Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering 0128212306, 9780128212301

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Woodhead Publishing Series in Biomaterials

Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering Edited by

Showkat Ahmad Bhawani Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia

Zoheb Karim MoRe Research AB, Sweden

Mohammad Jawaid Biocomposite Technology Laboratory, Institute of Tropical Forestry and Forest Products (INTROP), University Putra Malaysia (UPM), Serdang, Selangor, Malaysia

An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2021 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-821230-1 ISBN: 978-0-12-823218-7 For information on all Woodhead publications visit our website at https://www.elsevier.com/books-and-journals

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Dedication

The editors are honored to dedicate this book to Dr. Kishwar Usmani (Aunty of Dr. Mohammad Jawaid), who left this worldly life on January 14, 2021. Allah SWT forgive her small and big mistakes and Give her a Higher Place in Jannatul Firdous. Ameen.

Contributors

Aarzoo Department of Chemistry, School of Chemical and Life sciences, Jamia Hamdard, New Delhi, India Sadaf Afrin Department of Chemistry, Faculty of Sciences, Aligarh Muslim University, Aligarh, India Akil Ahmad School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia Asrar Ahmad Center for Sickle Cell Diseases, College of Medicine, Howard University, Washington, DC, United States Khalid M. Alotaibi Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia Mohammad Omaish Ansari Center of Nanotechnology, King Abdul Aziz University, Jeddah, Saudi Arabia Mohd Saquib Ansari Department of Biomedical Sciences, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, New Delhi, India M. Tarik Arafat Department of Biomedical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka, Bangladesh Maya Asyikin Mohamad Arif Faculty of Resource Science and Technology, University Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia Mohd Razip Asaruddin Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia Mohammad Ashfaq BS Abdur Rahman Crescent Institute of Science and Technology, Chennai, India Erkan T€ urker Baran University of Health Sciences Turkey, Institute of Health Sciences, Department of Biomaterials; University of Health Sciences Turkey, Institute of Health Sciences, Department of Tissue Engineering, Istanbul, Turkey

xiv

Contributors

Sunita Barik Department of Chemistry, Saraswati Degree Science College, Cuttack, Odisha, India Anant Narayan Bhatt Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, New Delhi, India Showkat Ahmad Bhawani Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia Anuradha Biswal Department of Chemistry, Veer Surendra Sai University of Technology, Sambalpur, Odisha, India Afzal Hussain Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Mohamad Nasir Mohamad Ibrahim School of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia Iqbal I.M. Ismail Department of Chemistry, Faculty of Science, King Abdul Aziz University, Jeddah, Saudi Arabia Zoheb Karim MoRe Research AB, Sweden Mohd Jahir Khan School of Biotechnology, Jawaharlal Nehru University, New Delhi, India Abhishek Kumar Department of Biomedical Sciences, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi; Institute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, New Delhi, India Vannessa Lawai Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia Zainab Ngaini Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia Mehvish Nisar Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia Mohammad Oves Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah, Saudi Arabia Md. Wahidur Rahman Department of Biomedical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka, Bangladesh

Contributors

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Mohd Rashid School of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia Chinmayi Rath Department of Chemistry, Saraswati Degree Science College, Cuttack, Odisha, India Mohd Ahmar Rauf Use-Inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory, Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, United States M. Samim Department of Chemistry, School of Chemical and Life sciences, Jamia Hamdard, New Delhi, India Rahul Kumar Sharma Department of Chemistry, Government Shyam Sundar Agrawal PG College, Sihora, Madhya Pradesh, India Laishram Rajendra Kumar Singh Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India Sarat K. Swain Department of Chemistry, Veer Surendra Sai University of Technology, Sambalpur, Odisha, India Aydin Tahmasebifar University of Health Sciences Turkey, Institute of Health Sciences, Department of Biomaterials; University of Health Sciences Turkey, Institute of Health Sciences, Department of Tissue Engineering, Istanbul, Turkey Abu Tariq Department of Chemistry, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Mohiuddin Khan Warsi Department of Biochemistry, College of Science, University of Jeddah, Jeddah, Saudi Arabia Asim Ali Yaqoob School of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia Bengi Yilmaz University of Health Sciences Turkey, Institute of Health Sciences, Department of Biomaterials; University of Health Sciences Turkey, Institute of Health Sciences, Department of Tissue Engineering, Istanbul, Turkey

