mechanism of action lies in the prevention of the attachment of viruses to target cells via electrostatic interactions with viral gp120 [66,67]. Carrageenan-based gel containing a mixture of lambda- and kappa-carrageenan (Carraguard) reached and was investigated in a phase III clinical trial on more than 3000 South African women, who were followed for up to 2 years. Although Carraguard was found to be safe for human use, unfortunately, its efficacy as a single microbicide in preventing vaginal transmission of HIV has not been confirmed [68]. The approach has been further exploited in investigations with carrageenan-based gels, namely a prototype zinc acetate carrageenan gel, designed and challenged in vivo on the macaques and mice infection model [69,70]. It is known that the zinc salts exhibit antiviral activity against a broad range of viruses, including HIV and HSV [71]. Therefore topical application of formulations containing low-dose zinc salts represents a promising strategy for preventing the sexual transmission of HSV-2 and potentially HIV, without the systemic use of antiretroviral drugs. The performed animal studies have proven the safety and high efficacy of formulations against both high-dose vaginal and rectal HSV-2 infections, respectively [69]. The formulation was further improved by addition of nonnucleoside reverse transcriptase inhibitor MIV-150 (PC-1005) and clinically tested on 20 healthy, HIV-negative, abstinent women. PC-100 used vaginally for 14 days was found to be well tolerated with low systemic levels of MIV-150 observed, while plasma zinc levels were unchanged. Postdose cervicovaginal lavages demonstrated both anti-HIV and anti-HPV activities, thus proving a platform for the novel formulation to be applied in prophylaxis of sexually transmitted viral infections [72]. Chitosan-based hydrogels are considered to be very attractive vehicles for vaginal drug delivery due to their lower pH (4 5), high water content, biodegradability, and pronounced mucoadhesiveness [73 75]. However, up to now, there have been a rather limited number of studies evaluating antimicrobial activities of chitosan hydrogels (without or with incorporated antimicrobial drug, either in a free or nanoparticle-encapsulated form) for the topical vaginal therapy of bacterial, fungal, and viral infections. Kandimalla et al. [37] investigated the efficacy of medium-molecular-weight (MMW) chitosan hydrogel incorporating metronidazole against P. aeruginosa biofilms for the possible treatment of recurrent bacterial vaginosis. The biofilms were treated with various concentrations of chitosan- and polycarbophil-based gels at pH values of 4 and 6.

320 Chapter 11 Interestingly, chitosan gels containing metronidazole were shown to be more effective in disrupting the integrity of P. aeruginosa biofilms at both pH values and a concentration of chitosan as low as 0.13% w/w. Polycarbophil-based hydrogel has shown comparable antibiofilm activity at pH 4 and at substantially increased polymer concentration (above 1%), while its efficacy was significantly reduced at pH 6. Since bacterial vaginosis is characterized with vaginal pH higher than 5, it is hypothesized that metronidazolecontaining chitosan gels could be more effective for the topical therapy of bacterial vaginosis than the commonly used polycarbophil gels. In another study, the 2% w/w chitosan hydrogels, prepared from LMW, MMW, or HMW chitosan and embedding miconazole or econazole within the polymer matrix, were assessed for the topical therapy of vulvovaginal candidiasis. The in vitro antifungal testing performed by applying the agar well diffusion method proved the efficacy of all tested gels containing antifungal drugs against C. albicans. The most pronounced inhibition zone was determined for miconazole embedded within the LMW chitosan gel (33.6 mm), followed by econazole LMW gel (31 mm), while the activities of miconazole within MMW and HMW chitosan gels were slightly less potent than miconazole in LMW chitosan. Surprisingly, the empty chitosan hydrogels (without the antimicrobial drugs) did not produce any zone of inhibition [76]. The authors suggested that the absence of the antifungal activities of the empty hydrogels could be favorable for vaginal administration; they raised concerns regarding a possible negative impact of chitosan on vaginal microflora. However, these results are not in agreement with other published findings and could be a consequence of the experimental setup and pH values of the gels, which varied between pH 3.8 and 5.4. Generally, the gels incorporating miconazole were of lower pH (4.5 4.8) than the corresponding empty gels (5.0 5.4), but exhibited higher pH values than the gels containing econazole (3.8 3.9). Very recently Perinelli et al. [77] evaluated the anti-Candida efficacy of 1% w/w LMW chitosan incorporated into 5.5% w/w hydroxypropylmethylcellulose (HPMC) gel, either as free polymer or assembled in nanoparticles (400 900 nm). While 1% w/w chitosan dispersion in buffer pH 4.5 displayed good activity against all tested Candida spp. strains, the chitosan nanoparticles in buffer pH 4.5 were ineffective against all tested C. albicans strains, whereas a comparable inhibition growth effect to 1% chitosan dispersion was observed against the tested non-albicans strains. Slightly higher activity of nanoparticles prepared at the ratio chitosan/sodium tripolyphosphate 12:1 was assumed to be a consequence of their smaller size as compared to the nanoparticles prepared at a 6:1 ratio. When 1% chitosan was incorporated into HPMC gel as free polymer, a variable degree of antifungal activity was obtained against all tested Candida spp. Overall, a slightly lower antifungal effect with respect to 1% chitosan dispersion was observed, which could be explained by the slow diffusion capacity compared to chitosan dispersion in buffer. The hydrogels incorporating 1% chitosan nanoparticles, in contrast to 1% chitosan nanoparticles

Hydrogels as intrinsic antimicrobials 321 in a form of liquid dispersion (active only on non-albicans strains), demonstrated activity against all tested Candida spp. strains. In addition to the hydrogels free of the antimicrobial drug, the authors also prepared hydrogels loaded with 0.75% w/w metronidazole aiming at the formulation with improved antimicrobial activity. Metronidazole has already been encapsulated in chitosan-containing liposomes (chitosomes) to achieve a dual effect of the mucoadhesive nanoformulation on both bacterial and fungal vaginal infections [78]. However, the presence of metronidazole in the hydrogels containing either 1% chitosan in a form of polymer or as nanoparticles dispersion has not increased the antimicrobial activity against all tested Candida spp. strains, proving that metronidazole has no intrinsic antiCandida effect on chitosan [77]. The search for hydrogels with intrinsic antimicrobial properties involved not only polymers of natural origin but also the plant extracts which could form hydrogels while exhibiting intrinsic antimicrobial activities. This approach represents a novel strategy for topical vaginal therapy. For instance, Sophora flavescens alkaloid gel has been investigated in vivo in a rat model for the therapy of aerobic vaginitis. In comparison to placebo, that is, Carbomer-based gel, the plant gel exhibited much higher antibacterial effects toward S. aureus and E. coli. Additionally, the number of vaginal lactobacilli significantly increased and the plant gel was well tolerated in contact with vaginal tissue. The finding is highly relevant considering the importance of maintaining the naturally occurring flora within the vaginal cavity. The authors suggested that the increased efficacy of the S. flavescens alkaloid gel was a result of the combined events involving high antimicrobial activity, immune regulation, and enhancement of lactobacilli growth supported by the moisture environment of the gel as well as its prolonged retention inside the vagina [79].

11.5 Periodontal diseases Periodontal disease is defined as a chronic infection caused by accumulation of bacteria in dental plaque which produces localized inflammation of the periodontium. It is commonly associated with the prevalence of Gram-positive aerobic and Gram-negative anaerobic bacteria and is highly prevalent in spite of increased efforts in the development of products for improved oral hygiene [80,81]. Biodegradable and injectable thermosensitive hydrogels are particularly attractive for the treatment of often unreachable periodontal pockets; thermosensitive hydrogels are liquefied at room temperature and form gel at the site of application (over 30 C) enabling sustained release of incorporated antimicrobial substance(s). Ji et al. [80] designed injectable 0.1% (w/w) chlorhexidine-loaded thermosensitive hydrogel using chitosan, quaternized chitosan, and α,β-glycerophosphate. Upon administering the

322 Chapter 11 hydrogel in liquid form, the gelation occurred at simulated in vivo condition (37 C) after only 6 minutes, further allowing sustained release of chlorhexidine over 18 hours. The formulation exhibited excellent inhibitory activity against periodontal pathogens, namely Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. The experiments clearly confirmed the crucial role of chitosan for antimicrobial activity of novel formulation; the MIC of chlorhexidine significantly decreased in the presence of chitosan solution and especially in the form of chitosan-based thermoresponsive hydrogel, thus proving the pronounced intrinsic antimicrobial effect of chitosan as well as quaternized chitosan. The inhibition zones obtained for the chitosanbased thermoresponsive gels revealed the strongest activity against A. actinomycetemcomitans, followed by P. intermedia and P. gingivalis. The great potential of the novel formulation for the local treatment of periodontal infections was further supported by the lack of acute toxicity in in vivo evaluation of the gel in rats. The effectiveness of chitosan, both as a vehicle for metronidazole in a form of hydrogel and as an active antimicrobial agent on its own, was confirmed in a clinical study involving 15 patients suffering from chronic periodontitis. Because of chitosan’s pronounced mucoadhesive properties as well as its antimicrobial activity, the required administration time for the 1% chitosan hydrogel was only twice a week; moreover, metronidazole could have been incorporated within the hydrogel at a concentration of 15% rather than the commonly used 25% (w/w). Patients were randomly divided into three groups. The first group received chitosan gel after scaling and root planning (SRP) treatment, while the second group received metronidazole-containing chitosan gel after SRP, and the third group (control) received only SRP treatment. The clinical parameters such as the probing depth, clinical attachment level, amount of gingival recession, plaque index, gingival index, and gingival bleeding time index were recorded at the baseline and at weeks 6, 12, and 24. The effect of gel formulations on the reduction of the gingival inflammation markers was found to be better than in the control group. In all groups, significant improvements were observed in clinical parameters between baseline and week 24. The reductions in probing depth values were 1.21 mm for blank chitosan hydrogel, 1.48 mm for metronidazole-containing chitosan gel, and 0.94 mm for control. The authors confirmed that chitosan itself as well as in combination with metronidazole are effective in the treatment of chronic periodontitis due to chitosan’s intrinsic antimicrobial properties [38]. We have provide a summarized overview of the relevant findings on chitosan-based hydrogels as intrinsic antimicrobials (Table 11.1). We included, in our opinion, the relevant references for readership to get an overview of the state of the art in this emerging field. We have to mention that we did not include the patent-protected work and opted to focus on scientifically proven and widely accessible research opus.

Table 11.1: Overview of chitosan-based hydrogels with intrinsic/synergistic antimicrobial activities. Target

Type of chitosan

Skin infections (wounds)

Chitosan hydrochloride Chitosan-grafted poly (acrylic)-co-(2-hydroxyethyl methacrylate) Chitosan from shrimp shells ($75% deacetylated)

Major findings

References

In vitro Bactericide effect In vitro Mica does not affect the antimicrobial activity of the hydrogel

[57] [59]

S aureus, P. aeruginosa

In vitro Antibacterial activities comparable to tetracycline and gentamycin

[60]

S. aureus, E. coli

In vitro Antimicrobial efficiency proportional to lincomycin content; increase in chitosan content reduces antimicrobial activities

[47]

Chitosan powder (80% 95% Silver deacetylated) nanoparticles

S. aureus, E. coli

Synergistic antibacterial effect of chitosan and silver nanoparticles; faster wound healing effect (rats)

[50]

HMW chitosan

Silver nanoparticles, gold nanoparticles

S. mutans, S. aureus, E. coli

In vitro, In vivo In vitro, In vivo

Plain chitosan gel prepared with 6% acetic acid is better than chitosan gels prepared with 4% acetic acid and embedding silver or gold nanoparticles; slower wound healing effect with gold nanoparticles in gel in vivo due to toxicity of the gold nanoparticles

[52]

LMW chitosan (85% deacetylated)

Zinc oxide nanoparticles

S. aureus, E. coli, P. aeruginosa, S. intermedius, S. hyicus

In vitro, In vivo

Negligible antibacterial effect of plain chitosan gel toward S. aureus and E. coli due to neutral pH of gel; significant increase in activity by incorporation of zinc nanoparticles; effectiveness confirmed in vivo against P. aeruginosa, S. aureus, and S. hyicus

[53]

Candida spp.

In vitro Antifungal activity varied among the tested Candida spp.; enhanced activity with chitosan nanoparticles in gel.

P. aeruginosa (biofilm)

In vitro Better antibiofilm activities in comparison to poly(acrylic)- [37] based gel at pH 6.

Mica

Aloe vera 1 silver nanoparticles Lincomycin

LMW chitosan MMW chitosan

Periodontal diseases

Study

E. coli S. aureus

O-carboxymethyl chitosan

Vaginal infections

Antimicrobials/ Bacteria/fungi nanoparticles/ plant extract

Metronidazole

In vitro Presence of quaternized chitosan inside the P. gingivalis, P. thermoresponsive gel significantly increased antibacterial intermedia, A. activity compared to pure chitosan actinomycetemcomitans

Chitosan (MW 108 kDa), quaternized chitosan HMW chitosan

Metronidazole

Clinical The significant reduction in probing depth values with blank chitosan gel and drug-loaded gel

[77]

[80]

[38]

A. actinomycetemcomitans, Actinobacillus actinomycetemcomitans; E. coli, Escherichia coli; P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia; P. aeruginosa, Pseudomonas aeruginosa; S. aureus, Staphylococcus aureus; S. hyicus, Staphylococcus hyicus; S. intermedius, Staphylococcus intermedius; S. mutans, Streptococcus mutans.

324 Chapter 11

11.6 Conclusion Hydrogels with intrinsic antimicrobial properties are gaining more attention with the increasing threat of antimicrobial resistance. Biopolymers used to form these hydrogels are attractive building blocks as they often exhibit multitarget properties besides their antimicrobial action; currently chitosan-based hydrogels are being widely studied for improved wound healing based on the ability of chitosan to act on enhanced healing and its intrinsic antimicrobial potential. However, other localized infections, such as genital ones, open up opportunities for novel strategic approaches and formulations. Those formulations bear high innovative potential and often comprise a delivery-system-in-hydrogel concept. Moreover, the opportunity to chemically modify the properties of natural origin polymers, tailoring of polymers, is one of the research lines emerging recently. So far, very limited concerns have been raised regarding the potential toxicity of the final formulations.

11.7 Perspectives Understanding the mechanisms behind polymers, including chitosan, exhibiting antimicrobial action would not only help us to optimize the formulation properties to maximize their efficacy but also address the possible resistance development. To achieve this, it is important to standardize the methodologies applied to evaluate and optimize the activity. Multidisciplinarity might be a shorter path to a better understanding of the mechanisms and optimization process. Tailoring the polymer properties would allow us to target specific infection sites as well as formulation properties. It is very clear that biomaterials with intrinsic antimicrobial properties are offering many opportunities in product development and their full promise is yet to be acknowledged.

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Hydrogels as intrinsic antimicrobials 327 [48] N.R. Sudarshan, D.G. Hoover, D. Knorr, Antibacterial action of chitosan, Food Technol. 6 (1992) 257 272. [49] K. Yang, Q. Han, B. Chen, Y. Zheng, K. Zhang, Q. Li, et al., Antimicrobial hydrogels: promising materials for medical application, Int. J. Nanomed. 13 (2018) 2217 2263. [50] Y. Xie, X. Liao, J. Zhang, F. Yang, Z. Fan, Novel chitosan hydrogels reinforced by silver nanoparticles with ultrahigh mechanical and high antibacterial properties for accelerating wound healing, Int. J. Biol. Macromol 119 (2018) 402 412. [51] Y. Zhou, Y. Zhai, L. Wang, L. Xu, M. Zhai, S. Wei, Radiation synthesis and characterization of nanosilver/gelatin/carboxymethyl chitosan hydrogel, Radiat. Phys. Chem 81 (2012) 553 560. [52] C. Samano-Valencia, G.A. Martinez-Castanon, F. Martinez-Gutierrez, F. Ruiz, J.F. Toro-Vazquez, J.A. Morales-Rueda, et al., Characterization and biocompatibility of chitosan gels with silver and gold nanoparticles, J. Nanomater (2014). Article ID 543419, 11 pp. [53] P.T. Sundheesh Kumar, V.K. Lakshmanan, T.V. Anilkumar, C. Ramya, P. Reshmi, A.G. Unnikrishnan, et al., Flexible and microporous chitosan hydrogel/nano ZnO composite bandages for wound dressing: in vitro and in vivo evaluation, ACS Appl. Mater. Interfaces 4 (2012) 2618 2629. [54] F. Wahid, J.J. Yin, D.D. Xue, H. Xue, Y.S. Lu, C. Zhong, et al., Synthesis and characterization of antibacterial carboxymethyl chitosan/ZnO nanocomposite hydrogels, Int. J. Biol. Macromol. 88 (2016) 273 279. [55] A.M. Alsharabasy, S.A. Moghannem, W.N. El-Mazny, Physical preparation of alginate/chitosan polyelectrolyte complexes for biomedical applications, J. Biomater. Appl. 30 (2016) 1071 1079. [56] N. Sukumar, T. Ramachandran, H. Kalaiarasi, S. Sengottuvelu, Characterization and in vivo evaluation of silk hydrogel with enhancement of dextrin, rhEGF, and alginate beads for diabetic Wistar Albino wounded rats, J. Text. Inst. 106 (2015) 133 140. [57] M.C. Straccia, G.G. d’Ayala, I. Romano, A. Oliva, P. Laurienzo, Alginate hydrogels coated with chitosan for wound dressing, Mar. Drugs 13 (2015) 2890 2908. [58] H. Chen, X. Xing, H. Tan, Y. Jia, T. Zhou, Y. Chen, et al., Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing, Mater. Sci. Eng. C 70 (2017) 287 295. [59] S. Noppakundilograt, K. Sonjaipanich, N. Thongchul, S. Kiatkamjornwong, Syntheses, characterization, and antibacterial activity of chitosan grafted hydrogels and associated mica-containing nanocomposite hydrogels, J. Appl. Polym. Sci 127 (2013) 4927 4938. [60] L.F. Go´mez Chabala, C.E. Echeverri Cuartas, M.E. London˜o Lo´pez, Release behavior and antibacterial activity of chitosan/alginate blends with Aloe vera and silver nanoparticles, Mar. Drugs 15 (2017) E328. [61] T. Hirase, E. Ruff, S. Surani, I. Ratnani, Topical application of platelet-rich plasma for diabetic foot ulcers: a systematic review, World J. Diabetes 15 (2018) 172 179. [62] S. Saghazadeh, C. Rinoldi, M. Schot, S.S. Kashaf, F. Sharifi, E. Jalilian, et al., Drug delivery systems and materials for wound healing applications, Adv. Drug Deliv. Rev 127 (2018) 138 166. [63] T.R. Nimal, G. Baranwal, M.C. Bavya, R. Biswas, R. Jayakumar, Anti-staphylococcal activity of injectable nano tigecycline/chitosan PRP composite hydrogel using Drosophila melanogaster model for infectious wounds, ACS Appl. Mater. Interfaces 8 (2016) 22074 22083. [64] R. Palmeira de Oliveira, A. Palmeira de Oliveira, J. Martinez de Oliveira, New strategies for local treatment of vaginal infections, Adv. Drug Deliv. Rev 92 (2015) 105 122. [65] J. Necas, L. Bartoskiova, Carrageenan: a review, Vet. Med. 58 (2013) 187 205. [66] S. Trapp, S.G. Turville, M. Robbiani, Slamming the door on unwanted guests: why preemptive strikes at the mucosa may be the best strategy against HIV, J. Leukoc. Biol. 80 (2006) 1076 1083. [67] V. Pirrone, B. Wigdahl, F.C. Krebs, The rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1, Antiviral Res. 90 (2011) 168 182. [68] S. Skoler-Karpoff, G. Ramjee, K. Ahmed, L. Altini, M.G. Plagianos, B. Friedland, et al., Efficacy of carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial, Lancet 372 (2008) 1977 1987.

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Further reading B.M. Holzapfel, J.C. Reichert, J.-T. Schantz, U. Gbureck, L. Rackwitz, U. No¨th, et al., How smart do biomaterials need to be? A translational science and clinical point of view, Adv. Drug Deliv. Rev 65 (2013) 581 603.

CHAPTER 12

The application of natural polymer-based hydrogels for agriculture Mohamed Mohamady Ghobashy* Radiation Research of Polymer chemistry department, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt

12.1 Introduction Agriculture is one of the greatest consumers of water in a time when there is a limited amount of water. The survival of plants by irrigation to prevent water stress is one of the most important limiting factors that have an effect on crop and fruit growth and productivity. Natural polymers could be candidates to enhance the water retention ability of soil. Natural polymers are biodegradable in nature where they are degraded in soil by microorganisms in ambient conditions, releasing enzymes capable of cleaving the linkage of macromolecules into small fragments. Natural polymers are very important in life processes; for example, DNA, proteins, peptides, and polysaccharides (carbohydrate). Natural polymers originate in nature and are found in two types of cells: prokaryotic cells and eukaryotic cells [1,2]. Natural polymers help maintain the balance within the ecosystem including soil, plants, air, and water [3]. Natural polymers have been developed for durability and resistance to all forms of degradation when copolymerized with synthetic hydrogel polymers. Polysaccharides are a class of natural polymer composed of sugar units mainly bonded together by glycosidic (1-4)-linked glucose units, with a general formula of (C6H10O5)n, 40 # n # 3000 [4]. Glycosidic linkages in the bond between the molecules in the nonbranching chain of sugar are usually of type 1:4 and in the branch chains are usually found to be of type 1:6, which are either alpha, beta, or alpha beta depending on the type of saccharin that binds them [5
7]. A synthetic polymer which is more oxygen permeable helps in forming agricultural polysaccharide hydrogel (APH) which provides a benefit to aerobic bacteria in soil [8,9]. Bacteria assist plants by increasing nutrient availability by providing fixed nitrogen [10,11].



corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00013-6 © 2020 Elsevier Inc. All rights reserved.

329

330 Chapter 12 The three most abundant polysaccharides are (1) cellulose, (2) starch, and (3) glycogen, which are also known as glucose polymers [12]. Cellulose, starch, and glycogen are strings of glucose molecules. Cellulose is the most abundant natural polymer on Earth and is found in the cell wall of green plants [13]. Because the glucose units are joined together differently in the three polymers (cellulose, starch, and glycogen), cellulose has different properties and shape than starch and glycogen [14]. The cellulose structure is a repeating structure of glucose units collected by other glucoses united in different orientations (180 degrees) from each other and forming β(1-4) glycoside linkages [15]. The structure of starch consists of both linear amylose and branch amylopectin that are repeat units of glucose. The glucose units are oriented in the same direction by α(1-4) glycoside linkage and branching every 24
30 glucose units takes place at C6, forming α(1-6) bonds. The enzymes that are capable of hydrolyzing starch are not capable of performing cellulose hydrolysis. Because the β configuration allows cellulose to form very long and straight chains, they interact with one another through hydrogen bonds that allow the formation of the strong fibril structure of cellulose [16]. Glycogen has a similar structure to amylopectin units in starch molecules but has a more branched and extensively compact structure than starch molecules [17]. The challenge in the field of agriculture is how to form acceptable soil conditioners that have a period of time before their biodegradation. For that the cooperation of natural polymers with synthetic hydrogel is needed. Hydrogel is a 3D cross-linked polyelectrolyte polymer that absorbs water on a large scale [18,19]. It is classified as a superabsorbent hydrogel (SAH) when it can absorb water to 100 times its own weight [20]. These characteristics and others, such as permeability, rigidity, and transparency make them candidates for as soil conditioners incorporated with natural polymers. The changing of copolymer composition in the soil conditioner synthesis leads to a broadly improvement of water availability and increased irrigation potency for plants, by enhancing the water-holding property of soil.

12.2 Classification of soil conditioner polysaccharide hydrogel Soil conditioner polysaccharide hydrogel (SCPH) is a cross-linked hydrophilic gel, which is commonly known as a superabsorbent and which can absorb a high amount of water in the swelling process; their dry and swollen states are shown in Fig. 12.1. This process is free swelling of freely absorbed water that leads to the dissociation of hydrophilic groups causing repulsion between charged groups and expands polymer coils. The free space between network chains is will sizes up by filled with several hundred water molecules. Before elaborating on the work of hydrogels as soil conditioners, it is important to understand how the hydrogel swells and holds water (Fig. 12.2). Water and swollen hydrogels are consider one factor that recommends them for use as soil conditioners by supporting their supplementary irrigation and nutrient-carrying ability, causing gradual

The application of natural polymer-based hydrogels for agriculture 331

Figure 12.1 The swelling process takes place when dry hydrogel comes into contact with water molecules.

1

Mobility process

4

Swelling process

Water-water vibrat ions

Pathway of diffusion Water molecules Ion-water vibrations

Pathway of diffusion

Deswelling Swelling Solid part

(100%)

Function group Covalent cross-linked hydrogel Liquid part

(99.9%)

Solid part

(0.1%)

Counter ion Pathway of diffusion Solid part

(90%) Solid part

Liquid part

(10%) Liquid part

2

Dissociation process

3

Hydrated process

Figure 12.2 The sequence of the four swelling processes.

(50%) (50%)

332 Chapter 12 water and nutrient release to the plants. The water-holding capacity is dependent on both the cross-linked density [21] and the number of hydrophilic groups [22]. The solubility power of water is gained by their free mobility by water
water vibrations. The water molecule is an attempt to dissolve hydrogel by cleave the strong covalent cross-linked by vibrational modes of the water molecule but it consequent success to cleavage of hydrogen bonds only. When hydrogels have hydrophilic function groups such as [COONa] in contact with water molecules the kinetic energy of water
water vibrations is transferred into water
ion vibrations (mobility process). The water vibration causes dissociation of [COONa] into COO2 and Na1 (dissociation process). The negative charges repel each other and are neutralized by water molecules and at the same time Na1 ions are hydrated (hydrated process). This is indicative of changes in the hydrogen bonding and allows the water molecules to become confined inside the pores of the network structure of the hydrogel (swelling process). Therefore the driving force for swelling is the presence of mobile polar molecules that are osmotically active [23]. Most of the hydrophilic hydrogels are destined for agricultural applications as soil conditioners and nutrient carriers, and can be applied when seeding or coated on the seed itself [24]. The swelling behavior of hydrogels in soil is affected by numerous factors, which are discussed below. It is important for adjustment of chemical and physical properties of soil to use polysaccharide hydrogels. The swelling/deswelling process is repeated without any substantial changes to the SAH ability to absorb water [25]. Hydrophilic soil conditioner hydrogel (SCH) can be categorized into three classes: (1) natural polymer, (2) natural polymer-based hydrogel, and (3) synthetic hydrogel. The first and second are discussed in detail in this chapter. The pathway to soil conditioners and fertilizer carriers is outlined in Fig. 12.3. Fig. 12.3 illustrates that the fundamental conditions should be considered in the preparation of soil conditioner. There are at least three factors to consider in selecting a hydrogel as a soil conditioner, it must be: (1) biodegradable, (2) superabsorbent, and (3) chemically covalent cross-linked.

Figure 12.3 At least three factors need to be considered in selecting the best hydrogel as a soil conditioner.

The application of natural polymer-based hydrogels for agriculture 333

12.2.1 Natural polymers Natural polymers are formed into hydrogel polymers by five gelation mechanisms, external gelation, internal gelation, inverse gelation, interfacial gelation, and multistep interrupted gelation [26]. Most soil conditioners are made using primarily polysaccharides (i.e., starches, chitosan, and celluloses). Polysaccharides are obtained from various sources including: 1. Plant origin—starch, cellulose, hemicellulose, psyllium, pectin, glucomannan, guar gum, locust bean gum, gum tragacanth, gum acacia, and agar; 2. Microbial origin—curdlan, levan, dextran, gellan, and xanthan; 3. Algal origin—alginate and carrageenan; 4. Fungal and marine animal origin—hyaluronic acid, chitosan, chitin, elsinan yeast glucans, pullulan, and scleroglucan. Polysaccharides exhibit a wide variety of unique physiological functions and chemical structures. In general, polysaccharides are inexpensive and many of their uses are more common in environmental applications. Depending on which monosaccharides are connected the polysaccharides take on a variety of structure forms and are classified as linear or highly branched polysaccharides. Polysaccharides can be classified into two classes: homo- and heteropolysaccharides that are made up of one type of monosaccharide unit or more than one type of monosaccharide unit, respectively. Another classification is the charge of the polysaccharide: some molecules are neutral and others possess a negative charge, such as the carboxylate or sulfate groups, and other are positive, such as chitosan. As well as polysaccharides being classified into two classes, they can be divided into aqueous or nonaqueous soluble. In agriculture, the latter calcification of polysaccharides is more appropriate and considered here. Water-soluble polysaccharide such as (guar) when it applied as soil conditioner is prevents surface crusting of soil [27]. Polysaccharides are hydrophilic and have an affinity for water solubility, so seed coating and controlled release of agrochemicals and fertilizers are more efficient [28,29]. Through ionotropic gelation, soluble polysaccharide of sodium-alginate is fabricated as beads loaded with an agrochemical formulation [30,31]. These beads are prepared with ionotropic gelation of polysaccharides such as agar, alginate, starch, with metal ions (Ca21, Ba21, and Al31) [32
34]. As mentioned in several literatures, insoluble polysaccharide has slower particulate degradation than soluble polysaccharide [35] like inulin, pullulan, and xylan. Therefore soluble polysaccharide offers advantages in agricultural applications.

334 Chapter 12 Also, chitosan is soluble in acidic aqueous media (1% acetic acid).1 Pure polysaccharide as a soil conditioner has been rarely studied but some research has dealt with polysaccharide hydrogel formation by cross-linker agents. In addition, the lack of use of polysaccharide alone as a soil conditioner is also due to the fast biodegradation of polysaccharides in soils under natural conditions [36]. In this regard, the research articles that deal with the native form of polysaccharide as soil containers stand out. In this respect, polysaccharide is almost hydride with synthetic hydrogels that are water soluble and nontoxic. In practice, is possible to prepare superabsorbent hydrogels based on polysaccharides for application in agriculture as soil conditioner using ionic cross-linking agents, this process called the ionic cross-linking process (ICP). For example, carboxymethyl cellulose (CMC)/ starch hydrogels [37] cross-linked used Al2(SO4)3  18H2O as ionic cross-linking agent. Another ICP is that carried out by Davidson et al. [38]. Davidson and coauthors prepared CMC hydrogel to use as a root-targeted delivery Vehicle (RTDV).2 The ICP is carried out by mixing CMC solution with the ionic cross-linking solution composed of calcium chloride, iron (II) chloride, and iron (III) chloride. The fertilizer (20:20:20) was released at about 80%
90% within 80 hours and CMC ionic was degraded over a 50-day period.

Synthetic hydrogels possess a biocompatibility property like natural polymers due to having a degree of flexibility like natural tissue and water absorption is very similar to natural polymers. Due to their benefits hydrogels have played a key role in the agricultural field. Hydrogels may be defined in different ways. The most common is the cross-linked polyelectrolyte network. The ability of hydrogels to absorb water is due to hydrophilic functional groups attached to the polymeric structure, while their resistance to dissolution arises from covalent cross-linkages between network chains that improve their mechanical stability.

This indicates that the native natural hydrogel is not able alone to resist the fast degradation and fast fertilizer released. Chitosan is covalently cross-linked using natural cross-linker of genipin which is much less toxic than glutaraldehyde [39]. Genipine is a cross-linker for polymers containing amino groups such as chitosan and gelatin, leading to covalent coupling [40
42]. Covalent chitosan hydrogel is more stable for longer periods of time than physical hydrogel [43]. This makes it better suited for bioremediation and encapsulation of fertilizers. The other method of forming covalently cross-linked chitosan hydrogels instant dialdehyde compounds such as glutaraldehyde is natural reagents like 1

2

The dissolving of chitosan takes placed by protonation of amino groups that are positively charged in the chitosan polymer chains. Thereby, they overcome the interaction force between chains that is replaced by a repulsion force. When solubilized, chitosan molecules can form weak intermolecular links through hydrophobic interactions between residual acetyl groups (from the mother chitin). The RTDV is a hydrogel that is loaded with nutrients and fertilizers that are supplemented to the plant’s root zone in a slow manner under a controlled system, and safely degrades in the soil after a period of time.

The application of natural polymer-based hydrogels for agriculture 335 genipine [44,45]. Chitosan beads capable of acting as a carrier for agrochemicals increase the time period for controlled release of bioactive materials (Atrazine) [46]. Another example of a polysaccharide used as an agrochemical carrier is gellan loaded with metribuzin substances for weed control in agriculture [47]. The disadvantage of polysaccharide hydrogel is mostly cross-linked by metal ions [48]. In most cases, crosslinking in this way is a reversible polymerization process, and the result is a physical hydrogel [49] which will degrade and dissolve after a short period of time [50]. Physical hydrogel is known as an “ionotropic hydrogel,” and calcium alginate is an example of a physical hydrogel [26]. The ionotropic process is an interaction of polyelectrolyte polymer with opposite ionic charges. This interaction is reversible, and can be decimated by changes in physical conditions, such as temperature, pH, ionic strength, or application of stress.

12.2.2 Natural polymer (polysaccharide)-based hydrogel The action of synthetic hydrogel when combined with natural polymers may involve the following: (1) increase the gelling power of polysaccharides in an aqueous environment allowing it to swell in good mechanical form; (2) sustain fertilizer release dependent on cross-linking density and enzymatic degradation of biodegradable hydrogel; (3) withstand breakdown of polysaccharides (degradation) in natural conditions; (4) increase the binding affinity of ions in soil; (5) exhibit resistance of chemicals and UV light; and (6) enhance the thermostability of the natural polymeric network. The preparation of superabsorbent hydrogel consisting of natural and synthetic hydrogels can take place by two main routes of polymerization reactions. Natural polymer reactions for preparing the polysaccharide-based hydrogels include: (1) direct cross-linking of hybrid natural polymers and synthetic monomers and/or polymers dissolving in an aqueous state; (2) grafting of vinyl monomers onto the surface of the powder of natural polymers in a solid state. The polymerization reaction that leads to a cross-linked hydrogel-based natural polymer is either chemical or physical. There is a lot of scientific literature available which demands soil conditioners from hybrid formulations of polysaccharides and synthetic hydrogels. Studies have also exhibited that slow or controlled release could be an alternative to overcoming normal fertilizer losses. The polysaccharide alone cannot easily produce cross-linked hydrogel with high stability, which is an essential condition for use as a soil conditioner and for control of nutrients. An alternative is cellulose starch derivatives used as a dual coating for improving the performance of slow-release urea by Qiao et al. [51]. They attempted to develop a duallayer slow-release urea fertilizer through coating the urea granules with ethyl cellulose (EC) as the inner layer and the starch-polyacrylamide (PAAm) as the outer layer. This exhibited a steady release behavior of urea for a period of over 96 hours. Another polymerization method is irradiation. Furthermore, irradiation of natural polymers leads to a chain session

336 Chapter 12 (degradation) process rather than a chain constructive (cross-linked) process. In order to produce stable cross-linked soil conditioner, natural polymers must be added to synthetic vinyl monomers. These include examples of synthetic monomers and polysaccharides with the versatility to form soil conditioners as demonstrated next. 12.2.2.1 Cellulosic derivative-based hydrogel designing concepts, properties, and perspectives for agricultural applications Excellent biocompatibility conforms to the principles of green chemistry and cellulose and cellulose derivatives have encouraged their use in agricultural applications [52]. Most cellulose derivatives do not occur naturally but are synthetically produced [53], however they remain biodegradable and biocompatible like their native natural source [54,55]. Cellulose is dissolved in ionic liquid like LiCl (5%
7%)/dimethylformamide and called cellulose dissolution solvent [56]. The main groups of cellulose derivatives are cellulose esters and cellulose ethers which provide different mechanical and physicochemical properties. The cellulose ester compounds such as cellulose acetate and methyl cellulose (MC) are insoluble in water, but may be well soluble in various organic solvents or ionic liquids depending on the amount of acetate groups in the polymer. Cellulose ethers give good transparent film-forming ability [57], which finds a variety of applications, such as classical material coatings, controlled-release systems, hydrophobic matrices, and semipermeable membranes for water treatment and pharmacy applications [58]. In agricultural applications cellulose ester it is not suitable for synthesizing soil conditioner-based hydrogels. In contrast, cellulose ethers presents good solubility, high chemical resistance, and nontoxic nature and are utilized in the manufacture of soil conditioner-based hydrogels. Cellulose ethers are generally highly hydrophilic and enhanced water-absorbent products when incorporated with synthetic hydrogel. The cellulose ether derivatives are more appropriate to agricultural applications due to being dissolved in a safe solvent (water). The most commonly used cellulose ethers are MC, EC, hydroxyethyl cellulose, CMC, sodium carboxymethyl cellulose (NaCMC), hydroxypropyl cellulose, and hydroxypropylmethyl cellulose. Cellulose ether-based hydrogels can be obtained by the polymerization of cellulosic materials mixed with synthetic monomers in their aqueous solutions. The polymerization can be induced by chemical or radiation methods. The cross-linking reaction between the cellulose ether chains and synthetic monomers in a water solution is described as radical polymerization. Radical polymerization activated by chemical agents (initiator) or water radiolysis induced by gamma irradiation may occur. Several literatures have reveal different methods for obtaining cellulose ether-based hydrogels, based on radiation and/ or other conventional methods. Also, the cross-linking could occur by absorption of a monomer into a cellulose powder (grafting) for use as water reservoirs for dry soils and simultaneous water release if necessary. The controlled release of water and loaded

The application of natural polymer-based hydrogels for agriculture 337 nutrients is important in agriculture. The most common mechanism of water and loaded nutrient release in superabsorbent hydrogel is by the diffusion mechanism. Grafting polymerization was carried out by Olad et al. to prepare slow-release nitrogen (N), phosphorus (P), and potassium (K) (NPK) fertilizer encapsulated by superabsorbent nanocomposite based on grafting the acrylic acid (AA) onto sulfonated carboxymethy cellulose in the presence of polyvinylpyrrolidone (PVP), silica nanoparticles, and NPK fertilizer compound [59]. Bortolin et al. [60] synthesized a series of hydrogels composed of PAAm, MC, and calcic montmorillonite (MMt) appropriate for the controlled release of a nitrogenated fertilizer (urea). The radical polymerization was processed using an initiator agent of sodium persulfateand two cross-linking agents such as N0 -N-methylene bisacrylamide and N,N,N0 , N0 -tetramethylethylenodyamine catalyzer were added. The presence of MMt clay in the hydrogel formulation improves the hydrogel properties by enhancing water absorption and mechanical resistance. In a general way, the presence of the clay mineral and the hydrogel releases the nutrient in a slower manner. The obtained SAH of PAAm-MC was hydrolyzed by NaOH and the controlled released of urea was examined after and before hydrolysis of the hydrogel. The result was that the release of urea is about 72 and 192 times slower for the hydrogel samples before and after hydrolyzing of the hydrogel samples, as compared with the urea without the presence of the hydrogel. Elbarbary and Ghobashy prepared NaCMC-based superabsorbent hydrogels of PVP by gamma radiation. The increased CMC content in PVP/CMC hydrogels increases the swelling and enhances the water-holding capability. Fast swelling of the hydrogels was obtained after 20 minutes of loading the hydrogel with three fertilizers of urea, monopotassium-phosphate, and NPK. The hydrogels showed a slow-release fertilizer (SRF) adsorption/desorption mechanism. The release rate of urea is much higher at 10 times than that of phosphate. After 3 days, urea released 60%, while phosphate released 10%
12%. Usually plants take up less than 50% of the available urea. Here the hydrogel released 60% of the urea in 3 days and retained 40% of the urea which was released over 9 days. The SRF, the high swelling, and the slow water retention behaviors of PVP/CMC hydrogels encourage their use as safer release systems for fertilizers and as soil conditioners in agricultural applications. The applicability of PVP/CMC hydrogels in the agricultural area shows a greater growth effect on corn (maize) plants. The growth of these plants in soil mixed with PVP/CMC hydrogel-loaded fertilizers is greater than in untreated soil. As shown above, several literatures have used sodium salt of carboxy methcellulose-based superabsorbent hydrogel as a fertilizer controller and soil conditioner due to their benefits such as low cost, hydrophilic groups allowing better binding of fertilizer, and degrading in a slower manner than other natural polymers.

338 Chapter 12 12.2.2.2 Starch derivative-based hydrogel designing concepts, properties, and perspectives for agricultural applications Starch-based superabsorbent hydrogel for agricultural applications is dealt with in several literatures. The SRF is important to reduce the loss of fertilizer from leaching. Xiao et al. [61] prepared urea-embedded starch-based PAAm superabsorbent hydrogel and studied the controlled release of urea. It was found that less than 15% of urea was released after 1 day and the release rate after 30 days exceeded 80%. Zhong et al. [62] prepared a water-retaining and controlled-release fertilizer (CRF) material based on sulfonated corn starch/poly(acrylic acid) embedded phosphate rock. Liu et al. [63] encapsulated urea with two layers of starch and the copolymers of AA and acrylamide, respectively. The polymerization successive carried out using an initiator of radical polymerization by ammonium persulfate. The urea was slowly released into the soil through the swollen network at about 10%, 15%, and 61% on the 2nd, 5th, and 30th days, respectively. Several literatures deal with the grafting of synthetic vinyl monomer such as acrylonitrile (An) onto starch to produce superabsorbent hydrogel [64
66]. Jyothi et al. [67] synthesized poly(acrylonitrile) grafted onto starch by a free radicalinitiated polymerization reaction. The urea fertilizer was loaded onto the grafted starch samples to obtain the dual benefit of sustained release and water retention properties. The slow release of urea was observed, where the urea was completely released over a period of 1108 days. The grafting process is carried out by a radical polymerization mechanism using an initiator. By heating the persulfate, initiator compounds are decomposed to produce radicals. The radical abstracts hydrogen from hydroxyl groups in starch molecules to form alkoxy radicals on the starch chains. Thus, this persulfate-saccharide redox system [68
70] provides active centers on the substrate to radically initiate polymerization of An, resulting in a graft copolymer of superabsorbent hydrogel.

12.3 The action of an agricultural superabsorbent hydrogel on chlorophyll content, soil texture, and fertilizer release mechanism When water comes into contact with land it is diffused and held in the spaces between soil particles. The soil becomes wet and when hydrogel is present it draws water into its network by osmosis. Chlorophyll is responsible for photosynthesis by combining water with carbon dioxide (CO2) forming carbohydrate and oxygen. Water abundance for plants increases their chlorophyll content and consequently increases the crop yield. Ghobashy et al. [71] prepared a highly porous superabsorbent hydrogel (SAH) of poly (acrylamide-gelatin) that has a positive effect as a fertilizer carrier and soil conditioner. The natural and synthetic polymer of SAH consists of gelatin (G) and acrylamide (Am)

The application of natural polymer-based hydrogels for agriculture 339

Figure 12.4 Cross-section of a bean leaf showing the location and concentration of the chlorophyll content of the whole chloroplast of the leaf; (A) chlorophyll remains in the center sandwiched inside the leaf (B).

which were polymerized and cross-linked by gamma radiation. The chemical modification of the obtained SAH increased the swelling degree from 220 to 720 g/g. Furthermore, Ghobashy and coauthors studied the effect of chemical modification on the release ability of three kinds of fertilizer (K, P, and urea) loaded on modified SAH samples. The obtained result indicates that the release character of these three fertilizers are slow, which is in agreement with the standard of SRFs of the Committee of European Normalization [72]. This could provide opportunities to diffuse nutrients to plants for a long time. Another important point was that plants in soil not containing SAH exhibited a significant decrease in chlorophyll content as compared to those cultivated with SAH in the soil (Fig. 12.4A and B). The effect of hydrogels on the soil is to provide further space for the flow of air and water infiltration. The soil texture in this case is ideal for planting. When the soil is dried out the hydrogel will release water rapidly from the interior polymer network into the exterior [73]. The water was stored inside a hydrogel network until needed and released back to the soil. Swollen hydrogel caused soil
water interactions and stability to the structure of the soil [74]. This is also observed for the leaching of nutrients and fertilizer added to agricultural soils. To reduce the leaching of fertilizers hydrogel is mixed into fertilizer preparations. The behavior of fertilized hydrogel makes it possible to maintain and release fertilizer in a slow manner. This leads to an increase in crop yield with a reduction in the required fertilizer due to reduced leaching. Loaded hydrogel with fertilizers has been shown to release at half the speed when fertilizer is placed in water alone [38,75,76]. Hydrogel as a slow-release

340 Chapter 12

Figure 12.5 A demonstration of the entire fertilizer release process from hydrogel.

method for water and dissolved fertilizers in soil is illustrated in Fig. 12.5. Usually the release mechanism of fertilizers is the diffusion mechanism which begins with a deswelling process where water is diffused and carries fertilizer through the porous network to the outside [77,78].3 The dissolution process for fertilizer comes quickly in the first few hours by dissolving the soluble fertilizer in the outer layer of the hydrogel (zone 1) when water contacts the wall of the hydrogel. Fertilizer release takes place successively for days (zone 2) and weeks (zone 3). The adsorption/desorption behavior of fertilizer is always observed and explains the SRF loaded inside the hydrogel. Almost 10% of the fertilizer remains in the bulk of the hydrogel which is strong bounded with functional groups.

12.4 Seed coating by hydrogel Nowadays, hydrogel is used as a seed coating with active substances such as insecticides and fungicides to improve seed growth and resistance against pathogens and pests in the juvenile stage, and to enhance seedling growth. Hydrogel enhanced the inherent capability of seed in soil and increased the attraction of water in proximity to the seed [79]. Fig. 12.6 show the effects of polyvinylpyrrolidone-poly(acrylamide) 3

The abounded mechanisms of fertilizer release are diffusion mechanism rather than osmotic pumping and convective release mechanisms.

The application of natural polymer-based hydrogels for agriculture 341

Figure 12.6 Seeds of watercress been placed in water (free hydrogel) and on polyvinylpyrrolidone-poly (acrylamide) (PVP-PAAm) hydrogel in a Petri dish.

(PVP-PAAm) hydrogel on the survival and germination of coated and uncoated watercress seeds. It is clear that, after five 5, the beneficial effect of PVP-PAAm hydrogel was greater than in the control sample. Hydrogel supplied seeds with both consistent moisture and oxygen necessary for germination metabolism and radical emergence and elongation [80
82]. Hydrogel is oxygen permeable, and in general the water uptake increased the oxygen permeability [83] and it is also known that seed germination requires oxygen and darkness.

12.5 Mode of action for polysaccharide hydrogel on multiple soil production processes (microorganism, nitrous oxide) and the effect of root
soil interaction on plant sprouting Soil is important for plant sprouting because it holds the roots and supplies them with water and necessary nutrients including essential minerals. It also provides support for plants and stores nutrients for plant survival in nature. The microorganisms are cause emission of nitrous oxide that effect on the planting, hydrogel has a predominant effect on N2O emission. In addition, to reach the high quality of a soil is needs critical levels of aggregate formation, facilitates cultivation, drainage and aeration, moisture-holding, reduces erosion of soil from oxygen and microorganisms, etc. Discussion here focuses on how the polysaccharide used as a soil conditioner is effective in preserving soil quality.

342 Chapter 12 1. Microorganisms inside soil help to adapt the ecosystem of the soil and are vitally important to the sustainability of the natural medium for the growth of plants. Thus microorganisms keep soils healthy and productive. Oxygenated soils are the prefer soil for most “aerobic” bacteria. Examples of aerobic bacteria include the Aerobacter genus which is widely distributed in soil and actinomycetes bacteria genus Streptomyces which gives soil its good “earthy” smell. Soil-associated aerobic bacteria decompose most polysaccharides into small fragments, usually sucrose and glucose units; this process is rapid in well-aerated soil [84,85]. Soils with these properties are called hydric soils, which are most suitable for plants with aerenchyma (internal spaces in stems and rhizomes) that allow atmospheric oxygen to be transported to the rooting zone [86]. Some polysaccharide-like chitin is stabilized by polysaccharides in soil for a long time, resulting from complexes with clays, metals, or humic acids which resist further decomposition [87,88]. The correlation between yields of corn and soil aggregation is very important; the yield of sugar beet is diminished when soil aeration falls below 12% [89]. The high concentration of polysaccharide soil conditioner is responsible for soil aggregation [90] and a good environment for anaerobic bacteria [91,92]. The increased cohesion force of soil results in not well-oxygenated soils which enables anaerobic bacteria to grow and also more complex microorganisms like fungi. In aggregated soil where oxygen is limited, known as hydric soils, this is the preferred soil for “anaerobic” bacteria. Most anaerobic soil is pathogenic bacteria and is able to kill off aerobic bacteria in the soil [93,94]. These soil bacteria have been determined to have negative consequences for plant growth and vigor via mechanisms that include phytotoxin and nutrient competition, and inhibition of Arbuscular mycorrhizal (AM) fungi.4 Anaerobic bacteria were surveyed for their ability to ferment 21 different polysaccharides including amylose, amylopectin, pectin, polygalacturonate, xylan, laminarin, guar gum, locust bean gum, gum ghatti, gum arabic, and gum tragacanth, Bifidobacterium, Peptostreptococcus, Ruminococcus, and Eubacterium species [95
99]. Bacteria dominate in tilled soils but they are only 20%
30% efficient at recycling carbon (C). Bacteria are higher in nitrogen (N) content (10%
30% nitrogen, 3
10 C:N ratio) than most microbes [100]. Polysaccharides also promoted microbial growth, and the microbes compete with the root for mineral nutrients and nitrogen. This could lead to a decrease in plant growth. 2. The nitrogen fertilizers such as ammonium (NH41) and nitrate (NO32) promote the emission of N2O that has a global warming contribution greater than that caused by CO2 emission [101]. The challenge now is to increase the retention of nitrogen (N) while mitigating methane (CH4) and N2O emission [102].5 4 5

Mitigation of CH4 and N2O emissions from soil is important to reduce global warming. Arbuscular mycorrhizal fungi increase soil nutrients and water absorption.