Editors’ biographies

Dr. Showkat Ahmad Bhawani is presently working as an Associate Professor at the Department of Chemistry, Faculty of Resource Science and Technology, UNIMAS Malaysia. He received his Ph.D. in Applied Analytical Chemistry from Aligarh Muslim University, Aligarh, India. He is working on the synthesis of molecular imprinting polymers for the removal/extraction of dyes, fungicides, and various natural products from environmental and biological samples. In addition to this, he is working on the development of new test methods and determining standard conditions for analysis (separation, isolation, and determination) of various analytes from environmental and biological samples. He is involved in the analysis of samples such as surfactants, amino acids, drugs, vitamins, sugars, and metal ions. He has edited two books and has written 12 book chapters, and he has published more than 40 papers in various journals. He is a lifetime member of the Asian Polymer Association and an editorial board member of several journals. Dr. Zoheb Karim worked as Senior Researcher at MoRe Research Ornskoldsvik AB. His current research is process-property-application correlation understanding of nanocellulose (crystals and fibers)-based assembled dimensional structures. Fabrications of functionally and structurally sound dimensional architecture for a predefined application using a best appropriate route and starting nanocellulose (required intrinsic properties) is another goal of his research. Recently, he patented a mechanism of metal ions removal by nanocellulose-based functionalized membranes with tailored pore-size which form metal ions-cluster on membranes and help to remove metal ions in high capacity (Patent application no. 1657849-9, AWAOATENT AB). Dr. Karim carried out his postdoctoral research at KTH Royal Institute of Technology Stockholm, Sweden, and before that he worked as a researcher at Lulea˚ University of Technology, Sweden. His research has been supported by some well-known funding agencies such as the European Commission (NanoSelect under the 7th Framework Programme), Kempe Foundation, Mistra Foundation (MisraTerraClean), R&D Council of Processum, etc. He has given various plenary and invited talks related to his research interest in various reputed institutes including the University of Oxford, Imperial College London, Indian Institute of Technology Delhi, University of Guelph, EMPA-Swiss Federal Laboratory of Material Sciences and Technology, etc. He attended various international and national conferences/symposiums/workshops/invited lecturers around the world to present his research works. He is a member of the International Association of Advanced Materials, the Technical Association of Pulp and Paper Industry (TAPPI), and Sveriges Ingenj€ orer. Moreover, he organized two workshops at Luea˚ University of Technology Lulea˚ in 2013 and 2014. He was a member of the International Committee of Biomaterials for Tomorrow, B4T (2018) organized by Mahatma Gandhi University, India.

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Editors’ biographies

At the same time, he was also a member of an international advisory committee of SafeBioPack (2018) organized by the Institute of Tropical Forestry and Forest Products (INTROP), University Putra Malaysia. Currently, he is also serving as an external expert/consultant of an Egypt-based company, inTEXive® (intexive.com). He has published about 31 peer-reviewed research articles of international repute and 10 book chapters with well-known publishers such as Elsevier, Springer Nature, CRC Press, TAPPI Press, and World Scientific. He also won the best paper award in 2012, awarded by Aufau Periodicals Publication USA. Dr. Mohammad Jawaid is currently working as a Senior Fellow (Professor) at Biocomposite Technology Laboratory, Institute of Tropical Forestry and Forest Products (INTROP), University Putra Malaysia (UPM), Serdang, Selangor, Malaysia, and also has been a Visiting Professor at the Department of Chemical Engineering, College of Engineering, King Saud University, Riyadh, Saudi Arabia since June 2013. He has more than 20 years of experience in teaching, research, and industries. His area of research interests includes hybrid composites, lignocellulosic reinforced/ filled polymer composites, and advanced materials: graphene/nanoclay/fire retardants, modification and treatment of lignocellulosic fibers and solid wood, biopolymers and biopolymers for packaging applications, nanocomposites and nanocellulose fibers, and polymer blends. So far, he has published 40 books, 65 book chapters, more than 350 peer-reviewed international journal papers, and several published review papers in “Top 25 Hottest Articles” in Science Direct during 2013–19. He also obtained two patents and six copyrights. He has Scopus H-index-55 (total citation 14171) and in Google scholar H-index-66 (total citation 20242). He is founding Series Editor of the Composite Science and Technology Book Series from Springer Nature, also Series Editor of Springer Proceedings in Materials, Springer Nature, and an International Advisory board member of the Springer Series on Polymer and Composite Materials. He worked as a guest editor on special issues of SN Applied Science, Frontiers in Sustainable Food Systems, Current Organic Synthesis and Current Analytical Chemistry, International Journal of Polymer Science, and IOP Conference Proceeding. He is also an Editorial Board Member of the Journal of Polymers and the Environment, Journal of Plastics Technology, Applied Science and Engineering Progress Journal, Journal of Asian Science, Technology and Innovation, and Recent Innovations in Chemical Engineering. In addition, he is a reviewer of several highimpact international peer-reviewed journals of Elsevier, Springer, Wiley, Saga, ACS, RSC, Frontiers, etc. Presently, he is supervising 12 Ph.D. students (five as Chairman, and seven as Member) and six Master’s students (one as Chairman, and five as Member) in the fields of hybrid composites, green composites, nanocomposites, natural fiber-reinforced composites, nanocellulose, etc. Twenty-six Ph.D. and 13 Master’s students graduated under his supervision in 2014–20. He has received several research grants at university, national, and international levels on polymer composites of around 3 million Malaysian ringgits (USD 700,000). He also delivered plenary and invited talks in international conferences related to composites in India, Turkey, Malaysia, Thailand, the United Kingdom, France, Saudi Arabia, Egypt, and China. Besides that, he is a member of technical committees of several national and international conferences on composites and material science. Recently, Dr. Mohammad

Editors’ biographies

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Jawaid received the Excellent Academic Award in the Category of International Grant-Universiti Putra Malaysia—2018 and also the Excellent Academic Staff Award in Category of industry High Impact Network during Community and Industry Network (ICAN 2019). Beside that he also won Gold Medal during JINM Showcase under Community and Industry Network Category at Universiti Putra Malaysia, Malaysia. He also received the Publons Peer Review Awards 2017, and 2018 (Materials Science), Certified Sentinel of Science Award Recipient—2016 (Materials Science) and 2019 (Materials Science and Cross Field). He is the winner of the Newton-Ungku Omar Coordination Fund: UK-Malaysia Research and Innovation Bridges Competition 2015. Recently he was recognized with a Fellow and Chartered Scientist Award from the Institute of Materials, Minerals and Mining (IOM), UK. He is also a lifetime member of the Asian Polymer Association, and the Malaysian Society for Engineering and Technology. He has professional membership of the American Chemical Society (ACS) and the Society of Plastics Engineers (SPE), USA.

Preface

This book provides solid, quantitative descriptions and reliable guidelines, reflecting the maturation and demand of the field and the development of new polysaccharide nanocomposites. It focuses on the different types of polysaccharide nanocomposites of cellulose, chitosan, alginate, etc. for gene delivery and tissue engineering. The book also covers polysaccharide hydrogels for tissue engineering and polysaccharide magnetic nanocomposites for gene delivery and highlights the most exciting applications in the field of gene delivery and tissue engineering. This book will be of interest to researchers working in the fields of material science, biomaterials, regenerative medicines, drug delivery, tissue engineering, polymer science/chemistry, and chemical engineering, and in the polymer industry. It will be useful for scientists working on polysaccharide nanocomposites for gene delivery and tissue engineering. The book will be very helpful for students in the development of new polysaccharide nanocomposites as well as graduates in polymer technology, biomedical science, and biotechnology. The two introductory chapters cover basic information about polysaccharides and nanocomposites, to provide a foundational understanding. The second section of this book covers chitosan and its derivatives-based dimensional frameworks as carriers for gene delivery, alginate- and hyaluronic-based hydrogels for tissue engineering, heparin- and cellulose-based nanocomposites for and dextran, pullulan, gellan, xanthan, and xanthun gum-based nanocomposites for tissue engineering applications. The last section describes polysaccharide-based 3D bioprinter inks for tissue engineering, polysaccharide-based nanocomposites for gene delivery, chitosan- and starch-based nanocomposites for gene delivery, and hyaluronic acid magnetic nanocomposites for gene delivery. Finally, we assure the readers that the information provided in this book can serve as a very important tool for anyone wishing to select/design polysaccharide-based nanocomposites to fulfil the requirements of gene delivery and tissue engineering applications. We are grateful to all the authors who contributed chapters to this book and who helped to turn our thoughts into reality. Lastly, we are grateful to the Elsevier team for their continuous support at every stage to make it possible to publish on time. Showkat Ahmad Bhawani Zoheb Karim Mohammad Jawaid

Polysaccharides Sadaf Afrina and Zoheb Karimb a Department of Chemistry, Faculty of Sciences, Aligarh Muslim University, Aligarh, India, bMoRe Research AB, Sweden

1.1

1

Introduction

Natural polysaccharides are generally constituted of simple carbohydrates with long chain molecules attached via glycosidic linkages. Carbohydrates are made up of aldehydes or ketones, and have multiples hydroxyl groups. They make up the majority of organic matter and play a crucial role in human life. Carbohydrates serve as energy stores, fuel, and various metabolic intermediates. In parallel, various sugars like ribose and deoxyribose form part of the structural framework of RNA and DNA. Polysaccharides are also present in the cell walls of bacteria and plants, and cellulose is the main constituent of plant cell walls [1, 2]. The most authentic classification performed for polysaccharides is based on degree of polymerization (DP). Polysaccharides may reach a DP of 105, whereas, by definition, the maximum DP for oligosaccharides is 10. Hence, by convention, compounds having a DP of 11 or more are designated polysaccharides. A monosaccharide is a monomer of saccharides, and a disaccharide is two conjugated monomers [1, 3]. Many studies are available related to the general introduction of polysaccharides, their chemical structures, chemical compositions, methods of isolation, types, etc. [4–7]. Hence, in this chapter, a very interesting aspect is explained: the nano-form of these polysaccharides, which is an area of emerging interest in scientific studies. Very few studies in the literature have been summarized related to nano-polysaccharides characterization, the isolation process, and their properties and applications. Consequently, this chapter explains the collective efforts and provides a summary of nano-polysaccharide understanding and their utilization in various applications.

1.2

Natural polysaccharide nanomaterials

Polysaccharides are molecules composed of monomer sugar units linked by glyosidic bonds; this bond is responsible for the attachment of monomers and forms polymers. They are the most abundant natural polymer on Earth [8, 9]. The amount of polysaccharide produced by plants per year exceeds by several orders of magnitude the synthetic production by the chemical industry [10]. Natural polysaccharides (Table 1.1) are synthesized to fulfill many different functions, such as energy storage in plants (i.e., starch), structural support of vegetal cells (i.e., cellulose), and gelling agents forming the intercellular matrix and containing several ions such as sodium, calcium, and Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00010-4 Copyright © 2021 Elsevier Inc. All rights reserved.