The application of natural polymer-based hydrogels for agriculture 343 Soil conditioner polysaccharide hydrogel retains most dissolved fertilizer nutrients, and can also decrease the nutrient need of agricultural soils, and reduce N leaching [103] and N2O emission [104
106]. 3. Swollen polysaccharide hydrogel increased the root
soil interactions and is a wellrooted activity. Soil is good for aeration but is not able to hold water, after irrigation or rainfall the pores are mainly filled with water, and the water escapes downward. Adding a soil conditioner polysaccharide hydrogel helps to hold water, thus creating favorable growing conditions for plant roots (Fig. 12.7). Fig. 12.8 show how the hydrogel is very tight to the roots enabling nutrients to be made available to the plant roots. Soil conditioner polysaccharide hydrogel adaptation of plant roots transports ions and nutrients faster. The root surface consists of plasma membrane containing phospholipids, proteins, and steroid molecules. Hydrogel improved the interaction between soil particles and roots by a hydrogen bond or dipole
dipole interaction that assists in improving the active transport of nutrients and

Figure 12.7 Effect of polysaccharide hydrogel on soil texture for plant growth: (A) water nonretention (drains out from the root zone); (B) the pores are filled with swollen hydrogel to aid water retention.

Figure 12.8 The hydrogel is very tight to the roots and increases the soil
root interaction.

344 Chapter 12 water to plants by negatively charged clay surfaces with anionic functional groups (Al31, Fe31, and Ca21). 4. The effect of plant sprouting using polysaccharide polymers as soil conditioners begins shortly after polysaccharide fermentation under the soil surface. In the previous section we outlined how soil conditioner lead to growth of anaerobic bacteria. These bacteria lead to polysaccharide fermentation into small molecules and small fragments. For example, starch is fermented under anaerobic conditions into α-amylase, pullulanase, α-D-glucosidase, sugar, and ethanol production and tolerance [107,108]. These fragments of sugars tend to be dominant in soils rich in plant remnants. The increased sugar content in the soil serves as food for any number of microorganisms. Depending on which microbes are present, this can have beneficial or harmful effects on the plant. Beneficial effects include all the activity that goes on in hot compost: nitrogen fixation, toxin decomposition, and nutrient production. These desired effects may be harmful, since fungal spores love to grow in such a media full of sugar so much so that they can turn that tree into dust. Sugar draws the water from roots, slowing down the process of transportation and hampering growth.

12.6 Polysaccharide hydrogels are significant in the control of plant disease The negative impact of fertilizers and herbicides on nontarget plants6 can lead to decreased crop yield. Consequently, there has been development of biodegradable polysaccharide hydrogel-based agrochemicals for effective and safe application in agricultural areas. For example, chitosan leading to formation of barriers triggers defense responses within the plant against invading pathogens [109]. Mercier et al. studied both polysaccharides of carrageenan and laminarin as elicitors7 of tobacco plant leaves compared with the activity of an elicitor of Phytophthora parasitica [110]. Polysaccharides in several literatures are used as elicitors [111
113], with especially chitosan being considered to be one of the most important as a plant defense booster [114]. Researchers have proved that polysaccharides (chitosane, chitine, etc.) elicit plant defense responses to a broad spectrum of phytopathogens, including plant viruses and fungi [112,115,116]. At present many experimental results have proved that chitosan can inhibit viral infections [117]. Treatment of bean with chitosan decreased the number of local necroses caused by alfalfa mosaic virus infection. It has been shown that chitosan inhibited 6

7

Nontarget plants growing in habitats adjacent to conventional fields may be exposed to herbicides by spray drift and also to excess fertilizers. Elicitors are chemical compounds; their tasks are to defend plants from pests, diseases, and synergistic organisms; chitosan is considered a natural elicitor.

The application of natural polymer-based hydrogels for agriculture 345 the infection caused by the bacteriophage, with the efficiency of inhibition of bacteriophage infection depending directly on the final concentration in the medium [118]. Major factors for suppressing phage infection by chitosan are phage particle inactivation and inhibition of reproduction at the cellular level. Evidently, chitosan may be used for induction of phago resistance in industrial microorganism cultures to prevent undesirable phagolysis caused by inoculum contamination by virulent bacteriophages or by spontaneous prophage induction in lysogenic cultures. Choudhary et al. [119] used Cu-chitosan nanoparticles to boost the defense responses and plant growth in corn (maize). It was found that Cu-chitosane nanoparticles were effective in plant growth and disease defense compared to blank samples of chitosan and CuSO4 individually.

12.7 A phytotoxicity test The absence of phytotoxicity of the polysaccharide hydrogel and the effects of the growing medium have been reported [120
122]. The assessment of the phytotoxicity of hydrogels is very important, to detect any decrease in growth. Plants treated with soil conditioner hydrogel increased the surface area and length of roots [123]. Seed coating can improve seed germination and seedling survival rate [124] In contrast, hydrogel applied to the root plug decreased Eucalyptus pilularis seedling mortality and enhanced survival of Corymbia citriodora subsp. variegata [125]. The application of a high dose (overdose) of hydrogel also caused seedling mortality [126
129]. Shooshtarian et al. [130] attributed a high seedling mortality rate in conditioner hydrogel to a reduction of free air resulting from the swelling of the hydrogel and the consequent reduction in soil aeration and decreased oxygen. Aeration is important because it enables the exit of CO2 produced by roots and microorganisms [131], and aeration allows soil to absorb oxygen that is essential for root development. The free space for good aeration and to enable the soil to retain water is directly proportional with the survival of the plant; these two conditions can achieved with hydrogel. However, when the problem is linked with aeration, the plant must first be removed and then the cultural medium (root zone) modified by larger particles in order to improve the drainage and porosity and lower the amount of aggregates [132]. The main point is that aeration management in cultural media is more sophisticated than water management. If a water shortage exists, it would be improved by irrigating. Ronald [133] recorded that seed germination was severely inhibited by the presence of a 1% cellulose xanthate solution. Although the cellulose xanthate solution was neutralized to pH 5 6.5 the germination of lettuce was reduced from 100% to 0%. Onion and chicory germination was reduced only from 93% to 78% and 90% to 63%, respectively. Hydrogel proved beneficial to the survival of seedlings. This reduced seedling mortality could be achieved using hydrogel. However, the application of STOCKOSORB AGRO

346 Chapter 12 (hydrogel) to the root system after lifting caused about a 19% higher survival rate compared to control variants. Regarding the fact that soil with fairly heavy texture possesses high capillary porosity rate and high moisture holding capacity, adding polymer to such soil does not cause effective changes in their aeration texture, even using large amounts creates a few problems related to increasing the capillary porosity in these soils. Hence, to solve the ventilation problem in these soils, lower quantities are recommended. However, in soils with light textures which do not have major problems in regard to drainage, adding polymer causes an increment in capillary porosity, the reason being linked to the properties of the hydrogel including it absorbing high moisture at a high rate.

12.8 Biodegradable hydrogel enables “nano” fertilizer release The loading of fertilizers in nanostructured forms was designed recently due to their improved crop yield, seed germination, photosynthetic activity, nitrogen stability, and increased carbohydrate content [134
137]. Hydrogel nanoparticles with another five types of nanostructured materials such as nanoclays, hydroxyapatite nanoparticles, mesoporous silica, and nanomaterials of carbon and other metal oxides are used as fertilizer carriers. The association of fertilizer with these nanomaterials brought about the new production of new nanofertilizers that are fully accessible to plants rather than conventional fertilizers [138]. Several literatures have suggested future directions toward the development and design of multifunctional nanomaterials, and construction of stimuli-responsive nanocarriers. Here we focus only on the features of using polymeric nanoparticles as nanocarriers of fertilizers. Nanoparticles of chitosan are an interesting material for use in controlled fertilizer release systems [139]. Nanofertilizers are soluble fertilizers loaded and/ or capsulated by nanoparticles of hydrogels, allowing for a slower release of nutrients into the soil. Nano fertilized biodegradable hydrogel slowly diffused into roots easily. The nanoparticle diffused into roots easily via symplastic and apoplastic pathways and translocated via xylem tissue to the aerial parts of the plants including the stems and leaves [140]. Entry of the nanoparticles through the cell wall depends on the pore diameter of the cell wall (5
20 nm) [141,142]. Hence, nanoparticles with diameter less than the pore size of the plant cell wall could easily enter through the cell wall and reach up to the plasma membrane [143,144]. This results in a noticeable difference in germination of sample lettuce seeds [145,146]. Nanofertilizers for promoting growth of plants should be using in low concentrations, as there can be significant health and environmental damage in high concentrations [147]. They enable biodegradable hydrogel nanoparticles to enter the plant, allowing their distribution in every organism related to the food chain. Compared to the other five nanomaterial carriers of fertilizer they are toxic to plants, animals, or humans

The application of natural polymer-based hydrogels for agriculture 347 at some exposure level, however this would not limit their use in various applications which are designed keeping in mind the critical exposure concentration, as discussed in most of the studies regarding the use of hydrogel nanoparticles as fertilizer carriers. Bortolin et al. [60] prepared a PAAm/MC/MMt nanocomposite hydrogel as a urea fertilizer carrier. The results indicated that the urea release was strongly controlled by the pH of the medium upon the hydrophilicity of the nanocarrier, and a hydrolysis treatment. The results showed that the release of urea was more prolonged, approximately 192 times more slowly, for the hydrolyzed hydrogel when compared to the control (pure urea). Additionally, according to the authors, these nanocomposites were the first carrier vehicles to release 90 g of urea per gram of dry hydrolyzed hydrogel used.

12.9 Biodegradable polysaccharide hydrogel offers “smart” fertilizer release The fertilizer carrier was developed to provide a critical sensing fertilizer that could release nutrients in a controlled manner. Recently the loading of fertilizer with smart hydrogel can overcome current limitations of conventional fertilizers [148
152]. Polymer-coated fertilizers (PCFs) are the most advanced CRF technologies [153]. This technique has relatively high costs. In order to enhancement the efficiency of fertilizer it is preferable to coat fertilizer with a smart biodegradable hydrogel (SBH). The fertilizer will have a better performance and be more advanced, such as becoming available for plants and roots for a long period of time, promoting efficient root uptake management which could be controlled by the amount of P, N, and K in the soil, and roots will have easy access to the fertilizer. The compaction will disappear and the crop yield will be increased. The challenges in the future of long-term plant growth include the synthesis of CRFs based on SBH. Smart hydrogels are suitable to release fertilizers and meet the practical requirements of plant growth, thus producing an economic crop yield and safe environment. The fertilizer release loaded into polymer can be controlled over other improvement options such as temperature, pH, and biological activity, as was described by Adams et al, [154], Oertli and Lunt [155], Broschat [156] Meurer et al. [157], and Kochba et al. [158]. Adams et al. [154] studied the critical effects of substrate moisture and temperature on macro- and micronutrient release of three PCF types: Polyon, Nutricote, and Osmocote. It was found that the effect of temperature on release fertilizer rate is more uniform across each Nutricote fertilizer than the other two fertilizers (Polyon and Nutricote). The fertilizer release rates for three PCF type nutrients were delayed by 20
40 days. The minimal fertilizer release was found at temperatures of 5 C
15 C and steady-state release was found in the mid-temperature range of 20 C
30 C.

348 Chapter 12 Oertli and Lunt [155] investigated inorganic fertilizer salt release rate in several elution and leaching experiments. It was found that the fertilizer release mechanism was a linear diffusion mechanism and was largely independent of the pH of the elutant and of the soil. With an increase in temperature from 10 C to 20 C the rate of release almost doubled. The release of NO32 and NH41 was faster than potassium and phosphates ions. Meurer et al. [157] synthesized pH-responsive biocompatible and nonphytotoxic poly (allylamine) hydrochloride microgel loaded with Fe31 ions, and a proof of concept for the microgel-loaded iron for iron-deficient cucumber plants. The result indicates that Fe31 ions

Figure 12.9 Outline of the significance of polysaccharide hydrogels in the agricultural field.

Figure 12.10 Aspects that encourage agricultural polysaccharide hydrogels to be more efficient.

The application of natural polymer-based hydrogels for agriculture 349 had strong binding to leaf surfaces and increases the chlorophyll content in leaves, confirming efficient delivery of ions. Research regarding CRFs based on SBH is still insufficient and should be developed more quickly.

12.10 Significance of polysaccharide hydrogel in the agricultural field Polysaccharide hydrogel has gained unique significance in agricultural applications (soil conditioner, fertilizer, and pesticide carriers). A wide range of advances have promoted the use of polysaccharide hydrogels due to their benefits (see Fig. 12.9). The aim of this chapter is to offer students, scientists, and professionals support to help them in developing APHs. In Fig. 12.10, aspects are outlined to encourage the development of more efficient APHs.

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343.

CHAPTER 13

Applications of natural polymer-based hydrogels in the food industry Hongbin Zhang1,*, Fei Zhang2 and Ronnie Yuan2 1

Shanghai Jiao Tong University, Shanghai, China 2DSM (China) Limited, Shanghai, China

Polymeric hydrogels are networks of polymer chains that are sometimes found as hydrocolloidal gels, in which water is the dispersion medium. Although hydrogels have not been generally defined, they are described as intermediate substances in an aqueous phase between solid and liquid and possess elastic (solid-like) and viscous (liquid-like) characteristics [1,2]. Hydrocolloids can be divided into thickeners and gelling agents, depending on the extent of molecular associations. For example, xanthan gum, a fermentation-derived hydrocolloid (biogum), is considered a thickener. Gellan gum, another common biogum, is classified as a gelling agent. Although xanthan gum is widely used in weakly gelled food, such as salad dressings, many hydrocolloids can form soft or firm hydrogels as gelling agents and/or major constituents in food; typical examples of these hydrocolloids are dairy dessert gels, jams, table jellies, some meat products, tofu, and noodles. Hydrogels formed by polysaccharides and proteins are consumed as food with a very long history in many countries, especially in Asia. For example, edible konjac gels have been utilized since the 2nd century BC as early as the Western Han Dynasty in ancient China. Tofu, a popular traditional gelled soy protein food in China, was allegedly invented by An Liu in the same period. Agar gels are a common food consumed for centuries in many areas of China and Japan (known as Kanten in Japanese). Hydrogels have received considerable attention in past decades due to their promising applications in tissue engineering, drug delivery, sustainable energy, and other traditional industries [3]. Food items that exist in the form of gels include jams, jellies, confectionery, desserts, and yogurt, which are composed of natural polymers as the main structure-forming ingredients [3]. This chapter mainly describes the functionality and applications of hydrocolloid gels in the food industry with a focus on understanding their gelation mechanisms and applications to different food systems.


corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00015-X © 2020 Elsevier Inc. All rights reserved.

357

358 Chapter 13 Polysaccharides and proteins are the two main categories of hydrocolloids that perform several functions, including stabilizing foams, emulsions, and dispersions; inhibiting ice and sugar crystal formation; texturization; and encapsulation. This chapter mainly focuses on polysaccharides that have a gelling nature and are used to construct three-dimensional networks in food systems by physical cross-linking. Commercially important gelling polysaccharides typically obtained from various plants or microorganisms are listed in Table 13.1. Gelling natural polymers with different chemical compositions, conformations, and chain structures can create distinct gel networks, which lead to diverse textures. A comparison of their relative gel textures is illustrated in Fig. 13.1. The types of texture are as follows: firm and brittle for low acyl (LA) gellan gum, agar, and κ-carrageenan; soft and flexible for gelatine, high acyl (HA) gellan gum and “synergistic” gels formed by mixtures of xanthan with plant polysaccharides, such as locust bean gum (LBG) or konjac glucomannan (KGM); and intermediate textures for gels made of alginate and pectin. Parameters, such as gelling agent level, ionic strength, pH, and temperature, influence gel formation and food properties in specific applications. The gelling mechanism of different polysaccharides and even different variants from the same polysaccharide varies with different dominant driving forces that stabilize the hydrogel network. The main molecular interactions in these hydrogels are hydrogen bonding, hydrophobic interactions, and ionic cross-linking. Either one of them or a combination of different intermolecular interactions contribute to hydrogel formation. Various types of junction zones are possibly formed depending on the polysaccharide type and gel-forming conditions (Fig. 13.2) [3]. Some polysaccharides form a gel upon heating and subsequent cooling (heat-set gels), such as for agar, carrageenan, gellan gum, and curdlan. Other polysaccharides form a gel at room temperature, involving specific types of cations, pH adjustment, or addition of certain cosolutes/chemicals, such as alginate, LA gellan gum, high methoxyl (HM) pectin, and KGM. Table 13.1: Main polysaccharides for constructing hydrogels and their origins. Botanical Plants Tree gum exudates Seeds Tubers

Starch, pectin, cellulose Gum Arabic (gum acacia), gum karaya, gum ghatti, gum tragacanth Guar gum, locust bean gum (LBG), tara gum, tamarind seed xyloglucan Konjac glucomannan

Algal Red seaweeds Brown seaweeds

Agar (agarose), carrageenan Alginate

Microbial

Xanthan gum, gellan gum, curdlan, dextran, hyaluronan (hyaluronic acid), bacterial cellulose

Animal

Chitosan, hyaluronan (hyaluronic acid)

Applications of natural polymer-based hydrogels in the food industry 359

Figure 13.1 Qualitative comparison of the texture of gels produced by different hydrocolloids. Reprinted with permission from P.A. Williams, G.O. Phillips, Introduction to food hydrocolloids, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of hydrocolloids, Woodhead Publishing Limited, 2009 [4].

Figure 13.2 Idealized junction zones in polysaccharide gels: (A) point cross-link, (B) extended block-like junction zone, (C) egg-box model for the junction zones in alginate and pectin gels [the calcium ions (eggs) link the blocks of the polysaccharide chains (egg-boxes) together], (D) double-helical junction zone, and (E) junction zone formed by aggregation of helical segments of the polysaccharide chains. Reprinted with permission from A. Nazir, A. Asghar, A. Aslam Maan, Chapter 13—food gels: gelling process and new applications, in: J. Ahmed, P. Ptaszek, S. Basu (Eds.), Advances in Food Rheology and Its Applications, Woodhead Publishing, 2017, pp. 335353 [3].

360 Chapter 13 Hydrogels or solutions can be classified depending on the physical structure of the biopolymer network as strong, weak, or pseudo gels [5]. The mechanical spectra of such gels obtained by rheological measurements are informative and useful for characterizing the gelling status of polysaccharide solutions and food systems. For “viscoelastic” materials of polysaccharide solutions and gels, the total stress in rheological measurement with lowamplitude oscillatory deformation can be resolved into an in-phase and out-of-phase component. The stress is then divided by the applied strain to determine storage modulus (G0 ), which characterizes the solid-like (elastic) response of the sample, and the loss modulus (Gv), which characterizes the liquid-like response. The variations in G0 and Gv with frequency (normally plotted on logarithmic axes) are known as the “mechanical spectrum” of the material, where η
is the complex viscosity and is often included in the spectrum [6]. The typical mechanical spectra for polysaccharide solutions, gels, and food based on these are shown in Fig. 13.3. For strong gels (Fig. 13.3C), solid-like characteristic

Figure 13.3 The four principles of mechanical spectra in low solid materials: (A) dilute solution, (B) entangled solution (pseudo gels), (C) strong gel, and (D) weak gels. Reprinted with permission from S. Kasapis, A. Bannikova, Chapter 2—Rheology and food microstructure, in: J. Ahmed, P. Ptaszek, S. Basu (Eds.), Advances in Food Rheology and Its Applications, Woodhead Publishing, 2017, pp. 746 [7].

Applications of natural polymer-based hydrogels in the food industry 361

Figure 13.4 Typical foods created with the assistance of polysaccharide hydrogels at different use levels.

(G0 ) predominates over liquid-like, viscous response (Gv, loss modulus) usually by at least an order of magnitude with slight changes in either modulus on varying frequency, and log η
decreases linearly as log ω increases with a slope close to 21. For “weak gels,” the elastic and viscous moduli have limited frequency dependence (Fig. 13.3D). Entangled polymer solutions or pseudo gels (also known as fluid gels) usually show mechanical spectra close to that in Fig. 13.3B, where G0 and Gv show strong frequency dependence with a crossover point at a specific frequency. For dilute solutions, Gv is higher than G0 across the entire frequency range (Fig. 13.3A). Food prepared using polysaccharide hydrogels can be categorized into fluid gels (pseudo gels), soft gels (weak gels), and hard gels (strong gels) (Fig. 13.4). Various juice beverages or protein drinks stabilized by fluid gels of polysaccharides, such as gellan gum at low concentrations (hundreds of ppm), typically have pseudo gel systems. The systems show characteristic fluid-like texture and excellent particle suspension stability due to possessing certain elastic modulus from the molecular network. Food, such as pudding and drinkable jelly, contains slightly higher levels of gelling hydrocolloids (a few thousand ppm) and are considered as soft gels with typical mechanical spectra of weak gels similar to that in Fig. 13.3D. The texture of food with high solid contents, such as jams and jelly candies with hard gel formats, is usually dominated by the strong gel network of hydrocolloids, such as pectin, gellan gum, and carrageenan with a dosage of approximately 1% or above. The materials of interest in this chapter are primarily hydrogels with typical food-related applications, constructed from various representative natural or semisynthetic gelling polymers, especially polysaccharide hydrocolloids, such as gellan gum, curdlan, carrageenan, agar, alginate, KGM, pectin, and cellulose derivatives, mainly methylcellulose

362 Chapter 13 (MC), hydroxypropyl methylcellulose (HPMC), and microcrystalline cellulose (MCC). This chapter does not focus on starches and protein-based gelling agents, such as gelatine, milk proteins, and soy proteins. The gelation mechanisms, properties, and functionalities of various typical hydrogels from polysaccharides and their effects on the rheological, structural, textural, and sensory properties of various food systems are discussed. Some emerging novel applications based on natural polymer hydrogels in the food industry, such as food for the elderly and 3D printed food, are also introduced.

13.1 Gellan gum Gellan gum is an anionic extracellular polysaccharide secreted by the microorganism Sphingomonas elodea and is available in substituted or unsubstituted forms, which are referred to as HA and LA gellan gum, respectively. The primary structure of gellan gum shown in Fig. 13.5 is composed of a linear tetrasaccharide repeat unit of two glucose, one glucuronate, and one rhamnose units [8,9]. HA gellan gum is the native polymer, which is produced with two acyl substituents present on the three-linked glucose (L-glyceryl) positioned at O (2) and acetyl at O (6). Every repeat unit roughly carries one glyceryl group, and only approximately half of the repeating units are acetylated [10]. Acyl groups present in the native polymer are removed by alkaline hydrolysis, which provides the H3CCOOH2C 1/2

n

Repeating unit of HA gellan gum

HOH2C HO O

O OH

CH2OH

OH

O

HO

O O

HO

CO2H

OH

OH

OH

O O H3 C

O

n Repeating unit of LA gellan gum

Figure 13.5 Primary structures of high and low acyl (LA) gellan gum with a tetrasaccharide repeating. The sites of attachment of glyceryl and acetyl substituents in high acyl (HA) gellan gum are indicated.

Applications of natural polymer-based hydrogels in the food industry 363 (A)

(B)

Carboxyl 5.64 nm

Glyceryl Acetyl

Figure 13.6 Double helix structure of deacylated gellan viewed (A) perpendicular to the helix axis (left) and (B) along the helix axis (right). The sites of attachment of glyceryl and acetyl substituents in high acyl (HA) gellan, relative to the position of the carboxyl group on the neighboring glucuronate residue, are indicated in (B). Reprinted with permission from R. Chandrasekaran, R.P. Millane, S. Arnott, E.D.T. Atkins, The crystal structure of gellan. Carbohydr. Res. 175 (1) (1988) 115 [13].

straight chain structure to LA gellan gum. Partially deacylated gellan was also investigated [11]. New gellan gum was launched by DSM hydrocolloids with a brand name of Gellaneer HS, of which the number of glyceryl and acetyl groups is between HA and LA gellan gum, while the gel properties are their combination [12]. The ordered structure of gellan in the solid state consists of a coaxial double helix (Fig. 13.6A), where each strand is a threefold, left-handed helix with a pitch of 5.64 nm. This structure results in helices stabilized by interchain associations involving glycerol groups, with the acetyl substituents positioned on the periphery of the helix (Fig. 13.6B). Gellan gum is the most recent addition to the range of gelling agents available commercially for food use with unique gelling properties and food applications. The gelation of gellan gum starts from its coilhelix conformation transition upon cooling. Many models explain the gel mechanism of gellan gum. Their overall consensus is that the driving forces are the hydrogen bonding and cation cross-linking among the ordered structure of aggregated helical segments, and the ordered junction is connected by disordered flexible polymer chains around junction zones [14]. The existence of an acetyl group in the periphery of the double helix can block the aggregation of the double helix, resulting in thermal reversibility but relatively weak gel texture for HA gellan gum. The glycerol group, which can participate in the hydrogen bonding formation within and between the participating strands can enhance the thermal stability of the double helix but will interrupt the cation-mediated aggregation, thus reducing the gel strength [6]. HA and LA gellan gum gels show distinct texture characteristics. LA gellan gum forms hard, nonelastic, and brittle gels, whereas HA gellan gum gels are soft, elastic, and nonbrittle. A comparison of the texture of HA and LA gellan gum gels through texture profile analysis is shown in Fig. 13.7. The components of food systems such as ion type, concentration, total soluble content, and pH significantly influence the strength of gellan gum gel [6]. In general, the gel strength increases with increasing ion concentration until

364 Chapter 13

Figure 13.7 Texture profile of high acyl (HA) () and low acyl (LA) (----) gellan gum gels measured on 1% gels at 70% strain. Reprinted with permission from G. Sworn, 9—Gellan gum, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 204227 [14].

reaching the maximum; further addition of ions weakens the gel. The efficiency of divalent cations is higher than that of monovalent cations, and the extent of this ion effect on gel strength for LA is more significant than HA. Apart from gel texture, a lot of different characteristics are observed between HA and LA gels. The addition of cations is not necessary for the formation of HA gellan gum gels, and their properties are less dependent on the concentration of ions in solution. However, LA should first interact with cations for gelation. HA gels typically set and melt between 70 C and 80 C and show good thermal reversibility [15]. While LA gellan gum shows thermal reversibility for monovalent mediated gel and significantly thermal syneresis, the melting temperature is higher than the setting temperature for divalent gel. Divalent cations are more efficient in promoting the gelation of LA gellan gum than monovalent ions. The gelling and setting temperatures of HA and LA increase with cation concentration. HA gellan gum gels perform better than LA on syneresis control [6,16]. Commercial LA gel has high transparency, whereas HA gel has a hazy appearance. An overview of the key properties of both the HA and LA forms is provided in Table 13.2. This finding is a useful reference from which existing food applications can be understood and new opportunities explored. Gellan gum is used commercially in various food applications due to its unique and versatile properties. These main applications include water-based gels, bakery applications, dairy food products, beverages, confections, and fruit applications [17]. The most common application of gellan gum is creating water-based gels, such as desserts and drinking gels. In dessert gels, LA gellan gum creates a firm, brittle texture with excellent clarity and heat

Applications of natural polymer-based hydrogels in the food industry 365 Table 13.2: Comparison of the key properties of high acyl (HA) and low acyl (LA) gellan gum [14] Properties

LA gellan gum

Hydration Sequestrants for aiding hydration Viscosity Gelling ions Setting temperature Melting Gel clarity Gel texture



. 80 C Yes Low Mono- or divalent or acid 25 C60 C No (except low ionic strength and in milk) Clear Firm, brittle

HA gellan gum . 70 C No High Not required 70 C80 C Yes Opaque Soft, elastic

Table 13.3: Typical food applications of gellan gum. Major food area

Typical products

Confectionery Jams and jellies Fruit preparation Water-based gels Elderly food Dairy products Beverages Encapsulation

Gummy or jelly candies, high chewy candy, and marshmallow Glazing agent, bakery fillings, and jellies Fruit jams for yogurt, fabricated fruit Dessert gels, aspics Pudding, mousse with desired texture for the elderly Ice cream, flavored milk, yogurt, milkshakes, and cheese Fruit, milk-based, plant protein-based, and alcoholic drinks Capsules, coating

stability, and its combination with HA or other hydrocolloids, such as LBG/xanthan gum blend, can create more diverse textures with improved syneresis control. Gellan gum is highly functional in bakery dry mixes, providing moisture retention and shelf-life extension in the final baked goods. LA gellan gum is employed to improve the bake stability for bakery fillings. Gellan gum, especially HA gellan gum, is functionally used in dairy products, including flavored milk, fortification plant protein, and yogurt, to enhance the suspension stability, control syneresis. and improve the texture. Gellan gum can overcome cloud and pulp settling in juices while providing a mouthfeel that is light and refreshing compared with other stabilizers. Gellan gum is used commercially in high-solids jelly confections, providing balanced textures between elasticity and brittleness with good flavor release. Gellan gum can also be used in some fruit applications, including low-solids jam and yogurt fruit preparations, to improve fluidic gel characteristics with good suspension stability and syneresis control. These typical food applications of gellan are summarized in Table 13.3.

13.2 Curdlan Curdlan, which is derived from “curdle” to describe its gelling behavior at high temperatures, is a natural polymer produced by microorganism fermentation. Curdlan has

366 Chapter 13

Figure 13.8 Chemical structure of curdlan.

been approved by the US Food and Drug Administration (FDA) as a food additive and has been registered in Korea, Taiwan, and Japan as dietary fiber [18]. In addition, curdlan shows strong bioactivities [19]. Curdlan is a high-molecular-weight polysaccharide characterized by repeating glucose subunits joined by β-linkage between the first and third carbons of the glucose ring (Fig. 13.8). The molecular weight of commercially available curdlan could exceed 2,000,000 Da [20]. In the solid state, curdlan exists in a triple helical structure as characterized by 13C NMR analysis. In its natural state curdlan is poorly crystalline [21], and has a granule form with a donut-shaped structure similar to that of starch. Curdlan forms a heat-set gel at both relatively high and low temperatures or during the neutralization or dialysis of the alkaline solution of curdlan. Curdlan powder is insoluble in cold water due to the existence of extensive intra/intermolecular hydrogen bonds, but it readily dissolves in alkaline solution and dimethylsulfoxide. High hydration occurs for curdlan powder in hot water (typically ca. 55 C) and further curdlan forms resilient hydrogels upon heating its aqueous suspension at elevated temperatures (80 C100 C). The resulting gel with a texture of the elasticity of gelatine and the rigidity of agar does not melt again upon either heating or cooling [20,22,23]. Fig. 13.9 shows a typical temperature dependence of storage modules G0 for a 2% suspension of curdlan during two heating and cooling cycles [19]. The gel (Gel I) formed by cooling an aqueous curdlan sol from ca. 55 C is thermally reversible or partially reversible, whereas the gel (Gel II) formed by heating this sol to high temperatures is thermally irreversible. Both gels experience a solgel transition (from sol to Gel I or Gel II). The rigidity of Gel II can be enhanced upon further cooling below 40 C, thus forming another gel state (Gel III). The gelgel transition from Gel II to Gel III is thermally reversible, which is concomitant with the formation of hydrogen bonds. The unique heat-gel properties of curdlan are derived from their helix structure transformation in the whole thermocycle [22]. Considering the chemical structure of curdlan, the gelation of native curdlan should be attributed to the hydrogen bonding along

Applications of natural polymer-based hydrogels in the food industry 367

Figure 13.9 Typical temperature dependence of storage modulus G0 for 2% suspension of curdlan during two heating and cooling cycles at 1 C/min. Reprinted with permission from Z. Cai, H. Zhang, Recent progress on curdlan provided by functionalization strategies. Food Hydrocoll. 68 (2017) 128135 [19].

with conceivable hydrophobic interaction. The formation of heat-induced hydrogels from native curdlan experiences unique solgel and gelgel transition that is rarely seen from many other polysaccharide hydrogels. An elaborate interpretation of structural change for curdlan gelation at different scenarios was proposed (Fig. 13.10). For a low-set gel (,55 C), the interior of the curdlan micelle is packed mostly with 7/1 single helical molecules and is partially occupied by triple helical molecules; in a high-set gel, the conformation of curdlan molecule changes to 6/1 triple helices, and curdlan micelle is occupied by molecules with a triple-stranded helix [24]. However, whether the conformation consists of exclusive single- or triple-helical structures or a combination of single- and triple-helical structures in curdlan gels remains disputed [19], which needs further exploration and clarification. Curdlan has a wide range of applications in various industries, especially in the food sector, and is an ingredient that functions as a gelling agent, thickener, stabilizer, and fat replacer (FR) in many products. Curdlan produces a retortable and freezable food gel, making possible the development of food products such as tofu noodles. Curdlan used in noodle dough reduces the leaching out of soluble ingredients and softening of noodles, resulting in clear soup broths. In freeze sweet goods, curdlan can improve the texture of cakes and the shape retention of ice creams. Curdlan can also be applied in meat products as fat mimetics (FMs) in fat-reduced formula, imparting fat mouthfeel and enhancing the water-holding capacity [25,26]. Considering the unique nature of heat stability, curdlan can imitate or

368 Chapter 13 Room temperature structure 7/1 Single helices + triple-stranded helix portions

w w w w w

w w w w

w ww w

Heat treatment (>120ºC)

w w w w

ww

w

High-temperature structure 6/1 Triple-stranded helices (Inter-chain displacement along the helical axis)

w

Drying in vacuum –H2O

w w

–H2O

w

and Slow cooling

w

22.65 Å

6/1 Triple-stranded helices

+H2O

w w

Moistening 18.12 Å

5.72 Å

w

w

Micelle diameter ca. 80 Å

(A)

(B)

(C)

Figure 13.10 Schematic representation of structural change between three forms of curdlan: (A) room temperature; (B) high-temperature structure at high humidity; (C) high-temperature structure at low humidity. Reprinted with permission from Reference N. Kasai, T. Harada, Ultrastructure of Curdlan, in: Fiber Diffraction Methods, American Chemical Society, 1980, vol. 141, pp. 363383 [24].

facilitate proteins in various food products, where gelling after heating is a distinctive feature that is mostly observed with protein-rich hydrocolloids. Curdlan contains high dietary fibers, which can also be used in the production of low-calorie food. As for the applications in confectioneries, curdlan lowers the oil uptake and moisture loss of donuts during deep-frying [27]. The abovementioned applications of curdlan, as well as its functions as a food additive or an ingredient in food product development are summarized in Table 13.4.

13.3 Carrageenan Carrageenan denotes a family of polysaccharides extracted from marine alga. It is a high-molecular-weight linear polysaccharide comprising repeating galactose units and 3,6-anhydrogalactose, which are both sulfated and nonsulfated and joined by alternating α-(1,3) and β-(1,4) glycosidic links (Fig. 13.11) [28]. Three basic types, namely, κ-, ι-, and λ-carrageenans, exist and may be obtained in relatively pure form or in a mixture with

Applications of natural polymer-based hydrogels in the food industry 369 Table 13.4: Food applications of curdlan [27]. Application

Function

Use level (%)

Noodle Kamaboko (boiled fish paste) Sausages, hams Processed cooked foods

Texture modifier Texture modifier Texture modifier, water holding Binding agent, improvement in moisture retention, and product yield Retention of shape Retention of moisture Retention of shape Gelling agent (stable against heating and freezingthawing) Gelling agent (stable against heating and freezingthawing)

0.21 0.21 0.21 0.22

Film formation Low-energy ingredient

110 30100

Processed rice cake Cakes Ice cream Jellies Fabricated foods Noodle-shaped tofu Processed tofu (frozen, retorted, and freeze-dried) Thin-layered gel food (frozen) Konjac-like gel food (frozen) Heat-resistant cheese food Edible films Dietetic foods

–O

3SO

OH O O O

O

O

n

OH OH κ-Carrageenans –

O3SO

OH O

O

O

O

O

n

OH –

OSO3

2 OS

OH

CH

HO

O

3

–

ι-Carrageenans

O

O

O

O OSO3–

HO

OSO3–

n

λ-Carrageenans

Figure 13.11 Repeating units of κ-, ι- and λ-carrageenans.

46 0.10.3 0.10.3 1.5 15

370 Chapter 13 Table 13.5: Gross structural difference between red seaweed polysaccharides [28]. Hydrocolloid

3,6-AnGalpa unitsb

Approximate sulfate content (%)

Agarose/agaran Furcellaran κ-Carrageenan ι-Carrageenan λ-Carrageenan

1 1 1 1 2

0 1620 1825 2534 3040

a

3,6-Anhydro-α-D-galactopyranosyl. λ-Carrageenan contains α-D-galactopyranosyl units in place of 3,6-AnGalp units.

b

another type(s). In all three types of carrageenan molecules, most sugar units have one or two sulfate groups esterified to a hydroxyl group at carbon atoms C2, C4 or C6 with sulfate contents ranging from 18% to 40%. The differences in the presence of 3,6-anhydro-Dgalactopyranosyl units and approximate sulfate half-ester contents for the three main types of carrageenan are provided in Table 13.5. Among the three types of carrageenan, κ- and ι- are the gelling ones, whereas λ-carrageenan is classified as nongelling. The mechanism of carrageenan hydrogel formation is related to the coilhelix conformation transition, which is shared by many other gelling natural polymers, such as gellan gum and agar. Gelation occurs upon cooling solutions of κ- and ι-carrageenan due to the formation of double or triple helices of restricted length. The linear helical portions then associate to form a rather firm, three-dimensional, and stable gel network in the presence of the appropriate cations (Fig. 13.12). As the carrageenan dispersion is heated, it does not hydrate fully until the temperature exceeds approximately 75 C80 C. During cooling, the solution shows a marked increase in the viscosity followed by gelation below 40 C50 C. The presence of these ions also increases the hydration, setting, and melting temperatures. For example, increasing the salt concentration significantly increased the gelling temperatures of κ- and ι-carrageenans (Fig. 13.13). Carrageenan possesses strong synergetic interactions with other hydrocolloids, such as dairy protein and galactomannans. Strong synergy is exhibited between κ-carrageenan and milk proteins. κ-Carrageenan not only forms a weak gel in the aqueous phase with existing salts but also interacts with positively charged amino acids in the surface of the κ-casein micelles (Fig. 13.14). The utilization of these interactions in dairy-based food systems is quite popular, such as in various flavored milk and fortified dairy drinks. Moreover, the galactose-free regions of LBG can associate with the regular helical structures that form within the κ-carrageenan network during cooling. Thus, hot solutions of κ-carrageenan/LBG forms strong elastic gels with low syneresis upon cooling below 50 C60 C. The maximum interaction and hence the peak rupture gel strength occur between the ratios of 60:40 and 40:60 of κ-carrageenan to LBG.

Applications of natural polymer-based hydrogels in the food industry 371

Figure 13.12 A representation of the hypothesized mechanism of gelation of κ- and ι-carrageenans. The polysaccharide chains in a hot solution are in a coiled state (A). As the solution cools, they intertwine in double-helical structures (B). On further cooling, the double helices are believed to nest together with the aid of K1 or Ca21 (C). Reprinted with permission from J.N. BeMiller, Carrageenan, in: J.N. BeMiller (Eds.) Carbohydrate Chemistry for Food Scientists, third ed., WPACIP, 2018 [28].

Gelling temperature (ºC)

80

1.0% kappa with potassium 1.0% iota with calcium

60

40

20 0

0.6 0.2 0.4 Gelling cation in solution (%)

0.8

Figure 13.13 Effect of cation (K1 or Ca21) concentration on gelling temperature for κ- and ι-carrageenans. Reprinted with permission from A.P. Imeson, 7-Carrageenan and furcellaran, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, 2nd ed., Woodhead Publishing, 2009, pp. 164185 [29].

372 Chapter 13

K+

Water gel reaction with K+ and Ca2+

K–

K–

Casein micelle

Figure 13.14 κ-Carrageenanκ-casein milk protein interaction. Reprinted with permission from A.P. Imeson, 7-Carrageenan and furcellaran, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, 2nd ed., Woodhead Publishing, 2009, pp. 164185 [29].

The composition and properties of carrageenan preparations depend on the species of carrageenans. Blending of gelling and nongelling types is commonly done to create the desired gel strength and texture. Sugar and food-grade salts are frequently added to standardize the gel or the viscosity characteristics for specific applications. The properties of the three types of carrageenans are summarized in a generalized form in Table 13.6. Commercial carrageenan can function as viscosity builders, gelling agents, and/or stabilizers in a wide range of applications (Table 13.7). They play an important role in modern-day formulations on texture improvement and cost reduction by providing diverse texture, structure, and physical stability in food products. In meat products, carrageenan enhances the quality and/or increases the cooked yield of poultry, ham, and sausage products. Water gels and cake glazes have used fast-gelling carrageenan for many years. Sauces, salad dressings, and dips utilize carrageenan to impart body, provide thickness, and stabilize emulsions. The utilization of carrageenan has been established in fluid dairy and dairy dessert products. The stabilization of cocoa and an additional mouthfeel can be attained using very small amounts of carrageenan due to protein reactivity. Whipped creams and toppings retain their stable form due to carrageenan. Carrageenan can assist with the stability of frozen dairy products by preventing whey separation and ice crystal formation in ice cream. Carrageenan is also used to create stable gels in puddings and pie fillings. These applications of carrageenans in various food applications are summarized in Table 13.7.

Applications of natural polymer-based hydrogels in the food industry 373 Table 13.6: Summary of properties of carrageenans [28]. Property

κ-Types

ι-Types

λ-Types

Soluble above 60 C Na1, K1, and Ca21 salt forms are insoluble Particles of Na1 salt form swell Particles of Na1 salt form swell. All salt forms are insoluble Soluble Soluble in hot solutions

Soluble above 60 C Soluble Na1 salt form soluble. Ca21 salt Soluble form produces thixotropic dispersions Insoluble

Soluble

Soluble Swell, but difficult solubility

Insoluble

Soluble in hot solutions

Soluble Soluble in hot solutions Soluble in hot solutions

Solubility In hot water In cold water

In cold milk

In hot milk In concentrated sugar solutions In concentrated salt solutions Gelation

Effects of cations on Strongest gels with K1 Types of gel formed Firm, brittle

Strongest gels with Ca21 Soft, elastic

Synergism with locust bean gum

High

High

Stable Undergoes hydrolysis when a solution of it is heated. Stable in gels Unstable

Stable Stable Undergoes hydrolysis when a Somewhat more solution of it is heated. Stable in stable gels Stable Unstable

High

Low

Nongelling Nongelling. Viscous solutions formed None

Stability At pH 7 and above In acidic systems

Of gels to freezethaw conditions Syneresis of gels

Nongelling

13.4 Agar Agar is a polysaccharide extracted from the cell walls of agarophyte algae. It is a gelling, thickening, and stabilizing food additive with a long history of use. As one of the first phycocolloids to be used, agar was the first approved generally recognized as safe additive by the FDA. The extraction process is built on gel syneresis, a phenomenon in which the free interstitial water is expelled from the gel network. This process can be achieved as a result of the association of polymer chains as promoted by a freezethaw cycle or mechanical pressure. The production process for agar is shown in Fig. 13.15. In the freezethaw process, the gel is slowly frozen to develop large ice crystals. Considering that the gel is rigid and

374 Chapter 13 Table 13.7: Typical products containing a carrageenan product [28]. Bakery products Cheesecake Cookies Fillings Bars Cereal Diet Granola Beverages Nutritional/diet Soy Confections Chewy candy Chocolate bars Fruit gushers Coffee creamer, nondairy Dairy products Bakery fillings Cheese dressings Cheesecake Chocolate milk Chowder Coffee cream Coffee creamer Cottage cheese, including low-fat Cream cheese Dips Evaporated milk Flan Frozen cheese Frozen cheese products Cheesecake Lasagna Pizza Frozen desserts Frozen novelties Frozen yogurt Ice creams, including fat-free Infant formulas Milkshakes

a

Meals ready to eat.

Nutritional/diet beverage Processed cheese Protein shake Puddings Yogurt, fat-free Whipped cream, pressurized Whipped toppings Whipping cream Egg substitute Frozen meals Gelled desserts Gel sticks and snacks Imitation crab meat, frozen Mayonnaise, low-fat Meat products Luncheon meats Sausages Turkey breast slices Weiners Meatless frankfurters Mixes, dry Breakfast drink Brownie Cake Cappuccino, instant Chocolate milk Chai tea latte Diet dairy Dips Hot cocoa Pancake Pie filling Pudding, including low-fat Soup MRE,a microwaveable Pudding snacks Salad dressing, low calorie Sauces Fudge Horseradish Stir fry Soups, low-fat, low-calorie Vegetable patties

Applications of natural polymer-based hydrogels in the food industry 375

Figure 13.15 Manufacturing process for agar [30].

inflexible, it cannot accommodate large ice crystals. Hence, the polysaccharide is concentrated into bundles, forming a sponge-like structure. When the block is thawed, the water is drained. Pressure may be used to increase the volume of expressed water, thus concentrating the agar to 10%12% [31]. The gel press process uses syneresis to dewater the gel, where a mechanical pressure of 510 kg/cm2 is applied progressively to the gel. The polymer chains disassociate, and free water is expressed, thereby increasing the agar concentration to approximately 20%. After concentrating, the agar strips or flakes are dried with hot air and ground to the appropriate mesh size, usually 80100 mesh (100150 μm). Agar has been assigned with a linear galactan configuration, but later small quantities of sulfate were identified within the macromolecule with a typical sulfate content of 1.5%2.5% [30]. A further study showed that agar has two components: a major fraction of a neutral firmly gelling polysaccharide called agarose and a minor fraction of a weakly gelling charged polymer called agaropectin. Agarose is a linear polymer composed of (13)-linked agarobiose units of galactopyranose that are (14)-linked to 3,6-anhydrogalactopyranose (Fig. 13.16) [32]. Agarose has a high molecular weight above 100,000 Da

376 Chapter 13

Figure 13.16 Structural unit of agarose.

that frequently surpasses 150,000 Da and a low sulfate content usually below 0.15%. Agaropectin is based on the same structural unit of agarobiose as agarose but with different side groups, including up to 8% sulfate groups and methyl and pyruvic acid acetyl groups [33]. It has a lower molecular weight, usually below 20,000 Da, typically 14,000 Da. The presence of these side groups prevents this polymer from adopting a regular structure. Hence, it does not contribute significantly to gel formation. Agar can form a thermally reversible gel upon cooling the hot solution, and this gel-forming ability makes it widely used in many practical applications as gelling agents and stabilizers in food, biochemistry or molecular biology, and industrial applications. The gelation of agar is exclusively dependent on the formation of hydrogen bonds, where random coils associate to form single and double helices upon cooling [34,35]. Following the phase separation process induced by helice aggregation, the microgels aggregate to form gels upon cooling. Fig. 13.17 elucidates this gelation process, where random coils associate during cooling to form helices followed by further aggregation of helices to provide the aggregated structures of agar gels. This transition temperature ranges from 30 C to 50 C, which is influenced by many parameters, including raw materials for agar production, agar concentration, and cooling rate [30]. Another very important property of agar is gel hysteresis, which is the difference between the gelling and melting temperatures. For example, the hysteresis value for Gelidium and low methyl ester Gracilaria agars is approximately 50 C60 C, but this value falls as the methyl ester content increases [36]. This thermal hysteresis imparts agar hydrogel with relatively higher heat stability compared with other thermal reversible gels, such as carrageenan gel. This phenomenon allows various advantageous applications of agar in food, requiring good stability at elevated temperature and high water activity. The main advantages of agar in different food applications are derived from the characteristic firm texture, heat tolerance of the gels, simple process of gelation without extra ions or solutes, stability in acidic conditions, and limited reactivity to other food components. These characteristics result in the high popularity for agar in many specific food applications, particularly where firm textured gels with good heat stability and moisture stabilization are required [37]. Some typical domains include water gels such as

Applications of natural polymer-based hydrogels in the food industry 377

Solution

Cool

Cool

Heat

Heat

Initial chain association

Final gel structure

Figure 13.17 Schematic illustration of the formation of agar hydrogel. Reprinted with permission from A. Imeson, Agar, in: A. Imeson (Ed.), Food Stabilisers, Thickeners and Gelling Agents, Blackwell Publishing Ltd., 2010 [30].

water dessert jellies, vegetable, meat and fish aspics, and artificial caviar, confectionery including sweets and candies, fruit jellies, nougat, candy fillings, piping gels, jellies and jams, and bakery products, such as icings and glazes for pastries, cakes, and donuts [30]. The addition of agar in yogurts to improve the texture attributes is widely employed. Moreover, its indigestable nature contributes to its untilization as sources of dietary fibers in healthy food. Another important agar application in food is for preparing culinary dishes traditional to certain cultures, such as Mitsumame, red bean jelly, and Tokoroten noodles in Japan [30].