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Table 1.1 Natural polysaccharides classified according to their origin. Origin

Polysaccharide

Plants Algae Animals Bacteria Fungi

Starch, cellulose, glucomannan, pectin, hemicellulose, gums, mucilage Agar, galactans, alginate, carrageenans Chitin, chitosan, hyaluronic acid, glycosaminoglycans, cellulose Dextran, levan, polygalactosamine, gellan, xanthan, cellulose Elsinan, chitin, chitosan, pullulan, yeast glucans

Reproduced with permission from Torres FG, Troncoso OP, Anissa P, Francesca G, Giuseppe B. Natural polysaccharide nanomaterials: an overview of their immunological properties. Int J Mol Sci 2019;20:5092–9.

magnesium (i.e., alginate in the brown algae). Some of them, such as cellulose, starch, chitin, and polysaccharides from seaweeds, are commercially important in several markets, ranging from paper production to food industry products. We will restrict our description to those polysaccharides and their interactions with the immune system. In this chapter, we shall discuss some of them in detail.

1.3

Polysaccharides in nano-form

It is well-known that controlling the size of polysaccharides is the biggest challenge in current scientific study. At the time of writing, there is no universally accepted size range that particles must fall within to be classified as microparticles or nanoparticles. However, many researchers classify particles smaller than 1 μm as nanoparticles and particles larger than 100 mm as macroparticles [11–23]. Nanoparticles are then a submicronic colloidal system, ranging in size from a few nanometers to 1 μm, and made of artificial or natural polymers [24]. Nanoscale typically has a greater surface area per mass. The nanometric size effect has led to intensive research in the area of nano-sized particles. The high surface area of nano-size particles opens various routes for novel interactive behavior with polymers/particles. Hence, this chapter will also discuss the novel interactive behaviors of nano-sized polysaccharides with other species. Various origins of polysaccharides are given in Table 1.1, thus in this chapter, the nano-forms of possible listed polysaccharides will be explained in detail [25].

1.4

Nano-starch

Starch is a polymer of carbohydrates consisting of numerous glucose units joined by glycoside bonds. Starch nanoparticles (St-NPs) have attracted much attention due to their unique properties that are different significantly their bulk material [26]. In a study, St-NPs were extracted from starch granules by means of physical or chemical treatments. Utilization of chemical treatments for preparation of St-NPs has received more attention than physical treatments [25]. Isolation of St-NPs has been reported in various scientific studies [27–29] and the fabricated nanoparticles are in the shape of 5–7 nm thick, 20–40 nm long, and 15–30 nm. In a study, St-NPs were synthesized

Polysaccharides

3

with native starch using a solvent displacement method modified with an aqueous alkaline medium as a solvent and ethanol as the organic phase [30], as shown in Fig. 1.1. Surface modifications of St-NPs have also been performed using various chemistries, in a study, surface of waxy maize starch isolated using H2SO4 hydrolysis was performed using two different reagents, namely, alkenyl succinic anhydride and phenyl isocyanate and various sophisticated techniques are employed for the characterization of modified St-NPs [31]. Furthermore, St-NPs with high specific area and surface hydroxyl groups have good potential for surface chemical modification to introduce any desired surface functionality. The accessible hydroxyl group content on the surface of St-NPs was reported to be around 14% of the total hydroxyl group in polymer structure, which is around 0.0025 mol/g of St-NPs [31, 32]. The main reported surface modification is grafting different reagents on the surface of the particles [33, 34] to prepare new material for different applications, especially in blends with polymers.

1.5

Nanocelluloses

Our group has been working with various kinds of functionalized nanocelluloses for many years. As discussed and reported in various scientific studies, the nano-form of cellulose exists mainly as crystalline cellulose (CNC) and cellulose nanofibers (CNFs) [22–24]. In our study, we have also used an integrated route of bioethanol for the isolation of crystalline cellulose having highly negative surface ζ-potential compared to sludge-based crystalline cellulose in acidic pH (pH 5.0) (Fig. 1.2) [14]. Surface modification of nanocellulose is performed using various chemistries, and very detailed report is published early [35]. In our lab, a green approach using enzyme hexokinase was studied for the introduction of phosphate (PO4 2 + ) groups on the surface of cellulosic fibers [21, 22]. Furthermore, in situ TEMPO functionalization of

Fig. 1.1 Transmission electron microscopy (TEM) of fabricated St-NPs: (A) the histogram represents the size distribution of fabricated nanoparticles; (B) using dynamic light scattering (DLS).

4

Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Fig. 1.2 Isolation routes of cellulose in nano-forms, crystalline cellulose (CNC), cellulose nanofibers (CNFs), and highly functional crystalline cellulose called bioethanol CNC (CNCBE) are reported in this chapter. Available functional groups on the surface of isolated celluloses are denoted on the right-hand side. Reproduced with permission from Mathew AP, Oksman K, Karim Z, Liu P, Khan SA, Naseri N. Process scale up and characterization of wood cellulose nanocrystals hydrolysed using bioethanol pilot plant. Ind Crop Prod 2014;58:212–19.

Fig. 1.3 In situ TEMPO functionalization of crystalline cellulose isolated using a bioethanol integrated route. High surface charge density and more negative ζ-potential indicate highly effective reactions.

CNCBE-based dimensional structures was also reported, where mild acid-based introduction of carboxylic groups (COO
) was performed to produce highly active and effective dimensional frameworks [11]. Fig. 1.3 represents the possible catalytic reaction for the in situ TEMPO oxidation of cellulose-based dimensional structures.

Polysaccharides

5

The functionalization of produced frameworks indicates the catalytic transformation of hydroxyl groups to carboxylic groups after mild acid reactions. Furthermore, more negative ζ-potential (
32 to
65 mV) and high density of carboxylic groups (36–321 mmol/kg) was reported after functionalization [11].