13.5 Alginate Alginates are derived from various species of brown seaweed, growing in the coasts of the North Atlantic, South America, and Asia. Alginate is a natural, high-molecular-weight polymer. It is the salt form of alginic acid with a degree of polymerization that usually ranges from 50 to 3000 after commercial processing, which is equivalent to molecular weights of approximately 10600 kDa. Alginic acid is a copolymer of the building blocks β-D-mannuronic acid (M) and its C-5 epimer, α-L-guluronic acid (G), which is linked together to form a linear polysaccharide with (1,4)-glycosidic bonds. The general structure

378 Chapter 13 –OOC

–OOC

OH

OH

O

O HO

O HO O OH

–OOC

M

O

M

–OOC

O

OH O

O

OH

OH

O O

O O

OH

O –OOC

OH G

n

M

–OOC

OH

O

O HO

n

–OOC

OH

G

OH

O

G

G

–OOC –OOC

OH

OH

O

O

OH O OH G

O

HO

O

–OOC

O

O

HO O OH

–OOC

OH

O

OH O O

–OOC

OH G

M

M

n

G

MMMMGMGGGGGMGMGGGGGGGGMMGMGMGGM M-block

G-block

G-block

MG-block

Figure 13.18 Block structures in alginate: M-blocks, G-blocks, and MG-blocks [alternating β-D-mannuronic acid (M) and α-L-guluronic acid (G)].

of alginate is shown in Fig. 13.18. Monomeric M- and G-residues in alginates are joined in sections consisting of homopolymeric M-blocks (-MMM-) and G-blocks (-GGG-) or heteropolymeric blocks of alternating M and G (-MGMG-). Alginates are produced as a range of salts by neutralizing alginic acid with different inorganic alkalis (Fig. 13.19). Among them, sodium alginate is predominantly used in food. Sodium alginate hydrates in cold or hot water and yields viscous solutions. Practically, hydration occurs with a dispersing agent such as sucrose and a nonsoluble solvent to avoid a large lump. The small particle of sodium alginate and high-speed shearing facilitate hydration. Apart from the thickening function of alginates in food, the most important property resulting from its block structure is the ability to form heat-stable hydrogel. In food applications, the primary alginate gel is constructed with calcium ions. Moreover, at slightly acid pH such as

Applications of natural polymer-based hydrogels in the food industry 379 Alginic acid Na2CO3

Na alginate*

K2CO3

K alginate*

NH4CO3

NH4 alginate*

CaCO3

Ca alginate

Propylene oxide

Propylene glycol alginate* * Water-soluble salts

Figure 13.19 Alginate salts produced from alginic acid for food use [38].

in fruit and jam applications, the alginate gel can be an acid-type gel or a mixed calcium/acid gel. G-blocks rather than M-blocks and MG-blocks participate in the junction zones by Ca21 cross-linking, which offers most of the gel strength [39]. The interaction between alginate and calcium ions is commonly visualized using the egg-box model, where Ca21 ions fit into the structural void in the G-blocks of alginate chains (Fig. 13.20). More than two G-blocks are joined in the lateral junction zones, implying modification of the original egg-box model (Fig. 13.20), where multilayer junction zones exist. The most important factors affecting the gelation of alginate and subsequently the gel properties are alginate concentration, chemical composition and sequence, the ratio between gelling and nongelling ions, and the presence of complexing agents, such as phosphates or citrate. The controlled interaction between sodium alginate and calcium salts yields coldsetting gels that are shear irreversible and heat stable. This control is possible through two fundamentally different methods: the diffusion method and the internal setting method. The diffusion method is characterized by letting a cross-linking ion (e.g., Ca21) diffuse from an outer reservoir into an alginate solution (Fig. 13.21A). Internal setting (sometimes also referred to as in situ gelation) differs from the former in that the Ca21 are released in a controlled fashion from an inert calcium source within the alginate solution (Fig. 13.21B). Alginates have been used in food applications for a long time and have been approved for various applications in several countries. As a cold-water-soluble biopolymer, alginate develops viscosity when hydrated in aqueous solutions and forms gels upon the addition of calcium. In practice, thickening and gelling occur when other ingredients in the product are in play. Typical food applications include reformed food, such as onion rings and olive fillings, cold-setting bakery cream fillings, and heat-stable bakery and fruit fillings. Table 13.8 categorizes some typical applications into four fields in terms of different functionality domains.

380 Chapter 13

Figure 13.20 Alginate gelation with Ca21 in the “egg-box model.” Reprinted with permission from H. Trond, G. Olav, F. Therese, O.A. Peder, L. Christian Klein, Alginates, in: A. Imeson (Ed.), Food Stabilisers, Thickeners and Gelling Agents, Blackwell Publishing Ltd., 2010 [38].

Figure 13.21 The two principal methods of the manufacturing of alginate gels: (A) diffusion setting; (B) internal gelling. Reprinted with permission from K.I. Draget, 29—Alginates, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 807828 [39].

Applications of natural polymer-based hydrogels in the food industry 381 Table 13.8: Alginate properties utilized in food products [38]. Gel forming

Thickening/water binding

Stabilizing

Film forming

Pet food Restructured fruit and vegetables Restructured fish and meat Puddings and desserts Cold prepared bakery creams Fruit preparations, fruit fillings Mousse Encapsulation, bead formation Tomato ketchup, tomato sauce Soups, sauces, cheese sauce Milkshakes Thickened cream Ice cream Mayonnaise Whipped cream Low-fat spread Salad dressing (PGA) Fruit juice (PGA) Beers, lagers (PGA) Baked goods Glazes for frozen meat and fish Film coatings for fresh meats Coatings for cakes and cookies Vegetable coating

13.6 Konjac glucomannan KGM is the main component of the tubers of konjac, which is a perennial plant of Araceae. Clinical studies have demonstrated that KGM can enhance health by lowering plasma cholesterol and improving carbohydrate metabolism, bowel movement, and colonic ecology [40,41]. KGM is a food additive (E425) that is widely used as a thickener and binder for gravies, soup, sauces, meat, and poultry. KGM can form gels in an alkaline environment or by combining with other hydrocolloids, such as xanthan gum or κ-carrageenan. These gels are quite important building blocks for many food applications, such as in jelly, noodles, tofu, and snacks [41]. KGM is a heteropolysaccharide composed of β-1,4-linked D-glucose (Glc) and D-mannose (Man) at a Glc/Man ratio of 1:1.6 as the main chain. The acetyl groups along the KGM backbone, which contribute to the solubility properties, are located every 919 sugar units at the C-6 (Fig. 13.22) [40]. The molecular weight of KGM ranges from 200 to 2000 kDa, which varies with cultivars, origin, processing method, and storage time [42]. KGM can form both thermal reversible and irreversible gels at different environments. In the presence of alkali, KGM forms a gel upon heating its solution, and the role of alkali is

382 Chapter 13

Figure 13.22 Chemical structure of konjac glucomannan (KGM).

believed to remove the acetyl groups. Fig. 13.23 shows the gelation kinetics for KGM with different degrees of acetylation (DA). As revealed here, the gelation time for KGM samples with higher DA needs a longer time than KGM samples with lower DA, evidencing that the gel mechanism is related to the deacetylation reaction and further polymer chain aggregation [22] This gelation mechanism is also supported by the peak at 1730 cm21 in the FTIR absorbance spectra of KGM films, which is attributed to the acetyl group [43] that disappears by alkali treatment. Konjac can also form thermally reversible gel by a synergetic interaction with other hydrocolloids, such as κ-carrageenan or xanthan gum. Fig. 13.24 shows the gel strength for 1% mixed gel with various KGM-to-carrageenan ratios in the presence of sugar at different concentrations. KGM is suitable for thickening, gelling, texturing, and water binding in various food applications. The alkali-treated konjac gel is strong, elastic, and thermally stable and has been used as a traditional dietary food in Japan, such as Konnyaku, for a long time. Synergistic gels produced by the combination of other hydrocolloids, such as xanthan gum and carrageenan, are the major products in the food industry as new types of healthy jellies [44]. Clinical studies have demonstrated that supplementing the diet with KGM significantly decreases plasma cholesterol and improves carbohydrate metabolism, bowel movement, and colonic ecology [41]. In addition, combination of KGM with starch was reported to manage the viscosity in food systems and to decrease blood glucose and cholesterol in mammals [45]. KGM can also be used to provide fat replacement properties in fat-free and low-fat meat products. Typical applications and functional uses of KGM are listed in Table 13.9.

Applications of natural polymer-based hydrogels in the food industry 383 (A) 4.4

log G′ (Pa)

3.4 Rs

Ac20

Ac21

Ac26

Ac27

Ac32

2.4 3.4 2.4 log G′ (Pa)

1.4

1.4 0.4

0.4

–0.6

0

50

100 Time (min)

150

200

–0.6 0

100

200

300

400

(B)

500 600 Time (min)

700

2100 2200 2300

Ac32

100

Gel time (min)

80

60

Ac27

Ac26

40

Ac21 20

Ac20

Rs 1

2

3 4 Acetate content (%)

5

6

Figure 13.23 (A) Time dependence of G0 of 2.0 wt.% KGM aqueous dispersions in the presence of Na2CO3 at 45 C. The degree of acetylation (DA) of each sample is shown in (B), the plot of the gel time at which G0 5 Gv against the DA of each sample. Reprinted with permission from K. Nishinari, H. Zhang, Recent advances in the understanding of heat set gelling polysaccharides. Trends Food Sci. Technol. 15 (6) (2004) 305312 [22].

13.7 Pectin Pectin is a natural heteropolymer and is present in all plant primary cell walls. Pectin can be used in diverse food applications, such as a gelling agent, emulsifier, stabilizer, glazing agent, and fat replacer (FR) [46,47]. As a kind of soluble dietary fiber and bioactive

384 Chapter 13 400 350

Gel strength (g)

300 250

Sugar concentration 0% 5% 10% 15%

200 150 100 50 7:3

6:4

5:5

4:6

3:7

2:8

KGM/Car ratio

Figure 13.24 Gel strength of mixtures of KGM/κ-carrageenan with various compositions. The total concentration of the mixtures is 1%. Data from S. Takigami, 32—Konjac mannan, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 889901 [44]. Table 13.9: Applications and functional uses of konjac glucomannan (KGM) [44]. Application

Function

Confectionery Jelly Yogurt Pudding Pasta Beverage Meat Edible film

Viscosity, texture improver, and moisture enhancer Gel strength, texture improver Fruit suspension, viscosity, and gelation Thickening, mouthfeel Water-holding capacity Fiber content, mouthfeel Bulking, fat replacer (FR), and moisture enhancer Water-soluble, water-insoluble

polymer, pectin has shown a number of functions, such as decreasing blood fat and combating various types of cancers, making it widely popular in the nutraceutical and pharmaceutical fields [46,48]. Although pectin was discovered more than 210 years ago, its chemical and structural properties and applications are still the subject of investigation due to its structural inhomogeneity and diverse functionalities. Pectin is a complex structural polysaccharide extracted from the cell walls of tremendously diverse plants. It is an anionic biopolymer with a backbone predominantly composed of α-1,4-linked galacturonic acid-based units (at least 65% by weight) in common [49]. This polymer also contains significant amounts of other sugars, such as L-rhamnose (Rha), D-arabinose (Ara), D-galactose (Gal), and another 13 different monosaccharides [50]. Pectin

Applications of natural polymer-based hydrogels in the food industry 385 (A)

(B) HG RGI

HG

HG

KEY 6-O Me GalA GalA

GalA RhA

Ara

RGI

Figure 13.25 Schematic model of the structure of pectin containing homogalacturonan (HG) and rhamnogalacturonan (RGI) regions: (A) contiguous model; (B) branched model based on an RGI core structure. Reprinted with permission from F. Naqash, F.A. Masoodi, S.A. Rather, S.M. Wani, A. Gani, Emerging concepts in the nutraceutical and functional properties of pectin—a review. Carbohydr. Polym. 168 (2017) 227239 [46].

chain structure is always referred to as an alternation of “smooth” and “hairy” regions along the entire length of the molecule. The smooth regions are represented by homogalacturonans (HGs), which form the linear backbone, and rhamnogalacturonans (RGIs) represent the hairy regions [51]. HG constitutes circa 65% of the pectin molecule, and the remainder is RGI. Two basic models are available on how these building blocks are arranged in pectin chains: an alternating HG and RGI model and an RGI backbone model (Fig. 13.25) [52]. The pectin properties are highly related to the degree of methyl esterification (DM) [49]. DM is defined as the percentage of GalA units esterified by methanol. Commercial pectins are classified as HM with DM .50% and low methoxyl (LM) with DM ,50%. DM is produced by acid, alkaline, and/or enzymatic deesterification of HM pectin natively existing in plant cell [53]. Amidated low methoxy pectin (ALMP) is another modified pectin obtained through high methoxyl pectin (HMP) aminolysis. The corresponding forms of galacturonic acid-based unit, free acid, methyl ester, or acid amide are shown in Fig. 13.26. Three pectins, namely, HMP, low methoxyl pectin (LMP), and ALMP, have different gelation mechanisms and therefore form gels with different properties. The gel characteristics are governed by their macromolecular attributes, such as the composition, size, and conformation of the polymers [54], which are varied among the three types of pectins. HMP gels are obtained at low pH in the presence of a high sucrose concentration

386 Chapter 13 OH

OCH3 OR OR

O

NH2 O

H

C

O

HO H H

OH O

Figure 13.26 Galacturonic acid, ester, and amide units found in pectin. Arrows indicate the potential for degradation by β-elimination in the ester form. Reprinted with permission from H.U. Endreß, S.H. Christensen, 12—pectins, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 274297 [49].

or similar cosolutes. A high sucrose concentration reduces water activity, facilitating chainchain interactions rather than chainsolvent interactions, whereas low pH protonated carboxylate residues reduce electrostatic repulsion [55]. Hydrogen bonds between pectin molecules are favored by the conformation of adjacent GalA units. A large number of hydrogen bonding among GalA confers significant thermodynamic stability to the gel. Hydrophobic interactions among the methyl ester group are essential for HMP gelation. Reportedly, the contribution of hydrophobic interactions to the free energy of junction zones in HMP with a DM of 70% is half that arising from hydrogen bonding [56]. Gel strength and setting temperature are influenced by pH, sugar types, contents, and processing parameters, such as cooling rate in gelation. In general, low pH and high-sugar content facilitate the formation of a strong hydrogel with fast gelation and high setting temperature, as partially demonstrated in Fig. 13.27. LMP gels are stabilized mainly by ionic cross-linkages via calcium bridges between two carboxylates from two different chains referred to as the “egg-box” model (Fig. 13.28). In general, egg-boxes formed between two neighboring chains are stabilized predominantly by electrostatic interactions, followed by hydrogen bonds and then by van der Waals interactions. For this reason, the availability of calcium ions is important and commonly governed by sequestrants either naturally present (e.g., citrate and other organic acid ions from fruit or milk) or added (commonly food-grade citrates, lactates, tartrates, or di- or polyphosphates). Reactivity to calcium is governed by the proportion and arrangement of

Applications of natural polymer-based hydrogels in the food industry 387 Rapid set (0.58%)

(B) 90 Temperature (ºC)

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80 Rapid

70 60 Slow

50 40

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Figure 13.27 (Left) Variation of gel strength of high methyl ester pectin with pH in a 65% sucrose gel (relative values); (right) variation of gel setting temperature in the same system. Reprinted with permission from H.U. Endreß, S.H. Christensen, 12—pectins, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 274297 [49].

Figure 13.28 The low methoxyl pectin (LMP) gelation mechanism is governed by ionic cross-linking via Ca21 between two carboxyl groups from two different chains in close proximity. Reprinted with permission from S.Y. Chan, W.S. Choo, D.J. Young, X.J. Loh, Pectin as a rheology modifier: origin, structure, commercial production and rheology. Carbohydr. Polym. 161 (2017) 118139 [47].

388 Chapter 13 100

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15

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40 50 Calcium added

60

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Figure 13.29 Variation of relative gel strength of low methyl ester pectin gels (conventional and amidated) with added calcium at 30% added sucrose and pH 3.0 with a citric acid/sodium citrate buffer system. Reprinted with permission from H.U. Endreß, S.H. Christensen, 12—pectins, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 274297 [49].

the carboxyl groups in the pectin chain. The reactivity increases with decreasing degree of esterification and is great but less controllable for gelling purposes if the arrangement of acid groups is less random, with blocks of de-esterified galacturonate units [49]. Gelation is favored by increased soluble solids but decreased by increasing pH or by increasing the level of sequestrant. However, a certain level of sequestrants, such as citrate, is essential to produce a practically workable gel system. With correct formulation, low methyl ester pectins can gel over a wide range of soluble solids (10%80%) and in either acidic or less acid-tasting products at a pH of a little below 3.0 to above 5.0. Amidated pectin is obtained by partial deesterification by ammonia, and that part of the ester groups is replaced by the amide groups. This process modifies the gelling properties in comparison with acid de-esterified pectins (LMP). The amide groups have a moderating influence and permit gelation over a wide range of calcium concentrations (Fig. 13.29) [49]. Pectin is primarily applied in food as a gelling agent and stabilizer. A wide range of pectins are developed following the specific food composition, manufacturing, and desired gel texture. Basically, pectin can control water in products and help create the desired texture. The traditional and main application of pectin is as a gelling agent in jams and jellies utilizing HM pectin to form gels at low pH and high-sugar levels and LM pectin to form gels at low sugar levels in the presence of calcium. One of the attractive features is that the pH at which pectin has desired gel texture and optimal stability matches the natural pH of fruit preserves. Compared with other hydrocolloids, this feature is unique to pectin. Another advantage is related to texture that is physically and also organoleptically optimal, and

Applications of natural polymer-based hydrogels in the food industry 389

Figure 13.30 The range of commercial nonamidated pectins with some typical applications. Reprinted with permission from H.U. Endreß, S.H. Christensen, 12—pectins, in: G.O. Phillips, P.A. Williams (Eds.), Handbook of Hydrocolloids, second ed., Woodhead Publishing, 2009, pp. 274297 [49].

finally, pectin gives an excellent flavor release due to its relatively low molecular weight when compared with other hydrocolloids. Pectin is used in recombined fruit beverages where HM pectin restores the mouthfeel to that of, for example, freshly squeezed orange. The stabilizing effect is seen in multiphase systems including emulsions, suspensions, foams, and acidified protein drinks. Fig. 13.30 offers an overview of food applications by using different pectins.

13.8 Cellulose-based polymers Cellulose, as the major constituent of most land plants, probably is the most abundant natural polymer with a linear chain of anhydroglucose with the β-O-glucopyranosyl structure (Fig. 13.31). Cellulose is the starting material for a wide range of modifications with uses both in the food industry and an even great variety of uses outside food application. Cellulosics introduced here are cellulose derivatives covering the range of modified celluloses approved as food additives with gelling properties. Key examples of the

390 Chapter 13

Figure 13.31 Structures of native cellulose, and cellulose derivatives of methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC).

structure of cellulose derivatives include MC and HPMC, as shown in Fig. 13.31. Carboxymethyl cellulose, which is a prevalent cellulose gum in food industry as a thickener and stabilizer, has received some concerns due to its negative influence on gut health [57], but it is not included in this part since it belongs to a nongelling polymer. Many commercially available MC and HPMC are water-soluble polymers. Commercial MC products have an average degree of substitution (DS) ranging from 1.5 to 2.0, and HPMC products have an average methyl group DS of 0.91.8 and hydroxypropyl groups DS of 0.11.0. The solubility of MC and HPMC cellulose ethers is highly reliant on the methyl and hydroxypropyl DS. Decreasing the level of substituent groups below a DS of 1.4 gives products wherein the solubility in water decreases. Increasing the substitution above a DS of 2.0 improves the solubility in polar organic solvents. Both MC and HPMC possess the rather unusual property of solubility in cold water and insolubility in hot water; thus, when a solution is heated, a three-dimensional gel structure is formed [58]. These heat-set gels are formed at elevated temperatures with high thermal reversibility, a property beneficial in many cooking applications. The most common grade of MC gels between 48 C and 64 C. By modifying production techniques and by altering the ratios of methyl and hydroxypropyl

Applications of natural polymer-based hydrogels in the food industry 391 substitutions, producing products wherein the thermal gelation temperature ranges from 50 C to 90 C and wherein the gel texture ranges from firm to rather mushy is possible. This gelling forces the heat transfer from convection to conduction, subsequently limiting the movement of the product and preventing boiling out of the filling during baking. The thermal gel structure also reduces moisture loss during heating [59]. The gelation mechanism is thought to be hydrophobic interactions among the methyl and hydroxypropyl groups induced at high temperature [6062]. Specifically, at low temperature, water molecules form hydrogen bonds with the hydroxyl groups along the MC chains and/or the cage-like structures surround the methyl groups of MC chains. Heating of such an MC solution will cause the destruction of (1) the hydrogen bonds and/or the cage structures and (2) the exposure of the hydrophobic regions of MC, forming the hydrophobic aggregates of MC. This hydrophobic association is the driving force stabilizing the three-dimensional hydrogel network at elevated temperature. This gelling behavior is reversible and reproducible. As shown in the dynamic modulus change with temperature for MC solution in Fig. 13.32, G0 and Gv increase upon heating the MC solution, resulting in a weak gel. After cooling, the gel reverts to the original-liquefied form, providing suitable product texture and mouthfeel. The rates of gelation and gel strength are strongly dependent on temperature, concentration, DS, and substantially on molecular weight [6062]. The major applications of these MC and HPMC are in the fields of binding and shape retention, film formation and barrier properties, and avoidance of boil-out and bursting at high temperatures [63]. MC and HPMC solutions gel and maintain a solid structure as the temperature increases. The thermal gellation properties of MC and HPMC can be used to bind and to give shape retention to products, wherein the ingredients themselves do not have particularly good binding properties. This process includes the categories, such as (A)

(B)

104 G'

Cooling

104 G'

103

Cooling

Heating 10

2

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0

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Figure 13.32 Storage modulus G0 and loss modulus Gv as a function of temperature in a heating to cooling thermal cycle: (A) for a 2.0 wt.% solution of MC(SM8000); (B) for a 2.8% solution MC(SM100). Reprinted with permission from Q. Wang, L. Li, Effects of molecular weight on thermoreversible gelation and gel elasticity of methylcellulose in aqueous solution. Carbohydr. Polym. 62 (3) (2005) 232238 [61].

392 Chapter 13 reformed vegetable products, for example, potato croquettes and waffles, onion rings, and the whole range of shaped soya protein and similar vegetarian products. The use of thermal gelation properties to inhibit boil-out is a common phenomenon for bakery filling. Principally, this method is also valid for sauces and other fillings, wherein boil-out needs to be avoided. MCC is another natural polymer derived from cellulose virtually used in all food segments, such as dairy, convenience, and low-moisture applications that require unique stabilization solutions or bulk filler properties to develop stable and palatable products [64]. MCC is prepared conventionally by mineral acid hydrolysis to hydrolyze the amorphous regions of cellulose resulting in crystalline fragments as shown in the schematics in Fig. 13.33. The chemical nature of the MCC is the cellulose which is glucan with a 1,4-β-glycosidic bond (Fig. 13.34). MCC can be dried to a pure, fine-particle form for powdered grade or coprocessed with a water-soluble polymer to deliver a colloidal form (Fig. 13.34).

Figure 13.33 Scheme of the common steps needed to produce microcrystalline cellulose (MCC) from cellulose source materials. Reprinted with permission from D. Trache, M.H. Hussin, C.T. Hui Chuin, S. Sabar, M.R.N. Fazita, O.F.A. Taiwo, et al., Microcrystalline cellulose: isolation, characterization and bio-composites application—a review. Int. J. Biol. Macromol. 93 (2016) 789804 [65].

Applications of natural polymer-based hydrogels in the food industry 393

OH O

HO

HOH2C

HOH2C O

O

Non reducing end

OH O O

HO OH

HO HOH2C

Cellobiose unit

O

HOH2C

OH O

O HO

O OH

HO

HOH2C

Anhydroglucopyranose unit

O O

Reducing end

Figure 13.34 Schematic presentation and chemical structure of microcrystalline cellulose. Reprinted with permission from J. Nsor-Atindana, M. Chen, H.D. Goff, F. Zhong, H.R. Sharif, Y. Li, Functionality and nutritional aspects of microcrystalline cellulose in food. Carbohydr. Polym. 172 (2017) 159174 [66].

Colloid MCC forms a three-dimensional network in water, providing efficient suspension and stabilizing functionality in various food products due to its weak gel characteristic. MCC can be coprocessed with carboxymethyl cellulose or other functional hydrocolloids, for example, alginate and pectin. Hydrated colloidal MCC forms a three-dimensional network based on the electrostatic repulsion of negatively charged cellulose crystals derived from the associated soluble hydrocolloid components. The functional properties based on these stable weak gel networks include ice crystal control, texture modification, emulsion stabilization, heat stability, foam stability, suspension of solids, and fat replacement. Rheology characterization reveals that the formed gel possesses certain gel-like properties with a high degree of thixotropy [64]. These properties impart a variety of desirable characteristics suitable for oil and water emulsions or dispersion-type food products. Most MCC/hydrocolloid systems are heat stable, and temperature changes have minimal or no effect on the functionality and viscosity of their dispersions. This property is extremely important in the preparation of heat-stable food products, especially when acids are present. The MCC/hydrocolloid products will resist extremely high temperatures including those used during baking, retorting, ultra-high temperature processing, and microwave heating,

394 Chapter 13 with minimal loss of viscosity, consistency, or color. MCCs are typically used in food applications, such as beverages with suspended solids or those needing high heat or acid stability, dairy drinks, whipping processing, ice creams, and fat replacements [64].

13.9 Emerging food applications of gelling natural polymers 13.9.1 Fluid gel A fluid gel is a colloidal system containing gelled particles dispersed in a nongelled continuous medium and constructed by applying shear to a biopolymer solution undergoing gelation [6769]. For example, low-acyl gellan gum (0.0250.25 wt.%) fluid gels containing NaCl (0.22 M) can be prepared by applying a short mechanical treatment once the gel phase transition temperature is estimated by oscillatory shear. A high-volume fraction of large gel-like polymeric domains dispersed in a polymer-poor continuous phase defines their microstructure (Fig. 13.35). They exhibit viscoelastic properties with a preponderance of the elastic over the viscous component under small-amplitude oscillatory shear and null frequency dependency of the elastic modulus, congruent with their microstructure (Fig. 13.35A) [70]. Many gelling polymers, such as gellan gum, carrageenan, agar, and whey protein, are used to create fluid gels and fluid gels in high-use levels can be used to prepare reduced-calorie or enhanced-satiety products or provide some kind of “lubricant” effect for a creamy

Figure 13.35 (A) Confocal scanning laser microscopy microphotograph at room temperature of 0.25 wt.% gellan fluid gel; (B) influence of low acyl (LA)-gellan gum concentration on the frequency dependence of the storage modulus (G0 ) (closed symbols) and the loss modulus (Gv) (open symbols) of fluid gels. NaCl concentration: 0.22 M. Temperature: 20 C. Reprinted with permission from M.C. Garcı´a, M.C. Alfaro, N. Calero, J. Mun˜oz, Influence of gellan gum concentration on the dynamic viscoelasticity and transient flow of fluid gels. Biochem. Eng. J. 55 (2) (2011), 7381 [70].

Applications of natural polymer-based hydrogels in the food industry 395 100

10

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100

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Figure 13.36 Frequency sweep for 0.06% carrageenan with milk after 15 min equilibrium at 5 C. From left to right: κ-carrageenan (4230), κ-carrageenan (4217), λ-carrageenan (4435). (K) G0 (Pa), (’) Gv (Pa), and (▲) η* (Pa/s). Reprinted with permission from A.B. Rodd, C.R. Davis, D.E. Dunstan, B.A. Forrest, D.V. Boger, Rheological characterisation of ‘weak gel’ carrageenan stabilised milks. Food Hydrocoll. 14 (5) (2000) 445454 [76].

mouthfeel [71,72]. Fluid gel based on low dosage of gelling polymers is a highly pseudoplastic weak gel network exhibiting elastic properties at rest and becoming liquid thin when shear is applied. Hydrocolloid polymers, such as carrageenan, gellan gum, and MCC, can form such fluid gels when they are directly used in beverage applications to stabilize the proteins, insoluble particles, and fibers without imparting heavy mouthfeel. In general, many milk proteins are stabilized by a specific electrostatic interaction of κ-carrageenan with κ-casein [73], along with the gel network from carrageenancarrageenan interactions [74]. Rheological measurement is a proved powerful technique to characterize mechanical attributes of the fluid gel or weak gel in food systems and reveal the structural information underlying it. A frequency sweep in oscillatory mode of the rheological model can be used to reveal the gel or solution status of a fluid. Gels typically show the following basic features: G0 . Gv over a large range of frequencies, and G0 is relatively independent of frequency [75]. Inspection of the data in Fig. 13.36 suggested a typical weak gel network for κ-carrageenan (4230 and 4217) with milk, whereas λ-carrageenan (4435) showed a solution behavior. Shear viscosityshear stress relationships are often applied to samples to characterize them in terms of their yield stress behavior and solution viscosity under large deformation conditions. The large critical viscosity at the structural point (yielding point) and significant hysteresis (Fig. 13.37) revealed κ-carrageenan (4230 and 4217) with milk tended to form a stronger fluid gel network than λ-carrageenan (4335). This phenomenon was consistent with numerous studies that only κ-carrageenan can specifically interact with milk protein resulting in enhanced suspension stability of dairy drinks, such as chocolate milk, with very low level use of carrageenan [77]. The weak gel networks of gellan gum were also intensively studied and widely used in food, such as fruit/vegetable beverages, flavored milk, and plant protein drinks. HA gellan

396 Chapter 13 100 Initial yield stress behavior Low stress structure rebuilding

10

Viscosity

Hysteresis

Viscosity (Pa.s)

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0.6 Pa.s

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0.038 Pa.s Structure point critical stress

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Figure 13.37 Schematics of structure evolution for a carrageenan-milk system at viscosity versus shear stress curves (left) and the diagram for 0.06% κ-carrageenan (4230), κ-carrageenan (4217), λ-carrageenan (4435) with milk after 15 min equilibrium (right). Reprinted with permission from A.B. Rodd, C.R. Davis, D.E. Dunstan, B.A. Forrest, D.V. Boger, Rheological characterisation of ‘weak gel’ carrageenan stabilised milks. Food Hydrocoll. 14 (5) (2000) 445454 [76].

gum at a use level of 300 ppm can dramatically increase the colloidal stability for carrot juice without causing heavy mouthfeel [78]. HA gellan gum can also be applied to stabilize emulsion due to its fluid gel nature that contributed the gel-like behavior of the continuous phase associated with the presence of HA gellan microgels/aggregates as a result of molecular entanglements [79,80]. The fluid gel of MCC is explored to stabilize various beverage systems, such as dairy drinks and beverage emulsion, because of the heat resistance of gel network and the amphipathy of micro/nanoparticles of cellulose aggregation of MCC [66,81].

13.9.2 Food for dysphagia management The group of elderly people (e.g., 65 1 years old) exhibits the fastest growth rate among all population segments. More than 700 million people will be over 65 years of age by 2020, and in many countries (e.g., Japan, Germany, and South Korea) about 5%10% of their populations will be over 80 years of age [82]. The requirements for food and nutrition by the elderly, particularly the very old and frail, demand urgent attention. It is proposed to divide the demands of elderly people into those linked to the oral experience (e.g., safe eating and sensory enjoyment) and those associated with the physiological changes of aging (e.g., changes in body composition and special nutritional needs) (Fig. 13.38) [82].

Applications of natural polymer-based hydrogels in the food industry 397

Mastication and swallowing dysfunctions

Loss of muscular body mass and bone mass

Need for texturemodified and special foods for the elderly Gradual loss in sensory perception and appetite

Other specific nutritional needs

Figure 13.38 Main aspects to be considered in the design of healthy foods for the elderly [82].

Mastication and swallowing functions deteriorate remarkably with age. Reduced difficulty during eating and inhibition of choking and aspiration during swallowing are critical to produce pleasant food textures for the elderly. Providing soft, palatable, and healthy texture-modified (TM) food for seniors, particularly those with masticatory/swallowing dysfunctions and/or needing special nutrition, is a major challenge for the food industry. Proteins, carbohydrates, and lipids in the different molecular sizes and aggregation formats are the main building blocks for these unique TM food products (Fig. 13.39) [83,84]. Some of them already constitute soft products (i.e., jellies, mousses, sauces, creams, and thick beverages) for dysphagia, and others are potentially to be used in the design of novel particulate food. It is widely accepted that the main textural parameters determined in soft gel-based TM food are hardness (hard to soft), adhesiveness (i.e., tendency of particles to adhere at their surfaces), and cohesiveness (ability to form a swallow-safe bolus in the mouth) [85]. Polysaccharide gels with diverse textures that are soft enough to be eaten by compression between the tongue and the hard palate without biting by the teeth are one of the preferable foods for patients with dysphagia. Hydrogels based on gellan gum were intensively studied and applied in building food, such as jellies and mousses, for elderly with chewing and swallowing difficulty. Based on designing hydrogels of gellan gum with different texture attributes, the viscoelasticity of the gels is considered to be quite important to ease the eating of jelly food for the elderly [86]. Gels consisting of LA gellan gum and psyllium seed gum have been compared with gels solely based on LA gellan gum in terms of mechanical properties, oral process profile, and sensory

398 Chapter 13

Figure 13.39 Some molecules and structural elements relevant in the design of soft texture-modified foods for the elderly (approximate scales). Reprinted with permission from J.M. Aguilera, D.J. Park, Texture-modified foods for the elderly: status, technology and opportunities. Trends Food Sci. Technol. 57 (2016) 156164 [82].

evaluation. Reportedly, the addition of psyllium seed gum improved the syneresis of LA gellan gum with alternation of its mechanical properties. The combined gel was less brittle than single LA gellan gum gel and showed high intensity of sensory cohesiveness. By correlating with the swallowing profile characterized by electromyography recording of the suprahyoid musculature, and the acoustic analysis of the swallowing sound, gels with high cohesiveness, performing similar to xanthan solution on facilitating swallowing, can reduce the risk of aspiration [86]. The time evolution of the gel texture at different cooking conditions should be given considerable attention because the mechanical properties of some natural polymer gels used to produce texture-modified food for the elderly varied with storage time, temperature, and making processes. Pudding is a widely used ready-to-serve diet food for the elderly. It is pointed out that some commercial puddings showed increased mechanical strength with storage time, especially at 40 C, possibly because of protein aggregation enhancement in the continuous phase during storage [87]. Gel strength of cold-set gels, such as agarose, gelatine, and gellan gum, becomes strong when the cooling rate is decreased. Therefore, this knowledge is important for practitioners when they make a gel. Making a gel in a short time is sometimes necessary, but too fast cooling leading to a very weak gel should not be forgotten, as shown in Fig. 13.40 for gellan gum gels formed at different cooling rates [89].

Applications of natural polymer-based hydrogels in the food industry 399

Figure 13.40 Stressstrain curve for 1.6 wt.% gellan gels prepared by slow (kept at 40 C for 20 h and then at room temperature) (solid curve) and rapid (B15 C/min) cooling (broken curve). Compression speed, 0.5 mm/s. Measurement temperature, 22 6 2 C. Gellan-4 and gellan-1 have different fractions. Self-standing gels of 1.6% gellan-4 could not be formed on rapid cooling and thus a stressstrain curve could not be observed. Reprinted with permission from Y. Nitta, M. Yoshimura, K. Nishinari, The effect of thermal history on the elasticity of K-type gellan gels. Carbohydr. Polym. 113 (2014) 189193 [88].

13.9.3 Jelly confections Among the various confectionary products, jelly or gummy candies are popular due to their unique soft and chewy textures and fresh flavors. Gummy or jelly candy is fabricated based on gelling natural polymers, such as gelatine, starch, pectin, gellan gum, and carrageenan, in the system containing less moisture (,18%20%) and a significant proportion of low-molecular-weight carbohydrates (e.g., sucrose and corn syrup). The primary structure and therefore texture of these gums are constructed based on various natural polymer stabilizers. These large proteins, gums, or polysaccharide molecules interact in a sugar solution to form a network that holds in the fluid sugar solution [90]. Usually gummy and jelly candy, which are constructed based on different natural polymers, show different texture characteristics. A jelly candy is made with anything other than gelatine—pectin, starch, agar, and gum acacia. Each has a different texture, but none of them is as highly elastic as gelatine. Gummy candy, as the name indicates, has a gummy texture which is not quite like chewing rubber bands or calamari but certainly more elastic

400 Chapter 13 than any other soft candies. Since Hans Riegel from Bonn, Germany, began creating a new form of candy based on gelatine in 1920, the iconic gummy bear, as one representative of the gummy candies, has been consumed in huge quantities globally. Gelatine arising from its firm, springy, resilient characteristics is assumed to be the only material that gives the gummy texture, and gummies made with gelatine have been around for quite a long time. Gelatine is derived from animal sources, usually pork or bovine. The animal origin of gelatine limits its use in certain diets, such as religious (Kosher and Halal) or vegetarian diets. In addition, the heat instability, where gelatine-based confections tend to be soft and sticky or melt under hot ambient conditions, that is, temperatures above approximately 40 C, is another concern for this traditional food additive used in gummy confections. As reported in US 6-586-032 B2, a gelatine-free gummy confection comprising the combination of gellan gum and carrageenan can provide a firm, resilient, gelatine-like texture in gelatine-free gummy confections. Notably, the type of gellan gum and carrageenan types and dosages are critical to producing the gummy texture [91]. Here, gellan gum can provide satisfactory hardness, whereas carrageenan imparts elasticity and cohesiveness. Numerous stabilizers are available to create different and unique textures for jelly or gummy candies, with consumers always looking for something unique from candy makers. Blending the different stabilizers allows to produce soft candies with textures that vary from the elastic texture of gelatine to the tender bite of pectin. Adding starch or pectin to gelatine reduces the elasticity of the gummy candy [92]. Another high-moisture-content candy is produced by using stabilizer mixtures including gellan gum, xanthan gum, and LBG with the assistance of collagen chasing. This jelly candy, with the brand name Kororo fruit jelly, shows an authentic fruit texture with balanced elasticity and brittleness. As revealed in the patent of EP3187053A1, the collagen chasing used to encapsulate the gummy candy body is critical for the high shelf-life stability of this high-moisture soft candy (20%30%) [93].

13.9.4 3D Printed food Three-dimensional (3D) printing, also referred to as additive manufacturing, is the creation of 3D objects by adding material layer-by-layer under computer control. This process is often used in aircraft, medical implants, automobiles, and even in fashion products to produce complex shapes that are difficult or impossible to achieve by other manufacturing techniques. Three-dimensional printing for food is a relatively nascent field with great potential, and the relevant research work has been increasing its focus on this field recently [94]. As shown in Fig. 13.41, the first step in the 3D food printing process is to set the code based on the virtual 3D model. After uploading the codes into a printer and choosing a preferred food recipe, the printing starts. Fabricated layers do not need to be completely

Applications of natural polymer-based hydrogels in the food industry 401 From idea to 3D printed cookie

3D modeling

G-code generation and interpretation

Oven

Printing

3D food printer

Figure 13.41 Overview of the 3D food printing process. Reprinted with permission from J. Sun, W. Zhou, D. Huang, 3D printing of food, in: Reference Module in Food Science, Elsevier, 2018 [94].

Figure 13.42 Selective laser sintering (left) and selective hot air sintering (right). Reprinted with permission from J. Sun, W. Zhou, D. Huang, 3D printing of food, in: Reference Module in Food Science, Elsevier, 2018 [94].

solidified but require sufficient rigidity and strength to support. A few technologies have been widely applied in direct food printing. Selective laser sintering/hot air sintering involves utilization of a sintering source (laser or hot air) to fuse powder particles and form a solid layer (Fig. 13.42). Hot melt extrusion/room temperature extrusion/hydrogel-forming extrusion employs movable extrusion to fabricate 3D modeled food (Fig. 13.43). This technology has been utilized to design printers, such as Choc Creator and Foodini [94], or print pasta by using classical recipes (durum wheat semolina and water) [95]. Binder jetting

402 Chapter 13

Figure 13.43 Hot melt extrusion (left) and room temperature extrusion (right). Reprinted with permission from J. Sun, W. Zhou, D. Huang, 3D printing of food, in: Reference Module in Food Science, Elsevier, 2018 [94].

Figure 13.44 Binder jetting and inkjet printing. Reprinted with permission from J. Sun, W. Zhou, D. Huang, 3D printing of food, in: Reference Module in Food Science, Elsevier, 2018 [94].

uses liquid streams to design the shapes across the powder beds [96], whereas inject printing dispenses a stream of droplets from a syringe-type printhead in a drop-on-demand way to deposit drops onto pizza bases, biscuits, and cupcakes (Fig. 13.44). The available materials for direct food printing can be classified into three categories: natively printable materials, nonprintable traditional food materials, and alternative ingredients. Hydrogel based on natural polymers is one kind of printable material, along with other food materials, such as cake frosting, cheese, hummus, and chocolate. Hydrocolloid polymers, such as cellulose derivative [97], carrageenan, xanthan gum, and starch [98], are added into some typical nonprintable food materials but consumed by people every day, like rice, meat, fruit, and vegetables. The hydrogel-forming extrusion hydrocolloid solutions can be extruded into a hardening/gel setting bath by using a syringe pipette, jet cutter, and similar apparatus. In this process, the polymer solution should present

Applications of natural polymer-based hydrogels in the food industry 403 viscoelastic characteristics first and then turn into self-supporting gels prior to the consecutive deposited layers. A 3D hydrogel extrusion technique to produce soft food based on agar and gelatine for elderly people with swallowing problems has also been reported [99]. In this work, a 3D edible gel printer to make soft food for the elderly was set up, and four kinds of soft foods prepared from agar and gelatine were printed by the 3D edible gel printer. As revealed, the viscosity of agar solution and other food inks is critical for the processability and the final texture properties of printed soft food. The combination of alginates of different guluronic/mannuronic acid ratios and pectin with high and low degrees of esterification has potential to reveal a new printable material for food structure design [100]. A 3D printing method was successfully developed based on the extrusion of bio-inks composed of a low-methoxylated pectin gel and embedded lettuce leaf cells [101]. Notably, land-plant cells encapsulated in pectin gels at high density can be 3D printed with good accuracy and reproducibility. The encapsulation of cells in bio-ink tended to decrease the mechanical and structural properties of the printed object in comparison with their reference results. This pioneering work demonstrates the potential of 3D printing with gelling natural polymers to produce 3D-printed cellular or particulate food.

13.9.5 Fat mimetics People’s pursuit of food products with reduced or low-fat content and food containing functional ingredients is more critical than ever before. Although many nutrition recommendations remain controversial, a consensus exists among health and nutrition professionals that the ratio of saturated fatty acids to poly- and monounsaturated fatty acids in the diet should be decreased, and the intake of dietary cholesterol should be limited. The effects of fat on cardiovascular disease are well documented, and they have been recognized as major factors in its etiology. Dietary treatment to prevent obesity and high blood cholesterol levels has proved an effective way to help prevent coronary heart disease [102]. For this reason, consumer demand for low-fat food has drawn huge attention from researchers for reducing the fat content of food. Problems of inferior organoleptic and physical properties in these products suggest the use of FRs to provide the desirable qualities. FRs are generally classified into fat substitutes and FMs. Fat substitutes are substances that have a chemical structure close to fats and similar physiochemical properties. FMs are macromolecules that have distinctly different chemical structures from fat. Typical FMs are natural carbohydrate polymers with diverse functional properties that mimic some of the characteristic physiochemical attributes and desirable eating qualities of fat [103]. Principally, these natural polymeric FMs incorporate water into a gel-type structure, resulting in lubrication and flow properties like those of fat. Some typical hydrocolloid gels, such as cellulosic and curdlan, are extensively studied and employed as FMs in various food systems.

404 Chapter 13 The effects of using curdlan as FMs on the mechanical and functional properties of meat products have been studied for a long time [26,104]. Curdlan has been found to increase the water-binding capacity and textural properties compared with a control in a study of duck muscle gels [105]. As revealed, curdlan played an important role of dietary fiber to enhance the functionality of meat products. A recent patent claimed a method regarding the formation of a meat-fat substitute containing the main ingredient curdlan, wherein the meatfat substitute has low calories, and the appearance and taste/texture of the original fat [106]. Curdlan can effectively improve the quality of nonfat sausage as FMs. It is reported that the viscoelastic properties of the nonfat sausage with the curdlan-based FM system were very close to those of the control (20% fat), especially when reheated [26]. All these findings indicate that curdlan can be an effective main ingredient in FMs for meat products [19]. MCC has been successfully used as FMs in some selected food systems. A significant amount of work has been conducted to reduce the amount of fat in ground meat, frozen desserts, and dairy and baked products. By virtue of its insoluble nature in water, the use of MCC as FMs in food systems has produced excellent results. It has been observed that MCC can effectively substitute 50% fat compared with standard product in a study on the effect of MCC as a FM on microstructure and sensory properties of fried beef patties [107]. This work also indicated that the sensory attributes of the MCC-based beef patties exhibited fat-like mouthfeel and were generally acceptable to the panelists. In the fried beef patties, heated samples with MCC had more juiciness than controls and had better fat-like mouthfeel. MCC is largely crystalline, with no net charge. It forms a particle gel network as an inert molecule and fills the gaps of the tight meat fiber network without causing any disturbance to the protein network during heating. In emulsified sausage, the cellulose microcrystals positively influence the mechanical properties of the product by enhancing the firmness of the final product due to its high compatibility in the meat matrix [108]. Apart from meat, MCC has been used to replace fats in emulsions, baked products, frozen desserts, mayonnaise, gravies, and sauce. For instance, soybean oil emulsion containing 60% oil has similar stability characteristics and rheological properties as compared to a 20% soybean oil emulsion containing 1%1.5% colloidal MCC [109]. Fat replacement with MCC also gives a rich creamy texture in low-fat sauces and dressings because the material is insoluble and can mimic fat texture and sensory perception [66].

13.10 Summary and outlook The natural polymer-based hydrogels from food hydrocolloids of polysaccharides introduced in this chapter are important and functional building blocks for both producing conventional food products and creating new trendy foods. The main functionalities of these food hydrocolloids, such as stabilization, texture modification, emulsification, and suspension, are highly dependent on their gelling natures. The full understanding of the

Applications of natural polymer-based hydrogels in the food industry 405 molecular structures, gelation mechanisms, properties, and functionalities of a variety of typical hydrogels from polysaccharides are prerequisite for food scientists to efficiently utilize them on food product innovation due to the complexity of these natural polymers from their diverse origins, manufacturing processes, varied molecular structures, and physicochemical properties. Modifications of molecular structure by adjusting the manufacturing or utilizing a mixture of existing gelling natural polymers to fit the current food product processes and improve their added value, are tactics to further expand these hydrogel applications in the food industry. In addition, microorganism-derived hydrocolloids can be produced in large quantities regardless of the deterioration of ecoenvironments or climates. Moreover, increasingly knowledgeable consumers continuously pursue food products with quality with respect to enhanced organoleptic, sensory properties, healthy image, and extended shelf-life. The clean label images of these natural polymers with gelling properties will play several important roles in the new revolution of the food industry. Their contributions to fat/sugar reduction in the diet demonstrated in this chapter reveal their power in establishing healthy food products. Their health benefits as one of the matrices of the “clean label” concept, which is already fully or partially accepted due to their dietary fiber attributes, will be another topic worthy of further studies. In addition, an on-going process has been observed to modify the chemical conformation of food hydrocolloids of polysaccharides to change their physical properties or processing availability.