1.6

Nano-alginate

Alginate is a naturally occurring anionic polymer typically obtained from brown seaweed. It has been extensively investigated and used for many biomedical applications due to its biocompatibility, low toxicity, relatively low cost, and mild gelation by addition of divalent cations such as Ca2+ [36]. Alginate gels are also promising for cell transplantation in tissue engineering, which aims to provide man-made tissue and organ replacements to patients who suffer the loss or failure of an organ or tissue [37]. In this approach, hydrogels are used to deliver cells to the desired site, provide a space for new tissue formation, and control the structure and function of the engineered tissue [38]. Alginate-based nanoparticles have been widely used in various applications [39, 40]. Two promising applications discussed in the literature are targeted drug delivery and tissue engineering, and due to their nontoxic nature, high surface area of nanoparticles (high loads of drugs and other active molecules), and effective biocompatibility, alginates in nano-forms are an effective candidate in these research fields [41–44]. In a study, a novel procedure for the fabrication of alginate nanoparticles in the aqueous phase of water-in-oil (w/o) nanoemulsion was developed and reported [45]. Moreover, the hybrid of alginate nanoparticles with metals nanoparticles/metals is also reported in the literature. In a study, stable nanoparticles of alginate were fabricated in mild acid conditions, and stability was further increased after crosslinking with zinc [46]. Fig. 1.4 provides a complete step-by-step production of alginate-zinc nanoparticles.

1.7

Nano-pectin

Pectin is a natural polysaccharide structurally composed of large amounts of poly-Dgalacturonic acid bonded via α-1,4-glycosidic linkage. According to its degree of methyl esterification, pectin can be classified as high methoxyl pectin or low methoxyl pectin, which yields some differences in its properties [47, 48]. Pectin has commonly been used as a gelling agent, a thickening agent, and a colloidal stabilizer in the food industry [49]. Its applications in the pharmaceutical industry have been increased in the last decade [50–52]. Nanoparticles of pectin are prepared using mechanical homogenization for dissolution enhancement of itraconazole [53] and further used for drug loading. It has been concluded that nanoemulsions were achieved when chloroform was used as an internal phase. The obtained emulsion was freeze-dried to obtain solid porous

Fig. 1.4 Step-by-step fabrication of alginate-zinc nanoparticles. Highly stable alginate nanoparticles were prepared using zinc-based cross-linking. Reproduced with permission from Pistone S, Qoragllu G, Smistad M, Hiorth. Formulation an preparation of stable cross-linked alginate-zinc nanoparticles in the presence of a monovalent salt. Soft Matter 2015;11:5765–74.

Polysaccharides

7

nanoparticles, and further various sophisticated analytical techniques were applied for characterizations. Scanning electron microscopy and optical images of produced nanoparticles are shown in Fig. 1.5. Furthermore, in a study, citrus-derived pectin nanoparticles were produced via the ionotropic gelation method, using magnesium (Mg2+) as the divalent cross-linker [54].

1.8

Nano-chitin and chitosan

The most abundant natural polymer available in the world is cellulose [22], with chitin and chitosan in second position [9]. The sources used for the isolation of chitin or chitosan are marine crustaceans, shrimp, and crabs. A study reported that about 1000 tons per year (70% of total) of these polymers comes from marine species. Chitosan is mainly produced by deacetylation or removing of acetyl groups from

Fig. 1.5 Optical images (left-hand column and middle column) and SEM images (right-hand column) of particles prepared from caprylic/capric triglyceride (CCT)-based and chloroform-based nanoemulsion using (A) high methoxy pectin, (B) amidated LM pectin with degree of esterification 29, and (C) low methoxy pectin (degree of esterification 38). Reproduced with permission from Burapapadh K, Takeuchi H, Sriamornsak, P. Development of pectin nanoparticles through mechanical homogenization for dissolution enhancement of itraconazole. Asian J Pharm Sci 2016;11:365–75.

8

Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

the chitin by alkali treatment [55]. However, deacetylation of chitin is almost never complete, and chitosan or deacetylated chitin still contains acetamide groups to some extent. Unlike cellulose, chitin and chitosan contain 5%–8% N2, which in chitin is in the form of acetylated amine groups and in chitosan is in the form of primary aliphatic amine groups; this makes chitin and chitosan suitable for typical reaction of amines [56]. However, chitosan is chemically more active than chitin due to the presence of primary and secondary hydroxyl groups on each deacetylated unit (Fig. 1.6). These reactive groups are readily subject to chemical modification to alter the mechanical and physical properties of chitosan. The synthesis of chitosan nanoparticles in the presence of sodium tripolyphosphate (TPP) as a reducing agent was discussed [57]. In another study, eco-friendly spherical chitin nanoparticles were synthesized using oxidative Oxone. This versatile-triple salt was effective in the cleavage of glycosidic bonds and etching out individualization of elementary fibrils to form chitin nanoparticles and selective oxidation occur at C6 hydroxyl of glucose molecule submit to form carboxylic acid [58]. In a very interesting study, the size of chitosan-TPP nano and microparticles has been controlled using various molar ratios of chitosan and TPP. Furthermore, the degree of acetylation (DA) is reported to be the second most influential parameter of tuning the size of produced nano/micro particles [59]. The sizes of produced nanoparticles are shown in Fig. 1.7. In the same study, in which very interesting results were discussed, the authors explored the influence of chitosan concentrations on the hydrodynamic radii of fabricated nanoparticles, as shown in Fig. 1.8. A high concentration of chitosan is responsible for the greater size of produced chitosan nanoparticles.

Fig. 1.6 Structure relationship between chitin and chitosan. Reproduced with permission from Islam S, Bhuiyan MAR, Islam MN. Chitin and chitosan; structure, properties and applications in biomedical engineering. J Polym Environ 2017;25:854–66.

Polysaccharides

9

Fig. 1.7 TEM micrographs of chitosan-TPP particles prepared from chitosan samples at varying DA and concentration with a NH2/PO4 molar ratio of 1.5 for DA 20% and 50%, and with a NH2/PO4 molar ratio of 1 for DA 35%: (A) DA 20% at 0.5 mg/mL chitosan concentration; (B) DA 20% at 2.5 mg/mL chitosan concentration; (C) DA 20% at 5 mg/mL chitosan concentration; (D) DA 35% at 0.5 mg/mL chitosan concentration; (E) DA 35% at 2.5 mg/mL chitosan concentration; (F) DA 35% at 5 mg/mL chitosan concentration; (G) DA 50% at 0.5 mg/mL chitosan concentration; (H) DA 50% at 2.5 mg/mL chitosan concentration; (I) DA 50% at 5 mg/mL chitosan concentration. Scale bars (A, D, G) ¼ 200 nm; (B, E, H) ¼ 500 nm; (C, F, I) ¼ 1 μm. Reproduced with permission from Sreekumar S, Goycoolea FM, Moerschbacher BM, RiveraRodriguez GR. Parameters influencing the size of chitosan-TPP nano and microparticles. Sci Rep 2018;8:4695.

Physical modifications of these polymers can be achieved by blending, which involves physical mixing of two or more polymers. It is one of the oldest and easiest modification approaches. Some common hydrophilic polymers that can be blended with chitosan to achieve oral drug delivery are poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), and poly (ethyl oxide) (PEO). Blending of chitosan and PVA improves the mechanical (tensile strength) and barrier properties (water vapor

10

Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Fig. 1.8 Influence of chitosan concentration on the average hydrodynamic diameter of particles prepared using DA 20% (black), DA 35% (red), and DA 50% (blue) with a NH2/PO4 ratio of 1.5 for DA 20% and 50%, and with a NH2/PO4 ratio. Reproduced with permission from Sreekumar S, Goycoolea FM, Moerschbacher BM, RiveraRodriguez GR. Parameters influencing the size of chitosan-TPP nano and microparticles. Sci Rep 2018;8:4695.

permeability) of chitosan films [60]. In this study, the blend improved the mechanical properties of fabricated composites (mainly tensile strength). Chemical modification is achieved by altering the functional groups in a compound. Chemical modification can be done in several ways, including chemical, radiation, photochemical, plasma-induced, and enzymatic grafting methods [61]. Chemical modification of chitosan results in the formation of several derivatives such as quaternized chitosan, thiolated chitosan, carboxylated chitosan, amphiphilic chitosan, chitosan with chelating agents, PEGylated chitosan, and lactose-modified chitosan. The primary amine (
NH2) groups of chitosan provide a reaction site for chemical modification to achieve various pharmaceutical applications [61], reacting with sulfates, citrates, and phosphates [62], which can enhance stability and drug encapsulation efficiency [62]. For example, to improve the solubility of chitosan in

Polysaccharides

11

intestinal media, N-trimethyl chitosan chloride (TMC), a quaternized chitosan, has been produced [63, 64]. The two forms of TMC, TMC 40 and TMC 60, enhance the intestinal permeation of hydrophilic macromolecular drugs.

1.9

Conclusion

In this chapter, a general overview of polysaccharides was provided. Furthermore, a new field of polysaccharides called “nano-polysaccharides” was elaborated in detail. Various methods for the production of polysaccharides nanoparticles were discussed and a special focus was given to the functionalization of these nanoparticles for various applications.

References [1] Bassas-Galia M, Follonier S, Pusnik M, Zinn M. Natural polymers: a source of inspiration. Bioresour Polym Biomed Appl 2017;31–64. [2] Gupta P, Nayak KK. Characteristics of protein-based biopolymer and its application. Polym Eng Sci 2015;55:485–98. [3] Ruiging X, Mark WG. Chemical synthesis of polysaccharide mimetics. Prog Polym Sci 2017;74:78–116. [4] Aspinall GO. Classification of polysaccharides. In: The polysaccharides. Elsevier; 1983. p. 1–9. [5] Hongbin Z, Fei Z, Juan W. Physically cross-lined hydrogels from polysaccharides prepared by freeze-thaw technique. React Funct Polym 2013;73:923–8. [6] Cheng S, Xie SJ, Carrillo JMY, Carroll B, Martin H, Cao PF, Dadmun MD, Sumpter BG, Novikov VN, Schweizer KS. Big effect of small nanoparticles: a shift in paradigm for polymer nanocomposites. ACS Nano 2017;11:752–9. [7] Balazs AC, Emrick T, Russell TP. Nanoparticle polymer composites: where two small worlds meet. Science 2006;314:1107–10. [8] Klemm D, Kramer F, Moritz S, Lindstr€om T, Ankerfors M, Gray D, Dorris A. Nanocelluloses: a new family of nature-based materials. Angew Chem Int Ed 2011;50:5438–66. [9] Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006;31:603–32. [10] Dumitriu S. Polysaccharides: structural diversity and functional versatility. 2nd ed. New York, NY, USA: Marcel Dekker; 2005. [11] Karim Z, Hakalahti M, Tammelin T, Mathew A, Oksman K. Effect of in situ TEMPO surface functionalization of nanocellulose membranes in the adsorption of metal ions form aqueous solution. RSC Adv 2017;7:5232–41. [12] Karim Z, Svedberg A, Lee KY, Khan MJ. Processing-structure-property correlation understanding of microfibrillated cellulose based dimensional structures for ferric ions removal. Sci Rep 2019;9:10277. [13] Karim Z, Svedberg A, Ayub S. Role of functional groups in the production of self assembled microfibrillated cellulose hybrid frameworks and influence on separation mechanism of dye from aqueous medium. Int J Biol Macromol 2020;155:1541–52.