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410 Chapter 13 [106] H.W. Chang, M.J. Tsai, Substitute for fat within meat and the forming method thereof. US Patent 9295277B2, 2016. [107] M. Gibis, V. Schuh, J. Weiss, Effects of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) as fat replacers on the microstructure and sensory characteristics of fried beef patties, Food Hydrocoll. 45 (2015) 236246. [108] V. Schuh, K. Allard, K. Herrmann, M. Gibis, R. Kohlus, J. Weiss, Impact of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) on functional characteristics of emulsified sausages, Meat Sci. 93 (2) (2013) 240247. [109] A. Imeson, Food Stabilisers, Thickeners and Gelling Agents, first ed., Wiley-Blackwell, Chichester, 2010.

Further Reading V.J. Morris, 1-Polysaccharides: their role in food microstructure, in: D.J. McClements (Ed.), Understanding and Controlling the Microstructure of Complex Foods, Woodhead Publishing, 2007, pp. 339.

CHAPTER 14

Application of polysaccharide-based hydrogels for water treatments Malihe Pooresmaeil1 and Hassan Namazi1,2,* 1

Research Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, Iran 2Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Science, Tabriz, Iran

14.1 Introduction Due to industrial development, great volumes of pollutants are generated and discharged into the environment, leading to serious problems. Among the contaminants, heavy metals and dyes are the most common and the most hazardous, as their ingestion can have a very long-lasting influence on the human organism. Various kinds of synthetic dyestuffs or heavy metals are seen in the effluents of wastewater. Dyes are colored-aromatic organic compounds and are generally classified into three groups: cationic or basic dyes, nonionic or disperse dyes, and anionic or acidic dyes. Cationic and anionic dyes are the most commonly used dyes in the textile industry due to their good solubility in water, easy application, and inexpensive characteristics [1,2]. Cationic dyes can easily interact with negatively charged cell membrane surfaces and enter cells. Entering the cationic dye into cells leads to serious health problems, hence more research has focused on the removal of cationic dye from water compared to anionic dyes [3]. Because water is one of the most essential human requirements for survival it is important to obtain good-quality water. Various methods have been used for water or wastewater treatment, including ion exchange, coagulation-flocculation, membrane filtration, activated sludge, electrochemical processes, flotation, biological treatment, adsorption, treatment with ozone, and reverse osmosis. Among these methods, adsorption is more popular due to its convenience, the simplicity of the process, the presence of various adsorbents, facile recovery, most economical short analysis time, and having no harmful by-products, such as ozone and free radicals [4
10]. The secondary waste, high cost, low efficiency, and high complexity of the other methods are disadvantages which limits their usage as water 

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00014-8 © 2020 Elsevier Inc. All rights reserved.

411

412 Chapter 14 treatment techniques. Adsorption is a process in which soluble substances are collected from the solution on a suitable interface without the production of any harmful by-products. Activated carbon (AC) is one of the most commonly used adsorbents for color or heavy metal adsorption from water or wastewater. Because of the relatively high cost of this adsorbent, today’s absorbents with low price, wide availability, and containing a large number of functional groups, such as polysaccharide-based adsorbents, are good candidates for this operation [4,11,12]. Polysaccharides are polymeric carbohydrate molecules composed of long chains of monosaccharide units with a large quantity of
OH,
CONH2,
SO3,
NH2, and
COOH functional groups. Polysaccharides have advantages such as nontoxicity, high efficiency, abundance, low cost, and environment-friendly properties [13
19]. The presence of the ionic functional groups in their structure led to the design of a new adsorption system based on these polymers. Different shapes of polysaccharides, including microspheres, membranes, gel, hydrogel, and films, have been prepared and their ability for the removal of pollutants from water and wastewater studied. This chapter provides detailed information about the use of polysaccharide-based hydrogels in water or wastewater treatment.

14.2 Hydrogels and their characterization Hydrogels are three-dimensional cross-linked polymer networks, which can absorb and retain a large amount of water, biological liquids, and solute molecules in their structure. In these materials, the porous network and higher water content provide the possibility to solute diffusing to the hydrogel structure. When dried, hydrogels shrink and recover their original volume [20
24]. Superabsorbent hydrogels are hydrogels that can absorb water in a short time as well as at quantities exceeding 100% of their mass. Hydrogels have the ability to be used in a wide range of applications such as wound dressings, hygienic products, contact lenses, gel actuators, artificial organs, horticulture, tissue engineering, drug-delivery systems, food, and agriculture [4,25
29]. There are several methods for hydrogel classification: ionic charge, type of cross-linking, and type of used monomer or polymer in their synthesis. By considering the surface charge, hydrogels can be classified as ionic or nonionic. The ionic types hydrogels with
CO22 or
SO32 functional groups are known as anionic hydrogels, and hydrogels with
NR31 functional groups are cationic hydrogels. Moreover, depending on the type of cross-linker used, hydrogels are classified into two main groups, for example, chemically cross-linked hydrogels or physically cross-linked hydrogels. In the chemically cross-linked hydrogels, commonly toxic cross-linkers are used but physical cross-linking avoids the use of toxic cross-linkers [24,30
34]. Finally, based on the type of constituent monomers or polymers, hydrogels are classified as natural (prepared from natural polymers) and synthetic hydrogels (prepared from synthetic monomer or polymers). Recently, due to the high adsorption capacities, the presence of a large number of

Application of polysaccharide-based hydrogels for water treatments 413 functional groups, reuse for continuous processes, and regeneration abilities, polysaccharidebased hydrogels have become a viable alternative for traditional adsorbents. Although crosslinking techniques have enhanced the mechanical properties of neat hydrogels, the poor mechanical strength of the neat hydrogels is their main drawback that must be resolved. Recently, researchers have focused on the addition of nanofillers into the polymer matrix to enhance their mechanical strength; carbon nanotubes, graphene, clay, bentonite, and montmorillonite are some of these nanofillers. Hydrogel nanocomposite is a result of the nanofiller addition to the polymer matrix which has the advantages of both parts [35,36].

14.2.1 Characterization of hydrogels There are several techniques, such as Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), swelling, X-ray diffraction (XRD), and the point of zero charge (pHpzc), to study hydrogel properties and their structures. In addition to the above techniques, for hydrogels used as an adsorbent, two-parameters, removal efficiency [R, Eq. (14.1)], and adsorption capacity [q, Eq. (14.2)] are determined to evaluate the adsorption efficiency of the designed adsorbent and adsorption process. R5

ðC0 2 Ce Þ 3 100 C0

(14.1)

V ðC0 2 Ce Þ Ms

(14.2)

qe 5

where C0 and Ce (mg/L), respectively, are the initial and equilibrium concentration of the pollutant in solution, V (mL) is the solution volume and Ms (g) is the mass of adsorbent. 14.2.1.1 Fourier transform infrared FT-IR is one of the most widely used analytical methods for study of presence of certain functional groups in compound structures and predicting the success of the reaction progress. Also, the formation of hydrogen bonding and covalent bonds in the system is elucidated with the FT-IR technique. In fact, the FT-IR technique can be used for approval of the successful cross-linking and preparation of hydrogels through shifting in some of the peak wave numbers and the appearance or disappearance of new peaks. 14.2.1.2 Scanning electron microscopy The morphology, structure, and porosity of adsorbents are important factors that determine the amount of pollutants removed. Hence, SEM is useful in adsorption studies. Moreover, the change in morphology by adding nanoparticles to the hydrogel network, which is an effective factor on the adsorption capacity, can be evaluated with SEM. For instance, it was observed that the addition of montmorillonite to the polymer matrix caused a more open,

414 Chapter 14 layered, and porous structure. The hydrogel composites with 0.4% and 0.6% showed a sponge-like morphology with small pore size, but hydrogel with a low percentage of clay (0.14.2%) showed a more porous structure with larger diameters. In the case of hydrogels with a larger pore size, it is expected that the permeability and diffusion rate of small molecules is higher, which is approved by the obtained adsorption capacity, that is, adsorption studies showed that H-0.2 has the highest absorption capacity compared to the other samples [11]. SEM images of chitosan-g-(N-vinyl pyrrolidone)/ montmorillonite hydrogel composites with different percentage of montmorillonite are shown in Fig. 14.1. 14.2.1.3 Swelling Swelling occurs when the water molecules are absorbed in the three-dimensional structure of hydrogels. To determine the swelling degree of the hydrogel, dry and clean hydrogels with known weights were soaked in an aqueous solution with defined pH and temperature. After the equilibrium time, excess water was eliminated from the surface using filter paper and the sample was rapidly weigh. Finally, the equilibrium degree of swelling (EDS) was calculated using Eq. (14.3). EDS 5

ðWs 2 Wi Þ Wi

(14.3)

In Eq. (14.3) Ws is the weight of the swelled hydrogel at equilibrium time and Wi is the weight of the dry hydrogel at the initial time. Diffusion of the water molecules to the hydrogel structure and swelling occurs due to three phenomena; first, swelling from the hydrogen bonds forming between the water molecules with the present hydrophilic groups in the hydrogel structure. Then water penetrates around the bound water and cage-like structures or clusters forms. Finally, more swelling is observed due to excess water entering the hydrogel network. Swelling percentages of hydrogels are affected by their chemical composition, ionic strength, cross-linking density, temperature, and pH. Any increases in cross-linking density lower the possibility of water penetration to the hydrogel network [11,37]. 14.2.1.4 X-ray diffraction XRD is a useful technique for the study of the crystalline structure of compounds such as hydrogels. Moreover, in the case of hydrogel nanocomposites, the addition of nanoparticles affects the crystallinity of compounds and makes its XRD different from a neat hydrogel, which is evidence of the success in the hydrogel nanocomposite preparation process. 14.2.1.5 Point of zero charge Since the surface charge of the adsorbent has a main effect on the quantity of pollutant adsorption, calculation of the pHpzc can provide useful information about the prediction of the success or otherwise of the adsorption process. pHpzc can be determined using the

Application of polysaccharide-based hydrogels for water treatments 415

Figure 14.1 Scanning electron microscopy (SEM) images of (A) H-1, (B) H-0.8, (C) H-0.6, (D) H-0.4, (E) H0.2, and (F) Chitosan. Reproduced with permission from A. Vanamudan, K. Bandwala, P. Pamidimukkala, Adsorption property of Rhodamine 6G onto chitosan-g-(N-vinyl pyrrolidone)/montmorillonite composite. Int. J. Biol. Macromol. 69 (2014) 506
513 [11].

batch equilibrium experiment. In this method, the adsorbent placed in the medium with different pH range, and the initial pH (pH0) and pH after the specific time (pHf) are measured. pHpzc is achieved from the curve intersecting the x-axis of the chart pH0
pHf versus pH0 [38].

416 Chapter 14

14.3 Chitosan Chitosan (CS) as a linear cationic biopolymer is the second most abundant natural biopolymer after cellulose. The structure of CS is similar to cellulose, the only difference being in the type of attached group to carbon number 2. The chemical structures of CS and cellulose are shown in Fig. 14.2. This polysaccharide is built from 4-linked-β-2-amino-2deoxy-glucopyranose residues, some of which are N-acetylated [37,39,40]. CS is extracted from the exoskeleton of insect and crustacean shells, algae, and fungal cellular walls. Moreover, CS can result from the full or partial strong alkaline deacetylation of chitin. The CS pKa and its molecular weight depend on the degree of deacetylation and can be changed during deacetylation. This cationic biopolymer has a pKa value of about 6.2
7. The advantage of CS with respect to other polysaccharides is the amine functional groups that provide the possibility for the preparation of various new materials with novel chemical properties. CS is nontoxic, biodegradable, widely available, highly effective, biocompatible, and inexpensive, it also has a high swelling and shrinkage index [38,41
44]. CS and its derivatives are used in numerous fields, such as solid-state batteries, paper finishing, biotechnology, biomedical research, drug delivery, agriculture, dietetic research, cosmetic industry, food industry, and textiles due to its biodegradability and

Figure 14.2 The chemical structures of chitosan (CS) and cellulose.

Application of polysaccharide-based hydrogels for water treatments 417 biocompatibility properties. Additionally, due to its physicochemical characteristics, high reactivity, chemical stability, high selectivity toward pollutants, and excellent chelation ability, CS was abundantly used for water treatment. In order to increase the adsorption capacity of neat CS, it can be modified through physical or chemical methods [45]. CS is not soluble in water, hence dilute mineral acids, such as nitric acid, hydrochloric acid, and phosphoric acid are used to dissolve it. CS has a flexible polymeric chain and antibacterial properties. Due to the presence of many
NH2 primary and secondary hydroxyl functional groups in the CS structure, intramolecular hydrogen bonds occur between them, which are visible in the XRD spectrum of pure CS as an XRD peak in 2θ 5 20 degrees. Despite the mentioned benefits, pure CS has some limitations such as weak mechanical properties, high solubility in acids, and easy gel formation or clumping. To overcome these limitations, various physical or chemical modifications such as composition with other materials including nanomaterials or polymer have been carried out [4,5,38,41,46,47]. Additionally, cross-linking of CS and hydrogel preparation increase its mechanical strength. Ethylene glycol, glycidyl ether, glutaric dialdehyde, epichlorohydrin (ECH), genipin, polyethylene glycol diglycidyl ether, tripolyphosphate (TPP), and sulfuric acid are conventional cross-linkers for the preparation of CS-based hydrogels. One of the advantages of ECH use as a cross-linker for CS-based hydrogel synthesis is the nonremoval of cationic amine function groups, which are the main adsorption site. Moreover, an advantage of TPP as a cross-linker is that there is no toxic effect despite the reinforcement of solids. Hence CS-based hydrogels as adsorbents have attracted increased attention because of their good mechanical strength, high accessibility, the presence of a high amount of amino and hydroxyl functional groups in the polymer structure, as well as their environment-friendly properties [6,48].

14.3.1 Chitosan-based hydrogels for the removal of dyes Metal-complex dyes are a type of industry dye which have both aromatic rings and toxic complex ions (such as Co21 or Cr31), hence, these dyes cause more toxicity compared to conventional dyes. Although these dyes can be removed by electro-coagulation or degradation, adsorption is preferable because of the easy facility and low cost. In the pollutant-removing process, the important issue is prevention of the creation of the secondary contamination in the pollutant absorption process. CS-based hydrogels are a good candidate for the adsorption of metal-complex dyes from water because they have both excellent abilities for metal ion bonding and good strength. Moreover, the composition of CS with inorganic materials such as hydroxyapatite [Ca10(PO4)6(OH)2, HA] enhance its mechanical strength and metal- and dye-removing efficiency. The batch equilibration method is commonly used for adsorption studies [42]. In the case of cationic dye removal, CS can only adsorb very small amounts of cationic dyes due to its cationic nature, hence,

418 Chapter 14 for obtaining a high adsorption capacity of cationic dyes with CS-based hydrogels, modification is necessary [20]. Malachite Green (MG) is a cationic dye with high solubility in water and is widely used in textile coloring, paper, and food industries. MG is harmful to reproductive organs and biological systems. Removal of MG has been studied using a series of composite hydrogels which were prepared through an in situ polymerization method using CS as the grafting backbone, attapulgite as the inorganic component, and acrylic acid (AA) and itaconic acid as the anionic monomers. The prepared hydrogel nanocomposites showed a high adsorption capacity; 2433 mg/g for the MG that it is related to the anionic characteristics of hydrogel composites which arise from the presence of anionic units in the hydrogel structure [49]. In other research, Vakili et al. [50] studied reactive blue 4 (RB4) adsorption with CS beads modified with 3-aminopropyl triethoxysilane (APTES), pure beads (317.23 mg/g), and modified beads (433.77 mg/g). APTES is a cationic organosilicon with terminal amine functional groups, hence this modification increases the amino group of beads, and as a result of this modification, anionic dye adsorption capacity is increased. Chatterjee et al. [51] investigated the adsorption performance of cetyltrimethyl ammonium bromide (CTAB)-impregnated chitosan beads (CS/CTAB beads), for the adsorption capacity of Congo Red (CR) as an anionic dye. The results showed that the CS/CTAB beads impregnated with 0.05% CTAB had higher CR adsorption capacity than CS beads for all of the initial concentrations of CR. The obtained results from these experiments clearly showed that the adsorption of CR onto CS beads might be due to the electrostatic interactions between CR molecules and amine functional groups of CS. Since the head group of CTAB molecules is cationic the integration of this molecule to the CS bead increases the cationic charge of adsorbent, and hence adsorption capacity is increased.

14.3.2 Chitosan-based hydrogels for the removal of heavy metals Heavy metals are one of the main classes of water pollutants that are toxic to human health. Toxic heavy metal ions enter into the environment through diverse industries. CS and its derivatives with abundant amino and hydroxyl groups on their backbones serve as chelation sites and hence are considered to be as an important adsorbent for heavy metal ions. The extent of metal adsorption using CS-based compounds is dependent on the degree of deacetylation, the nature of the metal ion, and solution properties [39,52]. Oxyanion pollutants such as vanadium(III, IV, and V) oxyanions are the most common materials, which leads to several harmful metalloids and metals entering into the water. The redox potential of the oxyanion is a determining factor on the level of their toxicity to humans. In an aqueous solution vanadium(V) is the most toxic common oxidation state of vanadium. Many reports have confirmed that vanadium oxyanions can be eliminated by adsorption on protonated chitosan flakes. Adsorption occurs due to electrostatic attraction interactions between the negative charge of the vanadium oxyanions and the positive charge of the CS.

Application of polysaccharide-based hydrogels for water treatments 419

Figure 14.3 Schematic of the possible mechanism for adsorption of vanadium oxyanions onto protonated chitosan flake and its recovery. Reproduced with permission from A. Padilla-Rodrı´guez, et al., Synthesis of protonated chitosan flakes for the removal of vanadium(III, IV and V) oxyanions from aqueous solutions. Microchem. J. 118 (2015) 1
11 [38].

The proposed mechanism for vanadium oxyanion adsorption and desorption on CS is shown in Fig. 14.3 [38]. The most commonly involved mechanism in heavy metal removal is ion exchange and the complexation mechanism. Since polymers contain one or more electron donor atoms such as S, N, P, and O they can eliminate the toxic heavy metals by the formation of coordinate bonds. Lead is one of the most toxic metals with fatal results for human. Lead entry into the food cycle through irrigation can lead to nervous system damage, poisoning, and cancer. Commonly, lead contamination results from battery products, leaching of ion-type RE mines, dyes, and other manufacturing industry products [37,52]. Numerous researches have been done on the possibility of lead removal. Recently the cooccurrence of F2 and Pb21 in water has been reported with hydrous zirconium oxide-impregnated CS beads. The results showed that this adsorbent could remove the lead and F2 ions through chelation with its amine-groups and an electrostatic interaction [53]. Copper is one of the essential nutrients for humans in trace amounts, but large doses can produce health problems such as gastrointestinal disturbance and kidney or liver failure [54]. Since Cu is another common water pollutant, CS/poly(vinylamine) composite beads have been synthesized and their ability for Cu21 removal investigated. The results showed that pH 4 lead to higher Cu21 sorption capacity. This behavior is due to the lower protonation degree of amine-groups at pH 4, compared to more acidic pHs. Ion-exchange and chelation between Cu21 ions and the sorbent surface were the proposed mechanism for Cu adsorption by this adsorbent [55].

420 Chapter 14 Arsenic (As) is a very toxic heavy metal. The predominant forms of As species in groundwater are arsenite (AsO332) and arsenate (AsO432), in which the As(III) is 10
60 times more toxic than As(V). Consequently, removing As(III) has become a great concern. Studies on the effects of As on human health showed that long-term intake of Ascontaminated water, even at trace concentrations, can lead to lethal diseases. Elwakeel and Guibal [56] designed a new hydrogel-based adsorbent using a CS biopolymer: CS/Cu(OH)2 and CS/CuO sorbents. The designed systems showed a high adsorption capacity in optimum conditions. The recycling of the sorbent was tested and its efficiency for five successive sorption/desorption cycles was approved. Due to the high volume of research on the study of the CS-based hydrogels in water treatment, only the results of a few cases are summarized in Table 14.1. Table 14.1: The adsorption capacity of chitosan (CS)-based hydrogels for the removal of various dyes and heavy metals from water. Maximum adsorption capacity (mg/g)

Dye or metal ions

References

Magnetic chitosan nanocomposite beads Poly(2-acrylamido-2-methylpropanesulfonic acid)/ chitosan hydrogel Poly(2-acrylamido-2-methylpropanesulfonic acid)/ chitosan hydrogel Poly(2-acrylamido-2-methylpropanesulfonic acid)/ chitosan hydrogel Poly(2-acrylamido-2-methylpropanesulfonic acid)/ chitosan hydrogel Magnetic chitosan composite composed of nanomagnetite, heulandite Magnetic chitosan composite composed of nanomagnetite, heulandite Chitosan-coated Fe3O4 (Fe@CS) beads Ion-imprinted chitosan-TiO2 bead Chitosan-g-poly (acrylic acid)/attapulgite (APT) composite CS bead CS reinforced with citric acid modified β-cyclodextrin Porous 3D network rectorite/CS gels Fe-cross-linked chitosan complex (Ch-Fe) N-(2-carboxybenzyl)-grafted chitosan bead N-(2-carboxybenzyl)-grafted chitosan bead N-(2-carboxybenzyl)-grafted chitosan bead N-(2-carboxybenzyl)-grafted chitosan bead Cross-linked chitosan
diethylenetriaminepentaacetic acid

20.408 46.1

MB Red dye

[5] [37]

74.3

MB

[37]

68.1

Cd(II)

[37]

22.5

Cr(III)

[37]

45.1

MB

[6]

149.2

[6]

70.57 10.97 1848

Methyl orange (MO) Pb21 Ni12 MB

[52] [57] [20]

6.18 498 162.6 295 308 381 208 175 192.3

As(III) Reactive Blue 49 MB Cr(VI) Cu(II) Ni(II) As(V) Cr(VI) Cr(VI)

[58] [59] [60] [61] [62] [62] [62] [62] [63]

Ionic liquid impregnated sulfate-cross-linked chitosan

250.90

Cr(VI)

[64]

Adsorbent

(Continued)

Application of polysaccharide-based hydrogels for water treatments 421 Table 14.1: (Continued)

Adsorbent Zr (IV)-immobilized cross-linked chitosan/bentonite composite Cross-linked chitosan/sepiolite composite Cross-linked chitosan/sepiolite composite Quaternized chitosan-coated bentonite Quaternized chitosan-coated bentonite Cross-linked beads of activated oil palm ash zeolite/ chitosan composite Cross-linked beads of activated oil palm ash zeolite/ chitosan composite Surfactant-modified chitosan beads Nano-ZnO/chitosan composite beads Chitosan cross-linked with trimesic acid Chitosan/CaCO3-silane nanocomposites Chitosan-Fe(OH)3 beads Chitosan-Fe(OH)3 beads Chitosan/Ag-hydroxyapatite nanocomposite beads Chitosan/Ag-hydroxyapatite nanocomposite beads Poly(vinyl alcohol) (PVA)/citric acid/chitosan beads Magnetic chitosan beads Cross-linked chitosan/β-cyclodextrin composite Chitosan/modified montmorillonite beads Chitosan/montmorillonite intercalated composite Chitosan-modified magnetic graphitized multiwalled carbon nanotubes (CNTs) Zr(IV)surface-immobilized cross-linked chitosan/ bentonite composite Xanthate-modified chitosan/poly(N-isopropylacrylamide) composite hydrogel Xanthate-modified chitosan/poly(N-isopropylacrylamide) composite hydrogel Xanthate-modified chitosan/poly(N-isopropylacrylamide) composite hydrogel CS/Fe-hydroxyapatite nanocomposite beads CS/Fe-hydroxyapatite nanocomposite beads Chitosan-based hydrogels Chitosan-based hydrogels Chitosan coated PVC beads Chitosan coated PVC beads Magnetic hydroxyapatite/chitosan composite Chitosan hydrogel beads

Maximum adsorption capacity (mg/g)

Dye or metal ions

References

438.6

MO

[65]

40.986 190.965

[66] [66]

66.6 847.5 199.20

MB Reactive orange 16 Cr(VI) Amido black 10B MB

270.27

AB29

[68]

125 189.44 129.53 33.90 445.32 314.45 40.11 127.61 41.5 294.11 392 5.6085 445.38 262.9

Cd21 Reactive Black 5 Cr(VI) Cu(II) CR MO Cu(II) Rhodamine B Cr(III) Cu(II) MO Reactive Red 120 Reactive red 136 CR

[69] [70] [71] [72] [73] [73] [74] [74] [75] [76] [77] [78] [79] [80]

418.

4Amido Black 10B

[81]

115.1

Cu(II)

[82]

172.0

Pb(II)

[82]

66.9

Ni(II)

[82]

1837 1324 190 235 87.9 120.5 112.36 76.9

Pb(II) MB Pd(II) Pt(IV) Cu(II) Ni(II) Ni(II) Crystal violet (CV)

[83] [83] [84] [84] [85] [85] [86] [87]

[67] [67] [68]

422 Chapter 14

14.4 Cellulose Cellulose, with a chemical formula of (C6H10O5)n, is the most abundant renewable biopolymer on Earth. The rate of cellulose production by nature is about 1011
1012 tons/ year. Cellulose can be produced from bacterial or plant sources. The molecular structure of bacterial cellulose (BC) is similar to plant cellulose. However, due to its high crystallinity, 3D network structure, and ultrafine nanosize, BC has several advantages compared to plant cellulose and is more suitable for adsorption applications. In this linear polysaccharide, Dglucose units are linked by β-(1-4)-glycosidic bonds to each other. Each monomer in the cellulose chain has three hydroxyl functional groups which are in the C2, C3 (secondary hydroxyl groups), and C6 (primary hydroxyl groups) positions. These three hydroxyl groups present two types of hydrogen bond: intramolecular hydrogen bonds and intermolecular hydrogen bonds. Cellulose is a tough, fibrous, and water-insoluble polymer because of its complex intermolecular and intramolecular hydrogen bonds [88
95]. Moreover, the presence of many hydroxyl groups in the cellulose structure and their high reactivity provide many possibilities for chemical modification reactions, hence there is a very modified form of cellulose. Esterification, halogenation, etherification, and oxidation are some of modification methods for cellulose modification. Carboxymethyl cellulose (CMC) is one of the most important anionic derivatives of cellulose, which is produced by partial substitution of the 2, 3, and 6 hydroxyl groups of cellulose with carboxymethyl groups as a result of esterification. Due to their biodegradation ability, solubility, and biocompatibility, CMC-based hydrogels have shown considerable potential use in wound healing, enzyme immobilization absorbents, and drug delivery [96
101]. Cellulose acetate is modified cellulose recognized as a type of cellulose ester and is another main derivative of cellulose. It is obtained from the acetylation of cellulose powder with acetic acid or acetic anhydride in the presence of sulfuric acid. Because of its high thermal, mechanical, and chemical features it has the ability to be used as an adsorbent [102,103]. In addition to the anionic derivative form of cellulose, cellulose can be modified to form cationic derivatives. Quaternary ammonium sodium salt is a cationic derivative of cellulose and is prepared by the homogeneous reaction of cellulose with 2,3-epoxypropyl trimethylammonium chloride, which is an effective adsorbent for anionic compounds [104].

14.4.1 Cellulose-based hydrogels for the removal of dyes The presence of the many hydroxyl groups and the biocompatibility and biodegradability of cellulose provide the possibility for cellulose-based hydrogel use in water treatment. Methylene blue (MB) is one of the most widely used cationic textile industry dyes, due to its low price. This dye can damage the ecosystem balance and affect the environment, it can also cause a heartbeat increase, shock, vomiting, jaundice, cyanosis, quadriplegia, and tissue necrosis in humans. MB is commonly used in coloring paper, dyeing cotton, as a

Application of polysaccharide-based hydrogels for water treatments 423 temporary hair colorant, and in wood [5,37]. Halouane et al. [36] prepared magnetic reduced graphene oxide (magnetic r-GO)-loaded cellulose hydrogels and investigated their ability for removing MB. First MP@cellulose particles were synthesized through the precipitation of magnetic particles in the presence of cellulose polysaccharides. In the next step, photopolymerization in the presence of poly(ethylene glycol) dimethacrylate yielded robust magnetic hydrogels. Cellulose was chosen as a coating because it is not only the most abundant natural polymer on Earth but it also has the ability to absorb hazardous dyes and heavy metal ions from wastewater. Moreover, magnetic r-GO nanohybrid induced magnetic properties to the designed system and enhanced its surface area. SEM images showed that the surface of the magnetic reduced GO-loaded hydrogel is rough and has a porous structure. The maximum adsorption capability obtained was 119 6 4 mg/g. Luo and Zhang [91] prepared maghemite (γ-Fe2O3) nanoparticles with a submerged circulation impinging stream reactor using FeCl3  6H2O and FeCl2  4H2O precipitation. Subsequently, oxidation and then blending of cellulose biopolymer with AC and Fe2O3 nanoparticles in defined condition formed cellulose beads (MCB
AC beads). The adsorption and desorption of the methyl orange (MO) and MB organic dyes using the synthesized beads were investigated to evaluate the ability of these two dyes to be removed by the prepared beads. Neither organic dye showed a pH effect on their adsorption capacity. This observation shows that the contribution of the encapsulated AC in dye removal is very high, and is not pH-sensitive. Moreover, the results showed that the adsorption capacity of MCB
AC beads for MO was higher than for MB. This revealed that in the negatively charged MO, binding with the beads through electrostatic interactions and hydrogen bonds was easier than MB. There was more adsorption of negatively charged organic dyes (MO) than positively charged MB, indicating a selective adsorption behavior for MCB
AC beads.

14.4.2 Cellulose-based hydrogels for the removal of heavy metals Because environmental protection is a vital current global problem, biosorption by using natural polysaccharides like cellulose and its derivatives has become a favorable method for heavy metal elimination. Microcrystalline cellulose (MCC) with good mechanical and chemical properties was recently applied as an adsorbent. However, the crystalline nature of MCC may limit its adsorption capacity for metals and other compounds. El-Naggar et al. [105] reported the synthesis of nanogels with free radical polymerization of acrylamide (AAm) and AA onto MCC using methylene bisacrylamide (MBA) as a cross-linking agent. The ability of the prepared system to remove cadmium as a heavy metal model from the aqueous medium was studied. Cadmium is a toxic heavy metal, which leads the kidney damage, increased blood pressure, renal disturbances, skeletal deformity, and bone lesions. Cadmium can enter the environment through wastewater from plating industries, phosphate fertilizers, pigments, Cd
Ni batteries, and alloys. The results of this research have shown that only a small amount of sample was enough to remove 97% of Cd(II) ions. The high

424 Chapter 14 adsorption of Cd(II) ions might be due to the rapid attraction between the negatively charged groups of nanogels and positively charged Cd ions. The removal of Pb21 from water is critical from the viewpoint of the environment and public health protection. Many researchers have focused on the design of new biosorbents with greater adsorption capacity for Pb21 removal. One of these reports described the preparation of magnetic cellulosebased nanocomposite beads (MCNBs). MCNBs were prepared by incorporating cellulose with carboxyl functionalized Fe3O4 nanoparticles and acid-activated bentonite. The prepared system showed a high adsorption capacity for Pb21. The high surface negative charges of beads facilitates the migration of positive lead ions to the negative region of the beads by electrostatic attraction. Moreover, the presence of carboxylic functional groups on the surfaces and inside of beads provides for the removal of Pb21 from the water through the coordination with carboxyl groups. Based on the above states, both physical adsorption and chemical adsorption play important roles in Pb21 removal. A schematic of the MCNB preparation, Pb21 adsorption, and desorption mechanism is shown in Fig. 14.4 [106]. Due to the large volume of research carried out on water treatment using cellulose-based hydrogels, only the results of a few cases are summarized in Table 14.2.

14.5 Starch Starch (ST) is one of the most abundant natural polysaccharides. There are many interesting research reports in the ST-based materials field because of its mechanical properties and biodegradability. Amylose and amylopectin are two homopolymers of D-glucose which ST is composed of. Amylose is a linear polymer with α-D-1,4-glycosidic bonds but amylopectin is a highly branched polymer with α-D-1,6-glycosidic linkages at the branching points in addition to α-D-1,4-glycosidic linkages in the main linear chain. The chemical structures of amylose and amylopectin are shown in Fig. 14.5. Depending on the source, it can contain different amounts of amylopectin and amylose, ranging from about 80%
90% amylopectin and 10%
20% amylose. The molecular weights of amylopectin and amylose have been valued to be about 107 Dalton and 105 Dalton, respectively [131
137]. Among the numerous polysaccharide-based adsorbents, ST and its derivatives are a cheap and environmentally safe adsorbent. Being abundant, biodegradable, and a renewable raw resource, with good chemically stability and high reactivity are some of the other advantages of ST. However, the poor processability and high brittleness and greater hydrophilicity of ST, which result from the presence of more hydroxyl groups, limit its use in some applications. To overcome these limitations ST modification is usually carried out [138,139]. The modification of ST can be performed using physical or chemical methods or a combination of these. Physical modification is achieved using hydrothermal processing (gelatinization). Chemical modification is done by the introduction of suitable functional

Application of polysaccharide-based hydrogels for water treatments 425

Figure 14.4 Schematic of the magnetic cellulose-based nanocomposite bead (MCNB) preparation, Pb21 adsorption, and desorption mechanism. Reproduced with permission from X. Luo, et al., Adsorptive removal of lead from water by the effective and reusable magnetic cellulose nanocomposite beads entrapping activated bentonite. Carbohydr. Polym. 151 (2016) 640
648 [106].

groups into the ST structure using derivatization reactions such as etherification, crosslinking, esterification, and grafting, or decomposition reactions such as acid or oxidation and enzymatic hydrolysis. Chemical modification is a frequently used method for improving ST properties [140]. Sodium trimetaphosphate, glutaraldehyde, phosphoryl chloride, ECH, sodium tripolyphosphate (STPP), a mixture of adipic acid and acetic anhydride, and vinyl chloride are commonly used cross-linkers for the preparation of ST-based hydrogel. ST-based hydrogels have gained increased attention because of their abundance, high swelling, nontoxicity, lower cost, raw material resources, good biodegradability, and biocompatibility. ST can be blended with synthetic polymers to obtain hydrogels with new properties.

426 Chapter 14 Table 14.2: The adsorption capacity of cellulose-based hydrogels for various dyes and heavy metals removal from water. Adsorbent Poly(itaconic acid/methacrylic acid)-graftednanocellulose/nanobentonite composite Graphene oxide (GO)/cellulose bead (GOCB) composites Carboxymethyl cellulose (CMC) immobilized Aspergillus fumigatus beads CMC-g-poly(2-(dimethylamino) ethyl methacrylate) hydrogel Polyvinyl alcohol/CMC hydrogels reinforced with graphene oxide and bentonite CMC hydrogel beads Cellulose nanocrystal-alginate hydrogel beads CdS quantum dots templated hydrogel Graphene oxide-cellulose nanowhiskers nanocomposite hydrogel Microgel based on nanocellulose and polyvinylamine Microgel based on nanocellulose and polyvinylamine Microgel based on nanocellulose and polyvinylamine Cross-linked graft copolymers of cellulose Cross-linked graft copolymers of cellulose Three-dimensional macroporous cellulose-based bioadsorbents Sepiolite/cellulose beads Microporous cationic hydrogel of hydroxypropyl cellulose CMC/organic montmorillonite nanocomposites Cellulose and gelatin-based composite hydrogels Carboxylated cellulose nanofibril-filled magnetic chitosan hydrogel beads Cellulose-based hydrogel Cellulose filament/poly(NIPAM-co-AAc) hydrogel Nanochitosan/sodium alginate (NaAlg)/ microcrystalline cellulose (MCC) bead Cellulose/CMC bioadsorbent Cellulose/CMC bioadsorbent Cellulose/CMC bioadsorbent Hemicellulose 2 chitosan biosorbent Hemicellulose 2 chitosan biosorbent Hemicellulose 2 chitosan biosorbent Polyaniline-coated magnetic CMC beads Lignocellulose-based composite hydrogel Graphene oxide/cellulose hydrogel Cellulose-based hydrogels

Maximum adsorption capacity (mg/g)

Dye or metal ions

References

350.8

Co(II)

[107]

30.090

MG

[108]

41.1

[109]

1825

Reactive brilliant red K-2BP MO

172.14

MB

[97]

82 255.5 137 122.5

MB MB Rhodamine B MB

[111] [112] [113] [114]

869.1

Congo red 4BS

[115]

1469.7

Acid red GR

[115]

1250.9

[115]

112.74 109.77 171.8

Reactive light yellow K-4G Ni(II) Cu(II) Ni21

314.47 2478

MG Anionic dye AO7

[118] [119]

171.37 49.1 171.0

CR Cu(II) Pb(II)

[120] [121] [122]

142.7 80.8 43.32

Cu(II) Pb(II) Cu(II)

[123] [124] [125]

558.9

Pb21

[126]

21

[126] [126] [127] [127] [127] [128] [127] [129] [130]

446.2 363.3 2.90 0.95 1.37 386.5 563.33 94.34 130

Cu Zn21 Pb21 Cu21 Ni21 U(VI) Cu21 Cu21 Cu(II)

[110]

[116] [116] [117]

Application of polysaccharide-based hydrogels for water treatments 427

Figure 14.5 The chemical structures of amylose and amylopectin.

Incorporating ST into synthetic hydrogel networks improves their swelling and adsorption capacity. ST-based hydrogels are used in various areas such as film packaging, drug delivery, tissue engineering, agriculture, food additives, and dye and heavy metal removal from water or wastewater [140
142].

14.5.1 Starch-based hydrogels for the removal of dyes A decolorization operation is generally used in wastewater treatment. ST-based hydrogels are low-cost adsorbents with good adsorption capacity, which can be used for removing various cationic or anionic dyes after modification with functional groups. For the removal of anionic Direct Red 81 dye, cationic ST-based hydrogel was prepared through graft copolymerization of N,N-diethylaminoethyl methacrylate with ST and then cross-linking with ECH. The prepared hydrogel showed a high adsorption capacity for the removing of Direct Red 81 as an anionic dye, compared to traditional adsorbents. The high adsorption capacity results from the cationic character of the substituted amino group in the N,Ndiethylaminoethyl methacrylate moiety of the hydrogel, which causes an electrostatic attraction between the cationic hydrogel and anionic dye [143]. With the growth of new biodegradable and biocompatible adsorbents based on ST, porous starch (PS) has received

428 Chapter 14 much interest because its preparation occurs without any toxic reagent. PS is prepared by hydrolyzing raw ST with ST enzymes (α-amylase or amyloglucosidase) under the ST gelatinization temperature in order to achieve a new adsorbing system with high adsorption capacity. Guo et al. [144] prepared cross-linked porous starch (CPS) in two steps. First, ST was cross-linked using ECH as a cross-linker. Second, hydrolyzing of the cross-linked ST with α-amylase obtained the CPS). Brunauer
Emmett
Teller and SEM analysis approved the porosity of CPS. The adsorption capacities of MB on native ST, PS, and CPS were obtained at 3.11, 7.26, and 8.33 mg/g, respectively, at the same conditions. It can be seen that CPS has a much higher adsorption capacity than native ST and a relatively higher adsorption capacity than PS. The results indicate that cross-linking and porosity enhanced the adsorption capacity of ST. Sharma et al. [145] prepared a starch/poly(alginic acid-clacrylamide) nanohydrogel using the copolymerization method and MBA as a cross-linker. The ability of the synthesized nanohydrogel studied for the removal of coomassie brilliant blue R-250 from aqueous solution. This nanohydrogel showed high efficiency for the removal of coomassie brilliant blue R-250 dye from an aqueous solution and the maximum adsorption capacity was obtained at 31.24 mg/g. Due to the cationic nature of the synthesized nanohydrogel, it is proposed that the attraction between cationic nanohydrogel and anionic dye is a main factor in dye adsorption.

14.5.2 Starch-based hydrogels for the removal of heavy metals Today’s heavy metal pollution is one of the most studied issues, due to its alarming rate of increase almost daily. Native ST is interacted through a physical or chemical interaction with a wide range of molecules, however, due to the absence of more of the chelating group in its structure, neat ST hydrogel has low metal adsorption capacity. To overcome this problem many researchers recently have been made to modify ST as a metal absorbent through introducing several functional groups such as carboxylate, AAm, xanthate, phosphate, acrylonitrile, and dithiocarbamate [146]. Carboxymethyl starch (CMS) is one of the most studied starch derivatives, in many fields. CMS is used in food, pharmaceuticals, cosmetics, and water treatment. Due to the solubility of CMS in the water medium, it is required to be stabilized, in order to be applied as an adsorbent. Cross-linking is the main method for this purpose. Anhydrides, phosphoryl oxychloride, ECH, dicarboxylic acids, and STPP are examples of the cross-linkers used for the preparation of CMS-based hydrogels. Use of cross-linked CMS hydrogel for the removal of Fe(II), Ca(II) Cu(II), Cd(II), and Pb (II) has been reported. Synthesized hydrogel has exhibited high metal cation adsorption efficiency; up to 98% for Fe(II), 96% for Ca(II), and above 99.7% for Cu(II), Pb(II), and Cd(II) [147]. Xing et al. [148] prepared cross-linked amphoteric starch containing tertiary amine and maleic (CCSM) and compared its ability for chromium(VI) removal with native starch. The results showed that the adsorption capacity of native starch was much lower than cross-linked amphoteric starch. Native starch, CCSM1, CCSM2, and CCSM3 showed

Application of polysaccharide-based hydrogels for water treatments 429 adsorption capacities of 1.7, 33.3, 26.3, and 22.8 mg/g, respectively. In all of the hydrogels, the DSs of quaternary ammonium cationic groups were 0.2 but the DSs of maleic anionic groups were 0.02, 0.04, and 0.07, respectively. The results of this modification indicated that the modification increased the adsorption capacity and may be related to the fact that in native starch the physical entrapment of the chromium(VI) ions only play a role, but in the case of CCSM a strong electrostatic attraction may be the reason for ion adsorption. Due to the high number of reports of water treatment using the starch-based hydrogels, only the results of a few cases are summarized in Table 14.3. Table 14.3: The adsorption capacity of starch-based hydrogels for various dyes and heavy metals removal from water.

Adsorbent Graphene oxide potato starch-based cross-linked biocomposite Starch/poly(N,N-diethylaminoethyl methacrylate) hydrogel Starch/cellulose nanowhiskers hydrogel composite Oxidized starch cross-linked chitosan/silica Oxidized starch cross-linked chitosan/silica Chitosan and starches-g-PAN cryobeads Chitosan and starches-g-PAN cryobeads Chitosan and starches-g-PAN cryobeads Highly cross-linked amphoteric starch Cross-linked porous starch (CPS) Bioinspired catecholamine/starch composites as superadsorbent Semi-IPN superabsorbentchitosan-starch hydrogel Cationic starch intercalated clay composite matrix Cross-linked starch polymer Porous rectorite/starch composites Porous rectorite/starch composites Starch-graft poly(acrylamide)/graphene oxide/ hydroxyapatite nanocomposite hydrogel Polyethylene-g-poly(acrylic acid)-co-starch/ organo-montmorillonite hydrogel Starch/SnO2 nanocomposite Semiinterpenetrating network hydrogel based on starch Starch/rice husk ash-based superabsorbent composite Polycarboxylated starch-based hydrogel Magnetic hydrogel beads based on poly(vinyl alcohol)/carboxymethyl starch-g-poly(vinyl imidazole)

Maximum adsorption capacity (mg/g)

Dye or metal ions References

500

MB

[149]

112

Direct Red 81

[143]

2050 67.2 94.4 100.6 83.25 74.01 141.9 9.46 2276

MB Direct Blue 71 Direct Red 31 Cu21 Ni21 Co21 Basic Green 4 MB MB

[150] [151] [151] [152] [152] [152] [139] [153] [154]

312.77 122.0 36.2 180.8 277.0 297

Direct Red 80 Brilliant blue X-BR CR Pb21 MB MG

[155] [156] [157] [158] [158] [159]

430

Pb(II)

[160]

192 388

Hg21 MG

[161] [162]

2225

MB

[163]

128.26 65.00

Cu21 Pb(II)

[164] [165]

(Continued)

430 Chapter 14 Table 14.3: (Continued)

Adsorbent Magnetic hydrogel beads based on poly(vinyl alcohol) (PVA)/carboxymethyl starch-g-poly(vinyl imidazole) Magnetic hydrogel beads based on PVA /carboxymethyl starch-g-poly(vinyl imidazole) Magnetic hydrogel beads based on PVA /carboxymethyl starch-g-poly(vinyl imidazole) Magnetic hydrogel beads based on PVA /carboxymethyl starch-g-poly(vinyl imidazole) Potato starch-acrylic-acid hydrogels Fe3O4@SiO2 starch-graft-poly(acrylic acid) nanocomposite hydrogel Cross-linked amphoteric starches Cross-linked cationic starch 2-Acrylamido-2-methylpropane-1-sulphonic acid (AMPS)/starch hydrogel AMPS/starch hydrogel Starch-based adsorbent Starch-based adsorbent Amino-functionalized starch/PAA hydrogel (NH2-starch/PAA) Cross-linked amino starch Cross-linked amino starch Cross-linked carboxymethyl starch Cross-linked carboxymethyl starch

Maximum adsorption capacity (mg/g)

Dye or metal ions References

83.60

Cu(II)

[165]

53.20

Cd(II)

[165]

83.66

CR

[165]

91.58

Crystal violet (CV)

[165]

576 80.64

MB CV

[166] [167]

49.26 1022 600

Cr(VI) Acid Orange 7 Basic violet

[168] [169] [170]

350 123.2 131.2 256.4

Co21 Pb(II) Hg(II) Cd(II)

[170] [171] [171] [172]

8.134 12.12 80 47

Cu(II) Cr(VI) Pb(II) Cd(II)

[173] [173] [174] [174]

14.6 Sodium alginate NaAlg is a water-soluble linear polyanionic polysaccharide obtained from marine brown. NaAlg is composed of alternating blocks of 1
4-linked α-L-guluronic acid and β-D-mannuronic acid residues arranged in a block-wise fashion and nonregular along the chain, as shown in Fig. 14.6. The ratio of these blocks depends on the source of the polymer [175,176]. NaAlg can be dissolved in either hot or cold water with strong stirring. This biopolymer is commonly used in the food industry to increase the viscous nature of liquids by playing an emulsifier role. The gel-forming ability of NaAlg results from the exchange of sodium ions from the guluronic acid portions with some of divalent cations (Ca21, Sr21, Ba21, etc.). In other words, the 3D network is formed as a result of the divalent cations binding to the α-Lguluronic acid blocks between two different chains [177].

Application of polysaccharide-based hydrogels for water treatments 431

Figure 14.6 The chemical structure of sodium alginate (NaAlg) biopolymer.

Because of the existence of more carboxylic groups, alginate beads are hydrophilic. Additionally they have high mechanical strength and good stability in operational conditions. Alginate-based hydrogels are widely used in the pharmaceutical industry for preparation of timed-release enteric coated tablets, and in wastewater treatment, the food industry, and agriculture. In order to obtain NaAlg-based hydrogels with high adsorption capacity, modification is necessary. NaAlg can be modified through ionic cross-linking, chemical cross-linking, and grafting polymerization reaction resulting in a new material with improved properties [178,179].

14.6.1 Alginate-based hydrogels for the removal of dyes Alginate has good adsorption capacity toward various pollutants which arise from the presence of many carboxylic and hydroxyl functional groups in its structure. Due to the high ability of composites containing alginate in water treatment, there are many reports which have studied the use of alginate-containing compounds as an effective adsorbent for water treatment. The use of alginate as an adsorbent provides selective adsorption of organic molecules due to the possibility of an interaction with the negative carboxylate groups on alginate. Li et al. [180] prepared novel magnetic alginate beads and investigated their ability for the removal of cationic and anionic dyes. At first, CoFe2O4 submicrospheres were prepared, in the following polydopamine (PDA) was deposited on the CoFe2O4 submicrospheres, through dopamine oxidative polymerization. Finally, the resulting CoFe2O4
PDA was mixed with NaAlg and the mixture was added dropwise to the Ca21 ion solution and hydrogel beads were obtained. The prepared composite beads showed the advantages of NaAlg, PDA (a large number of functional groups), and CoFe2O4 (fast and easy magnetic separation). The results showed that the prepared beads presented lower adsorption ability for anionic dyes, while more than 95% of cationic dyes were removed in the same experimental conditions. This observation may be related to the

432 Chapter 14 difference in the chemical structures of dye molecules that lead to different adsorption capacities. In fact, cationic dyes due to the electrostatic interaction could be easily adsorbed using anionic polymers. Alginate could be modified using several compounds, or it could be blended with other synthetic or natural biopolymers to improve its anionic dye adsorption ability. For instance, novel blend hydrogel beads were prepared using the NaAlg and poly(N-vinyl-2-pyrrolidon) (NaAlg/PVP hydrogel bead) by gelation method into a calcium chloride solution. The ability of the synthesized system was compared with pure NaAlg beads for the removal of anionic dyes such as reactive red 120 (RR), cibacron brilliant red 3B-A (CBR), and remazol brilliant blue R (RBB). The effect of pH on the adsorption capacity of dyes was studied at pHs of 1.2
5.0. The adsorption of dyes was negligible for NaAlg hydrogel beads, whereas the highest amount of adsorption was obtained at pH 1.2 for blended NaAlg/PVP hydrogel beads. The high electrostatic attraction between the positively charged adsorbent (positive charge on the nitrogen atom of PVP groups) and the anionic dye molecules could be the main reason for this observation [181].

14.6.2 Alginate-based hydrogels for the removal of heavy metals Chromium is the 16th most toxic material, and is widely used in the electroplating, cement, leather tanning, dyeing, wood preservatives, metal processing, paint and pigments, steel fabrication, textile, and canning industries. Discharge of wastewater effluents of these industries produces a large amount of chromium in the environment, Cr(III) and Cr(VI). Whereas Cr(III) is necessary for humans in glucose metabolism in trace amounts, most of the hexavalent-containing compounds are toxic and could be linked to lung cancer [182]. Many researchers have been working on the design of new adsorbents with high adsorption capacity to remove Cr(VI) due to its highly toxic nature. Yan et al. [183] designed a novel adsorbent for Cr(VI) removal using NaAlg beads as a functional platform in which PDA
PEI hollow cavity composites were dispersed in the inside NaAlg and then PEI was grafted onto the surface through a cross-linking reaction. The designed system showed a 524.7 mg/g adsorption capacity. It is proposed that the efficient removal of Cr(VI) is due to the improved interior/ surface accessibility of reactive sites as a result of the modification. Due to the good adsorption of Cu21 and Pb21 in acidic pH using cationic polymers as a new adsorbent; alginate/polyethyleneimine hydrogel was designed and its ability for Cu21 and Pb21 ion removal was studied. The biosorbents modified with amine-groups exhibit high adsorption capacity for Cu21 and Pb21 ions. In fact, the amine-groups of polyethyleneimine portions on the adsorbent easily can be protonated in acidic solutions, and thus capture Cu21 and Pb21 ions by electrostatic attraction and ion exchange [184]. Due to the high volume of research on the study of alginate-based hydrogels in water treatment, only the results of a few cases are summarized in Table 14.4.

Application of polysaccharide-based hydrogels for water treatments 433 Table 14.4: The adsorption capacity of alginate-based hydrogels for various dyes and heavy metals removal from water. Adsorbent Calcium alginate beads impregnated with nanogoethite Mesoporous activated carbon-alginate beads Porous hectorite clay-alginate composite beads Magnetic alginate/oxidized multiwalled carbon nanotube composites Alginate/Polyamidoamine (PAMAM) dendrimer-Halloysite beads Alginate/PAMAM dendrimer-Halloysite beads Iron(II) cross-linked chitin-based gel beads Zirconium oxide immobilized alginate beads Zirconium oxide immobilized alginate beads Zirconium oxide immobilized alginate beads Sodium alginate (NaAlg)-based cross-linked beads Hydrogel beads of PVA-NaAlg-chitosan (CS)-montmorillonite Iron-oxide modified sericite alginate beads Iron-oxide modified sericite alginate beads Modified cross-linked cellulose/NaAlg with polyethyleneimine Modified cross-linked cellulose/NaAlg with polyethyleneimine Modified cross-linked cellulose/NaAlg with polyethyleneimine Zinc oxide modified clay over alginate beads NaAlg/poly(N-vinyl-2-pyrrolidone) blend hydrogel beads NaAlg/poly(N-vinyl-2-pyrrolidone) blend hydrogel beads NaAlg/poly(N-vinyl-2-pyrrolidone) blend hydrogel beads Alginate/calix [4] arenes-modified graphene oxide nanocomposite beads Activated carbon
alginate composite material Calcium alginate/activated carbon composite beads

Maximum adsorption capacity (mg/g)

Dye or metal ions

References

181.1

CR

[177]

230 785.45 905.5

MB MB MB

[25] [185] [186]

113.64

MG

[187]

61.73 128.5 32.3 28.5 69.9 2042

Sunset yellow FCF MO As(III) As(V) Cu(II) Pb21

[187] [188] [189] [189] [189] [190]

137.2

MB

[191]

21.61 133.73 177.1

As(V) Pb(II) Cu(II)

[192] [192] [193]

110.2

Zn(II)

[193]

234.2

Pb(II)

[193]

546.89 116.8

CR Reactive red-120

[194] [181]

73.3

[181]

170.36

Cibacron brilliant red 3B-A Remazol brilliant blue R (RBB) MB

15.7 66.7

Pb(II) As

[196] [197]

55.3

[181] [195]

14.7 κ-Carrageenan Carrageenans are sulfonated polysaccharides which are obtained by alkaline extraction of Rhodophyceae red seaweeds. Depending on the extraction condition and type of source, carrageenans contain around 25,000 galactose repeating units. Carrageenans composed of D-galactose residues are linked alternately in 3-linked-β-D-galactopyranose and

434 Chapter 14

Figure 14.7 The chemical structure of κ-carrageenan (κ-car) polysaccharide.

4-linked-α-D-galactopyranose units. Based on the position of the ester sulfate groups and substitution degree on their free hydroxyl groups, they are classified into three groups: iotacarrageenan, kappa-carrageenan, and lambda-carrageenan [143,198,199]. The structure of κ-car is shown in Fig. 14.7. κ-Car is a very important commercial form of carrageenan and is composed of alternating α-(1-3)-D-galactose-4-sulfate and β-(1-4)-3,6-anhydro-D-galactose. This polymer is a negatively charged polysaccharide with one sulfate group per repeating disaccharide group, hence it is a good biocompatible adsorbent candidate for cationic dye removal. κ-Car is a nontoxic, biocompatible, biodegradable, more hydrophilic polymer. The hydrophilicity of κ-car results from the presence of numerous hydroxyl groups and highly ionic sulfate anion in its structure. Due to this advantage, it is used as a cost-effective stabilizer, binder, thickener, and texture and moisture retainer and modifier. Moreover, this polymer has positive cardiovascular effects such as reducing cholesterol and blood pressure and has been applied as a potential antitumor medicine, antihyperlipidemic, immunomodulator, and anticoagulant [3,199
201]. κ-Car can form hydrogels through the cross-linking. The ability for hydrogel forming is due to physical cross-linking of the sulfonate anions with K1 or polycationic polyelectrolytes (especially CS). Additionally, it can be cross-linked by using nontoxic genipin as a chemical cross-linker. Hydrogel-based κ-car is a good choice for water treatment but the poor gel strength and low environmental stability are limiting factor of κ-car using in water treatment. The blending of κ-car with other functional polymers is one of the solutions to this problem and produces a new adsorbent with beneficial properties [30]. Karimi et al. [12] prepared magnetic carrageenan/CS hydrogel through in situ syntheses of magnetite and reported the successful cross-linking of CS using carrageenan. A schematic of the crosslinking is shown in Fig. 14.8.

14.7.1 κ-Car-based for the removal of dyes κ-Car is an anionic biopolymer, hence it is expected to have a high adsorption capacity toward cationic dyes. MB is a commonly used cationic dye due to its high solubility

Application of polysaccharide-based hydrogels for water treatments 435

Figure 14.8 Scheme of magnetic chitosan
carrageenan complex preparation. Reproduced with permission from M. H. Karimi, et al., Ionically crosslinked magnetic chitosan/κ-carrageenan bioadsorbents for removal of anionic eriochrome black-T. Int. J. Biol. Macromol. 113 (2018) 361
375[12].

properties. Liu et al. [202] prepared new composite beads based on CMC/κ-carrageenan (κ-car)/activated montmorillonite and studied their ability for MB removal, due to the harmful properties of MB even in trace concentrations. The results showed that the nanocomposite with CMC:κ-car (1:1) had the highest removal (%) of MB. These results showed that both κ-car and CMC participated in the MB adsorption and -COOH and -SO3H groups of CMC and κ-car play an important role in the adsorption process. The adsorption of MB on this adsorbent occurred through two mechanisms. First, a strong electrostatic interaction between positively charged MB and negatively charged groups on the adsorbent, and second hydrogen bond formation between imine groups of MB molecules (RCH 5 NR) and the reactive OH groups of the used polymers. In the other research, nanocomposite hydrogel based on κ-car and AA was prepared by embedding nanosilver chloride and its ability for the adsorption of anionic (CR, MO) and cationic [crystal violet (CV) and Rhodamine B] dyes from aqueous solutions was assessed. The results showed that the nanocomposite hydrogel has good absorption capacity for cationic dyes in comparison to anionic dyes, which is related to the anionic surfaces of the nanocomposite hydrogel. Also, CV can be removed better than Rhodamine B, which is related to the presence of an anionic carboxylate group in the Rhodamine B structure [1].

14.7.2 κ-Car-based hydrogels in heavy metal removing Groundwater is a natural source of drinking water. Usually, the main problem with this water is the high concentrations of heavy metals and Ca, Fe, and Mg salts, which lead to an increase in the toxicity and hardness of the water. The existence of -SO3H and -OH functional groups in the κ-car structure could cause the chelation and removal of metal

436 Chapter 14 Table 14.5: Effect of carrageenan modification on metal removal efficiency. R% Sorbent type CR Car-CNC Car-CNF Car-TC-CNF

Cu

21

72 66 86 91

21

Pb

60 58 76 83

Ca21

Mg21

Fe21

40 15 49 57

53 45 44 71

55 29 45 63

Reproduced with permission from Ali, K.A., et al., Development of carrageenan modified with nanocellulose-based materials in removing of Cu2 1 , Pb2 1 , Ca2 1 , Mg2 1 , and Fe2 1 . Int. J. Environ. Sci. Technol. 2018 [203].

cations from water and wastewater. To improve the ability of κ-car to remove metal cations from water, modification it useful. Ali et al. [203] prepared a κ-car -based bead and modified it with three types of cellulosic derivatives [cellulose nanocrystals (CNCs), cellulose nanofibers (CNFs), and tricarboxy cellulose nanofibers (TC-CNFs)] and compared their ability in removing Cu21, Pb21, Ca21, Mg21, and Fe21. The result of this study is given in Table 14.5. The results showed that hydrogel with a higher amount of carboxylic groups showed better metal cation-removing efficiency, due to the number of carboxylate and hydroxyl groups: TC-CNF . CNF . CNC. Lead is the most toxic metal ion leading to health problems, and hence in one research graft copolymers of polyacrylamide and κ-car were prepared using ceric ammonium nitrate as an initiator. Because of the higher functionality of κ-car, the grafting reaction of κ-car and AAm yields microgel. Microgel with optimum characteristics was used for Pb21 ion removal from an aqueous solution. Adsorption experiments were performed at a pH range of 2
8. By increasing pH from 2 to 5 the biosorption capacity was increased and the maximum adsorption capacity was observed at pH 5. On the other hand, with a further increase in pH from 5 to 8 the adsorption capacity decreased. This behavior shows that binding of the Pb21 ions through an ion-exchange mechanism may be the reason for the observed adsorption capacity. The decrease in adsorption capacity with a pH increase was related to the precipitation of Pb21 ions as Pb(OH)2. By using the prepared system the maximum capacity was obtained at 19.60 mg/g in optimum conditions [204]. Due to the high number of research reports on the study of κ-car-based hydrogels in water treatment, only the results of a few cases are summarized in Table 14.6.

14.8 Effect of various factors on the adsorption capacity 14.8.1 Temperature The temperature of the solution is one of the main effective parameters for the adsorption of heavy metal or dyes, therefore a decrease or increase in temperature will change the

Application of polysaccharide-based hydrogels for water treatments 437 Table 14.6: The adsorption capacity of κ-carrageenan (κ-car)-based hydrogels for various dyes and heavy metals removal from water. Adsorbent Carrageenan/multiwalled carbon nanotube hybrid hydrogel Magnetic kappa-carrageenan/poly(vinyl alcohol) (PVA) nanocomposite hydrogels Kappa-carrageenan/poly(glycidyl methacrylate) hydrogel beads Ionically cross-linked magnetic chitosan/κ-carrageenan (κ-car) Chitosan-cross-linked κ-carrageenan Magnetic and chitosan-cross-linked κ-carrageenan CarAlg/MMt nanocomposite hydrogels Magnetic and K1-cross-linked kappa-carrageenan nanocomposite beads Carrageenan-based hydrogel nanocomposites containing laponite RD Kappa-carrageenan biopolymer-based nanocomposite hydrogel Kappa-carrageenan-g-polyacrylic acid/TiO2
NH2 hydrogel nanocomposite Carrageenan-based nanocomposite containing montmorillonite Kappa-carrageenan wet beads Carrageenan dried beads Kappa-carrageenan/acrylic acid (AA) hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel Kappa-carrageenan/AA hydrogel

Maximum adsorption capacity (mg/g)

Dye or metal ions

References

118

CV

[35]

78.2

CV

[30]

166.62

MB

[205]

280

[12]

130.4 123.1 85 84.7

Eriochrome black-T MB MB CV CV

[206] [206] [175] [201]

79.8

CV

[31]

344

MB

[207]

833

MG

[208]

46.4

CV

[209]

52 44 172 202 202 216 221 230 239 244

CV CV Fe Pb Mn Zn Cu Sr Cd Al

[210] [210] [211] [211] [211] [211] [211] [211] [211] [211]

equilibrium adsorption capacity. In most metal ions, an increase in the solution temperature decreased the adsorption capacity. This observation is seen from the fact that the increase in temperature increases the mobility of metal ions, hence desorption of metal occurs. Apart from the exothermic adsorption of metal ions in some works, metal adsorption occurred in an endothermic nature. The explanation for this behavior is that more active sites with lower resistance for metal ions with an increase in temperature lead to more adsorption. In the case of dyes, any change in the temperature changes the mobility and solubility of dyes and, depending on the dye and absorbent nature, both endothermic and exothermic behaviors are observed [212].

438 Chapter 14

14.8.2 Adsorbent dosage The optimum adsorbent dosage is a key parameter, which affects the amount of adsorbed adsorbate. The surface area increases with increasing adsorbent dosage. In order to avoid consuming an excess amount of adsorbent, the finding an optimal dosage is necessary. Several research groups have investigated the influence of adsorbent dosage on dye or metal removal by varying the adsorbent dosage concentration. Commonly, the removal efficiency increases by increasing the amount of adsorbent dosage, due to the increasing number of accessible active sites of adsorbent. However, after a certain adsorbent dosage, the adsorption capacity remains constant. This constant is generally due to the presence of a large number of accessible surface-active groups compared to the adsorbate amount [213,214].

14.8.3 pH pH can have a very important role in the entire adsorption process. The effect of pH on the adsorption process is dependent on the chemical structure of the adsorbent and pollutant and the optimum pH is determined based on these two parameters. Varying the solution pH changes the adsorption capacity by changing the ionization degree and surface charge of the adsorbent. For instance, study of the pH effect on the adsorption of the AR 37 and AB 25 dyes using a chitosan bead in pH ranging from 2 to 12 showed that the adsorption of AR 37 and AB 25 onto cross-linked chitosan beads drastically decreased with increasing pH. The protonation of amine-groups in acidic pH and the creation of an electrostatic attraction between adsorption sites and anionic dye is a reason for the higher adsorption in acidic pH. However, in an alkaline medium more OH2 ions will compete with the anionic sulfonic groups of AR 37 and AB 25 to access the adsorption sites of chitosan beads, hence the adsorption capacity is decreased [47]. Moreover, in many cases, the removal of one metal ion from an aqueous solution is very pH dependent. Generally, the adsorption of cations is favored at pH . pHPZC, while the adsorption of anions is favored at pH , pHPZC. pHPZC is the pH value at the pHpzc [20].

14.8.4 Time A study of the plot of the adsorbate adsorption against time shows that the adsorption process commonly occurs in three stages: first, rapid adsorption occurs, subsequent adsorption becomes slow, and finally, adsorption reaches the equilibrium state and remains constant. The high adsorption capacity in the first step could be related to the rapid attachment of adsorbate molecules to the surface adsorbent through surface mass transfer. The second step is slower and probably related to occupying the available external sites in the first step. Finally, the accessible adsorption sites become rarer in the third step, causing the equilibrium state [214].

Application of polysaccharide-based hydrogels for water treatments 439

14.8.5 Adsorbate initial concentration The initial adsorbate concentration is another influencing factor which affects the adsorption capacity. An increase in the initial adsorbate concentration increases the dye removal capacity. In fact, in solution with a higher dye concentration, there are higher concentration gradients at the hydrogel
solvent interface that cause the dye to easily moving into the hydrogel from the dye solution and resulting in high removal capacity [37].

14.9 Adsorption kinetics Adsorption kinetics is the study of the adsorption process rate. The controlling step and mechanism of the adsorption process are determined by study of the adsorption kinetics. Adsorption commonly occurs in three steps: (1) migration of adsorbate molecules to the outer surface of adsorbent, (2) diffusion of adsorbate to the boundary layer, and (3) finally diffusion from the adsorbent surface into internal adsorbent sites via pore diffusion [206]. In order to study the adsorption kinetics, the pollutant concentration in supernatants is determined with using a UV
vis spectrophotometer at λmax of dye after different time intervals. The pseudo-first-order (PFO) kinetics, pseudo-second-order (PSO) kinetics, and intramolecular diffusion kinetics models are commonly used models for study of the rate and mechanism of the adsorption process. The fitness of the adsorption mechanism with each of the models is determined by the coefficient of determination (R2) [52].

14.9.1 Pseudo-first-order model PFO kinetics is known as the Lagergren rate equation. In this model, the difference between the amount of adsorbed adsorbate molecules on adsorbents at equilibrium adsorption time and a defined time is determined by the adsorption process rate [206]. Eq. (14.4) is used for determination of the adsorption process in accordance with PFO kinetics model. Logðqe 2 qt Þ 5 Logqe 2

k1 t 2:303

(14.4)

In Eq. (14.4) qt (mg/g) is the amount of adsorption capacity at time t (min), k1 is the rate constant of the PFO kinetic model (min21) and qe (mg/g) is the amount of absorption capacity at equilibrium time (min). The plot of log(qe 2 qt) versus t gives a straight line and confirms the suitability of the PFO kinetic model for adsorption kinetics. In this model, adsorption depends only on the nature of the adsorbates [6,215].

440 Chapter 14

14.9.2 Pseudo-second-order model The PSO kinetics model reaction equation is known as the Ho and McKayrate equation. In this model, the sharing or exchanging electrons between adsorbent and adsorbate is supposed to be a rate-limiting step [206]. A linear form of the PSO kinetic model can be expressed by Eq. (14.5). t 1 t 5 1 qt k2 q2e qe

(14.5)

where qt (mg/g) is the amount of adsorption capacity at time t (min), qe (mg/g) is the amount of adsorption capacity at equilibrium time (min), and k2 (g/mg/min) is the rate constant of the PSO kinetic model. The adsorption at equilibrium time (qe) and rate constant (k2) can be obtained from the slope and intercept, respectively. The plot of t/qt against t gives a linear relationship and suggests the fitness of the PSO kinetics with the adsorption mechanism. Accordance with PSO kinetics proposes that the adsorption process is governed by chemisorption [6].

14.9.3 Intraparticle diffusion model While the PFO and PSO kinetic models are two effective models for determination of the adsorption process mechanism, these two models do not elucidate the diffusion mechanism of adsorbate molecules on adsorbents. The intraparticle diffusion model is an appropriate model to study the adsorption process. The diffusion mechanism of adsorbate on adsorbents can be seen by the intraparticle diffusion model proposed by Weber and Morris [206] (Eq. 14.6): qt 5 kid t0:14:5

(14.6)

where kid (mg/g/min20.5) is the intraparticle diffusion rate constant. If the plot of qt versus t0.5 gives a single straight line passing through the origin, the sorption process is controlled only by intraparticle diffusion, otherwise it is controlled by both interfacial and intraparticle diffusion where in this condition kid for each phase is obtained from the slope of linear plots in each part. The higher values of kid 1 compared to kid 2 suggest that the adsorption of adsorbate in the initial phase arose on external binding sites on the adsorbent, while diffusion to these sites is relatively rapid. By occupying the external active sites, dye molecules have a tendency to enter into the adsorbent. Hence, the adsorption shifts to the second phase of adsorption, where mainly in this state, adsorption occurs within the interior of pores [6]. Kinetics of the CV adsorption by kappa-carrageenan-g-poly(acrylamide)/ sepiolite nanocomposite hydrogels were studied by Mahdavinia et al. and the rate constant and mechanism of adsorption were investigated with PFO, PSO, and intraparticle diffusion kinetic models. The obtained parameters for the PFO and PSO rate for CV adsorption onto

Application of polysaccharide-based hydrogels for water treatments 441 Table 14.7: The pseudo-first-order (PFO) and pseudo-second-order (PSO) rate parameters of crystal violet (CV) adsorption onto nanocomposites. First-order kinetics H NH5 NH10

Second-order kinetics 3

21

2

qe1 (mg/g)

qe, Exp. (mg/g)

k1 3 10 (min )

R

33.1 30.9 17.8

20.4 29.8 34.4

0.39 1.6 1.6

0.8853 0.7873 0.8786

qe2 (mg/g) k2 3 103 (g/mg/min) 20.4 31.5 35.6

0.87 1.34 2.13

R2 0.9529 0.9878 0.9972

Reproduced with permission from G.R. Mahdavinia, A. Asgari, Synthesis of kappa-carrageenan-g-poly(acrylamide)/sepiolite nanocomposite hydrogels and adsorption of cationic dye. Polym. Bull. 70 (8) (2013) 2451
2470 [32]

Figure 14.9 Intraparticle diffusion kinetics of crystal violet (CV) dye adsorption onto kappa-carrageenan-g-poly (acrylamide)/sepiolite nanocomposite hydrogels. Reproduced with permission from G.R. Mahdavinia, A. Asgari, Synthesis of kappa-carrageenan-g-poly(acrylamide)/sepiolite nanocomposite hydrogels and adsorption of cationic dye. Polym. Bull. 70 (8) (2013) 2451
2470 [32].

nanocomposites are given in Table 14.7. From the obtained results (high correlation coefficient R2 . 0.95 for PSO) it can be concluded that the CV adsorption kinetic for all nanocomposites has the best fitting with PSO and adsorption occurs through the chemisorption process. Fig. 14.9 shows the plotting of qt versus t0.5 as is clear from Fig. 14.9, the diffusion plot of CV onto nanocomposites occurred in a multilinear fashion containing two linear parts. The results are given in Table 14.7. In all of the nanocomposites, the kid values for second linear sections are smaller than the first section. This observation shows that the dye adsorption at the first section is faster than the second section, which could be related to the ease of availability of high adsorption centers at initial times [32].

442 Chapter 14

14.10 Adsorption isotherm The study of the adsorption equilibrium isotherm models is essential for determination of the type of interactive behavior between adsorbents and solutes and provides valuable information to assess the adsorption capacity of an adsorbent. For the isotherm study, the adsorbent is added into the solution of pollutant with different initial concentrations. After achieving the adsorption equilibrium, the equilibrium concentration of a pollutant in the aqueous phase is measured. There are many isotherm models to describe the equilibrium relationship between the adsorbent and adsorbate, such as the Temkin, Sips, Freundlich, and Langmuir isotherm models. The Freundlich and Langmuir isotherm models are commonly used models to study the equilibrium of the removal process for the determination of the maximum adsorption capacity. The correlation of the equilibrium data with each one of the equations shows its match with the absorption process and determines the interaction type between the adsorbate and adsorbent [13,48].

14.10.1 Freundlich isotherm Eq. (14.7) shows the linear form of the Freundlich isotherm model. In this isotherm model, it supposes that the adsorption occurs within sites that are heterogeneous in energy. 1 Lnqe 5 LnKf 1 LnCe n

(14.7)

In the above equation, qe is the equilibrium adsorption capacity of the adsorbent (mg/g), Kf is the Freundlich parameters and represents the measure of adsorption capacity [(mg/g)(L/ mg)n], Ce is the equilibrium concentration of the solute in the solution (mg/L), and 1/n is the Freundlich parameter and shows the adsorption intensity. If 1/n is less than 1.0, the adsorption intensity is favorable over the entire range of the studied concentration. However, if 1/n is more than 1.0, the adsorption intensity is favorable at a higher concentration and much less at a lower concentration. By drawing the chart of Ln qe versus Ln Ce the qe, KF, n, and Ce can be determined. The Freundlich model is in accordance with the adsorbent with the heterogeneous site and multilayer adsorption process [38,160].

14.10.2 Langmuir isotherm In the Langmuir isotherm, all adsorption sites are equivalent in energy. Eq. (14.8) is the linear form of the Langmuir isotherm model and is used for the study of the agreement or disagreement of the absorption process with this model. Linear regression of Ce/qe to Ce, in which 1/qm is the slope and 1/KLqm is the intercept, was used for determination of the Langmuir isotherm parameters.

Application of polysaccharide-based hydrogels for water treatments 443 Ce 1 Ce 5 1 bqm qe qm

(14.8)

In this model, Ce is the concentration of solute in solution at equilibrium time (mg/L), qe is the amount of adsorption capacity at equilibrium time (mg/g), qm is the maximum adsorption capacity (mg/g), and b is the affinity of solute to the adsorbent that it is related to the adsorption free energy (L/mg). The higher amount of b shows that the adsorption requires less free energy. Accordance of the adsorption with the Langmuir isotherm model shows that the adsorbent surface is homogeneous or uniform and adsorption occurs as a monolayer. The shape of the Langmuir isotherm is identified with the dimensionless RL parameter (Eq. 14.9). RL 5

1 1 1 bC0

(14.9)

where b is the Langmuir parameter (mg/g) and C0 is the initial concentration of the solute (mg/L). The result depends on the shape of the isotherm: R 5 0 indicates that the adsorption is irreversible, R 5 1 linear, R . 1 unfavorable, and 1 , RL . 0 indicates that conditions are favorable for adsorption [38]. Irani et al. [160] prepared polyethylene-g-poly (acrylic acid)-co-starch/organomontmorillonite hydrogel to remove Pb(II) from the aqueous solution. The Langmuir and Freundlich isotherm plots of Pb(II) adsorption are shown in Fig. 14.10.

Figure 14.10 Freundlich (A) and Langmuir (B) isotherm plots of Pb(II) adsorption onto the polyethylene-g-poly (acrylic acid)-co-starch/organo-montmorillonite hydrogel. Reproduced with permission from M. Irani, et al., Synthesis of linear low-density polyethylene-g-poly (acrylic acid)-co-starch/organo-montmorillonite hydrogel composite as an adsorbent for removal of Pb(II) from aqueous solutions. J. Environ. Sci. 27 (2015) 9
20 [160].

444 Chapter 14 As it is clear from the plots, the Langmuir model with a correlation factor (R2 5 0.96) has a good accordance with experimental data in comparison with Freundlich model (R2 5 0.90) in the removal process of Pb(II). Compatibility with the Langmuir model indicates the monolayer coverage of lead ions on the adsorbent. The value of RL was obtained in the range of 0
1 which confirms the favorable uptake of Pb(II).

14.11 Reusability of adsorbent The reusability and regeneration ability of an adsorbent could decrease the pollutant removal costs and provide economical water treatment processes. Desorption studies are a good technique to verify the reversibility of the adsorption process and the reusability of adsorbent. For this purpose, after the first dye removal by the adsorbent, it is eluted and washed successively, then dried and desorption studies are carried out on it. The adsorption
desorption cycles are repeated several times with the same adsorbent. Reusability is usually determined during definite consecutive cycles under optimum conditions, and before each cycle a fresh dye solution is used. The number of the adsorption process with adsorption capacity near to the initial value determine the number of continuous adsorption and stripping cycles which adsorbent could be used without decrease in its efficiency. The higher number of the reusable cycles for an adsorption system lead to the more decrease in the process cost, less creation of secondary wastes, and finally better elucidating the adsorption mechanism [41,52,59,216].

14.12 Conclusion Heavy metal ions and dyes are examples of pollutants which when disposed of in the environment cause to serious problems to microorganisms and humans. To eliminate these hazards materials, several methods have been developed. Adsorption is a popular method for the removal of heavy metal ions or dyes due to advantages such as ease of the process, low cost, and the prevention of secondary waste production. Various adsorbents have been assessed for pollutant removal. Polysaccharide-based hydrogels are a good candidate for water treatment through the adsorption method, because of their biocompatibility, low cost, degradability, and having more functional groups in their structure. This chapter has revealed the significant progress in the use of biosorbents based on chitosan, cellulose, starch, κ-car, NaAlg, as well as their modified forms. These adsorbents are low cost, widely available, and potentially effective for the purification of water or wastewater contaminated with heavy metals or dyes. Moreover, the effects of several parameters on the adsorption capacity, adsorption kinetics, and adsorption isotherm have been presented.

Application of polysaccharide-based hydrogels for water treatments 445

14.13 Acknowledgments The authors gratefully acknowledge the University of Tabriz and Research Center for Pharmaceutical Nanotechnology (RCPN) of the Tabriz University of Medical Science for financial support for this research.

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134.

CHAPTER 15

Historical view of hydrogel characterization Nour Elhouda Ben Ammar1,*, Mohamed Barbouche2 and Ahmed Hichem Hamzaoui1 1

Laboratory of Useful Materials Valuation, National Center of Research in Material Science (CNRSM), Soliman, Tunisia 2Laboratory of Nanomaterials and Systems for Renewable Energy, Research and Technologies Centre of Energy, Hammam-Lif, Tunisia

15.1 Introduction Hydrogels are defined as superabsorbent polymers (SAPs) which are moderately crosslinked. They are characterized by a 3-D hydrophilic network which makes them able to absorb and conserve considerable amounts of aqueous fluids even under a certain amount of heat or pressure. In fact, hydrogels were previously called “hydrophilic gels” and outlined as polymeric materials having a three-dimensional network and presenting in their structure some hydrophilic groups as mentioned by Klement in 1960 [1]. The first cross-linked network material that appeared in the literature and has been described by its typical hydrogel properties, especially its high water affinity, was a polyhydroxyethyl methacrylate hydrogel developed in 1960 [2]. Later they were referred to as SAPs. This name was attributed first to poly(acrylic acid)based hydrogels developed by Weaver in the 1970s for use as feminine towels [3]. After that and for more than 45 years, hydrogels experienced a lot of changes and were used in a multitude of applications as mentioned in previous chapters dependent on their properties. Hydrogel polymer network origins fall into three main categories, synthetic polymers, natural polymers, and hybrid materials with both synthetic and natural elements. Synthetic polymers that have been commonly employed in hydrogel studies incorporate poly(ethylene oxide) [although sometimes poly(ethylene glycol), abbreviated as PEG, when the molecular mass is below 20,000 g/mol], poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol) (PVA), and poly(acrylamide) (PAAm). Natural polymer hydrogels consist of proteins, such as collagen and silk, denatured proteins, such as gelatin, and polysaccharides, such as agar and alginate. Both synthetic and natural gels can be multicomponent, either as


corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00017-3 © 2020 Elsevier Inc. All rights reserved.

459

460 Chapter 15 grafted block copolymers or as interpenetrating networks of two or more independent polymers. Another mechanism for generating hybrid multicomponent materials is the grafting of a biological recognition motif, such as the arginineglycineaspartic acid cell recognition sequence found in many cell-adhesive proteins such as integrins to a synthetic polymer in order to improve cellular biocompatibility with the gel network. Synthetic polymers are known to have poor biocompatibility and biodegradability, while natural polymers have weak mechanical properties. Therefore creating hybrid systems improves biocompatibility and increases the mechanical strength of gels network. The first striking property of hydrogels is their water retention capacity; it has been essential to evaluate this capacity since their first appearance. The evaluation of water uptake is the principal assay to be performed on hydrogel samples, as it can be a measure for many of their properties: cross-linking degree, mechanical properties, degradation rate, etc. For many gels, the evaluation of swelling and swollen state stability is the simplest, cheapest, and most accurate way to discriminate between cross-linked gels and noncrosslinked original polymers. These techniques have been used with natural and synthetic hydrogels and, given their simplicity and low cost, their use persists despite the development of other characterization tools. In the following, we describe some methods used for water uptake measurement including determination of the gel fraction, water retention, and release capacity. These measurements will allow the determination of their structural and physicochemical properties. After that, we describe the history of network structure characterization, and discuss the determination of porosity and estimation of hydrogel heterogeneity.

15.1.1 Gel fraction study In order to determine the gel content of a given material, a few methods have been proposed. According to the first one, one measures the insoluble portion in a dried sample after immersion in distilled water for 16 hours [4], 48 hours [5], or a week [6] at room temperature, while changing the water every 24 hours. The sample should be prepared at a diluted concentration (B1%) to ensure that the hydrogel is completely dispersed in the water. The gel fraction is then calculated as follows: % Gel 5

Wg 3 100 Ws

(15.1)

where Wg is the mass of the dry gel after synthesis and before swelling and Ws is the mass of dry gel after removal of nonreactive species. The second proposed method to determine gel fraction was used by Plungpongpan et al. on polyvinylpyrrolidone (PVP)/methyl hydroxyethyl cellulose blended hydrogels [7]. After weighing the dried hydrogels, they are put into a 4 3 4 cm sieve. This is immersed in hot

Historical view of hydrogel characterization 461 distilled water at 80 C for 1 hour to remove the soluble impurities in the aqueous phase. The hydrogels are then collected and immersed in ethanol for 15 minutes to remove organic impurities. The samples are finally dried in an oven at 60 C for 48 hours and then weighed to determine the gel content defined by the following equation: % Gel fraction 5

wd 2 ws 3 100 wi 2 ws

(15.2)

where wd is the weight of the dried gel after extraction, ws is the weight of the sieve, and wi is the initial weight of the dried gel

15.1.2 Swelling ratio measurement Determination of water retention capacity is primordial to evaluate the hydrogel properties. The first method that can be used is Japanese Industrial Method K8150, which was used to measure the swelling of hydrogels [5]. It consists of immersing the dry hydrogel in distilled water for 48 hours at room temperature with magnetic stirring. After swelling, it is filtered using a 30 mesh stainless steel net (681 μm). The swelling is calculated as follows:
 wf 2 wi S5 3 100 (15.3) wi where Wf and Wi are the weight of swollen and dry gels, respectively. Another alternative for measuring the degree of hydration is based on the dispersion of the dry hydrogel (0.050.1 g) in sufficient water (2530 mL) for 48 hours at room temperature [4]. The mixture is then centrifuged to obtain the hydrated hydrogel layers. The free water is removed and the swelling can be calculated as follows [8]: % Swelling 5

B 3 100 C

(15.4)

where C is the weight of hydrogel obtained after drying and B is the weight of the insoluble part after extraction with water. Another method to measure the swelling ratio is based on Japanese Industrial Standard K7223 [4]. The dry gel is immersed in distilled water for 16 hours at room temperature. After swelling, the hydrogel is filtered using a 149-μm stainless steel screen. The swelling is calculated according to Eq. (15.4).

15.1.3 Weight loss measurement The monitoring of hydrogel dehydration is an important parameter as it makes it possible to manage the release of water or of active substances retained inside the network. This can be done by gravimetric measurement. The hydrated gel is filtered to remove excess water, its

462 Chapter 15 mass is measured, and then it is put back in a constant temperature oven to follow the evolution of the deswelling percentage as a function of time according to Eq. (15.5) [9]:
 mf 3 100 (15.5) % deswelling 5 mi where mi is the initial weight and mf is the final weight.

15.2 Comparative study between previous and current characterization techniques 15.2.1 Determination of bounded and free water The nature of water in hydrogels may be important in controlling the solute diffusional mechanism in swelling-controlled release systems, as reported by Cheon et al. [2], and through hydrogel membranes, as reported by Li et al. [10]. The physical properties of water inside hydrogels change because of the extensive hydrogen bonding and polar interactions with the polymer. This change controls the solute diffusion and partition coefficients. One of the structural models of water proposed by Khare and Peppas [11] indicates the presence of three different classes of water in any hydrogel. In fact, when a dry hydrogel begins to absorb water, the first water molecules entering the matrix will hydrate the most polar, hydrophilic groups, leading to “primary bound water.” As the polar groups are hydrated, the network swells, and exposes hydrophobic groups, which also interact with water molecules, leading to hydrophobically bound water, or “secondary bound water.” Primary and secondary bound water are often combined and simply called “total bound water.” The network will then imbibe additional water, due to the osmotic force toward infinite dilution. This additional swelling is opposed by the covalent or physical cross-links, leading to an elastic network retraction force. Thus, the hydrogel will reach an equilibrium swelling level. The additional swelling water that is imbibed after the ionic, polar, and hydrophobic groups is called “free water,” and is assumed to fill the space between the network chains, the center of larger pores, macropores, or voids. In order to determine the nature of water linking within the network, many techniques have been proposed. The simplest way to estimate it is using the solute dosage. This method is based on the use of small molecular solutes. When solute molecules or probes are used, the labeled solution is equilibrated with the hydrogel, and the concentration of the solute molecule in the gel at equilibrium is measured. This method assumes that only the free water in the gel can dissolve the considered solute, and the free water content can be calculated from the amount of imbibed solute molecule and the measured molecule concentration in the external solution. The bound water is then obtained by measuring the difference between the total water content of the hydrogel and the calculated free water content [12,13].

Historical view of hydrogel characterization 463 This method is clearly much simpler than current methods, but its accuracy depends on the solubility of the solute in the water and the nonintervention of the structural water in the solubilization process. It should be noted also that this method assumes that the solute does not interact with the gel matrix chains. Nowadays, many other techniques are used and seem to be more efficient in the determination of water interaction with hydrogel network, for example, the use of pulsed Hnuclear magnetic resonance (H-NMR). This instrument operates by subjecting the sample to a radio frequency pulse in the presence of a magnetic field [14,15]. Since the solid-state hydrogen will return to equilibrium much more rapidly than those in the liquid state, the instrument can give direct information on bound and free water content by measuring the T2 relaxation time. Ben Ammar et al. [16] used pulsed H-NMR to determine the limit of bound water in PVP/agar hydrogel. Fig. 15.1 illustrates the measurement of the relaxation time as a function of the percentage of hydration for a PVP/agar hydrogel. It is clear that T2 remains constant up to a value close to 20% of hydration. Beyond this value, a considerable evolution of T2 is observed, eventually stabilizing after 150% hydration. In fact, at hydration rates of less than 20%, the total amount of water added will be retained by means of hydrogen bonds or polar interactions, so that the relaxation times do not exceed 0.5 ms, almost equal to the relaxation time of the dry hydrogel. The added water then behaves like structural water linked to the network, also called primary bound water. Beyond 20%, the value of T2 begins to grow rapidly, translating a significant

Figure 15.1 Effect of polyvinylpyrrolidone concentration on the hydrogel relaxation time.

464 Chapter 15 freedom of movement of the water molecules in the network and called secondary bound water. As a result, the connection of the water with the network weakens more and more and approaches the behavior of free water or bulk water [16]. A volume of 20% characterizes the limit of bounded water in the structure. Another used technique is differential scanning calorimetry (DSC). It is known that DSC monitors the gross phase changes of water in polymers. When a hydrogel sample is cooled below 0 C, the free and interracial water in hydrogel network freeze but the bound water remains in the nonfrozen state, since it is strongly associated with the polymer chains. DSC measures the freezable water, and the remaining water in the concentrated polymer phase is unfrozen. This unfrozen water fraction is herein termed the bound water fraction. The peak at lowest temperature in the DSC thermogram of a hydrogel is attributed to the freezing of interracial water at a temperature significantly lower than 0 C. The second fusion peak which appears at 0 C is due to the free water portion of the hydrogel water [11]. Khare and Peppas used DSC to study water in ionic copolymeric hydrogel networks of poly (2-hydroxyethyl methacrylate-co-methacrylic acid) and poly(2-hydroxyethyl methacrylateco-acrylic acid) [11]. Also, Kyritsis et al. used this technique to study polymerwater interactions in poly(hydroxyethyl acrylate) hydrogels coupled with dielectric, calorimetric, and sorption isotherm measurements [13], etc. Then the bound water is obtained by the difference in the measured total water content of the hydrogel test specimen, and the calculated free water content [17].

15.2.2 Hydrogel network characterization Properties of superabsorbent hydrogels, whether natural or synthetic, depend mainly on the network parameters. For this purpose, it is very important to determine the degree of cross-linking, the molecular weight between cross-links, and the size of the pores to be able to interpret the behavior of the synthesized material. Many methods were developed since the appearance of hydrogels to enable network parameter characterization. Because by controlling the degree of cross-linking, it is possible to tune the property of the material and optimize it for many different applications getting theoretically, in this way, a wide spectrum of applications starting from the same original polymer to modified and enhanced polymers. 15.2.2.1 Cross-link density Previously, and in order to estimate the cross-linking degree, Charlesby based his experiments on solgel analyses and proposed an equation designated by the CharlesbyPinner equation, which, based on a simple solgel analysis, allows determining

Historical view of hydrogel characterization 465 the quotient of the degradation and cross-linking [18] without being able to determine the exact cross-linking density. This method was used on cross-linked hydrogels using the gamma radiation technique and the proposed equation is as follow [19]: s1

pffiffi p0 2 s5 1 q0 uw;0 D q0

(15.6)

where s is the soil fraction s 5 1 2 gel; p0 is the average chain splitting per unit of monomer and per unit of dose; q0 is the proportion of cross-linked monomer unit per unit of dose; uw,0 is the average degree of polymerization of the initial mass; D is the irradiation dose or concentration of the cross-linking agent. pffiffi s 1 s 5 f ð1=DÞ gives an increasing curve with a horizontal and we can   asymptote  determine p0 =q0 by drawing the tangent to this curve. p0 =q0 corresponds to the intersection of this tangent with the x-axis. This equation has limitations since it is only valid for polymers whose molecular weight distribution is known. For this, another more generalized equation has been proposed by Olejniczak et al. [20], which makes it possible to trace the relationship between the soil fraction and the dose, but this time in a straight line. This new formula is called the OlejniczakRosiakCharlesby equation:

 pffiffi p0 Dv 1 Dgel p0 s1 s5 1 22 (15.7) q0 q0 Dv 1 D where Dv is the virtual dose, which is needed to change the molecular weight distribution of the polymer from a random distribution to its current distribution. This dose is an indicator of some existing deviations in the real system resulting from Charlesby’s proposal and linked to the distribution of the molecular mass and to the random formation of crosslinking nodes. Dv may be determined using the following equation:
 4 1 1 Dv 5 2 3q0 2u1 u2

(15.8)

where u1 is the weight and u2 is the average molecular weight. Dgel is the gelation dose, which corresponds to the dose at appearance of the first insoluble fraction of the polymer. Given the presence of several unknowns while having only two variables: the fraction sol (s) and the dose (D), it is imperative to pass through appropriate software in order to determine the values of Dv, Dgel, q0, and p0.

466 Chapter 15 This modified equation of Olejniczak makes it possible to plot the results in the form of a straight line, thus to obtain Dgel and p0 =q0 more easily. This equation has been confirmed by the analysis of the experimental results of several polymers including PVP cross-linked by gamma irradiation. The disadvantage of this method is that it considers that the cross-linking and degradation depend only on the irradiation dose or the concentration of the cross-linking agent in the case of chemical synthesis, without taking into account the flexibility and mobility of the polymer [for example in the case of poly(ethylene glycol)] [21]. In fact, the structure of the polymer, especially the flexibility of the polymer chain, is in close relationship with the probability of cross-linking and scission reactions and the cross-linking process, for example, the value of cross-linking increased with increasing cross-linking temperature as increasing temperature may result in increasing mobility of the polymer chain. In general, the cross-linking behavior was characterized by the relationship between sol fraction and radiation dose. In this context, Wanxi et al. [22] added another parameter to Charlesby’s equation which is exponent β of the dose. We thus obtain the ZhangSunQian equation:
  pffiffi 1 P0 β 1 (15.9) D D s1 s 5 q0 u1 q0 where P0 is a constant; β 5 0.206 1 0.02 Tg; β is a function of the glass transition temperature that characterizes the flexibility of the polymer. This equation was proved on many polymers such as polystyrene, PVA, natural rubber, etc. [22]. This equation is applicable over a wide range of flexible and rigid polymers, but its limit is that it always assumes random cross-linking and is only valid at a certain level of the total cross-linking process [23]. Another option to determine the exact value of cross-link density is based on the hydration experiments, basically simple and costless. In fact, Uzun et al. used the hydration experiment to determine the network structure and its cross-linking density in order to control the cross-linking of dimethylaminoethyl methacrylate in the presence of ethylene glycol dimethacrylate [24]. All measurements were made at room temperature. Each experiment was performed when the sample reached constant moisture content. Its mass was used to calculate the volume fraction of the polymer υ2m and the equilibrium degree of hydration Q using the following equation as mentioned also in Abd Alla et al.’s work on superabsorbent hydrogels based on tara gum/acrylic acid [25]. ! 1 11ρ    (15.10) Q5 5 υ2m ρw 1=w 2 1

Historical view of hydrogel characterization 467 The swelling coefficient Q is of fundamental importance since it defines many of the properties that are critical in hydrogels, including, but not limited to, the mechanical and transport properties. The swelling coefficient differs based on the polymer backbone chemistry and can vary widely between different polymers. ρ and ρw are the densities of dry gel and water, respectively. w is the mass of the gel in the relaxed or hydrated state for the determination of υ2m. Hence, the estimation of the cross-linking degree based on the solgel method makes it possible to obtain approximate values without being able to determine the exact experimental values. For this reason, nowadays researchers opt for determination of the cross-linking degree more precisely by rheological and mechanical measurements. In fact, the rheological properties are very dependent on the types of structure present in the system (association, entanglement, cross-links). Polymer solutions are essentially viscous at low frequencies. At high frequencies, the elasticity dominates (G0 . Gv). This corresponds to a Maxwell type behavior with a single relaxation time which can be determined from the point of intersection of the curves G0 and Gv. This relaxation time increases with concentration. For cross-linked microgel dispersions, G0 and Gv are almost independent of oscillation frequency [26]. This technique was used to evaluate the effect of cross-linking degree on the elasticity of PVP-based hydrogels [27] and to characterize the lattice structure in acrylic acid/locust bean gum hydrogels (AAcNa/LBG) [28], collagen-PVP-based hydrogels [29], and several other hydrogels [19,25]. In order to determine the cross-linking degree, it is necessary to start with a deformation sweep test at constant deformation rate and temperature, followed by a frequency sweep test at constant temperature and amplitude of deformation imposed. The estimation of the linear viscoelastic region using these tests allows a precise determination of the elastic modulus G0 and viscous modulus Gv. Indeed, the effective cross-link density ve is related to the elasticity modulus G0 by the following equation [28]. ρ ve 5   0 A ρ=GR RTðυ2r Þ2=3 ðυ2m Þ1=3

(15.11)

where ρ is the density of the polymer, υ2m is the volume fraction of the cross-linked polymer in equilibrium with the hydrated gel, and υ2r is the volume fraction of the polymer at the relaxed state and may be determined using Eq. (15.10). The prefactor A is equal to 1 for an affine network and 12/ϕ for a phantom network. The forced junction theory indicates that a real polymer network has properties closer to those of the phantom network model and that the number of branches from a cross-linking site is ϕ 5 3 [30,31]. Another technique to determine the effective cross-link density ve is based on uniaxial compression tests. The elastic properties and compression modulus of the hydrogel may be determined using this instrument. Before the start of each measurement, the gel is kept in

468 Chapter 15 distilled water at room temperature until it reaches its hydration equilibrium. Directly before the experiment, it is superficially dried with filter paper. Subsequently, the samples are cut into small cubes and the dimensions of each cube are fixed in the device before the start of each measurement. The uniaxial compression test makes it possible to estimate the amplitude of the deformation. The stress values (σ) were determined using Eq. (15.12) [32]: σ5

F A

(15.12)

where F is the force, and A the cross-section of the analyzed sample. The parameters generated by the instrument are force and displacement. This information is then converted into the compression modulus (G), using Eq. (15.13) [33] σ5

G λ 2 λ2

(15.13)

where λ 2 λ2 is the relative strain of the sample, and λ is the strain and is calculated from Eq. (15.14). λ5

ΔL L0

(15.14)

where ΔL is the deformation of the sample and L0 its initial height [30]. To determine thevalue of the compression modulus or elasticity G, we draw the curve    F 5 f 1= λ 2 λ2 for the initial stages of deformation. The value of G is calculated from the slope of the curves. Indeed, by using the values of the compression modulus determined during the uniaxial compression test, and based on the statistical theory of the elastic modulus of elasticity relating to a homogeneous network of Gaussian chains [25], the cross-link density can be determined using the following equation: ve 5

Gm ARTðυ2r Þ2=3 ðυ2m Þ1=3

(15.15)

where ρ is the density of the polymer, the perfactor A, equal 1 for an affine network and 12/ϕ for a phantom network. 15.2.2.2 Molecular weight between cross-links Several theories have been proposed to calculate the average molecular weight between the cross-linking nodes. Based only on hydration experiments it was easy to calculate this parameter. In fact, in the case of highly swollen polymers, the constrained junction theory

Historical view of hydrogel characterization 469 indicates that a real network has properties close to those of the phantom model. The following equation uses the phantom network model and is valid for natural or nonionic structures [34]. 2=3 1=3

ð1 2 2=φÞV1 v2m v2r Mc 5 vðlnð1 2 v2m Þ 1 v2m 1 χv22m Þ

(15.16)

where Mc is the average molecular weight of the network chains, v2m is the volume fraction of the cross-linked polymer in the hydrated state, v2r is the volume fraction of the cross-linked polymer in the relaxed state, that is, after cross-linking and before the start of hydration experiments, V1 is the molar volume of the solvent, χ is the solventpolymer interaction parameter, ϕ is the functionality, and v is the specific volume of the polymer. The solventpolymer interaction parameter χ of hydrogel is calculated using Eq. (15.17) [31]. χ5

1 v2m 1 2 3

(15.17)

The volume fraction of the polymer v2m is calculated using Eq. (15.10). On the other hand, based on rheological measurements, the average molecular weight between cross-links Mc of a cross-linked structure is directly dependent on the effective cross-link density according to the following equation: Mc 5

ρ ve

(15.18)

which can be obtained from the results of the rheological measurements using Eqs. (15.11) and (15.18). These same equations may be used also to determine Mc from uniaxial compression tests. 15.2.2.3 Mesh size The mesh size (ξ) defines the space between the macromolecular chains in a cross-linked network. It is usually characterized by the correlation length, or the distance between two adjacent cross-links [35]. This parameter may be also determined using rheological measurements or uniaxial compression tests by Eq. (15.19). 21=3 ξ 5 v2m




1=2 Mc Cn 2 l Mr

(15.19)

Cn in the previous equation is the Flory characteristic ratio or the rigidity factor of the hydrogel [36], l is the carboncarbon length (154 μm), and Mr is the molecular weight of the monomeric unit.