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[14] Mathew AP, Oksman K, Karim Z, Liu P, Khan SA, Naseri N. Process scale up and characterization of wood cellulose nanocrystals hydrolysed using bioethanol pilot plant. Ind Crop Prod 2014;58:212–9. [15] Karim Z, Mathew AP, Mouzon J, Oksman K. Nanoporous membranes with cellulose nanocrystals as functional entity in chitosan: removal of dyes from water. Carbohydr Polym 2014;112:668–76. [16] Karim Z, Claudpierre S, Grahn M, Oksman K, Mathew AP. Nanocellulose based functional membranes for water cleaning: tailoring of mechanical properties, porosity and metal ion capture. J Membr Sci 2016;514:418–28. [17] Karim Z, Afrin S, Husain Q, Danish R. Necessity of enzymatic hydrolysis for production and functionalization of nanocelluloses. Crit Rev Biotechnol 2017;6:1–16. [18] Afrin S, Karim Z. Green catalytic approach for isolation and surface modification of nanocellulose: necessity of enzymes over chemicals. ChemBioEng 2017;4:289–303. [19] Karim Z, Afrin S. Nanocellulose as novel supportive functional material for growth and development of cells. Cell Dev Biol 2015;4:2–7. [20] Chunliang Z, Yapei Z, Ruitao C, Keying L, Juanjuan L, Xingyu J. Manufacture of hydrophobic nanocomposite films with high printability. ACS Sustain Chem Eng 2019;7:15404–12. [21] Onwumere J, Piatek J, Budnyak TM, Chen J, Mathew AP, Karim Z, Slabon A. CelluPhot: hybrid cellulose-bismuth oxybromide membrane for pollutant removal. ACS Appl Mater Interfaces 2020;12:42891–901. [22] Karim Z, Monti A, Barcaro G, Svedberg A, Ansari MA, Afrin S. Enhanced sieving of cellulosic microfibers membranes via tuning of interlayer spacing. Environ Sci Nano 2020;7:2941–52. [23] Karim Z, Svedberg A. Controlled retention and drainage of microfibrillated cellulose in continuous paper production. New J Chem 2020;44:13796–806. [24] Liaw KL, Wang YC, Huang KR, Lee JY, Lai CS. Advanced polyimide materials: syntheses, physical properties and applications. Prog Polym Sci 2012;37:907–74. [25] Sihem BH, Albert M, Christian P, Sami B. Starch nanoparticles formation via high power ultrasonication. Carbohydr Polym 2013;92:1625–32. [26] Suk FC, Suh CPS, Hiang T. Size controlled synthesis of starch nanoparticles by a simple nanoprecipitation method. Carbohydr Polym 2011;86:1817–9. [27] Canxin C, Benxi W, Zhengyu J, Yaoqi T. Facile method for fluorescent labeling of starch nanocrystal. ACS Sustain Chem Eng 2017;5:3751–61. [28] Jinfeng L, Fayin Y, Lin L, Yun Z, Guohua Z. Joint effects of granule size and degree of substitution on octenyl succinated sweet potato starch granules as pickering emulsion stabilizers. J Agri Food Chem 2018;66:4541–50. [29] Yongbing Z, Jong GS, Young ML. Polyimides containing aliphatic/alicyclic segments in the chains. Prog Polym Sci 2019;92:35–88. [30] Hebeish A, El-Rafie MH, EL-Sheikhr MA, El-Naggar ME. Ultrafine characteristics of starch nanoparticles prepared using native starch with and without surfactant. J Inorg Organomet Polym Mater 2014;24:515–24. [31] Helene A, Sonia MB, Mohamed NB, Alain D. Surface chemical indication of waxy maize starch nanocrystals. Langmuir 2005;21:2425–33. [32] Dufresne A. Crystalline starch based nanoparticles. Curr Opin Colloid Interface Sci 2014;19:397–408. [33] LeCorre D, Bras J, Dufresne A. Starch nanoparticles: a review. Biomacromolecules 2010;11:1139–53.