470 Chapter 15

Figure 15.2 Evolution of network parameters.

A comparative study between solgel, rheological, and mechanical compression tests used to characterize network parameters was done on PVP/agar hydrogels, according to Ben Ammar et al. [16]. Fig. 15.2 illustrates the difference between the average molecular weight value, the effective cross-link density, and the mesh size determined by the solgel method and those calculated from the rheological and mechanical experiments. As illustrated in Fig. 15.2, the magnitude of molecular weight between cross-links Mc and the effective cross-linking degree ve determined from mechanical and rheological experiments are close to each other. The differences were attributed to practical difficulties in the preparation of samples. In fact, hydrogel has a soft structure and it is not easy to cut samples into perfectly smooth cubic pieces for mechanical analysis. In addition, in order to execute the uniaxial compression tests, the initial force was chosen as 1 N to avoid the nonhomogeneity of the sample surface, and as a consequence, strain started before reaching this force is not considered and could affect the perfect similarity of results of mechanical analysis compared to rheological measurements. On the other hand, the rheological analyses were independent of the size and shape of the gel, providing more reproducible results. As for the solgel network characterization technique, a large difference between the values determined by this method and those by rheological and mechanical measurements was observed. In fact, this method is based only on a gravimetric measurement of the hydration capacity of hydrogels, which explains the large difference between the values

Historical view of hydrogel characterization 471 determined by this method and those by rheological and mechanical measurements. The significant difference in Mc value is mainly explained by the measurement of the swelling degree itself, because simple gravimetric measurements of swollen hydrogel are done after superficial drying of the sample with filter paper. This approach may lead to weighing errors which explain the Mc ’s values. This method is most appropriate in the case of a lack of advanced equipment, since it allows the estimation of the order of magnitude of network parameters and to compare the effective cross-linking degree between different samples based only on gravimetric measurements. Meanwhile dynamic rheological measurement seems to be the most accurate method because of its independence from the shape and size of samples. This allows having reproducible results. In addition, it makes it easy to determine the linear viscoelastic region with which one can determine the exact value of G0 by doing a simple strain sweep test followed by a frequency sweep test. Moreover, the values of ξ are in agreement with the observations by scanning electron microscopy (SEM). As far as the uniaxial compression test is concerned, the obtained values are close to those determined by rheological measurements. The difference between these values is mainly due to practical complications in sample preparation, which means that this method could be convenient for dealing with solid gel structures. Fig. 15.3 illustrates SEM images of previously studied hydrogels [16]. It is obvious that the order of magnitude of the pores is similar to those determined by rheological and mechanical measurements, but not the solgel characterization technique, which confirms moreover the conclusions carried out from this comparative study.

Figure 15.3 SEM images of polyvinylpyrrolidone/agar hydrogels shown at different magnifications (A) 10 μm and (B) 50 μm. SEM, scanning electron microscopy.

472 Chapter 15 15.2.2.4 Porosity of hydrogels Pores may be formed in hydrogels by phase separation during synthesis, or they may exist as smaller pores within the network. The average pore size, the pore size distribution, and the pore interconnections are important factors of a hydrogel matrix that are often difficult to quantify. These factors are most influenced by the composition and cross-link density of the hydrogel polymer network. Pore-size distributions of hydrogels depend on the cross-link nature of the hydrogel itself and are strongly affected by three factors. The first is the concentration of the chemical cross-links of the polymer strands in the case of covalent links. This concentration is determined by the initial ratio of cross-linker to monomer. The second parameter is the concentration of the physical entanglements of the polymer strands. This concentration is determined by the initial concentration of all polymerizable monomers in the aqueous solution. The final one is the net charge of the polyelectrolyte hydrogel, determined by the initial concentration of the cationic and/or anionic monomer. These three factors can be quantified using the composition of the hydrogel, that is, by the nominal concentrations of monomer and cross-linker. Most techniques to investigate the porosity of hydrogels are limited because they require the pore solvent and/or temperature to be altered, causing the gel to shrink, swell, or require mathematical manipulation and assumption, which may introduce unwanted artifacts. In fact, porosity is a morphological feature of a material that can be simply described as the presence of a void cavity inside the bulk. It is useful to control the porosity in many devices for a wide variety of applications, such as optimal cell migration in hydrogel-based scaffolds or tunable lode/release of macromolecules, and is defined by the following equation: Porosity% 5

Vpores 3 100 Vbulk 1 Vpores

(15.20)

In a sample, pores can show different morphologies: they can be closed, open as a blind end, or interconnected, again divided into cavities and throats. These porosities have been studied and evaluated in many papers in the past decades using various techniques, such as theoretical methods, like the unit cube analysis, mass technique, Archimedes method, and liquid displacement method. These analyses are commonly coupled with optical and electronic microscopy. Other interesting techniques include mercury porosimetry, based on Washburn’s equation, with the inconvenience of being a destructive assay, gas pycnometry, gas adsorption (that can be issued using different procedures such as small quantity adsorption, monolayer and multilayer adsorption), liquid extrusion porosity, an assay that permits to evaluate the sample’s permeability too, and capillary flow porosity, again a test

Historical view of hydrogel characterization 473 based on Washburn’s equation. Furthermore, another important assay is micro-computed tomography, also called X-ray microtomography, a relatively new imaging technique, simply described as a nondestructive high-resolution radiography, capable of qualitative and quantitative assays on samples and evaluation of their pore interconnections. Between the quantitative assays that can be performed, micro-CT can give information on average pore size, pore size distribution, pore interconnection, strut/wall thicknesses, and anisotropy/ isotropy of the sample (in the sense of presence/absence of preferential orientation of the pores). It remains an expensive technique both in terms of money and time [37,38]. Microscopy techniques can be used in thousands of different assays involving hydrogels. They are both involved in qualitative and quantitative tests, from simple morphological assessing of a material’s properties to more complex biocompatibility assays. Briefly, by microscopy techniques, topography and surface morphology can be assessed. These techniques can be divided into many classes, by increasing the magnification power: optical microscopy, SEM, transmittance electronic microscopy (TEM), tunneling microscopy (STM), atomic force microscopy, and environmental scanning electronic microscopy [38]. The porous structure of a hydrogel is also affected by the properties of the surrounding solution, especially by dissolved ionic solutes (Donnan effects) and by dissolved uncharged solutes which partition unevenly between the gel phase and the solution phase (osmotic effects). The amount of water in a hydrogel, that is, the volume fraction of water, and its free versus bound water “character” determine the absorption (or partitioning) and diffusion of solutes through the hydrogel. Labeled molecular probes of a known range of molecular weights (MW) were previously used to estimate pore sizes in hydrogels [17]. For example, fluorescein-labeled dextrans were used to calculate porosity [39]. Probe solute permeation is a useful method for characterizing pores and their interconnections in hydrogels [40]. The probe solute size and shape, its relative hydrophilic and hydrophobic character, and the availability of “free” water molecules to hydrate and dissolve the solute molecules are important factors governing solute permeation and must be taken into account.

15.2.3 Inhomogeneity of hydrogels Hydrogels are interesting materials for a variety of pharmaceutical and hygienic applications. Although the swelling behavior of polymer networks and the elasticity of such systems appear to be understood quite well, there are still questions concerning the structure of real networks and the structure development during the gelation process. In contrast to model networks with constant length of network chains between cross-links, real networks exhibit a wide distribution of chain lengths between network junction points. Furthermore, a variety of network defects, such as dangling ends, elastically ineffective loops, and

474 Chapter 15 cross-link agglomerations, are known to occur. These defects change the effective crosslinking density of the networks and thus influence their elastic properties and swelling behavior [41]. Unfortunately, the exact nature and quantity of network defects are very difficult to evaluate experimentally. In contrast to ideal gels with a homogeneous distribution of cross-links, real gels always exhibit an inhomogeneous cross-link density distribution, known as the spatial gel inhomogeneity [42]. Indeed, cross-linking junctions, necessary for obtaining an infinite three-dimensional network, render the gels intrinsically inhomogeneous. The inhomogeneous gel microstructure has a major effect on its properties, as well as on the interaction between the water and polymer network, which is not easy to explain and needs to be well understood. The gel inhomogeneities are of considerable interest and importance in attempts to characterize such materials physically. From a practical point of view, spatial inhomogeneity is undesirable because it dramatically reduces the optical clarity and strength of gels, which are properties closely connected with many industrial applications such as contact lenses, super absorbents, etc. On 1971, Richards investigated the inhomogeneity parameter of polyacrylamide gels [43]. She used N,N0 -methylenebisacrylamide (BIS) as a cross-linker, and used a spectrophotometer at 550 nm to measure the absorbance of the gels in order to collect information about the gel turbidity compared to a water solution. If the absorbance is significant, it rises rapidly at a lower wavelength, suggesting that scattering is the dominant attenuation process. After polymerization, the gel resembles a three-dimensional network immersed in a solvent, composed of PAAm chains joined together by cross-links by the BIS residue. Therefore if the network was perfect, there would be no free chain ends, loops, entanglements, or free BIS residues in the solution. For an ideal gel network, Richard supposed that the end-to-end distance for a growing chain follows a Gaussian distribution and that each BIS residue is connected to its four nearest neighbors in which the BIS residues are distributed randomly in space by a Poisson distribution [43]. Thus for an ideal gel:

 2M 0:589 3 1 C 3=2 T0 5 (15.21) N β C 12C where T0 is the total monomer concentration in an ideal gel, C is the proportion by weight of the cross-linker, N is Avogadro’s number, and M is the molecular weight of the monomer. The concept of an ideal gel divides gels into two types. Crumpled gels in which T . T0 and clustered gels in which T , T0. In a clustered gel, there are insufficient monomers to connect the cross-linker residues to their nearest neighbors. The monomer chains must either extend beyond their equilibrium mean end-to-end distance or the distribution of

Historical view of hydrogel characterization 475 cross-linker molecules must become nonrandom and clustered, which seems to be more probable. And in the case of crumpled gels, there is an excess of monomers and either the chains must be less extended or they must connect more distant cross-linker residue. Such a gel might be expected to relax back to a state resembling an ideal gel if allowed to swell to equilibrium in solvent. In the absence of entanglements, or similar effects, the volume would increase by a factor T/T0, though in practice a lower ratio is probable. It is unlikely that an ideal gels exist, but one might expect gels for which T is close to or below T0 to exhibit clustering and gels for which T is close to or above T0 to exhibit the swelling effect mentioned previously. No clear line of demarcation between crumpled and clustered gels should be apparent but the properties of a gel should show a steady transition as T decreases. In the case of dilute and swollen gels, the theory described by Flory gives the relation [36]: q 2q

2=3


 T 21 5v C

(15.22)

where v is the partial specific volume of the gel matrix and q is the volume expansion factor. For crumpled gels, the volume expansion factor should approach T/T0 but for clustered gels, it must depend on the statistical properties of clusters. In other words, a clustered gel would swell as if it were an ideal gel with the same total concentration but a reduced value of C [43]. These assumptions concerning the swelling properties of crumpled and clustered gels are admittedly drastic and simplistic; nevertheless, they may provide a qualitative picture of the behavior of real gels and their inhomogeneity features in the 1970s. After that, since the gel inhomogeneity is closely connected to the spatial concentration fluctuations, scattering methods such as inelastic light scattering and small-angle neutron scattering were used starting in the 1980s [44] to understand local conformation and dynamics of chains. Then, small-angle X-ray scattering was also employed to investigate the spatial inhomogeneities [45]. In fact, gel inhomogeneity can be manifested by comparing the scattering intensities from the gel and from a semidilute solution of the same polymer at the same concentration. The scattering intensity from gels is always larger than that from the polymer solution. The excess scattering over the scattering from the polymer solution is related to the degree of the inhomogeneities in gels [46]. As mentioned in Grube et al.’s work on hydrogels synthesized by thiol-ene polymerization [47], the inhomogeneity of hydrogels can be determined using dynamic light scattering. The time-averaged scattering intensities, hIiT, and the time-averaged intensity correlation functions (ICF) should be determined for a multitude of gel samples by randomly moving the cuvette rotation/translation unit before each run. In order to divide the ensemble-averaged scattering

476 Chapter 15 intensity, hIiE, the proposed method given by Joosten et al. and Shibayama of the partial heterodyne [48] proposed to use the time-averaged scattering intensities measured at the different positions, in its two components hI iE 5 hIF iT 1 hIC iE

(15.23)

where hIF iT is the time average of the fluctuating component arising from dynamic, liquidlike concentration fluctuations. This contribution is essentially ergodic. The static part, hIC iE , is due to spatial inhomogeneities possibly resulting from the cross-linking process. The latter is the quantity of interest to characterize the microstructure of the gels. From the time-averaged ICF measured at each position, apparent diffusion coefficients DA are estimated according to Eq. (15.24) [49]: DA 5 2

1 @ lim lnðg2T ðq; τ Þ 2 1Þ 2q2 τ-0 @τ

(15.24)

  where q 5 4πn=λ0 sinðθ=2Þ is the amplitude of the scattering vector with θ being the scattering angle, λ0 is the wavelength of the incident light in a vacuum, and n is the refractive index of the medium. For different sample positions, generally different values of DA were obtained, which came with different local scattering intensities hIiT. The relationship between DA and the cooperative diffusion coefficient, D, is proposed as follows [48]:
 hIF iT DA (15.25) D5 22 hI iT By plotting hI iT =DA versus hI iT , the data formed essentially a straight line, from whose slope and intercept the fluctuating component of the scattering intensity, hIF iT , as well as D were obtained. A sufficiently large number of data points (measurements at different positions) and perceptible variations of scattering intensity with position were, of course, needed in order to define the line with adequate accuracy and as a consequence determine hIC iE attributed to inhomogeneity [47].

15.3 Conclusion Recently, many hydrogel-based networks have been designed and tailored to meet the needs of different applications. The favorable property of these hydrogels is their ability to swell when put in contact with an aqueous solution. This property is related not only to the physicochemical structure of the hydrogel, but also its distribution and organization in space. Thus, the characterization of this conformation becomes essential to be able to interpret and predict the behavior of the hydrogel in a precise environment, and under

Historical view of hydrogel characterization 477 various circumstances. This chapter demonstrates the literature concerning techniques to evaluate water interaction with hydrogel networks, especially swelling capacity and gel fraction data. It also discussed previously used technologies to characterize water bounding into the hydrogel network compared to currently adopted techniques. The story of network structure characterization, means, the effective cross-link density, the average molecular weight between cross-links, and mesh size was also developed, starting with a hypothesis and equations to more precise and sophisticated techniques. A comparison between previous and currently used techniques was outlined. Finally, an estimation of inhomogeneity and porosity of hydrogels was discussed.

References [1] W. Klement, Hydrophilic gels for biological use r 1960 Nature Publishing Group, 1960. [2] S. Cheon, I. Keun, K. Park, Hydrogels for delivery of bioactive agents: a historical perspective, Adv. Drug Deliv. Rev. 65 (2013) 1720. Available from: https://doi.org/10.1016/j.addr.2012.07.015. [3] M. Fusayoshi, Trends in the development of superabsorbent polymers for diapers, Superabsorbent Polym., ACS Sympos, American Chemical Society, 1994, pp. 8898. Available from: https://doi.org/10.1021/bk1994-0573.ch007. [4] T. Katayama, M. Nakauma, S. Todoriki, G.O. Phillips, M. Tada, Radiation-induced polymerization of gum arabic (Acacia senegal) in aqueous solution, Food Hydrocoll. 20 (2006) 983989. Available from: https://doi.org/10.1016/j.foodhyd.2005.11.004. [5] N. Nagasawa, T. Yagi, T. Kume, F. Yoshii, Radiation crosslinking of carboxymethyl starch, Carbohydr. Polym. 58 (2004) 109113. Available from: https://doi.org/10.1016/j.carbpol.2004.04.021. [6] S. Benamer, M. Mahlous, A. Boukrif, B. Mansouri, S.L. Youcef, Synthesis and characterisation of hydrogels based on poly(vinyl pyrrolidone), Nucl. Instrum. Methods Phys. Res., Sect. B 248 (2006) 284290. Available from: https://doi.org/10.1016/j.nimb.2006.04.072. [7] K. Plungpongpan, K. Koyanukkul, A. Kaewvilai, N. Nootsuwan, P. Kewsuwan, A. Laobuthee, Preparation of PVP/MHEC blended hydrogels via gamma irradiation and their calcium ion uptaking and releasing ability, Energy Procedia 34 (2013) 775781. Available from: https://doi.org/10.1016/j. egypro.2013.06.813. [8] C. Sandeep, H.S.L. Kanupriya, Hydrogels: a smart drug delivery system, Int. J. Res. Pharm. Chem. 2 (2012) 603614. [9] H. Aoki, S. Al-Assaf, T. Katayama, G.O. Phillips, Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMt): Part 2—Mechanism of the maturation process, Food Hydrocoll. 21 (2007) 329337. Available from: https://doi.org/10.1016/j. foodhyd.2006.04.002. [10] X. Li, Q. Li, X. Xu, Y. Su, Q. Yue, B. Gao, Characterization, swelling and slow-release properties of a new controlled release fertilizer based on wheat straw cellulose hydrogel, J. Taiwan Inst. Chem. Eng. 60 (2016) 564572. Available from: https://doi.org/10.1016/j.jtice.2015.10.027. [11] A.R. Khare, N.A. Peppas, Investigation of hydrogel water in polyelectrolyte gels using differential scanning calorimetry, Polymer (Guildf.) 34 (1993) 47364739. Available from: https://doi.org/10.1016/ 0032-3861(93)90710-R. [12] L.H. Yahia, History and applications of hydrogels, J. Biomed. Sci. 4 (2015) 123. Available from: https://doi.org/10.4172/2254-609X.100013. [13] A. Kyritsis, P. Pissis, J.L. Go´mez Ribelles, M. Monleo´n Pradas, Polymer-water interactions in poly (hydroxyethyl acrylate) hydrogels studied by dielectric, calorimetric and sorption isotherm measurements, Polym. Gels Networks 3 (1995) 445469.

478 Chapter 15 [14] W. Kuhn, Investigation of Molecular Dynamics in Crosslinked Polymers Using NMR Methods-Crosslinking, Aging, Filler-Matrix Interactions, 4th Qingdao Intl. Rubber Plastic Forum, Qingdao, China, 2008. [15] M. Fratricova, P. Schwarzer, et al., 1H-NMR relaxation study of cross-linking and aging pro cesses in polyurethane coatings, KGK 59 (2006). ,http://cat.inist.fr/?aModele 5 afficheN& cpsidt 5 17795774.. [16] N.E. Ben Ammar, T. Saied, M. Barbouche, F. Hosni, A.H. Hamzaoui, M. Sen, ¸ A comparative study between three different methods of hydrogel network characterization: effect of composition on the crosslinking properties using solgel, rheological and mechanical analyses, Polym. Bull. (2017). Available from: https://doi.org/10.1007/s00289-017-2239-0. [17] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 64 (2012) 1823. Available from: https://doi.org/10.1016/j.addr.2012.09.010. [18] A. Charlesby, S.H. Pinner, Analysis of the solubility behaviour of irradiated polyethylene and other polymers, Proc. R. Soc. A Math. Phys. Eng. Sci. 249 (1959) 367386. Available from: https://doi.org/ 10.1098/rspa.1959.0030. [19] M. Sen, ¸ H. Hayrabolulu, P. Ta¸skın, M. Torun, M. Demeter, M. Cutrubinis, et al., Radiation induced degradation of xanthan gum in the solid state, Radiat. Phys. Chem. 124 (2016) 225229. Available from: https://doi.org/10.1016/j.radphyschem.2015.10.005. [20] C.A. Olejniczak, J. Rosiak, Gel/dose curves for polymers undergoing simultaneous cross-linking and scission, Int. J. Radiat. Appl. Instrum. Part C Radiat. Phys. Chem. 38 (1991) 113118. Available from: https://doi.org/10.1016/1359-0197(91)90052-4. [21] L.F. Miranda, A.B. Luga˜o, L.D.B. Machado, L.V. Ramanathan, Crosslinking and degradation of PVP hydrogels as a function of dose and PVP concentration, Radiat. Phys. Chem. 55 (1999) 709712. Available from: https://doi.org/10.1016/S0969-806X(99)00216-9. [22] Z. Wanxi, H. Tianbai, S. Jiazhen, Q. Baogong, A general equation for the relationship between sol fraction and radiation dose in radiation crosslinking, Int. J. Radiat. Appl. Instrum. Part C Radiat. Phys. Chem. 33 (1989) 581584. Available from: https://doi.org/10.1016/j.ydbio.2003.10.018. [23] K. Makuuchi, Critical review of radiation processing of hydrogel and polysaccharide, Radiat. Phys. Chem. 79 (2010) 267271. Available from: https://doi.org/10.1016/j.radphyschem.2009.10.011. [24] C. Uzun, M. Hassnisaber, M. Sen, ¸ O. Gu¨ven, Enhancement and control of cross-linking of dimethylaminoethyl methacrylate irradiated at low dose rate in the presence of ethylene glycol dimethacrylate, Nucl. Instrum. Methods Phys. Res., Sect. B 208 (2003) 242246. Available from: https:// doi.org/10.1016/S0168-583X(03)01112-1. [25] S.G. Abd Alla, M. Sen, A.W.M. El-Naggar, Swelling and mechanical properties of superabsorbent hydrogels based on Tara gum/acrylic acid synthesized by gamma radiation, Carbohydr. Polym. 89 (2012) 478485. Available from: https://doi.org/10.1016/j.carbpol.2012.03.031. [26] A. Omari, R. Tabary, D. Rousseau, F.L. Calderon, J. Monteil, G. Chauveteau, Soft water-soluble microgel dispersions: structure and rheology, J. Colloid Interface Sci. 302 (2006) 537546. Available from: https:// doi.org/10.1016/j.jcis.2006.07.006. [27] G.J.M. Fechine, J.A.G. Barros, M.R. Alcaˆntara, L.H. Catalani, Fluorescence polarization and rheological studies of the poly(N-vinyl-2-pyrrolidone) hydrogels produced by UV radiation, Polymer (Guildf.) 47 (2006) 26292633. Available from: https://doi.org/10.1016/j.polymer.2006.02.001. [28] M. Sen, ¸ H. Hayrabolulu, Radiation synthesis and characterisation of the network structure of natural/ synthetic double-network superabsorbent polymers, Radiat. Phys. Chem. 81 (2012) 13781382. Available from: https://doi.org/10.1016/j.radphyschem.2011.11.069. [29] M. Demeter, M. Virgolici, C. Vancea, A. Scarisoreanu, M. Georgiana, A. Kaya, et al., Network structure studies on γirradiated collagenPVP superabsorbent hydrogels, Radiat. Phys. Chem. 131 (2017) 5159. Available from: https://doi.org/10.1016/j.radphyschem.2016.09.029. [30] M. Sen, ¸ O. Agu¸s, A. Safrany, Controlling of pore size and distribution of PDMAEMA hydrogels prepared by gamma rays, Radiat. Phys. Chem. 76 (2007) 13421346. Available from: https://doi.org/10.1016/ j.radphyschem.2007.02.028.

Historical view of hydrogel characterization 479 [31] J.E. Mark, Molecular aspects of rubberlike elasticity, Die Angew. Makromol. Chem. 202 (1992) 130. Available from: https://doi.org/10.1002/apmc.1992.052020101. [32] R.C.M. de Paula, J.F. Rodrigues, Composition and rheological properties of cashew tree gum, the exudate polysaccharide from Anacardium occidentale L, Carbohydr. Polym. 26 (1995) 177181. Available from: https://doi.org/10.1016/0144-8617(95)00006-S. [33] M.L. Oyen, Mechanical characterisation of hydrogel materials, Int. Mater. Rev. 59 (2014) 4459. Available from: https://doi.org/10.1179/1743280413Y.0000000022. [34] N. Mahmudi, M. Sen, ¸ S. Rendevski, O. Gu¨ven, Radiation synthesis of low swelling acrylamide based hydrogels and determination of average molecular weight between cross-links, Nucl. Instrum. Methods Phys. Res., Sect. B 265 (2007) 375378. Available from: https://doi.org/10.1016/j.nimb.2007.09.007. [35] A.S. Hickey, N.A. Peppas, Mesh size and diffusive characteristics of semicrystalline poly(vinyl alcohol) membranes prepared by freezing/thawing techniques, J. Membr. Sci. 107 (1995) 229237. Available from: https://doi.org/10.1016/0376-7388(95)00119-0. [36] P.J. Flory, Y.-I. Tatara, The elastic free energy and the elastic equation of state: elongation and swelling of polydimethylsiloxane networks, J. Polym. Sci. Polym. Chem. Ed. 13 (1975) 683702. Available from: https://doi.org/10.1002/pol.1975.180130403. [37] H.R. Lin, Y.J. Yen, Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies, J. Biomed. Mater. Res. Part B Appl. Biomater. 71 (2004) 5265. Available from: https://doi.org/10.1002/jbm.b.30065. [38] K.L. Spiller, S.J. Laurencin, D. Charlton, S.A. Maher, A.M. Lowman, Superporous hydrogels for cartilage repair: evaluation of the morphological and mechanical properties, Acta Biomater. 4 (2008) 1725. Available from: https://doi.org/10.1016/j.actbio.2007.09.001. [39] L.C. Dong, A.S. Hoffman, Q. Yan, Dextran permeation through poly(N-isopropylacrylamide) hydrogels, J. Biomater. Sci. Polym. Ed. 5 (1994) 473484. Available from: https://doi.org/10.1163/ 156856294X00158. [40] N. Annabi, J.W. Nichol, X. Zhong, C. Ji, S. Koshy, A. Khademhosseini, et al., Controlling the porosity and microarchitecture of hydrogels for tissue engineering, Tissue Eng. Part B Rev. 16 (2010) 371383. Available from: https://doi.org/10.1089/ten.teb.2009.0639. [41] B. Lindemann, U.P. Schro¨der, W. Oppermann, Influence of the cross-linker reactivity on the formation of inhomogeneities in hydrogels, Macromolecules 30 (1997) 40734077. Available from: https://doi.org/ 10.1021/ma961870x. [42] I. Yazici, O. Okay, Spatial inhomogeneity in poly(acrylic acid) hydrogels, Polymer (Guildf.) 46 (2005) 25952602. Available from: https://doi.org/10.1016/j.polymer.2005.01.079. [43] E.G. Richards, C.J. Temple, Some properties of polyacrylamide gels, Nat. Publ. Gr. 232 (1971) 1617. Available from: https://doi.org/10.1038/229560a0. [44] J. Bastide, S. Candau, L. Leibler, S. Cedex, U.L. Pasteur, Z. De Saulcy, et al., Osmotic deswelling of gels by polymer solutions, Macromolecules 14 (1981) 719. [45] O. Okay, General properties of hydrogels, Hydrogel Sens. Actuat. 6 (2009) 115. Available from: https:// doi.org/10.1007/978-3-540-75645-3. [46] N. Orakdogen, M.Y. Kizilay, O. Okay, Suppression of inhomogeneities in hydrogels formed by freeradical crosslinking copolymerization, Polymer (Guildf.) 46 (2005) 1140711415. Available from: https://doi.org/10.1016/j.polymer.2005.09.082. [47] S. Grube, W. Oppermann, Inhomogeneity in hydrogels synthesized by thiol-ene polymerization, Macromolecules 46 (2013) 19481955. Available from: https://doi.org/10.1021/ma302520p. [48] J.G.H. Joosten, J.L. McCarthy, P.N. Pusey, Dynamic and static light scattering by aqueous polyacrylamide gels, Macromolecules 24 (1991) 66906699. Available from: https://doi.org/10.1021/ ma00025a021. [49] S. Haefner, M. Rohn, P. Frank, G. Paschew, M. Elstner, A. Richter, Improved PNIPAAm-hydrogel photopatterning by process optimisation with respect to UV light sources and oxygen content, Gels 2 (2016) 10. Available from: https://doi.org/10.3390/gels2010010.

CHAPTER 16

Characterization tools and techniques of hydrogels Sayan Ganguly1, Poushali Das2 and Narayan Ch. Das1,2,* 1 2

Rubber Technology Centre, Indian Institute of Technology, Kharagpur, India School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur, India

Abbreviations , r02 . μ-CT 3D BC C60 CCD CDs Cn CNTs DUT FH GQDs IPN l LCST OCTSM PAAm PEG PNIPAM PTFE PVA SA SANS SAXS SR SWCNTs USANS VPT θ ξ


root mean square distance between two consecutive cross-linking points microcomputed tomography three-dimensional bacterial cellulose buckminsterfullerene charge-coupled device carbon dots characteristics ratio proposed by Flory carbon nanotubes device under test fluorescent hydrogel graphene quantum dots interpenetrating polymeric network bond length lower critical solution temperature optical coherence tomography-based spherical microindentation poly(acrylamide) poly(ethylene glycol) poly(N-isopropylacrylamide) poly(tetrafluoroethylene) poly(vinyl alcohol) sodium alginate small-angle neutron scattering small-angle X-ray scattering swelling ratio single-walled carbon nanotubes ultra small-angle neutron scattering volume phase transition bond angle distance between two consecutive cross-linking points

corresponding author

Hydrogels Based on Natural Polymers. DOI: https://doi.org/10.1016/B978-0-12-816421-1.00016-1 © 2020 Elsevier Inc. All rights reserved.

481

482 Chapter 16

16.1 Introduction Hydrogels are a three-dimensional (3D) cross-linked, insoluble mass consisting of hydrophilic polymeric chains which are quite susceptible to volume alteration against external stimuli. The properties of hydrogels are always of concern to scientists because of their mode and area of applications. The hydrophilicity of hydrogels appears to be due to their polar pendant groups inherited from precursor monomers. Cross-linking of hydrogels is classified in various aspects; most commonly they are covalent, ionic, and physical hydrogels [1]. Experimentally, hydrogels can be evaluated by their flow behavior against external pressure. This flow property of materials is termed rheology. As a matter of fact, it has already been proved that a dilute solution of polymer in water acts with Newtonian flowability, whereas in a gelled system the flow property is slightly different. From a rheological point of view hydrogels are basically a combination of elastic and viscous attributes. Hence, to assess a hydrogel; their flow behavior analysis is a common characterization. The most prominent use of hydrogels is in the biomaterial field because hydrogels mimic the soft tissues and muscles of animals. For the last 30 years, a large number of hydrophilic polymers have also served in this area. Natural polymers like alginate, starch, agarose, gelatin, collagen, carrageenan, and other polysaccharides had been explored. In synthetic hydrogel fabrication, the first hydrogel was reported by Wichterle and Lim in 1960 [2]. With commercial success in soft materials research, hydrogels are the most promising candidate and can be regulated by means of external stimuli, namely, pH, temperature, ionic strength, electric pulse, light, solvent, enzymes, and a saline environment [36]. In 1980, Lim and Sun developed calcium ion cross-linked alginate hydrogel beads for microcapsule formation [7]. Yannas et al. first fabricated burn dressing/healing hydrogel skins from collagen [8]. The research results based on improvement of hydrogel technology can be sorted into several areas [9]. First-generation hydrogels basically emphasized the cross-linking methods. The initial research was directed at the improvement of water uptake behavior, desirable mechanical properties, and swelled gel dimensional integrity. After that, in the 1970s, research was channelized to the stimuli-responsive behaviors and the mode of applications. In this phase, most hydrogels were loaded with small molecules which were subjected to leaching out from the gel matrix by means of their external stimuli response. In this domain, drug delivery, fertilizer release, and other dye releases had immense significance [10]. The third-generation hydrogels were comprised of stereo-complex formation-assisted gelation. These are poly(ethylene glycol) (PEG)PLA physical interactions [11,12].

16.2 Hydrogels: microstructureproperty relationship Before going on to the main segment of this discussion, one basic query has to be answered. Why do we need to uncover the microstructure and hydrogel properties before

Characterization tools and techniques of hydrogels 483 discussing the characterization of hydrogels? The answer can be given as to evaluate the practical life span and usability of hydrogels. There are various kinds of hydrogels, based on their origin, synthesis methods, mode of application, type of polymeric network, and cross-linking methods. The most common hydrogels are chemical cross-linked hydrogels. The hydrogel network, which is liable to cover the whole 3D network of hydrogel, can be categorized as synthetic polymeric, natural, and a combination of these [13]. The most common synthetic polymers used to fabricate hydrogels are PEG or polyethylene oxide, polyacrylamide (PAAm), polysaccharides, poly(hydroxyethyl methacrylate), poly(vinyl alcohol) (PVA), etc. [14,15]. Natural polymer-based hydrogels are normally fabricated from collagen [16], silk [17], gelatin [18], and polysaccharides [19]. The most common commercially available polysaccharides are agar [20], alginate [21], carrageenan [22], starch [5,23], and psyllium [24,25]. For chemically cross-linked hydrogels these polymers are used as graftable polymers. This mechanism of fabricating hybrid hydrogels forms IPN-type hydrogels. In the case of physical hydrogels, the most significant distinction with chemical hydrogels is the physical entanglement of macromolecular chains. Fig. 16.1 illustrates the hypothetical formation of chemical and physical hydrogels for chemical gels and covalent anchoring at the specific intersection points of macromolecular chains, where physical hydrogels consist of entanglement or interactions throughout a domain not in a specific point. For alginate-based hydrogels ionic cross-linking is a common phenomenon.

Figure 16.1 (A) Ideal cross-linked 3D assembly with tetra-functional linkages. (B) Nonideal cross-linked network including chain ends and loops. (C) Ideal cross-linked double network gel. (D) Physically entangled network. (E) Helix formation in network. (F) Alginate-like network with ionic linkages between adjacent chains. 3D, Three-dimensional.

484 Chapter 16 Cross-linking points and the density of cross-linking points in a hydrogel matrix impact significantly on the mechanical viscoelastic or flow property of the hydrogels. At a molecular level hydrogels are also considered as porous materials. The pores present in hydrogel house water molecules when they are subjected immersed in aqueous media. The porous morphology of hydrogels is an outcome of internal cross-linking. Porosity in the hydrogel matrix is directly related to the water uptake behavior of hydrogels, called swelling behavior, this can be carried out dynamically or in equilibrium. Moreover, the gradual leaching of water molecules from hydrogel matrix is called a desoiling or poroelastic relaxation experiment. The swelling ratio (SR) or swelling coefficient is defined as the ratio of weight of swollen gel to dry gel. SR 5

Vswollen Vdry

(16.1)

The swelling ratio has utmost significance to define various properties which are critical in the gel matrix, viz. mechanical toughness and transport properties. The swelling ratio differs on the type of polymers and the hydrophilicity of the monomers taken for fabricating hydrogels. For example, poly(HEMA) shows for relatively low swelling whereas hydrogels made of acrylic acid monomer has immense soiling ratio. SR 5

Vpolymer 1 5 Q Vpolymer 1 Vwater;initial 1 Vwater;imbibed

(16.2)

As shown in Eq. (16.2) water uptake is dependent on two condition, the initial water to be imbibed and the surplus volume of water penetration during swelling. The microstructural attributes of the hydrogel matrix can also be evaluated by another scaling parameter called cross-link density. Fig. 16.2 is a graphical representation of the macromolecular change among the junction points. The average molecular weight between two adjacent cross-linking points is Mc, which is related to the SR by the following relation.

Figure 16.2 Microstructural parameters of hydrogels.

Characterization tools and techniques of hydrogels 485 This relation is applicable for moderate to high swelling (superabsorbent) hydrogels (SR or Q . 10); 3=5

SR or Q 5 βM c

(16.3)

where β is a constant related to the specific volume of the polymer, the main interaction parameter between polymer and solvent molecules and the molar volume of water. Again the SR is related to the 3D network mesh size, sometimes called the pore diameter, that is, the distance between two consecutive cross-linking points (ξ) in the spatial (3D) network. ξ 5 Q1=3 ðr 20 Þ1=2

(16.4)

where ,r02 . is the root mean square distance between two consecutive cross-linking points. ,r02 . can be written as, ðr 20 Þ1=2 5 lðCn Nb Þ2

(16.5)

where l is the bond length, Nb stands for the number of bonds, and Cn implies the characteristics ratio proposed by Flory. Cn generally lies in the range of 510 which corroborates deviations from ideality for freely rotating chains in the hydrogel mass. An approximation to evaluate the value of Cn for freely movable macromolecular chains is: CN D

1 1 cosθ 1 2 cosθ

(16.6)

where θ is the bond angle between consecutive segments. The mesh size (ξ) is basically a result of the gelation chemistry associated with the cross-link density, temperature, environmental pH, and other external stimuli. The pore size of a hydrogel also influences its mechanical and structural properties and adsorbent nature. As the mechanical properties of hydrogel are quite poor compared to other viscoelastic polymer composites, to improve their structural properties become the primary requirement to commercialize a hydrogel system. Generally, in hydrogel internal microstructure, the dimensional stability of any hydrogel system is related to the cross-link density. A highly dense network hydrogel with high crossing density provides high gel strength and good dimensional stability but the water uptake behavior is sacrificed due to high cross-link density. Thus, depending on the mode of application, the water uptake behavior and cross-link density are optimized. For physical hydrogels the macromolecular chain entanglement occurs due to hydrophobic interactions between the blocks present in the polymer. In the domain of physical hydrogels, the major two classes are thermoreversible and rheo-reversible.

486 Chapter 16

16.3 Mechanical characterizations of hydrogels 16.3.1 Uniaxial tensile testing As hydrogels are soft materials they lack mechanical robustness. The mechanical properties have significance in evaluating the extent of failure and longevity of hydrogels. In recent times, the most common method to evaluate the mechanical properties of hydrogels was by uniaxial tensile experimentation, which is sometimes called strip extensiometry. These methods are widely accepted by the scientific community to determine the mechanical toughness and ultimate tensile strength of various hydrogels. Moreover, uniaxial tensile testing infers a mechanical toughening mechanism for anisotropic filler-embedded nanocomposite hydrogels. Uniaxial tensile testing for soft/light samples is generally carried out by holding the terminals of specimens between two grips to ensure there is no slippage. The terminals of the hydrogels are physically covered by rough surfaced papers. Fig. 16.3 shows pictorial illustrations of several conventional techniques applied to carry out the mechanical testing of soft materials. In the case of extensiometry, two major classifications are available, one is strip extensiometry and the other is ring extensiometry. These experimentations result in a typical stressstrain plot for hydrogels. From the stressstrain plot one can obtain the finite values of Young’s modulus, stress, and ultimate tensile stress [26]. In addition to this extensiometry, the viscoelastic nature of hydrogels also can be determined. In terms of rheology these are called the creep test and stress relaxation experiment. Though this is a destructive experiment, it has immense significance due to nontime-consuming data collection and multidimensional explanations related to microstructure and morphology. For biological samples, hydrogels are a major concern because of their flexibility, noncytotoxicity, tuneable mechanical properties, and tissue mimetic features.

Figure 16.3 (A) Uniaxial extensiometry; (B) ring extensiometry; (C) compression test; (D) hydraulic bulging test; (E) indentation test.

Characterization tools and techniques of hydrogels 487

16.3.2 Compressive testing The compressive toughness testing of hydrogel is another destructive experiment for hydrogels to evaluate their mechanical properties. This testing is generally carried out by placing the hydrogel specimen between parallel plates followed by vertical pressing. A pictorial illustration of compressive experimentation has been given in Fig. 16.3C. The platens used for compressive experimentation are normally nonporous and smooth as per their surface morphology. But sometimes a single porous platen also has been utilized for multiphase gel materials. For multiphase materials, if there is any leaching out of fluids from the gel matrix, the porous platen will resolve that situation by permeation. The application of external pressure with respect to strain gives the compressive stressstrain plot, which infers the typical load-bearing and compressive fracture toughness of soft materials. Moreover, from such a data acquisition technique several theoretical model fittings also have been implemented. Comparing the versatility and popularity points of view between extensiometry and compression, the majority of research uses compressive experimentations. This is due to the easy sample preparation and unconditional restrictions over the specimen geometry. In general, extensiometry demands more precise sample dimensions and as good as possible sample surface smoothness; whereas in the case of compressive stress measurement there are no such restrictions as this testing requires only flat surfaces for even distribution of external compressive stress throughout the gel matrix. Such compressive mode-enabled testing also has significance over the judgment of nanocomposite hydrogels. Nanocomposite hydrogels are a special class of hydrogel where nano-inclusions have been entrapped inside the hydrogel matrix in order to improve the hydrogel properties in a synergistic fashion. Most popularly, nano-reinforcement incorporated hydrogels have been nurtured because of their load-bearing and fatigue performances. Recently, in situ nanoclay-based biomimetic adhesive type polymerizable monomers have been used to enable the fabrication of tough hydrogels [27]. The extent of stress transfer and load-bearing response of hydrogels reflects their compressive cyclic loadingunload plots.

16.3.3 Bulge experiment Prior to rupture, tough hydrogels go through a process called necking. In this domain, the strain hardening phenomenon has marked theoretical inference. Strain hardening can be assessed by a hydraulic bulge experiment for hydrogel sheets under biaxial tensile force. In this test both the stress and strain can be evaluated, whereas for typical tensile testing the experiment is restricted to uniform strain. A bulge test of hydrogels reflects their plastic flow behavior. The sample architecture for a bulge test is circular in shape (diaphragmlike), tightly held at the outer fringe of the sample holder, followed by uniform stretching via an external force applied laterally. One restriction to the specimen preparation is that

488 Chapter 16 the thickness to bulge diameter ratio should be small enough to eradicate the bending effect of hydrogels during clamping. In the early years, researchers used this test for thin metal films [28], but, in 1987, polyimide film was first experimented with via a hydraulic bulge test [29]. The displacement after the external force is measured by a charge couple detectors (CCD) camera laser probe. The typical formula for interpretation of external applied pressure and displacement relation can be written as: P5

C1 σ 0 t C2 Et h 1 4 h3 2 a a

(16.7)

where σ0, t, and a correspond to the residual stress, film thickness, and membrane halflength, respectively. For half-length assumption, the radius is taken into consideration, whereas, for rectangular specimens, the half-length is considered as the shortest length/ dimension of the rectangle. h is the extent of deflection after application of stress. E is Young’s modulus of the hydrogel and C1, C2 are the constants depending upon the finite element analysis and Poisson’s ratio, respectively. Mitchell et al. first accumulated the C1 and C2 values calculated by various researchers. The brief data for the calculated constant values have been tabulated in Table 16.1. Maier-Schneider et al. evaluated the values of the constants by utilizing a minimization strategy [30]. In general, two typical types of loading configurations have been standardized in bulge testing; “deflection into the orifice” and “deflection away from the orifice”. This test was first popularized by Xiang et al. in 2005 when they proposed mathematical discussions of rectangular hydrogel membrane [31]. Moreover, they also provided information regarding the elasticity, hysteresis, and toughness of the membrane sheets. Thus it could be inferred that besides uniaxial tensile testing, the bulge/deflection experiment also has great significance in hydrogel characterizations to evaluate the tissue mimetic nature of soft materials. Table 16.1: Calculated C1 and C2 values obtained from pressuredisplacement models Bulge geometry

C1

C2(1 2 υ)a

Circular

4.0 4.0 4.0 3.04 3.04 3.41 3.39 3.45 1.55 2.0

2.67 2.67(1.026 1 0.233υ)21 (7 2 υ)/3 1.473(1 2 0.272υ) 1.473(1 2 0.272υ) 1.37(1.075 2 0.292υ) (0.8 1 0.062υ)3 1.994(1 2 0.271υ) [30/(1 1 υ)][0.035 2 (16/(800 2 89υ))] 8/[6(1 1 υ)]

Square

Rectangular a

υ 5 Poisson’s ratio.

Characterization tools and techniques of hydrogels 489

16.3.4 Indentation test The indentation test is another crucial mechanical restructure characterization of soft materials. It is actually a single-point compression test where the surface deflection has been quantified (Fig. 16.3E). In this experiment, a force displacement transducer is connected to an indentation probe. This arrangement results in a forcedisplacement plot from which the elastic modulus of the materials (here hydrogel) can be calculated. One thing that should be mentioned is that indentation tip/probe geometry has utmost importance in assessing material rigidity. Recently, many scientific modifications via software precision have made this technique more attractive to the academic and research community. The data obtained after indentation are normally the critical stress of indentation, amount/depth of indentation, stress relaxation, and sample rigidity. These data have been acquired also in nanosized domains [32]. The mode of applications of such testing has been implemented in gel coating and soft materials attached to a hard surface. The indentation test shows superiority over conventional mechanical characterizations in terms of very low time consumption, online, and real-time data acquisition. The localized failure behavior is ideally evaluated from this experiment. However the disadvantage to this set up is it is not as desirable for cell-assembled hydrogels. This is due to the restriction of this experiment to a sterile environment. To overcome this limitation, a modified version of indentation has been implemented where sterile environment-based testing can be carried out without any difficulties. The name of the modified version is long-focal microscopy-based spherical microindentation which was adopted to carry forward the research based on online 3D cell-impregnated hydrogels. This technique was first proposed by Liu and Ju to calculate the mechanical properties of egg shell membranes [33]. Hydrogel thickness for this testing has to be kept below 1 mm. In this instrument, there are two parts, that is, the sample holder consisting of a spherical pin pointed indenter and the image acquisition set up. The hydrogel sample is attached radially by means of a circular clamping system with an inner diameter of 20 mm. The hydrogel is point-indented by a 4-mm PTFE sphere, 316L stainless steel, and 440 stainless steel sphere. These probe materials were chosen after initial assessment of hardness of the gel surface. The hydrogel to indenter diameter ratio was kept constant at 5.0 throughout the process. For 3D cell-impregnated hydrogels, the whole system is kept in sterile phosphate buffer saline medium at 37 C, 5% CO2. This environment severely impacts the mechanical properties of hydrogels. The image-capturing device consists of a long-distance objective microscope and computer-controlled CCD camera. The displacement of gel membrane is measured by a computer-controlled stage micrometer. The viscoelasticity of the specimens was evaluated indirectly by deformation of the hydrogels. In addition, another type of indentation method also has been adopted, called optical coherence tomography-based spherical microindentation (OCTSM). The theory behind this

490 Chapter 16 method is Hertz contact theory, which is performed by means of a spherical ball. OCTSM is a noninvasive technique followed by backscattering of light passing through the specimen. In brief, the setup consists of two typical light beams; one passed through the specimen and the other passes without any scattering as a reference beam. The backscattering of light is detected by a photodetector which develops a photograph of the cross-sectional microstructure. The depth of penetration/indentation is reflected over the sample thickness and geometry of the specimen. To be precise, indentation is a type of localized compression test which records the load to depth with time as a triparameter compromisation. The obtained value from indentation is the reduced modulus or ER, which can be written as, ER 5

E 1 2 γ2

(16.8)

where γ is Poisson’s ratio. Indentation for soft materials is classified as macroscale indentation (“mm” scale) and micro/nanoscale indentation (“μm” or “nm” scale). In recent years, nanoscale indentation has become a very promising strategy to investigate the mechanical properties of hydrogels [34].

16.4 Rheology 16.4.1 Viscoelasticity and microstructure Viscoelasticity deals with the flow and deformational behavior of materials. The term is adopted from the Greek work “rheos” meaning “the river” or “flowing.” This technique has been used by various industries related to paints, adhesives, paper, packaging, cosmetics, foodstuffs, pharmaceutics, polymer product making, surface technology, and even in the glass/ceramic and metal industries. The basic term “viscoelastic” consists of two subwords indicating viscous dominating over elastic. This implies the time-dependent deformational behavior of materials. The viscoelasticity of a material is highly dependent on the external force applied to the material of interest. The mechanical response of hydrogels normally shows time-dependent exponential curves which have close resemblance to elastomers. Hydrogel consists of a huge amount of water which acts as a plasticizer for hydrogels. Soft hydrogels with elastomer-like behavior are welcomed nowadays for applying in soft artificial tissues, muscle mimetic devices, tissue actuations, artificial skin, and mussel mimetic electronic membranes [3537]. When external shear is applied to a hydrogel specimen there are several consecutive phenomena resulting which can be summarized as an initial acceptance of external force, initiating mobility in macrochains followed by stress dissipation of polymer chains over time resulting in a gradual decaying/sigmoidal curve.

Characterization tools and techniques of hydrogels 491 There are some specific characteristics to such chain mobility and deformations which are categorized as “creep” and “stress relaxation.”

16.4.2 Creep behavior Creep is time-dependent deformational change or strain (γ) monitored in a constant stress (σ). Fig. 16.4A and B show a typical creep curve for viscoelastic fluids. The creep compliance, J(t) is the ratio of shear strain to applied shear stress. If creep compliance has been measured over a very short or long period of time, it shows negligible time dependency. At a very small time scale the polymer molecular chain mobility is much higher which implies an unrelaxed state of the system, as designated by Junrelaxaed in Fig. 16.4C. In contrast, at a longer time scale the molecular reorganization is better, and has been termed as the relaxed state of the system, Jrelaxed. For the assessment of product performance in long-term applications, creep study is highly significant. The data obtained from creep study reflect the materials’ recovery after being subjected to a fixed load. Ganguly et al. performed a creep study for nanocomposite hydrogels where the impregnated filler was anisotropic clay tactoids [25]. Clay is a highly abundant ceramic material. It has several applications besides hydrogels, such as solid support for catalyst, rheology modifier, and air impermeable coating [3843]. In their work they showed enhancement in elasticity after incorporation of clay nanofillers into a hydrogel matrix. Clay-incorporated hydrogels

Figure 16.4 Model plots for creep (AC) and stress relaxation (DF) of viscoelastic materials.

492 Chapter 16

Figure 16.5 Creep (left) and strain recovery (right) of clay-reinforced nanocomposite hydrogels [25]. r 2018. Reproduced with permission from Elsevier.

showed better elasticity and minimization of residual strain after a creep experiment as shown in Fig. 16.5. In another experiment, ionic cross-linked PVA hydrogels were also tested in a creep experiment [44]. They showed that PVA with Fe31 cross-linked hydrogels generally had 23% viscoelastic deformation. In low cross-linked systems, molecular deformation is quite easy because of the low level of elasticity. However, in the case of high cross-linked systems, the spring back action is much better, implying a better viscoelastic response. For high cross-linking systems the macromolecular chains are deformed under an external load. When external forces are applied, the slippage of polymeric chains is inhibited due to the interchain attachment (either physical bonding or chemical bonding). Thus, this experiment can be done to discover the longevity of soft materials under constant load. Viscoelasticity is therefore affected by cross-linking in the gel matrix as well as fillerpolymer chain attachment (physisorption).

16.4.3 Stress relaxation Stress relaxation is another time-dependent phenomenon where a finite amount of strain has been kept constant. This experiment is depicted graphically in Fig. 16.4DF. When a material is subjected to deform up to a certain deformation, there is a molecular chain relaxation inside the matrix due to exponential stress dissipation. Such stress dissipation contracts the specimen after a finite time and the remaining part which is not returned back to the exact initial position is called the residual strain. If this deformation and sparing back action happen instantaneously, then the phenomenon is called “elastic.” But in the case of hydrogels, the spring back action is time-dependent and the time decay of stress has a

Characterization tools and techniques of hydrogels 493 resemblance to the molecular relaxation. In Fig. 16.4F the shear modulus G(t) has been illustrated in relaxed and unrelaxed states. If one tallies the creep compliance and the stress relaxation modulus, a reciprocal relationship is seen.

16.4.4 Dynamic mechanical behavior The quantitative estimation of viscoelastic and rheological properties is best evaluated by means of dynamic mechanical testing. This is a frequency-based experiment. This oscillatory test gives the measure of complex shear modulus, which is given in Eq. (16.9):


σ G 5 G 1 iG 5
γ


0

00

(16.9)

where G0 and Gv correspond to the elastic (storage or real part) and viscous (loss or imaginary part) shear modulus, respectively. G
is the complex shear modulus. In the dynamic mode, that is, the oscillatory mode, the complex strain/deformation can be evaluated as


γ 5 γ0 expðiωtÞ

(16.10)

were ω and γ 0 indicate the angular frequency and initial strain value, respectively. If the Maxwell model is taken into consideration, then the viscoelasticity can be implemented by calculating the first derivative of strain rate and the equation will be:








dγ 1 dσ σ 5 1 G dt dt Gθ

(16.11)

where θ is the characteristic time constant and is closely related to η/G. η is the viscosity of the polymer system. After calculating this equation, the linear form of the equation after integration is


σ Gω2 θ2 ωθG 1i
5 2 2 γ 1 1 ω2 θ2 11ω θ

(16.12)

or


σ 5 σ0 expðiωt 1 δÞ

(16.13)

where δ is the angular phase lag having a maximum amplitude of σ0. Now, after comparing the aforementioned equations, it can be summed up that 0

G5

Gω2 θ2 1 1 ω2 θ2

(16.14)

494 Chapter 16 and 00

G 5

Gωθ 1 1 ω 2 θ2

(16.15)

Eqs. (16.7) and (16.8) are characteristic equations for calculating the elastic (storage) and viscous (loss) modulus for the specimens, respectively. Another thing also can be evaluated from the analysis, which is a damping parameter or dissipation factor, or to be precise “tan δ.” “Tan δ” is the ratio of viscous to elastic modulus. Tan δ can be numerically written as tanδ 5

G00 1 5 0 ωθ G

(16.16)

“tan δ” is a measure of dissipation energy after the application of oscillatory forces to a polymer matrix. This dissipation energy is solely in terms of heat. In the case of rheology, low temperature and high frequency are related in experimental conditions. At low temperature the hydrogels have a rigid type of system, rather than “glassy.” However, after increasing the temperature the molecular chains’ mobility becomes reduced, so that the system shows a glassy to rubbery transition. This results in a gradual decrement in elastic modulus and an increment in viscous modulus. Similarly, at high frequency, the polymer chains are incapable of reorienting, thus there is no chain relaxation. Improper chain relaxation hinders the segmental mobility resulting in enhancement of the elastic modulus. However, at a relatively lower frequency, the relaxation time required for polymer chains is enough that the stress dissipation is facile in this case. This implies a comparative lowering of the elastic modulus as lower frequency which is related to the dynamic mechanical behavior at high temperature. The damping parameter is the measure of how much energy has been dissipated during oscillatory testing. As “tan δ” is proportional to the loss modulus of the polymer system, this means the damping character of the specimen will be greater if “tan δ” increases. From a typical “tan δ” plot the glass transition temperature (Tg) can be evaluated. In terms of “tan δ,” the glass transition temperature can be defined as that temperature where the damping factor shows an apex value in the plot. Moreover, several other temperature-assisted transitions also can be defined by this analysis. The most significant transition that occurred at the highest temperature is called the “α-transition.” Other subsidiary temperature-based transitions also take place during this experiment, and are called “β-transitions.” “β-transitions” have relatively lower temperature transitions with respect to “α-transitions.” If the inherent cross-linking is uniform in nature, then the “α-transitions” are more prominent and the broad peak of Tg can be observed. The rheological testing of hydrogels is carried out normally in a swollen state, as in the swollen state the hydrogels behave like soft materials with sufficient rubber-like flexibility. In addition to the dynamic mechanical system, equipment for such cases includes the

Characterization tools and techniques of hydrogels 495

Figure 16.6 Rheological properties of gelatin hydrogel (Gel-H), gelatin/BC composite hydrogel (Gel/BC-H), and magnetic composite hydrogel (Mag-H) [45]. r 2018. Reproduced with permission from Elsevier.

parallel plate rheometer. Herein, researchers can tune the desired rheological conditions; such as strain sweep, temperature sweep, and viscosity evaluation. The distinguishable difference between elastic and loss moduli implies the gel state of the materials. Gelatin and bacterial cellulose (BC)-based hydrogels have been evaluated to assess their elastic and loss moduli [45]. They showed that a very small amount of BC did not significantly improve the elasticity of hydrogel (Fig. 16.6). Dextrin-based hydrogels have been tested previously and showed a tan δ vs. time plot depicting the “gel point” [46]. The gel point is the particular point where a gradual decrement in tan δ curves in various frequencies converge to meet. Thus, for hydrogels, synthesis parameters like gel time estimation are significant because they have a direct relationship to the gel strength. Moreover, another trustworthy method to estimate the gel point is “cross-over point evaluation.” The crossover point is the point where elastic and loss moduli alter their values/trends. That means in a pre-gel state, the loss modulus is dominant over the elastic one, whereas after gelation the trend shows the opposite. This implies a comparative enhancement in elasticity after gelation cannot be ruled out. Cross-over point estimation has been estimated in several literatures [47,48]. Nanofiller-based semi-IPN hydrogels also showed similar rheological behaviors. Clay and graphene are two of the most used fillers in the hydrogel field. When anisotropic fillers are incorporated inside hydrogels, the gel rheology showed much better thixotropic characteristics. Clay-based hydrogels with high gel strength have been described elsewhere [49,50]. Graphene-reinforced hydrogels also show high gel strength during oscillatory shear force experiments [51]. The gel strength is a quantitative parameter to estimate the gel dimensional stability of hydrogels in dynamic/oscillatory conditions. Graphene is a unique filler which has immense ability to adhere polymer chains by means of physisorption. This enables delayed network rupturing with increasing graphene content. Fig. 16.7 is an illustrated experiment of this work.

496 Chapter 16

Figure 16.7 (A) Rheological behavior of the hydrogel in a frequency sweep experiment. (B) Rheological behavior of the hydrogel in a shear stress sweep experiment. (C) Elastic modulus to loss modulus ratio versus graphene concentration in hydrogel (corresponds to the gel strength or elasticity of the nanocomposite hydrogels). (D) Dependency of the effective concentration of elastic chains with graphene concentration. The plot shows enhancement of cross-linking points and effective concentration of chains with increasing graphene content [51]. r 2018. Reproduced with permission from Elsevier.

For double-network hydrogels, the flow property was also calculated by Liu et al. [52]. Double-network hydrogels are another special class of hydrogel where two intertwined polymeric networks are cross-linked. Generally, double-network hydrogels are also called full-interpenetrating polymer networks (full-IPN). PVA/PAAm and sodium alginate (SA) have been gelled together and rheologically characterized. Single-network hydrogels are greatly inferior to double-network hydrogels. Fluorescent injectable hydrogels were developed by another research group with a self-healable feature characterized by a rheological experiment [53].

Characterization tools and techniques of hydrogels 497

34°C G',G''×10a(Pa)

P-3: a=4

P-2: a=2

26°C

P-1: a=0 1E–3

10

21°C 20

30 40 Temperature (°C)

50

60

Figure 16.8 Temperature dependence of storage and loss modulus and their crossover. Crossover point designates the gel time [54]. r 2007. Reproduced with permission from Elsevier.

In addition to gel time, gel temperature also can be estimated from rheology assessment. Gel temperature is the critical temperature where the gelation initiates or takes place. As per the flow behavior of materials, elastic modulus corresponds to the gel state, whereas for liquid state, loss modulus is the most dominant factor. Tang et al. showed how to evaluate the gel temperature from a rheological study [54]. As per their study (Fig. 16.8), initially the pre-gel state showed both elastic modulus and loss modulus values, where elastic modulus is less dominant. In fluid form, the polymer chains are more labile to flow after application of external shear forces. However, when temperature was applied to the system, the initiator disintegrated to fragments followed by a free radical gelation process. As the polymer/monomer mix is thermally insulating in nature, the gain of activation energy from external temperature would be slightly delayed. Thus, for thermal or redox initiators, there will be an induction period for initiators to propagate gelation. As per the revealed data, it is clear that the gel temperature has a direct relation to the added polymer phase. In that report, the authors added chitosan as the polymer phase. With increasing polymer phase the gel temperature was increased. This is an outcome of intermolecular H-bonding. Initially hydrophilic polymer itself has a tendency to self-assemble over a long range. When they poured it into protic media, a competitive H-bond formation took place between the solvent front and polymer chains. Thus to form an intertwined microstructure, the intermolecular microstructure has to scission before homogeneous gelation, resulting in delayed gelation. Higher elastic modulus implies better gel strength. Sometimes a typical hydrophobic association between the polymer chains cannot be ruled out. Similar types of mechanistic approach have already been proposed elsewhere [55].

498 Chapter 16

16.5 Small-angle X-ray scattering technique X-ray was first discovered by Wilhelm Conrad Rontgen and earned him the Nobel Prize in physics. In 1920 P. Krishnamurti reported Small-angle X-ray scattering (SAXS) on amorphous materials which were basically colloidal systems [56]. After that, there was not no really significant research into SAXS. The main difficulty in measuring the scattering of X-rays after bombardment with samples is the distinguishable intensity measurement between background X-ray and scattered beams. Since then, several designs have been created to improve the collimation system. Collimation is related to the beam size and slit/pinhole tuning. There are various designs of collimators. The major ones are the pinhole (slit) collimation system, Kratky collimation system, and BonseHart channel cut collimation system (for very small angles). Synchrotron X-ray has a very high photon count, resulting in a high-intensity 1D plot. Furthermore, the tunability of X-ray wavelength across a drift close to the K or L edge of a component has made SAXS a practicable approach for structure and morphology evaluations of a particular system in the presence of other interacting materials [57,58]. The interactions between X-rays and the present particulate materials are briefly depicted in Fig. 16.9. SAXS can be utilized to assess a large variety of materials in which at least

Figure 16.9 Illustration of typical nanoprobes and their range of analysis. SAS, Small-angle scattering; USAS, ultrasmall-angle scattering.

Characterization tools and techniques of hydrogels 499 biphasic systems have been mostly nurtured. In the case of multiphase systems, a variation in electron density affects the drastic scattering intensities of the specimens. In the case of laboratory X-ray sources, the incident photon count (or flux) lies in the range of 103108 photons/s. However, in synchrotron-based systems, the flux generally lies around 10111013 photons/s. There are some specific beamlines with superiority in photon flux at around 1014 photons/s, like BL19LXU in the Spring-8 synchrotron. A similar type of flux is also found in SACLA, Japan, the European XFEL, and LCLS in the United States. Another very important aspect in this situation is background noice and parasitic scattering. Parasitic scattering is unwanted scattering which is intensity loss for SAXS evaluation. To be more precise, the SAXS detector has a step-scanning Geiger counter and photographic film simulators. Nowadays, most laboratories have replaced this type of detectors with 2D gasfilled wire detectors [59]. CCDs are another class of detector besides these 2D gas phase detectors [60]. The MYTHEN detector is a better and problem-free detector but is less cost-effective, which follows direct photon-counting irrespective of other factors [61]. Other modified 2D detectors are the PILATUS [62], EIGER [63], Medipix. and PIXcel detectors [64]. Ionic cross-linked hydrogels can be evaluated by SAXS. When alginate has been crosslinked by Ca21 ions, the junction points are more prominent as per Guinier approximation which was better assessed under a cross-sectioned system. The cross-sectional radius of gyration (Rg) can be evaluated by Eq. (16.17) I ðqÞ  expðR2g q2 =2Þ

(16.17)

Kim et al. performed analysis based on such ionic cross-linked alginated hydrogel beads where they proposed that ionic cross-linked hydrogels are stiff and rigid due to their extensive network formation inside the alginate matrix [65]. Ionic cross-linking among alginate chains are proposed as an “egg-box” model formation. Thus, in the case of ionotropic gelation, SAXS has great significance in assessing the network system, which has a direct relationship on the water uptake behavior. SAXS also can be compared with the flow feature rather than the rheological behavior. A stiffer network results in higher elastic modulus, that is, high gel strength (ratio of elastic to loss shear modulus). In another work, biocompatible PEG hydrogels were evaluated under SAXS study [66]. As per this study, PEG cross-linked by diacrylamide has an immense effect on their junction points which was carried out in in situ SAXS measurement. The in situ SAXS experiment is shown in Fig. 16.10. As per this figure, it can be seen that initially there was no such strong peak at the precursor solution, but after a few seconds, a rising peak was obtained. This peak is called the correlation peak, and has been attributed to the formation of a gel state. In addition, the peak shift was also seen in a higher q range, which implied a smaller length scale. The strong correlation peak is also attributed to the 3D network-like

500 Chapter 16

Figure 16.10 SAXS curves showing in situ polymerization of a PEG-diacrylate precursor solution [66]. r 2010. SAXS, Small-angle X-ray scattering. Reproduced with permission from the American Chemical Society.

morphology. In the case of a dense network system, the juncture points behave as scattering centers. In the case of a swollen network system, a similar kind of experiment was also carried out. When PEG was cross-linked in the presence of diacrylate, the macrochains of the PEG network were restricted to a perfect conformation, whereas, after water uptake, the PEG chains were more liable to rearrange themselves inside the gel matrix. Moreover, the cross-link and degree of polymerization also can be estimated by this SAXS study. Nanocomposite hydrogels with clay as a reinforcement are another area of superabsorbent hydrogels. In this context, polyacrylate (PAA) and nanoclay have been blended homogeneously in order to integrate their structures and properties [67]. In the case of a high clay concentration, interparticle repulsive interaction is more dominant for moderatemolecular-weight PAA. SAXS also gives an idea of the size and distribution of pores inside a porous hydrogel matrix. For semi-IPN type nanocomposites the gel strength, mechanical robustness, and filler dispersion are evaluated and discussed elsewhere [49]. In Ref. [49], nanoclay was in situ gelled by polymethacrylic acid and SA. Such semi-IPN hydrogels become more mechanically robust when anisotropic nanofillers are as reinforcement. As per the SAXS study, the filler distribution showed an improvement in scattering intensity. As nanoclay has an immense surface area, it has the opportunity to anchor to the hydrophilic polymer chains inside the hydrogel matrix. Nanocomposite hydrogels have a dual contribution in the scattering phenomenon; one is their mass fractal and the other is their surface fractals. Two such physical terms are quite significant in SAXS study.

Characterization tools and techniques of hydrogels 501 After obeying the simple power law equation, the SAXS spectra infer the combination of surface and mass fractals for nanocomposite hydrogels. As per the rule of Guinier and Porod, the determining factor is the power law exponent. The value of the power law exponent can give an idea of the microstructure and special configuration of hydrophilic network systems. In the case of in situ hydrogels, the nanoclay itself has relatively small hydrodynamic size or radius of gyration. The radius of gyration is the quantitative parameter of SAXS spectra which can imply the particle agglomeration character. Inside nanoclay confined in hydrogels, the agglomerated feature is more prominent due to the physisorption of polymer chains inside the gel matrix. A high agglomeration of such hydrophilic clusters enhances the degree of agglomeration, resulting in inferior mechanical and rheological properties. The most significant model related to this system is the HurdSchmidt model [68,69].

16.6 Small-angle neutron scattering The neutron is a subatomic particle with a mass of 1839 times that of an electron (1.674928 3 10227 kg), a magnetic moment of 29.6491783 3 10227 J/T, and a 15- minute lifetime. As per the wave particle duality, a neutron has both a particle as well as a wavelike nature. Thus it has a proneness to reflect, scatter, refract, and diffract after interacting with other particles/matter. First, it should be taking into consideration that during smallangle neutron scattering (SANS) experimentation, particle and neutron collisions occur in two ways; nuclear scattering and interactions with unpaired electrons. Interactions with unpaired electrons are corroborated as an enhancement in the magnetic moment. This is called magnetic scattering. The most common assumption is elastic scattering of neutrons after particle-to-neutron collisions. A brief illustration has been depicted in Fig. 16.11 for SANS set up. When a neutron collides with the nucleus, the scattering is dependent upon the interaction potential between the neutron and nucleus, which has a very small domain (10215 m).

Figure 16.11 Instrument and component design for typical SANS facility. SANS, Small-angle neutron scattering.

502 Chapter 16 This is a much shorter distance than a neutron wave (10210 m), resulting in the nucleus scattering. The spherically symmetrical scattered wave function will be: b ψi 5 2 e2ikz r

(16.18)

where z and k represent the distance from the nucleus and wave, respectively. b is the scattering length of nucleus and r is the distance between the neutron and nucleus. The “ 2 ” sign is due to the repulsive force of interactions. The scattering length is significantly dependent over the isotopes. As an example, 1H and 2H (or D) have some discrepancy in their scattering lengths. The scattering length of normal hydrogen is drastically distinguishable from D. Thus hydrogen replacement during a SANS experiment is a commonly adopted technique for analysis, and is called “contrast variation.” Scattering cross-section is another term for SANS experiments. Scattering cross-section is a hypothetical nomenclature corroborating how “big” the nucleus seems to the incident neutron. It is evaluated as the ratio of the neutron count after scattering per unit time to neutron flux. SANS analysis is related to the particles present in a phase or density difference between the phases. In this context, contrast variation is another technique for complex systems. Fig. 16.12 is a schematic representation of the contrast variation technique adopted by various hypotheses. PVA hydrogels are the most practiced area of neutron scattering analysis. As the degree of hydration of PVA affects the crystallinity, thus a great amount of anisotropy can be observed in this system. SANS is an ideal nondestructive tool to investigate the anisotropic character inside PVA gels where the structural alterations are monitored at a scale of 100 nm [70]. Ultra-SANS (USANS) was also performed to estimate the wide range of crystallinity in PVA hydrogels. The thermally induced anisotropy can be corroborated by a combination of SANS and USANS, as reported elsewhere [71,72].

Figure 16.12 Contrast variation measurement for SANS study. SANS, Small-angle neutron scattering.

Characterization tools and techniques of hydrogels 503 Volume phase transition (VPT) is another area of hydrogels (thermoreversible hydrogels), where macromolecular chains are prone to arrange themselves to withstand the gelled structure. In this context, poly(N-isopropylacrylamide) (PNIPAM) hydrogels are the most significant. PNIPAM hydrogels show discriminating temperature-responsive characteristics in a roughly ambient temperature window [7375]. Fig. 16.13 is the SANS intensity versus scattering vector plot for PINIPAM hydrogels at various temperatures [75]. The figure shows swelling and collapsing of hydrogels under temperature alterations. Such VPT was also observed in another report [76]. As shown in these curves, there was a distinct peak in the low q region. This gave the idea of concentration variation of macromolecular chains. The homogeneity in hydrogel systems can be evaluated here after assessing the peak. In the case of a swollen system the hydrogel is quite phase separated, in the order of a few nanometers. The peak is the proper indication of microscopic gel domains acting as the scatterer in a SANS experiment. Water molecules imbibed into hydrogel matrices work as spacer molecules. Thus, from a microscopic point of view, the hydrogel in a swollen state is an inhomogeneous system which has been scattered by incident neutrons. Similar phenomena can be attributed to the presence of various stimuli including pH, temperature, and other small molecules [77,78].

Figure 16.13 SANS spectra of PNIPAM hydrogel in a wide temperature window with temperature reversible behavior [75]. r 2011. PNIPAM, poly(N-isopropylacrylamide). Reproduced with permission from The Society of Polymer Science, Japan (SPSJ), Nature-Springer.

504 Chapter 16

16.7 Fluorescent behaviors of hydrogels Very recently, fluorescent hydrogel (FH) has drawn attention in soft materials research. FHs are special hydrogels which are most easily fabricate after the addition of fluorescent substances inside a hydrogel matrix. FHs have a wide range of applications in tissue engineering and cell differentiation works. Their inherent fluorescence character means they are illuminated from inside of the body resulting in significant in vivo applications [79]. In addition, they also have important characteristics in the enhancement of toughness and rubber-like elasticity of hydrogels. Polymer hydrogels are normally very soft and sometimes inferior in terms of their applicability. The best and most cost-effective way to prepare such hydrogels is to incorporate fluorescent carbon dot (CD)/graphene quantum dot-reinforced hydrogels. Usually carbon is known as a black material, and, until some recent research, it was difficult for it to be soluble in water and it unveiled a high luminescence property. However, only the classical bulk carbon was associated with the black material because when its size was reduced to the nanometer scale its physical and chemical properties were drastically changed from the macroscopic material [8084]. The best known carbon nanomaterial could make several shapes including buckminsterfullerene (C60) [82], graphene [85], CNTs [83,86], nanodiamonds [81], carbon nanofibers [80,87], and CDs [8890], which were recently discovered. Similar to its widespread famous older cousins like fullerene, CNTs, and graphene, the most recent form of nano-carbon, the CDs, is motivating intensive research efforts in its own right. CDs were discovered accidently during the purification of SWCNTs fabricated from arc-discharge soot by Xu et al. [91]. CDs are a new-fangled family member of carbon nano-structured materials that contain quasispherical, discrete nanoparticles with sizes less than 10 nm [92,93]. The size and surface chemistry are important for the fluorescent carbon materials as the surface of the nanosized carbon material is comprised of sp2 and sp3 carbons, oxygen, and nitrogen chemical groups, and postmodified functional groups (Fig. 16.14). CDs mainly include carbon nanodots (CNDs), graphene quantum dots (GQDs), and polymer dots (PDs) [94]. GQDs [95] are anisotropic and hold one or a few layers of graphene and linked functional groups on their edges. The lateral dimensions of GQDs are larger than their height. CNDs are spherical in shape and categorized into carbon nanoparticles without crystal lattice, and carbon quantum dots a with crystal lattice. PDs are aggregated or cross-linked polymers derived from monomers or linear polymers. Due to the variety of CDs, there are many fabrication approaches for CDs, mainly divided into two groups: the top-down and bottom-up techniques. The top-down method is realized by cleaving or breaking down carbonaceous materials by means of a chemical, physical, or electrochemical approach. The latter involves carbonization of small molecules, pyrolysis, or stepwise chemical fusion of small organic molecules. Scientists are making efforts in this field due to the superior

Characterization tools and techniques of hydrogels 505

Figure 16.14 Three categories of fluorescent CDs are GQDs, CNDs, and PDs [94]. r 2015. CDs, Carbon dots; CNDs, carbon nanodots; GQDs, graphene quantum dots; PDs, polymer dots. Reproduced with permission from Springer.

optical properties of CDs in comparison with other carbonaceous materials, as well as their cost-effectiveness, ease of synthesis, large-scale production, tunable surface properties, biocompatibility, etc. [92,9699]. This variety of features empowers them in numerous applications such as sensing, bio-labeling, catalysis, anticounterfeiting, and as drug carriers and energy devices [51,99104]. These CDs combine a number of auspicious attributes of semiconductor quantum dots in terms of photoluminescence dependent on excitation wavelength and size, long-time photostability, and ease of bioconjugation while not having the problem of intrinsic toxicity or elemental scarcity and unnecessary harsh, tedious, expensive, or ineffective synthesis steps [105,106]. Ruiz-Palomero et al. reported fluorescent nanocellulose-based hydrogels for laccase-sensing applications [107]. The improvement in FH physical features is multidimensional. The main characterizations which make FHs significantly distinguishable are their mechanical, thermal, and optical properties. Most commonly hydrogels are a cross-linked polymer mass which is insoluble in solvents. These cross-links are either chemical or physical. However, when CDs are incorporated into gels, two types of cross-linking can be seen: chemical and physical. Some researches however have been carried out where chemical and physical cross-linking were attributed in hydrogels to improve their excellent toughness. PAAm and CD-based hydrogel with 650% elongation has been reported elsewhere [108]. Such hydrogels are superior in terms of their stress dissipation. Similar work was also reported by Zhu et al. associated with a self-healing characteristic [109]. The optical properties of FHs are primarily their UV-visible spectra. An agar-CD composite was prepared and evaluated

506 Chapter 16 by UV spectra. They reported a sharp peak for FHs in which approximately 75% of the total fluorescent behavior came from CDs [110]. Incorporation of CDs in ionic liquid is another way to drastically improve their luminescent property and gel strength [111]. Such hydrogels are desirable in their sensing applications, biomedical diagnosis, and controlledrelease behavior. Controlled release is a diffusion-based phenomenon. In these ionic liquidbased hydrogels small molecular imbibition can be tuned by adjusting their molecular interactions. Similar FHs with diffusion-based drug-release behavior were also reported by other researchers [112]. The temperature-responsive behavior of thermoresponsive hydrogels was reported in another research work [113]. The optical property of FHs was evaluated in this work. As normal, CDs are almost indifferent in their photoluminescent (PL) behavior when temperature has been kept as a function. But surprisingly, an anomalous trend has been noticed when thermoresponsive FH was tested against temperature variation. The PL behavior of CD-based PINIPAM hydrogels showed a downward trend with an increment in temperature. This decrement in PL intensity has been accounted for by an enhancement in scattering points in PINIPAM hydrogels just above the LCST. FHs were also reported by the addition of fluorescent molecules inside the gel matrix. Chitosan, PVA, and 9anthraldehyde were reported as a gelled mass which was tested under FL microscopy [114]. Again these data showed similar results as the gel phase acts as an incident laser-scattering center. Here also the sol phase or pregel state showed better FL behavior than the gel state. This result could also be indirect proof of gelation. In another reported work, the authors prepared hyperbranched poly(amidoamine) and oxidized alginate-based hydrogels which were injectable and self-healable in nature [53].

16.8 Microcomputed tomography Microcomputed tomography (μ-CT) is an extra transmission image technique. In this instrument, incident X-ray beams are coming from an X-ray generator and traverses through assemble followed by recording of the transmitted are scattered beams to the detector. In the detector we obtain images called radiographs. The specimen for analysis can be rotated or tilted to several degrees. The mode of rotation can lie in the range of 180 or 360 generating a series of projection images. The projection images are processed using software to evaluate the internal structure, morphology, microstructure, and void-filler inclusions present in the specimen. This technique can result in horizontal and vertical assessment of the projected sample. In brief, the micro-CT process can be elaborated/ performed in four consecutive steps. First is the extra generation, second the transmission of X-ray beams throughout the sample, third is the desired rotation of the sample to acquire a series of projection images, and lastly reconstruction of the projected images or a radiograph by computer software. Fig. 16.15 illustrates graphically the micro-CT process.

Characterization tools and techniques of hydrogels 507

Figure 16.15 Micro-CT graphical illustration. CT, Computed tomography.

Figure 16.16 Basic anatomy of an X-ray source used in micro-CT. CT, Computed tomography.

First we discuss the X-ray source. For CT systems, fixed beams are generated by directing electrons produced in a cathode. The material of the cathode is normally tungsten or copper. The target cathode emits X-ray beams which fall onto the sample. Fig. 16.16 illustrates the basic anatomy of an X-ray source. The beam size has significance in the radiograph and resolution; the finer the electron beam, the smaller the spot size of the X-rays and we obtain a better-resolution radiograph. The emitted X-rays have the specific shape of the projection. In general, the shape is likely to be a cone where the origination point is the spot on the target and the beam diverges in a conical shape. The second phase of the process is the absorption of X-ray beams in the sample. The exceptions are classified as partial absorption and differential absorption. Partial absorption corresponds to the photons observed in the specimen during the process. The differential absorption varies depending on the contrast. If there is no differential absorption the result comes out as a homogeneous gray level. The unabsorbed X-rays are recorded by the detector. In the third phase, rotation of the sample is very important. In this phase, the incident X-rays judge the total specimen in various dimensions and angles. This provides a better microstructure assessment and improved cross-sectional radiographs.

508 Chapter 16 The testing is mostly done in the case of porous gel samples. For porous gels, preparation freeze-drying has been chosen for uniform pore size. Hydroxyethyl methacrylate (HEMA)based aero gel has been characterized by micro-CT to assess the pore size and microstructure [115]. The electron microscopic images of the porous morphology of aerogels were initially shown. The main disadvantage of scanning electron microscopic images is their restriction to quantitative estimation of porosity and void volume present inside the matrix. Thus micro-CT is the ideal choice in such cases where quantitative requirement of porosity and pore volume is mandatory. Micro-CT also showed the interconnectivity of pores. As the porosity is a fundamental quality to evaluate aerogels and other porous type materials, micro-CT could correlate the other physical features. Thus, in an indirect fashion, micro-CT could explain the strength, compressibility, swelling behavior, kinetics, small molecular imbibition, and other diffusion-based data reduction. The homogeneous pore size in the horizontal as well vertical direction of a specimen also has been performed in another work [22].

16.9 Electrical characterizations General polymers are insulating in nature. However, for the scenario of hydrogel research the situation is different to an extent. Polyelectrolyte hydrogels are slightly different in their external stimuli responsiveness. In order to impose electro-conductivity in hydrogels, generally two strategies are adopted; one is using conducting polymer as an electron carrier and another is using conducting inclusions as an electron transporter. The second method is much better because of the ease of synthesis, fabrication, and simple purification methods. Such hydrogels are capable of controlled release of small molecules, namely, drugs, enzymes, and other endogenous chemicals. That is why these types of devices are called “intelligent” drug-delivery systems. Precise control of drugs in the human body is very important as an excess/inadequate proportion of drug could be detrimental for better physiological fitness. Now the question is why electro-stimulation is becoming so important in technology research? Electro-stimulation has a precise magnitude of current which can influence controlled release of small molecules even in in vivo systems—sensationally called iontophoresis and electroporation. This device is installed as a transdermal and dermal delivery unit [116]. This is not only limited to the area of laboratory research, it is also marketed with the trade name of Iontocaine, which is a medical unit for pulsatile release of lignocaine by iontophoresis. When a patient needs to be administered drugs, this small iontophoretic device can be attached to his/her arm as a dermal patch with the prescribed format of medications. Electrodes are normally attached to the conducting patch and the electrode terminals are connected to form a complete circuit for electro-stimulation. This drug-delivery technique reduces the requirement for surgical or invasive treatments. In an electro-responsive system, the hydrogels behave differently. Most often it is seen as a volume change, that is, swelling. Moreover, there are several observed physical changes to

Characterization tools and techniques of hydrogels 509 hydrogels, including deswelling, bending, and sometimes shape recovery behaviors. This behavior of hydrogels can result in them being used as actuators. Hydrogel bending is monitored in the case of mechanical devices, for example, valves, artificial limbs, soft robotics, molecular machines, and biomedical implanted devices [117]. Another method of interest is electro-conducting hydrogels with conductive inclusion or fillers. This area is based on a special theory of an electronic transportation phenomenon inside the hydrogel matrix [118]. These foreign inclusions liable for electronic transportation are called conductive fillers [119121]. The conductive fillers are mostly in nano dimensions. Nanofillers with electronic conduction are supported by the theory of electrical percolation [122124]. Electrical percolation is a special physical hypothetical phenomenon where a minimum number of conducting 3D networks, like pathways, are formed in the fabricated nanofiller-based hydrogels. Nanofiller-based hydrogels are called nanocomposite hydrogels. Percolation threshold is an effective term to designate a conducting hydrogel which reveals the quantitative estimation of required filler to gain an electric flow inside a hydrogel matrix. An IPN-based conducting gel coating was also reported which implied foreign inclusion enhanced electrical conductivity [125127]. For electrical responsiveness, the hydrogels were tested under diffident setups. The designs are depicted in Fig. 16.17. The device under test (DUT) is normally placed either in a phosphate buffer system or saline media. The environment of the electrodes applied should be in the conducting region in such a fashion that there will not be any interruption to the current flowing to the conducting patches. The electrodes applied are normally platinum or carbon. Sometimes noncontacting mode electrodes are also applied where the environment of the DUT has been kept in conducting media, such as electrovalent salts. For polyelectrolyte gel special when external electric field has been applied gel shows volume change. Polyelectrolyte gels are categorized as ionic as well as cationic gels; where ionic gels generally collapse or shrink at the anode end and the cationic gels shrink at the cathode end. This volume change of polyelectrolyte hydrogels is generally diffusion

Figure 16.17 Experimental setups of various electro-responsive drug-delivery systems.

510 Chapter 16 controlled. This negative volume change is described as hydrogel swelling. Such anisotropy in this hydrogel phenomenon can be monitored visually. Fluctuations in the gel dimensions are seen even in very low-magnitude electric currents. The volume change becomes significant when the externally applied voltage is high. However, the swelling is not always linear or proportional to the externally applied field. At a higher voltage when the gels start to the swell up to a certain degree, the resistivity of the intrinsic charges increases because of the decrease in free water present in the hydrogel matrix. Gong et al. proposed that the maximum amount of swelling is dependent on the charge transport throughout the hydrogel matrix, and not solely dependent on the externally applied voltage [128]. As per their hypothesis to evaluate the electrical property of hydrogel it is more appropriate to calculate the charge rather than the externally applied voltage. When the external electric field is off, the external environmental fluid molecules imbibes into the gel matrix. Hence this can be called a switch “onoff” phenomenon, which is dependent on the external electric field. The cyclic “onoff” experiment also has significance in calculating the gel strength and the longevity of the hydrogel specimens in service life. The electro-responsive character of polyelectrolyte hydrogels is dependent on many factors such as the experimental setup, composition of hydrogel, charge density, degree of cross-linking, pendant chains, time of swelling equilibrium, and the extent of hydrophilicity present in the matrix. Beside these, several divisions are also reported. Tanaka et al. reported a negative volume change to PAAm gel in a wateracetone mixture [129]. It was proposed that this volume change can be understood by means of a phase transition of the gel matrix. The phase transition is an accumulative effect of several competitive forces. The significant competitive forces are positive osmotic pressure of the counter ions, negative pressure due to interpolymer affinity, and rubber-like elasticity of the polymer spatial network. For electro-induced characterization the deswelling and swelling take place simultaneously when the gel deswells at one electrode at the same time the gel as swelling at the other electrode in the device. Such a volume change obeys three basic principles: first is the stress gradient generated in the hydrogel after application of an external electric field; second is localized pH variation in close proximity to the electrodes; and last is electro-osmosis of water molecules associated with the electrophoresis phenomenon. Another electro-induced responsive system has been reported by Kwon et al. [130]. They named the system electro-induced gel erosion. This theory proposed the erosion rather than any kind of deswelling after application of an electric field. When water-soluble monomers and polymers are mixed and interact chemically or by means of any kind of physical interactions, water-insoluble 3D polymeric networks have been formed gradually. Surface erosion from hydrogels occurs when one end of the hydrogel specimen is attached to an electrode and the other end is in a noncontact position keeping the distance of 1 cm apart in between them. When an electric field is applied, the free end starts to erode and produces lots of ions in the environment. Erosion from the gel surface results in disruption of

Characterization tools and techniques of hydrogels 511 physical and ionic cross-linking between the polymeric segments followed by leaching of drug molecules from the gel matrix. Because of this erosion, mass loss of the gel sample is observed and follows the zero-order release kinetics. This process continues until 80% of the initial mass has been lost from the matrix. This also ruptures the dimensional stability and shape of the cell matrix. However, there are several disadvantages to electro-responsive gels as drug carriers. These limitations include typically delayed release and response times, gel fatigue, and a nonlinear relationship between cumulative release (% release) and applied current. However, the slow release can be overcome by minimizing the amount of gel. Hydrogel microspheres or injectable hydrogels can be tuned to achieve the best electrostimulation results. Though electro-responsive drug-delivery devices are immense area of interest in the biomedical field, this field has not been fully explored in the pharmaceutical market.

16.10 Conclusion Hydrogels are classified as soft materials by materials scientists. In this chapter, the extensive testing of soft polymeric hydrogels has been illustrated. Porous hydrogels have some drawbacks due to improper stress distribution inside the matrix. Thus, characterization-based discussions have the utmost significance in the area of hydrogels. The wide areas of hydrogel applications need initial quality control before market promotion. The required physicomechanical features can be obtained after proper characterizations of hydrogels. Porous hydrogels are extensively used in superabsorbent-like applications. Toughness hydrogels have high importance in biomedical fields and some specific bio-glue applications. Moreover, very recently, conducting hydrogels also have gained immense interest in electric pulsatile drug delivery, thus electrical characterizations of hydrogel also had been discussed in this chapter. To summarize, the vital experimental tools and their related characterizations are the choice of materials scientists before the recommendations of any materials.

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acetic anhydride, 425
427 Acrylamide, 44, 106
107, 125
126, 135
136 3-(Acrylamido)-phenylboronic acid (3-APBA) molecules, 202 Acrylic acid (AA), 417
418 Acrylic acid/bacterial cellulose (AA/BC) hydrogels, 182, 182f 4-[(4-Acryloyloxyphenyl) azo] benzoic acid (AOPAB), 207 Activated carbon (AC), 411
412 Addition reaction, 99
101 Additives and nanoparticles, 239
240 Adipic acid, 425
427 Adipose-derived stem cells (ASCs), 287
288 Adsorbent, reusability of, 444 Adsorption, 411
412 Adsorption capacity, various factors on, 436
439 adsorbate initial concentration, 439 adsorbent dosage, 438 pH, 438 temperature, 436
437 time, 438 Adsorption isotherm, 442
444 Freundlich isotherm, 442 Langmuir isotherm, 442
444 Adsorption kinetics, 439
441 intraparticle diffusion model, 440
441 pseudo-first-order (PFO) model, 439

pseudo-second-order (PSO) model, 440 Aerobic bacteria, 342 Agar, 373
377 manufacturing process for, 375f Agar-CD composite, 505
506 Agar/chitosan thin films, 252 Agaropectin, 375
376 Agarose, 375
376 -based hydrogels, 163 structural unit of, 376f Agricultural polysaccharide hydrogel (APH), 329 Agriculture, natural polymer-based hydrogels for, 329 agricultural superabsorbent hydrogel on chlorophyll content, 338
340 on fertilizer release mechanism, 338
340 on soil texture, 338
340 biodegradable hydrogel and “nano” fertilizer release, 346
347 biodegradable polysaccharide hydrogel and “smart” fertilizer release, 347
349 phytotoxicity test, 345
346 polysaccharide hydrogels in control of plant disease, 344
345 mode of action for, 341
344 significance of, 349 root
soil interaction on plant sprouting, effect of, 341
344

519

seed coating by hydrogel, 340
341 soil conditioner polysaccharide hydrogel (SCPH), 330 natural polymer (polysaccharide)-based hydrogel, 335
338 natural polymers, 333
335 Agriculture and horticulture, 43
44 Aldehyde, natural polymer-based hydrogels prepared using, 96t Aldehyde hyaluronic acid (A-HA), 93 Alginate, 21, 29, 157, 256, 277, 377
380 -based anisotropic capillary hydrogels seeded with bone marrow stromal cells (BMSCs), 294f egg-box model, 378
379, 380f ionically cross-linked hydrogels based on, 59
63 manufacturing of, 380f properties of, 174t, 381t Alginate-based hydrogels, 156
158, 483
484 Alginate/carboxymethyl cellulose (CMC) blend hydrogel bead, 59
62 Alginate/gelatine-blended hydrogels, 252 Alginic acid, 427
429 Alginic acid microcapsule, 3
4 Alkylation of chitosan (ACS), 73
74

520 Index α-L-guluronic acid, 377
378 α-transition, 494 Amidated low methoxy pectin (ALMP), 385
386 3-Aminopropyl triethoxysilane (APTES), 417
418 5-Aminosalicylic acid (5-ASA), 199 Amphotericin B-based biohydrogel, 260 Amphoteric ions with inherent antibacterial properties, 253
254, 255f Amylase, 18
19 Amylopectin, 18
19, 424 chemical structures of, 427f Amylose, 424 chemical structures of, 427f “Anaerobic” bacteria, 342 Anhydroglucose units (AGU), 161 Anionic hydrogels, 412
413 Antibacterial (KIGAKI)3
NH2 peptide, 253 Antibacterial agent-containing hydrogels, 258
259 Antibacterial properties, hydrogels with, 252
259 Antibiotics, 258 Antifungal properties, 260
262 Antiinflammatory properties, 262
263 Antimicrobial resistance, 309 Antioxidant properties, hydrogels with, 247
252 Antiviral properties, 260 Applications and developments of natural polymer-based hydrogels, 175f Arbuscular mycorrhizal (AM), 342 Arsenic (As), 420 Aspergillus fumigates, 262 Atom transfer radical polymerization (ATPR), 206 Autonomous healing concept, 237f 2,20 -Azobis (2-amidinopropane) (AAPH), 250

B Bacterial cellulose (BC), 180f, 290, 422 -based hydrogels, 494
495 properties of, 174t Bacterial nanocellulose (BNC), 278
279 Bacterial vaginosis, 312
313 Basic fibroblast growth factor (bFGF), 293 Benzaldehyde-modified PEG, 233 β-chitin/nanosilver scaffolds, 256
257 β-D-mannuronic acid (M), 377
378 β-sheet peptides (PP), 176
177 β-transitions, 494 Bifidobacterium, 342 Bioactive substances, 292
293 Biocompatibility, 3
4 Biodegradable hydrogel, 5 and “nano” fertilizer release, 346
347 and “smart” fertilizer release, 347
349 Biogum, 357 Biohydrogels, 247 propertiesof. See Biological properties of hydrogels Biological extract-loaded hydrogels, 259 Biological properties of hydrogels, 247 antibacterial properties, 252
259 antibacterial agent-containing hydrogels, 258
259 hydrogel with inherent antibacterial activity, 252
254 nanocomposite hydrogels with antibacterial activity, 254
258 antifungal properties, 260
262 antiinflammatory properties, 262
263 antioxidant properties, 247
252 antiviral properties, 260 Biological sulfated polysaccharides, 299

BL19LXU, 498
499 Bone marrow stromal cells (BMSCs), 294 Bone morphogenetic protein (BMP-2), 298 Bone tissue engineering tissue engineering scaffolds in, 296
298 Bonse
Hart channel cut collimation system, 498 Bounded and free water, determination of, 462
464 Brain-derived neurotrophic factor (BDNF), 294 Brain tissue tissue engineering scaffolds in, 294
295 Buckminsterfullerene (C60), 504
505 Bulge experiment, 487
488 Bulk hydrogels, 8 Bulk polymerization, 153 Bulk water, 151
152 Butanedioldiglycidylether (BDDE), 95
96, 97f, 98f, 99

C Cadmium, 423
424 Calcium alginate, 285 Calixarenes, 231 Candida albicans, 260, 262, 312 Carbon dots (CDs), 504
505 Carbon nanodots (CNDs), 504
505 Carbon nanofibers, 504
505 Carbon nanomaterial, 504
505 N-Carboxyethyl chitosan (CEC) polymer, 183f Carboxylatednanocrystalline cellulose (CNCC), 179, 210 Carboxymethyl cellulose (CMC), 120
121, 162, 259, 334, 422
based hydrogel, 126
128 Carboxymethyl chitosan (CMCh), 93, 173
174 N-Carboxymethyl chitosan, 313
314 O-Carboxymethyl chitosan, 314

Index Carboxymethyl starch (CMS), 428
429 Carrageenan, 123
124, 135
136, 319, 368
372, 433
436 products containing, 374t properties of, 373t Carrageenan-based hydrogels, 158 Carraguard, 319 Cartilage tissue engineering cellulose-based hydrogels for, 298
299 Catechol-modified four-arm PEG (cPEG), 233 Cationic hydrogels, 412
413 Cefazoline, 137 Cell-cross-linking, 157 Cell delivery system, hydrogelmediated, 293 Cell transplantation, 294 Cellulose, 17
18, 30, 177
179, 278
279, 329
330 -based pH-responsive hydrogels, 173
174 -based polymers, 389
394 -coated magnets, 186 composites, 76
77 derivatives, 74
76 properties of, 174t structure of, 18f, 161
162, 286
299, 422
424 Cellulose-based hydrogels, 161
162, 422
424 in bone tissue, 296
298 in brain tissue, 294
295 in cartilaginous tissue, 298
299 for dyes removal, 422
423, 426t for heavy metals removal, 423
424, 426t in myocardial engineering, 292
293 in stem cells, 286
288 in urinary system, 296 in vascular regeneration, 288
291 Cellulose acetate, 422 Cellulose dissolution solvent, 336
337 Cellulose ether-based hydrogels, 336
337

Cellulose-grafted polymers, 184
185 Cellulose hydrogel beads (CHBs), 178f Cellulose nanocrystals (CNCs), 194
195 pH-responsive mechanism of, 175f Cellulosic derivative-based hydrogel, 336
337 Cement, compressive strength of, 143
144 Cerium oxide nanoparticle
alginate nanocomposite hydrogel, 250
251 Cerium oxide nanoparticles, 250
252, 251f Characteristics of hydrogels, 38
40 mechanical properties, 39
40 cross-linking, 40 porosity and permeation, 39
40 physical and chemical properties, 38
39 swelling and solubility, 38
39 rheology, 40 Characterization, of hydrogels, 412
415 Fourier transform infrared, 413 point of zero charge, 414
415 scanning electron microscopy (SEM), 413
414 swelling, 414 X-ray diffraction (XRD), 414 Characterization tools and techniques, 481 electrical characterizations, 508
511 fluorescent behaviors of hydrogels, 504
506 mechanical characterizations of hydrogels, 486
490 bulge experiment, 487
488 compressive testing, 487 indentation test, 489
490 uniaxial tensile testing, 486

521

microcomputed tomography (μ-CT), 506
508 microstructure
property relationship, 482
485 rheology, 490
497 creep behavior, 491
492 dynamic mechanical behavior, 493
497 stress relaxation, 492
493 viscoelasticity and microstructure, 490
491 small-angle neutron scattering (SANS), 501
503 small-angle X-ray scattering (SAXS) technique, 498
501 Charge couple detectors (CCD) camera laser probe, 487
488, 498
499 Charlesby
Pinner equation, 464
465 Charlesby’s equation, 466 Chemical cross-linking methods, 92
109 addition reaction, 99
101 click chemistry, 101
105 condensation reaction, 106 enzyme-catalyzed cross-linking, 107
108 epoxide-based cross-linking, 95
99 free radical polymerization, 106
107 miscellaneous, 108
109 Schiff base reaction, 92
95, 92f, 93t Chemical hydrogels, 4, 32
35 enzymatic reaction, crosslinking by, 35, 35t free-radical polymerization, 34 functional groups, cross-linking by, 33
34 by condensation reaction, 33
34 cross-linking with aldehydes/ dihydrazide/Schiff-base formation, 33
34 Michael addition reaction, 33 high-energy radiation, crosslinking by, 34
35

522 Index Chemical hydrogels (Continued) UV light, cross-linking by, 34 Chemically covalent cross-linked hydrogels, 332, 334
335 Chemically cross-linked hydrogels, 412
413 Chemical-responsive hydrogel, 5
7 Chemical stimuli-responsive hydrogels, 41
44 glucose-sensitive hydrogels, 42
44 natural hydrogels, applications of. See Natural hydrogels, applications of pH-responsive hydrogels, 41
42 Chitin, 22 structure of, 22f Chitosan (CS), 22, 122, 128
129, 173
174, 182
183, 256
257, 277, 310, 313
318, 323t, 334
335 -based hydrogels, 29, 154
156, 233, 280f, 313
318, 416
421 for dyes removal, 417
418, 420t for heavy metals removal, 418
421, 420t chemical structures of, 416f intrinsic antimicrobial properties of, 310
313 ionically cross-linked hydrogels based on, 63
64 properties of, 174t structure of, 22f Chitosan derivatives, hydrophobic association hydrogels derived from, 72
74 Chitosan/glycerophosphate hydrogel, 70
71 Chitosan
gold nanoparticle hydrogels, 315 Chitosan-graft-poly(acrylic acid)/ cellulose nanofibril hydrogel composite, 176, 176f Chitosan
pectin composite hydrogel, 258

Chitosan
silica hybrid (CSH) hydrogel enhanced by HA (HA-CSH), 280
281 Chlorophyll, 338, 339f Chondruscrispus, 123
124 Chromium, 432 Chrysanthemum instagram, 143 Ciprofloxacin-loaded hydrogels, 258 Citric acid, 108
109 Citric acid-based thermoresponsive hydrogel, 252 Classification, of hydrogels, 4
10, 26f based on the formation mechanism of threedimensional network structure, 4 chemical hydrogels, 4 physical hydrogels, 4 based on their degradability, 5 biodegradable hydrogels, 5 nonbiodegradable hydrogels, 5 based on their functions in practical applications, 8
10 in situ hydrogels, 8
9 molecularly imprinted hydrogels, 9 nanohydrogels, 9
10 porous hydrogels, 10 based on their responsiveness to external stimuli, 5
8 environmentally responsive hydrogels, 5
7 environmentally unresponsive hydrogels, 8 based on their size, 8 bulk hydrogels, 8 microhydrogels, 8 based on their sources, 4 natural polymers, 4 synthetic hydrogels, 4 Clay-based hydrogels, 494
495 Clay-incorporated hydrogels, 491
492 Click chemistry, 101
105 60 Co γ-ray irradiation, 125
133, 137, 143
144

Cold-set gels, 397
398 Collagen, 165
167, 275
276, 278 and gelatin, 23
24, 27 Collagen/cellulose hydrogel beads (CCHBs), 177
179, 178f Collagen type 1/chitosan hydrogel, 263 Collimation system, 498 Compressive testing, 486f, 487 Condensation reaction, 33
34, 106 Conducting hydrogels, 511 Conductive fillers, 509 Congo Red (CR), 417
418 Conjugated addition reaction. See Michael addition reaction Controlled-release fertilizer (CRF), 338 Copaiba oil nanoemulsions, 262
263 Copolymeric hydrogels, 37, 152 Copolymerization technique, 106
107 Copolymer worms, 176
177 Copper, 419 Copper-catalyzed azide-alkyne cycloaddition (CuAAC), 101, 102f Copper ion cross-linked nanofibrillated cellulose hydrogel, 59 Cord blood mononuclear cell (CBMNC) transplantation therapy, 293 Correlation peak, 498 Corymbiacitriodora, 345 Cosmetics, 44 Covalent cross-linking, 155 Covalent interactions, self-healing mediated by, 226
228 Creep behavior, 491
492 Creep test, 486 Cross-link density, 464
468, 484
485 Cross-linked HEMA hydrogel, 3
4 Cross-linked hydrogels, 3 Cross-linked network, 3, 119 Cross-linked porous starch (CPS), 427
428

Index Cross-linked silicone-based materials, 235 Cross-linking of polymers, 223 Crown-ethers, 231 Crystallization, 31 Cu-chitosan nanoparticles, 344
345 Cucurbiturils, 231 Cucurbit[8]uril (CB[8]) gel, 231
233 CuO nanoparticles, 258 Curdlan, 365
368 chemical structure of, 366f food applications of, 369t CyamopsistetragonolobusLinn., 21 Cyclodextrins (CDs), 104
105, 231

D Deacetylation (DD), 71 Defect filling stent, 296 Deferoxaminemesylate (DFA), 79 Degree of methyl esterification (DM), 385 Desoiling, 484 Device under test (DUT), 509 Dextran, 30, 250 properties of, 174t Dextran and gelatin (DEXGEL), 285 Dextran/chitosan hydrogel formulations, 260 Dextrin-based hydrogels, 494
495 Dicarboxylic acids, 108
109 Didiol
borax complex network, 185
186 Diels
Alder reaction, 64
67, 102
104, 104f, 234
235 Differential scanning calorimetry (DSC), 39, 464 2,6-Dihydroxynaphthalene, 209
210 N-(3,4-Dihydroxyphenethyl) acrylamide (DOPAm), 188 Dihydroxyphenylalanine (DOPA)modified PEG, 229 2,20 -Dimethoxy-2-phenyl acetophenone, 106
107

N-(3-Dimethylaminopropyl)-N0 ethylcarbodiimide hydrochloride, 93 N,N-(3-Dimethylaminopropyl)-Nethyl carbodiimide (EDC), 106 Direct Red 81 dye, removing of, 427
428 Dithiothreitol, 215
216 Donnan effects, 473 Double-network (DN) hydrogel, 64
67, 281
282, 496 Doxorubicin, 182
183, 193
194 Doxorubicin hydrochloride, 215
216 Drug delivery, 42
43, 137
138 Drug-loaded nf-BC/SA hybrid hydrogels, 212f, 213f, 214f Dual cross-linked hydrogels, 201, 202f Dyes removal cellulose-based hydrogels, 422
423, 426t chitosan (CS)-based hydrogels, 417
418, 420t κ-carrageenan, 434
435, 437t sodium alginate (NaAlg), 431
432, 433t starch (ST) based hydrogels, 427
428, 429t Dynamic mechanical behavior, 493
497 Dysphagia management, food for, 396
398

E Econazole, 260
261 “Egg-box” model, 59 EIGER detectors, 498
499 Electrical characterizations, 508
511 Electrically responsive hydrogels, 41 Electric field, 5
7 Electro-conducting hydrogels, 509, 511 Electro-induced gel erosion, 510
511 Electron beam (EB) accelerator, 120

523

Electroporation, 508
509 Electroresponsive hydrogel, 289
290 Electrostatic interactions, 226
228 between natural polyelectrolytes with opposite charges, 53
59 blending of natural polyelectrolyte solutions, 54
55 electrostatic screened complexation by salt, 55 semidissolution acidification sol
gel transition, 55
59 Electrostatic screened complexation by salt, 55 Elicitors, 344 Embryoid bodies (EBs), 75
76 Embryo stem cells, 286
287 Environmentally responsive hydrogels, 5
7 Environmentally unresponsive hydrogels, 8 Enzymatic reaction, cross-linking by, 35, 35t Enzyme-catalyzed cross-linking, 107
108 Enzyme-mimetic electron transfer reactions, 251
252 Epichlorohydrin (ECH), 95
99, 97f, 98f, 417 Epoxide-based cross-linking, 95
99 Equilibrium degree of swelling (EDS), 414 Equilibrium swelling ratio (ESR), 179 Escherichia coli, 253, 314
317 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide, 165 Ethyl cellulose (EC), 335
336 2,20 -(Ethylenedioxy)diethanethiol (DEG), 104
105 Ethyleneglycoldiglycidylether (EGDE), 95
96, 97f, 98f, 99 Eubacterium, 342 Eucalyptus pilularis, 345 Eucheuma, 123
124

524 Index Eucheumadenticulatum, 123
124 European XFEL, 498
499 Extensiometry, 486
487 Extracellular matrix (ECM), 278
279 content of, 275f

F Fat mimetics (FMs), 367
368, 403
404 Fat replacer (FR), 403 Fe-catechol gel, 229 Fibrin, 28 Fibrin gel, 298 Fibroblast-derived matrix (FDM), 288f First-generation hydrogels, 482 Fluid gels, 360
361, 394
396 Fluorescent behaviors of hydrogels, 504
506 Fluorescent hydrogel (FH), 504
506 Fluorescent injectable hydrogels, 496 Fmoc-based cationic hydrogel, 254 Food industry, natural polymerbased hydrogels in, 357
410 agar, 373
377 alginate, 377
380 carrageenan, 368
372 cellulose-based polymers, 389
394 curdlan, 365
368 dysphagia management, food for, 396
398 fat mimetics (FMs), 403
404 gellan gum, 362
365 gelling natural polymers, food applications of, 394
404 fluid gel, 394
396 jelly confections, 399
400 konjacglucomannan (KGM), 381
382 pectin, 383
389 3D printed food, 400
403 2-Formylphenylboronic acid, 261
262 Fourier transform infrared, 413

Free-radical polymerization, 34, 106
107, 157 Free water, 151
152, 462 Freundlich isotherm model, 442 Full-interpenetrating polymer networks (full-IPN), 249, 496 Functionalized natural polymers, 194

G Gamma radiation technique, 464
465 γ-ray irradiation, 125
127, 125f, 129
131, 133
136 Gaussian chains, 468 Gelatin, 23
24, 400 -based hydrogels, 494
495 properties of, 174t Gel fraction study, 460
461 Gel inhomogeneity, 473
475 Gellan gum, 357, 362
365, 397
398 -based poly(lactide-co-glycolide) nanoparticle-loaded system, 259 food applications of, 365t key properties of, 365t Gel
sol transition, 5
7 Genipin, 108 Genipine, 334
335 Gentamicin-loaded hydrogels, 258
259 Glucan, properties of, 174t Glucose polymers, 329
330 Glucose-responsive hydrogels based on natural polymers, 202
204 Glucose-sensitive hydrogels, 42
44 applications of natural hydrogels, 42
44 agriculture and horticulture, 43
44 biomedical applications, 42
43 cosmetics, 44 hygiene products, 44 separation technology, 44 tissue engineering, 43 optical fibers, 203f

Glutaraldehyde, 93, 425
427 -cross-linked collagen hydrogel scaffolds, 296 Glycerophosphate (GP), 70
71 Glycidyl methacrylate (GMA), 207 Glycogen, 329
330 Gold nanoparticles, 257 Grafting, 120, 124
125, 135
136 Graphene oxide (GO), 205
206 Graphene quantum dots (GQDs), 504
505 Graphene-reinforced hydrogels, 494
495 Groundwater, 435
436 Guaran. See Guar gum Guar gum, 21 structure of, 21f Guava leaf extract-loaded hydrogels, 252 Gummy candy, 399
400 GY785, 299

H Hard gels, 361 Healing agents, 224 Heavy metals adsorption, 187f removal cellulose-based hydrogels, 423
424, 426t chitosan (CS)-based hydrogels, 418
421, 420t κ-carrageenan, 435
436, 437t sodium alginate (NaAlg), 432, 433t starch (ST) based hydrogels, 428
429, 429t Hertz contact theory, 489
490 Heteropolysaccharides, 333 High-energy radiation, crosslinking by, 34
35 High internal phase emulsions (HIPEs), 187
188 High methoxyl pectin (HMP), 385
386 Historical view of hydrogel characterization, 457 bounded and free water, determination of, 462
464 gel fraction study, 460
461

Index hydrogel network characterization, 464
473 cross-link density, 464
468 mesh size, 469
471 molecular weight between cross-links, 468
469 porosity of hydrogels, 472
473 inhomogeneity of hydrogels, 473
476 swelling ratio measurement, 461 weight loss measurement, 461
462 Homogalacturonans (HGs), 384
385 Homogeneous network of Gaussian chains, 468 Homopolymeric hydrogel, 37, 152 Homopolysaccharides, 333 Honey, 259 Host
guest interactions, 231
233 Human bone marrow
derived MSCs, 288 Human embryonic stem (HES), 75
76 Human nasal chondrocyte (HNC)based cartilage engineering, 290 Human umbilical vein endothelial cells (HUVECs), 196 Hurd
Schmidt model, 500
501 Hyaluronic acid (HA), 22
23, 93, 123, 123f, 297 structure of, 24f Hyaluronic acid (HA) gellan gum, 362
365, 395
396 Hyaluronic acid-based hydrogels, 28
29, 101
102, 133
135, 159 Hyaluronic acid/cellulose nanocrystal bionanocomposite hydrogel (HA-CNCs), 287 Hydraulic bulging test, 486f Hydric soils, 342 Hydrocolloids, 357, 359f, 361
362, 402
405 Hydrogen bonding, 32, 230
231 formation of hydrogels via, 77
80

effect of hydrogen bonding on the properties of physical hydrogels, 79
80 physical hydrogels, 77
78 Hydrogen peroxide, 251
252 Hydrophilic gels, 459 Hydrophilic groups/segments, 3 Hydrophilic hydrogels, 332 Hydrophilicity, 3
4 Hydrophilic polymers, 3D structure of, 284 Hydrophobic association hydrogels, 74
77 based on chitosan, 69
74 chitosan/glycerophosphate hydrogel, 70
71 hydrophobic association hydrogels derived from chitosan derivatives, 72
74 derived from cellulose composites, 76
77 derived from cellulose derivatives, 74
76 Hydrophobic associations, 69, 228
229 Hydrophobized polysaccharides, 32 Hydroxyapatite (HA), 280
281 Hydroxyethylcellulose-based hydrogels, 263 2-Hydroxyethyl methacrylate, 24
25 Hydroxyethyl methacrylate (HEMA)-based aero gel, 508 Hydroxypropyl cellulose-acrylic acid (HPC-AA), 181 Hydroxypropyl methylcellulose (HPMC), 190
192, 191f, 361
362, 390
392 Hygiene products, 44

I Iminoboronate
chitosan-based hydrogels, 261
262, 261f Indentation test, 486f, 489
490 Inflammation-targeting hydrogel microfibers, 263

525

Inherent antibacterial activity, hydrogel with, 252
254, 263 Inhomogeneity of hydrogels, 473
476 In situ hydrogels, 8
9 Insulin drug-release testing, 181 “Intelligent” drug-delivery systems, 508
509 Intelligent hydrogels. See Environmentally responsive hydrogels Intensity correlation functions (ICF), 475
476 Interpenetrating network, 38 Intraparticle diffusion model, 440
441 Inulin, 20 structure of, 20f Ionically cross-linked (IC) hydrogels, 59
67 based on alginate, 59
63 based on chitosan, 63
64 reversible double-network hydrogel, 64
67 Ionic cross-linking process (ICP), 157, 334 Ionic interaction, 31
32 Ionic type hydrogels, 412
413 Ionotropic hydrogel, 334
335 Iontophoresis, 508
509 ι-carrageenans, 368
370 IPN-based conducting gel coating, 509 IPN-type hydrogels, 483
484 Islet-like clusters (ILCSs), 285 Itaconic acid, 417
418

J Japanese Industrial Method K8150, 461 Jelly candy, 399
400 Jelly confections, 399
400

K κ-carrageenan, 368
370, 433
436 chemical structure of, 434f for dyes removal, 434
435, 437t

526 Index κ-carrageenan (Continued) for heavy metals removal, 435
436, 437t Kappaphycusalvarezii, 123
124 Keratin-based hydrogels, 165 Konjacglucomannan (KGM), 381
382 applications and functional uses of, 384t chemical structure of, 382f Kratky collimation system, 498

L Lactobacillus rhamnosusATCC 53103, 182 LA gellan gum, 362
365, 397
398 λ-carrageenans, 368
370 Laminaria hyperborean, 122 Langmuir isotherm model, 442
444 LCLS in the United States, 498
499 Lead, 419 Lessonia, 122 D-Leucine-phenylalaninephenylalanine, 258 Light-responsive cotton fibers, 206 Light-responsive hydrogels based on natural polymers, 204
208 Lignin, 19
20 -based hydrogels, 167
168 Liquid hydrogels, 37 Locust bean gum (LBG), 358, 370 Lower critical solution temperature (LCST), 5
7, 192 Low methoxyl pectin (LMP), 385
388, 387f Lysyl oxidase, 107
108

M Macroporousbilayered tubular chitosan
gelatin scaffolds, 291f Magnetic cellulose-based nanocomposite beads (MCNBs), 423
424, 425f

Magnetic cellulose microspheres (MCMs), 189
190 Magnetic cellulose
TiO2 nanocomposite microspheres (MCTiMs), 189
190, 191f Magnetic chitosan
cellulose hydrogels, 187f Magnetic CMChitoCar beads, synthesis of, 214f Magnetic field-sensitive hydrogel, 5
7 Magnetic-responsive natural polymer-based hydrogels, 186
192 natural polymer and magnets grafted into polymer networks, 189
192 natural polymers as coatings for magnets, 186
188 Magnetic scattering, 501 Magnetic γ-Fe2O3 nanoparticles, 187 Malachite Green (MG), 417
418 Maturation (heat-induced aggregation), 32 Maxwell model, 467, 493 MCC/hydrocolloid products, 393
394 MCCM, 187 Mechanical characterizations of hydrogels, 486
490 bulge experiment, 487
488 compressive testing, 487 indentation test, 489
490 uniaxial tensile testing, 486 Medipix detectors, 498
499 Mesenchymal stem cells (MSCs), 285, 288 Mesh size, 469
471 Metal and metal-oxide nanoparticles, 254 antibacterial mechanisms of, 256f Metal-complex dyes, 417
418 Metal ion adsorption, 140
141 Metal
ligand coordination complexation, 229 Metallo-supramolecule, 229

Methacrylic anhydride (MA)modified hydroxypropyl cellulose (HPC), 192
193, 193f (Methacryloyloxy) ethyl carboxyethyl chitosan (MAOECECS), 101 Methylcellulose (MC), 192, 361
362, 390
392 Methylcellulose (MC) hydrogel, 210, 287
288 Methylene bisacrylamide (MBA), 106
107, 423
424 N,N0 -Methylenebisacrylamide, 474 Methylene blue (MB), 422
423, 434
435 Metronidazole, 320
321 Michael addition reaction, 33, 99
101, 100f Micrangium, formation of, 283 Microcapsules, 237
238 Microcomputed tomography (μ-CT), 506
508 Microcrystalline cellulose (MCC), 361
362, 392
394, 392f, 393f, 404, 423
424 Microhydrogels, 8 Microorganism, 341
343 Microscopy techniques, 473 Microstructure
property relationship, 482
485 Microvascular networks, 239 Molecularly imprinted hydrogels, 9 Molecular weight between crosslinks, 468
469 Mono-responsive hydrogels, 209 Multipolymeric hydrogels, 152 Multistimulation-responsive hydrogels based on natural polymers, 209
217 Myocardial engineering, tissue engineering scaffolds in, 292
293 MYTHEN detector, 498
499

N Nanoclay, 500
501 Nanocomposite beads (MCNBs), 423
424

Index Nanocomposite hydrogels, 487, 500
501 creep study for, 491
492 with antibacterial activity, 254
258, 263 Nanocrystalline cellulose (NCC), 176
177 Nanodiamonds, 504
505 Nanofertilizers, 346
347 Nanofiller-based hydrogels, 509 Nanofiller-based semi-IPN hydrogels, 494
495 Nanofillers with electric conduction, 509 Nanohydrogels, 9
10 Nanoparticles, 239
240, 313
315 “Nano” fertilizer release, biodegradable hydrogel and, 346
347 Naphthyl-functionalized cellulose (HEC-Np), 209
210 Natural hydrogels, applications of, 42
44 agriculture and horticulture, 43
44 biomedical applications, 42
43 drug delivery, 42
43 wound dressing, 42 cosmetics, 44 hygiene products, 44 separation technology, 44 tissue engineering, 43 Natural hydrogels, classification of, 25
38 based on the nature of crosslinking, 30
35 chemical hydrogels, 32
35 physical hydrogels, 31
32 basis of charge, 36 basis of composition, 37
38 copolymeric hydrogel, 37 homopolymeric hydrogel, 37 interpenetrating network, 38 semiinterpenetrating network, 37 basis of configuration of polymer chain, 37 on the basis of physical state, 36
37 liquid hydrogels, 37

semisolid hydrogels, 36 solid hydrogels, 36 hydrogels from proteins, 26
28 collagen and gelatin hydrogels, 27 fibrin, 28 silk proteins/fibroin, 27
28 polysaccharide-based natural hydrogels, 28
30 alginate hydrogels, 29 cellulose, 30 chitosan hydrogels, 29 dextran, 30 hyaluronic acid hydrogels, 28
29 Natural polymer-based hydrogels, 3
4, 152, 335
338, 459
460 cellulosic derivative-based hydrogel, 336
337 prepared using aldehyde, 96t prepared using epoxy crosslinking agents, 99t starch derivative-based hydrogel, 338 Natural polymers, 17
24, 333
335 based on proteins, 23
24 collagen and gelatin, 23
24 based on proteins, 23
24 collagen and gelatin, 23
24 polysaccharides from animal origin, 22
23 chitin and chitosan, 22 hyaluronic acid, 23 xanthan gums, 22 polysaccharides of plant origin, 17
21 alginate, 21 cellulose, 17
18 guar gum, 21 inulin, 20 lignin, 19
20 starch (amylose and amylopectin), 18
19 self-healing properties of hydrogels based on. See Self-healing hydrogels Network characterization, of hydrogel, 464
473

527

cross-link density, 464
468 mesh size, 469
471 molecular weight between crosslinks, 468
469 porosity of hydrogels, 472
473 Newtonian mechanical behavior, 3 N-hydroxysuccinimide (NHS), 106 N-isopropylacrylamide (NIPAm), 215 Nitrogen fertilizers, 342
343 Nitrous oxide, 341
343 Nonbiodegradable hydrogels, 5 Noncovalent interactions, selfhealing mediated by, 225
233 electrostatic interactions, 226
228 host
guest interactions, 231
233 hydrogen bonding, 230
231 hydrophobic associations, 228
229 metal
ligand coordination complexation, 229 Nondestructive high-resolution radiography, 472
473 Nuclear magnetic resonance (NMR), 39 Nutricote, 347

O Olejniczak
Rosiak
Charlesby equation, 465 Optical coherence tomographybased spherical microindentation (OCTSM), 489
490 Oscillatory shear test, 240 Osmocote, 347 Oxa-Michael addition, 101 Oxanorbornadiene (OB) cycloaddition reaction, 102, 103f Oxetanes (OXE), 236 Oxidative stress, 247
248 Oxime reaction, 102, 105f Oxolanes (OXO), 236 Oxyanion pollutant, 418
419 Oxygen-insensitive free radicals, 235
236

528 Index P PAAm-MC, 337 PAM-GO-gelatin hydrogels, 205
206, 205f Pectin, 383
389, 385f Peptide-based materials, 226 Peptides with antibacterial peptides, 253 Peptostreptococcus, 342 Percolation threshold, 509 Periodontal diseases, chitosanbased hydrogels for, 321
323, 323t Phase transformation, 292
293 Phenylboronic acids (PBA), 202 Phosphoryl chloride, 425
427 Photocatalysis, 257 Photo-cross-linked methacrylated dextran, 259 Photodynamic therapy (PDT), 210
211 Photoluminescence (PL), 210, 506 Photopolymerizable chitosan derivatives, 101 Photoresponsive hydrogels, 5
7, 41 Photosensitizer (PS), 210
211, 212f pH-responsive hydrogels, 5
7, 41
42 pH-responsive natural polymerbased hydrogels, 173
186 modified natural polymer network-introduced pH responses, 179
186 with properties enhanced by natural polymers, 176
179 pH-responsive shape memory materials, 184f Physical hydrogels, 4, 31
32 crystallization, 31 hydrogen bonding, 32 hydrophobized polysaccharides, 32 ionic interaction, 31
32 maturation (heat-induced aggregation), 32 stereocomplex formation, 31 Physical hydrogels based on natural polymers, 49

crystallization cross-link, formation of the hydrogels via, 67
69 formation of hydrogels via hydrogen bonding, 77
80 effect of hydrogen bonding on the properties of physical hydrogels, 79
80 physical hydrogels, formation of, 77
78 hydrophobic association hydrogels, 74
77 derived from cellulose composites, 76
77 derived from cellulose derivatives, 74
76 hydrophobic association hydrogels based on chitosan, 69
74 chitosan/glycerophosphate hydrogel, 70
71 derived from chitosan derivatives, 72
74 ionic interaction, 53
67 formation of hydrogels via electrostatic interaction between natural polyelectrolytes with opposite charges, 53
59 formation of ionically cross-linked hydrogels based on natural polymers, 59
67 Physically cross-linked hydrogels, 412
413 Physical stimuli-responsive hydrogels, 41 electrically responsive hydrogels, 41 photoresponsive hydrogels, 41 temperature-responsive hydrogels, 41 Phytophthoraparasitica, 344 Phytotoxicity test, 345
346 PILATUS detectors, 498
499 Pinhole (slit) collimation system, 498 PINIPAM hydrogels, 506 PIXcel detectors, 498
499

Plant sprouting using polysaccharide polymers, 344 Platelet-reach plasma (PRP), 318 Pluronic
α-cyclodextrin supramolecular hydrogels, 259 Point of zero charge, 414
415 Poisson distribution, 474 Poly(2-hydroxyethyl methacrylateco-acrylic acid), 464 Poly(2-hydroxyethyl methacrylate-comethacrylic acid), 464 Poly(acrylamide), 249 Poly(acrylic acid) (pAA) polymer, 226 Poly(caprolactone) (PCL), 240 Poly(DOPAm-coPFOEA), 188 Poly(ethylene glycol) (PEG), 252
253 PEG-dialdehyde, 93 PEG
PLA physical interactions, 482 Poly(ethylene glycol)
poly (ε-caprolactone)-based polyurethane (PECU), 184
185 Poly(HEMA), 484 Poly(hydroxyethyl acrylate) hydrogels, 464 Poly(lactic-co-glycolic acid) (PLGA), 275 Poly(L-glutamic acid)-grafthydroxyethyl methacrylate hydrogels, 259 Poly(L-glutamic acid-2hydroxylethyl methacrylate) (PGH), 181 Poly(L-glutamic acid-2hydroxylethyl methacrylate)/ hydroxypropyl celluloseacrylic acid (PGH/HPCAA) microgel, 181f Poly(N-isopropylacrylamide) (PNIPAM) hydrogels, 179, 195, 503, 503f

Index Poly(N-isopropylacrylamide)/ethyl cellulose (PNIPAM/EC) core
shell microspheres, 198
199, 198f, 199f Poly(N-isopropylacrylamide) hydrogels, 5
7 Poly(N-isopropylacrylamide-cobutyl methacrylate) (PNB) nanogels, 196 Poly(N-vinylcaprolactam) (PNVCL), 194
195 Poly(tannic acid)-based hydrogels, 248
249, 249f Poly(vinyl alcohol) (PVA), 491
492 Poly(vinyl alcohol) (PVA)-borax hydrogels, 185
186, 185f Polyacrylamide (PAAm), 64
67 Polyacrylamide (PAM) network, 205
206 Polyacrylamide gels, 474 Polyacrylate (PAA), 500
501 Polyacrylic acid, 4 Polycaprolactone (PCL), 275 Polycarbophil-based hydrogel, 319
320 Polydimethylsiloxane-modified chitosan (PMSC), 279 PMSC covalent amphiphilic polymer networks (CAPNs) self-assembling procedure of, 279f Polydopamine (PDA), 431
432 Polyelectrolyte complex (PEC), 53
54 Polyelectrolyte gels, 509
510 Polyelectrolyte hydrogels, 508
509 Polyelectrolyte solutions, blending of, 54
55 Polyethyleneglycoldiglycidylether (PEGDE), 95
96, 97f, 98f, 99 Polyethylene polyamine (PPA), 216
217 Polyethylene polyamine (PPA)/ gelatin hydrogel, 217f Polyglucuronide hydrogel, 297 Polyglycolic acid (PGA), 275

Polyimide film, 487
488 Polylactic acid (PLA), 275 Polymer-coated fertilizers (PCFs), 347 Polymer dots (PDs), 504
505 Polymer-grafted light-responsive cotton fibers, 207f Polymer hydrogels, 504 Polymers, natural, 4 Polymethacrylic acid, 500
501 Polymethylmethacrylate (PMMA) film, 240 Poly-N-isopropylacrylamide, 252 Polyon, 347 Polyphenols, 247
248 Polysaccharide-based hydrogels, 140 Polysaccharide-based hydrogels, radiation preparation of, 124
136 carboxymethyl cellulose
based hydrogel, 126
128 carrageenan, 135
136 chitosan-based hydrogel, 128
129 hyaluronic acid
based hydrogels, 133
135 potential applications, 136
144 biomedical materials, 137
140 metal ion adsorption, 140
141 super water absorbent, 142
143 use as an additive to improve the compressive strength of cement, 143
144 sodium alginate
based hydrogel, 129
133 starch-based hydrogel, 125
126 Polysaccharide-based hydrogels for water treatments, 411
456 adsorbent, reusability of, 444 adsorption capacity, various factors on, 436
439 adsorbate initial concentration, 439 adsorbent dosage, 438 pH, 438 temperature, 436
437 time, 438

529

adsorption isotherm, 442
444 Freundlich isotherm, 442 Langmuir isotherm, 442
444 adsorption kinetics, 439
441 intraparticle diffusion model, 440
441 pseudo-first-order (PFO) model, 439 pseudo-second-order (PSO) model, 440 cellulose-based hydrogels, 422
424 for dyes removal, 422
423, 426t for heavy metals removal, 423
424, 426t characterization, of hydrogels, 412
415 Fourier transform infrared, 413 point of zero charge, 414
415 scanning electron microscopy (SEM), 413
414 swelling, 414 X-ray diffraction (XRD), 414 chitosan (CS)-based hydrogels, 416
421 for dyes removal, 417
418, 420t for heavy metals removal, 418
421, 420t κ-carrageenan, 433
436 for dyes removal, 434
435, 437t for heavy metals removal, 435
436, 437t sodium alginate (NaAlg), 430
432 for dyes removal, 431
432, 433t for heavy metals removal, 432, 433t starch (ST) based hydrogels, 424
429 for dyes removal, 427
428, 429t for heavy metals removal, 428
429, 429t Polysaccharide-based natural hydrogels, 28
30

530 Index Polysaccharide-based natural hydrogels (Continued) preparation methods, 28
30 alginate hydrogels, 29 cellulose, 30 chitosan hydrogels, 29 dextran, 30 hyaluronic acid hydrogels, 28
29 Polysaccharide gels, idealized junction zones in, 359f Polysaccharide/hyaluronic acid copolymers, 296 Polysaccharide hydrogel mode of action for, on multiple soil production processes, 341
344 phytotoxicity test, 345
346 in plant disease control, 344
345 significance of, in agricultural field, 349 Polysaccharides, 256, 275
276, 278
279, 329, 357 from animal origin, 22
23 chitin and chitosan, 22 hyaluronic acid, 23 xanthan gums, 22 for constructing hydrogels and their origins, 358t hydrogels from, 154
162 alginate-based hydrogels, 156
158 carrageenan-based hydrogels, 158 cellulose-based hydrogels, 161
162 chitosan-based hydrogels, 154
156 hyaluronic acid
based hydrogels, 159 starch-based hydrogels, 159
161 of plant origin, 17
21 alginate, 21 cellulose, 17
18 guar gum, 21 inulin, 20 lignin, 19
20 starch (amylose and amylopectin), 18
19

structures of, 120
124 carrageenan, 123
124 chitosan, 122 hyaluronic acid, 123 sodium alginate, 122 sodiumcarboxymethyl cellulose, 120
121 starch, 120 Polythiophene-g-poly (dimethylaminoethyl methacrylate)-doped methylcellulose (MC) hydrogel, 210 Polyurethane (PU), 275 Polyvinyl alcohol (PVA), 275 Polyvinyl alcohols (PVA) hydrogel, 209
210, 230, 230f, 280
281, 502 Polyvinylpyrrolidone-poly (acrylamide) (PVP-PAAm) hydrogel, 340
341, 340f, 496 Poroelastic relaxation experiment, 484 Porosity and permeation, 39
40 Porosity of hydrogels, 472
473 Porous hydrogels, 10, 511 Porous scaffolds, 284 Porous starch (PS), 427
428 Positively charged polypeptides/ poly(ethylene glycol) (PEG) hydrogel, interaction between, 254f Potassium persulfate, 106
107 Preparation, of hydrogel, 153
154 Pressure-sensitive hydrogel, 5
7 Primary bound water, 38
39, 462
464 Properties of natural polymers, 174t Property of hydrogels, 460 Proteins, hydrogels from, 26
28, 163
167 collagen-based hydrogels, 165
167 keratin-based hydrogels, 165 preparation methods, 27
28 collagen and gelatin hydrogels, 27 fibrin, 28

silk proteins/fibroin, 27
28 silk-based hydrogels, 164 Proteins, natural polymers based on, 23
24 collagen and gelatin, 23
24 Pseudo-first-order (PFO) model, 439 Pseudomonas aeruginosa, 253, 256
257, 315
316 Pseudo-second-order (PSO) model, 440 Pudding, 397
398 Pulsed H-NMR, 463

Q Quaternary ammonium compound, 253 Quaternized tunicate cellulose nanocrystals (Q-TCNCs), 201

R Radiation preparation of polysaccharide-based hydrogels, 124
136 carboxymethyl cellulose
based hydrogel, 126
128 carrageenan, 135
136 chitosan-based hydrogel, 128
129 hyaluronic acid
based hydrogels, 133
135 sodium alginate
based hydrogel, 129
133 starch-based hydrogel, 125
126 Reactive blue 4 (RB4) adsorption, 417
418 Redox/pH stimuli-responsive degradable Salecan-g-SSpoly(IA-co-HEMA) hydrogel, 215
216 Relaxed state of the system, 491
492 Residual strain, 492
493 Reswelling, 5
7 Retro-Diels
Alder reaction, 102, 234 Reversible double-network hydrogel, 64
67

Index Rhamnogalacturonans (RGIs), 384
385 Rheology, 482, 490
497 characterization, 393
394 creep behavior, 491
492 dynamic mechanical behavior, 493
497 stress relaxation, 492
493 viscoelasticity and microstructure, 490
491 Rhizopusoryzae, 262 Rhodamine B, 434
435 Ring extensiometry, 486, 486f Rod-like particles (RLPs), 176
177 Root-targeted delivery Vehicle (RTDV), 334 (R/S)-ketoprofen, 262 Ruminococcus, 342

S Saccharomyces cerevisiae, 253 SACLA, Japan, 498
499 Salecan-g-SS-PIH hydrogels, 216f Salt-responsive hydrogels based on natural polymers, 199
201 Scaffolds binding of growth factors with, 285
286 for tissue engineering, 273
275, 283 Scaling and root planning (SRP), 322 Scanning electron microscopy (SEM), 413
414, 471 Schiff-base formation, 33 Schiff base reaction, 92
95, 92f natural polymer-based hydrogels prepared using, 93t with cross-linking agents, 94f Secondary bound water, 38
39, 462
464 Seed cells in tissue engineering strategies, 294 Seed coating by hydrogel, 340
341 Self-healing, pH-responsive hydrogels and, 185
186

Self-healing hydrogels, 223 characterization of, 240
241 design, 224 microstructure, design of, 236
240 additives and nanoparticles, 239
240 microcapsules, 237
238 microvascular networks, 239 self-healing mediated by covalent interactions, 226
228 self-healing mediated by noncovalent interactions, 225
233 electrostatic interactions, 226
228 host
guest interactions, 231
233 hydrogen bonding, 230
231 hydrophobic associations, 228
229 metal
ligand coordination complexation, 229 Self-healing implantable devices, 224 Semibound water, 151
152 Semidissolution acidification sol
gel transition, 55
59 Semiinterpenetrating network, 37 Semiinterpenetrating polymer network (SIPN), 195, 195f Semi-IPN hydrogels, 500
501 Semisolid hydrogels, 36 Separation technology, 44 Short-chain saturated aliphatic polyesters, 275 Silanehydroxypropyl methylcellulose (SiHPMC), 299 Silk-based hydrogels, 164 Silk/calcium silicate/sodium alginate (SCS) composite scaffolds, 278f Silk proteins/fibroin, 27
28 Silver nanoparticles, antibacterial activity of, 254, 256 Single-network hydrogels, 496

531

Skin therapy, antimicrobial hydrogels for, 313
318, 323t Slow-release fertilizer (SRF), 337
338 Small-angle neutron scattering (SANS), 501
503 Small-angle X-ray scattering (SAXS) technique, 498
501 Smart biodegradable hydrogel (SBH), 347 “Smart” fertilizer release, biodegradable hydrogel and, 347
349 Smart hydrogels. See Environmentally responsive hydrogels Smart materials, 310 Sodium alginate (NaAlg), 93, 122, 123f, 277, 430
432, 496, 500
501
based hydrogel, 129
133, 258
259 chemical structure of, 431f for dyes removal, 431
432, 433t for heavy metals removal, 432, 433t Sodium alginate/CMC hydrogels, 131
133 Sodium carboxymethyl cellulose (NaCMC), 120
121, 179 Sodium carboxymethylcellulose (NaCMC)-based hydrogels, 199
200 Sodium dodecyl sulfate (SDS), 228
229 Sodium trimetaphosphate, 425
427 Sodium tripolyphosphate (STPP), 425
427 Soft gels, 361 Soft-tissue engineering, scaffolds for, 139
140 Soil conditioner hydrogel (SCH), 332 Soil conditioner polysaccharide hydrogel (SCPH), 330

532 Index Soil conditioner polysaccharide hydrogel (SCPH) (Continued) natural polymer (polysaccharide)-based hydrogel, 335
338 cellulosic derivative-based hydrogel, 336
337 starch derivative-based hydrogel, 338 natural polymers, 333
335 Sol
gel analysis, 464
465, 467, 470 Sol
gel conversion, 4 Sol
gel transition, 8
9 Solid hydrogels, 36 Solute dosage, 462 Solvent
polymer interaction parameter, 468
469 Sophoraflavescens alkaloid gel, 321 Spatial gel inhomogeneity, 473
474 Sphingomonas elodea, 362
363 Stabilizers, 400 Staphylococcus aureus, 256
257, 314
317 Staphylococcus hyicus, 315
316 Staphylococcus intermedius, 315
316 Staphylococcus saprophyticus, 259 Starch (ST) based hydrogels, 125
126, 159
161, 424
429 for dyes removal, 427
428, 429t for heavy metals removal, 428
429, 429t Starch, 18
19, 120, 329
330 properties of, 174t structure of, 19f Starch derivative-based hydrogel, 338 Stem cells, 286
287 application of cellulose-based hydrogel in, 286
288 transplantation, 294 Stereocomplex formation, 31 Stimuli-responsive hydrogels, 40
44 chemical stimuli, 41
44

glucose-sensitive hydrogels, 42
44 pH-responsive hydrogels, 41
42 physical stimuli, 41 electrically responsive hydrogels, 41 photoresponsive hydrogels, 41 temperature-responsive hydrogels, 41 Stimuli-responsive natural polymer-based hydrogels, 173 glucose-responsive hydrogels, 202
204 light-responsive hydrogels, 204
208 magnetic-responsive hydrogels, 186
192 multistimulation-responsive hydrogels, 209
217 pH-responsive hydrogels, 173
186 salt-responsive hydrogels, 199
201 thermal-responsive hydrogels, 192
199 Strain-promoted azide-alkyne cycloaddition reaction (SPAAC), 102, 103f Stress relaxation, 492
493 Stress relaxation experiment, 486 Stretched hydrogels, 65f Strip extensiometry, 486 N-Succinyl-chitosan (S-CS), 93, 313
314 Sulfated glycosaminoglycans, 299 Superabsorbent hydrogel (SAH), 330, 338
340 Superabsorbent polymers (SAPs), 459 Superoxide, 251
252 Super water absorbent, 142
143 Supramolecular hydrogel, preparation of, 209f Swelling, 414 behavior, 484 coefficient, 467 processes, 331f and solubility, 38
39

Swelling ratio (SR), 484
485 measurement, 461 Synchrotron X-ray, 498 Synthetic hydrogels, 4, 334 Synthetic materials, 275 Synthetic polymers, 277, 286, 459
460

T Tan δ, 494 Tannic acid, 248 Temperature-responsive culture dishes, 287f Temperature-responsive hydrogels, 41 Temperature-sensitive cellulose, 74 Terbinafine hydrochloride, 261 tert-butylhydroperoxide (tertBOOH), 250 3,30 ,5,50 -Tetramethylbenzidine (TMB), 251
252 Texture, types of, 358 Texture-modified (TM) food, 397 Theophylline, 137 Thermal-responsive natural polymer-based hydrogels, 192
199 natural polymer impacts on thermal-responsive behaviors of, 192
194 performance improvement of, 194
199 Thermo-responsive hydrogel, 5
7 Thermoresponsive phase transition, 157 Thermo-responsive poly(Nisopropylacrylamide)nanocrystalline cellulose (PNIPAAm-CNC) hybrid hydrogels, 197f Thermosensitive hydrogels, 321
322 Thermoswitchable polymeric photosensitizer (T-PPS), 210
211 Thiolated chitosan, 313
314 cross-linked with maleic acidgrafted dextran, 259

Index Thiol-ene “click” chemistry, 104
105 Thiol-ene reaction, 102, 105f Thiol
Michael type reactions, 104
105 Third-generation hydrogels, 482 Thiuram disulfide moieties, 236 3D printed food, 400
403, 401f Three-dimensional (3D) scaffolds creation of, architectural and mechanical challenges associated with, 274f Time dependence, 225 Tissue culture polystyrene (TCPS) dishes, 287
288 Tissue engineering, 43, 273 cellulose-based hydrogel scaffold, 286
299 in bone tissue, 296
298 in brain tissue, 294
295 in cartilaginous tissue, 298
299 in myocardial engineering, 292
293 in stem cells, 286
288 in urinary system, 296 in vascular regeneration, 288
291 common application of, 273 properties of hydrogel in, 277
286 cell adhesion property, 278
279 growth factor binding, 285
286 high porosity and internal connectivity, 282
284 mechanical property, 279
282 proteolytic degradation, 284
285 scaffolds used in, 273
275 Toughness hydrogels, 511 Trans-cyclooctene functional group, 102
104 Trans-ferulic acid, 250

Transforming growth factor-β1 (TGF-β1), 285 Transglutaminase, 107
108 Traumatic brain injury (TBI), 294 N,N,N-Trimethyl chitosan, 313
314 Trithiocarbonate (TTC), 235
236 Trivalent metal ions, 59
62 Tyrosinase, 107
108

U Ultra-SANS (USANS), 502 Uniaxial compression tests, 467
468 Uniaxial tensile testing, 486 Upper critical solution temperature (UCST), 192 Ureido-pyrimi-dinone (Upy) dimers, 80 Urinary system, tissue engineering scaffolds in, 296 UV light, cross-linking by, 34

V Vacuum freeze-drying method, 289 Vaginal therapy, chitosan-based hydrogels for, 318
321, 323t Vanadium, 418
419 Vancomycin-loaded hydrogels, 259 Vanillin, 93, 95 Vascular regeneration, tissue engineering scaffolds in, 288
291 Vinyl chloride, 425
427 Vinyl-functionalized nanogels (NGs), 176
177 4-Vinyl-phenylboronic acid (VPBA), 202
204 Viral infections, 260 Viscoelasticity, 491
492 and microstructure, 490
491

533

Volume phase transition (VPT), 503 Volume phase transition temperature (VPTT), 5
7

W Washburn’s equation, 472
473 Water in hydrogels, 151
152 Water treatments, polysaccharidebased hydrogels for. See Polysaccharide-based hydrogels for water treatments Weight loss measurement, 461
462 Wound dressing, 42, 139 Wound therapy, antimicrobial hydrogels for, 313
318

X Xanthan gum, 22, 357 structure of, 23f Xanthomonas campestris, 22 X-ray, 498 X-ray diffraction (XRD), 414 X-ray microtomography, 472
473 Xylan, 215 Xylan-based P(NIPAm-g-AA) hydrogel copolymer networks drug-delivery behaviors and cell proliferation of, 215f Xylan-type hemicellulose-based hydrogel, 208f Xylan-type hemicellulose-graftphotoisomericazobenzene hydrogels, 208f

Z Zhang
Sun
Qian equation, 466 Zinc oxide nanoparticles, 257
258, 315
316 Zinc oxidenanorods, 316