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[34] Corre DL, Angellier CH. Preparation and application of starch nanoparticles for nanocomposites: a review. React Funct Polym 2014;85:97–120. [35] Habibi Y. Key advances in the chemical modification of nanocelluloses. Chem Soc Rev 2014;43:1519–42. [36] Gombotz WR, Wee SF. Protein release from alginate matrices. Adv Drug Deliv Rev 1998;31:267–85. [37] Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev 2001;101:1869–79. [38] Hay ID, Rehman ZU, Ghafoor A, Rehm BHA. Bacterial biosynthesis of alginates. J Chem Technol Biotechnol 2010;85:752–9. [39] Ahmad N, Roland R, Meritxell L, Conxita S, Rauzah H, Hairul AT. Influence of nonionic branched-chain alkyl glycosides on a model nano-emulsion for drug delivery systems. Colloids Surf B Biointerfaces 2014;115:267–74. [40] Pan H, Lijie Y, Jian X, Dejun S. Preparation of highly stable concentrated W/O nanoemulsions by PIC method at elevated temperature. Colloids Surf A Physicochem Eng Asp 2014;447:97–102. [41] Guo KW. Current relevant nanotechnologies for the food industry. Wiley; 2015. p. 623–36. [42] Nasr FH, Sepideh K. Design, characterization and in vitro evaluation of novel shell crosslinked poly(butylene adipate)-co-N-succinyl chitosan nanogels containing loteprednol etabonate: a new system for therapeutic effect enhancement via controlled drug delivery. Eur J Med Chem 2015;102:132–42. [43] Venkatesan J, Sukumaran A, Se-Kwon K, Min S. Seaweed polysaccharide-based nanoparticles: preparation and applications for drug delivery. Polymers 2016;8:30. [44] Bagal-Kestwal DR, Kestwal RM, Chiang BH. Bio-based nanomaterials and their bionanocomposites. vol. 17. Wiley; 2016. p. 255–330. [45] Machado AHE, Lundberg D, Ribeiro AJ, Veiga FJ, Lindman B, Miguel MG, Olsson U. Preparation of calciulm alginate nanoparticles using water-in-oil (w/o) nanoemulsion. Langmuir 2012;28:4131–41. [46] Pistone S, Qoragllu G, Smistad M, Hiorth. Formulation an preparation of stable crosslinked alginate-zinc nanoparticles in the presence of a monovalent salt. Soft Matter 2015;11:5765–74. [47] Mohnen D. Pectin structure and biosynthesis. Curr Opin Plant Biol 2008;11:266–77. [48] Sriamornsak P. Application of pectin in oral drug delivery. Expert Opin Drug Deliv 2011;8:1009–23. [49] Van-Buren JP. Function of pectin in plant tissue structure and firmness. In: Walter RH, editor. The chemistry and technology of pectin. New York: Academic Press; 1991. p. 1–17. [50] Piriyaprasarth S, Sriamornsak P. Flocculating and suspending properties of commercial citrus pectin and pectin extracted from pomelo (Citrus maxima) peel. Carbohydr Polym 2011;83:561–8. [51] Sriamornsak P, Thirawong Y, Weerapol D. Swelling and erosion of pectin matrix tablets and their impact on drug release behaviour. Eur J Pharm Biopharm 2007;67:211–9. [52] Sungthongjeen S, Sriamornsak P, Pitaksuteepong T. Effect of degree of esterification of pectin and calcium amount on drug release from pectin-based matrix tablets. AAPS Pharm Sci Tech 2004;5, E9. [53] Burapapadh K, Takeuchi H, Sriamornsak P. Development of pectin nanoparticles through mechanical homogenization for dissolution enhancement of itraconazole. Asian J Pharm Sci 2016;11:365–75.

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[54] Jacob EM, Borah A, Jindal A, Pillai S. Synthesis and characterization of citrus-derived pectin nanoparticles based their degree of esterification. J Mater Res 2020;35:1514–22. [55] Yeul YS, Rayalu SS. Unprecedented chitin and chitosan: a chemical overview. J Polym Environ 2013;21:606–14. [56] Kurita K. Controlled functionalization of polysaccharides chitin. Prog Polym Sci 2001;26:1921–71. [57] Anand M, Sathyapriya P, Maruthupandy M, Beevi AH. Synthesis of chitosan nanoparticles by TPP and potential larvicidal applications. Front Lab Med 2018;2:72–8. [58] Luong JHT, Gedanken A. Eco-friendly and facile preparation of spherical chitin nanoparticles. ChemistrySelect 2018;3:10787–91. [59] Sreekumar S, Goycoolea FM, Moerschbacher BM, Rivera-Rodriguez GR. Parameters influencing the size of chitosan-TPP nano and microparticles. Sci Rep 2018;8:4695. [60] Park SY, Jun ST, Marsh KS. Physical properties of PVOH/chitosan-blended films cast from different solvents. Food Hydrocoll 2001;15:499–502. [61] Dambies L, Vincent T, Domard A, Guibal E. Preparation of chitosan gel beads by ionotropic molybdate gelation. Biomacromolecules 2001;2:1198–205. [62] Al-Qadi S, Grenha A, Carrio´n-Recio D, Seijo B, Remun˜a´n-Lo´pez C. Microencapsulated chitosan nanoparticles for pulmonary protein delivery: in vivo evaluation of insulinloaded formulations. J Control Release 2012;157:383–90. [63] Thanou MM, Kotze AF, Scharringhausen T, Lueßen HL, De-Boer AG, Verhoef JC, Junginger HE. Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers. J Control Release 2000;64:15–25. [64] Shukla SK, Mishra AK, Arotiba OA, Mamba BB. Chitosan-based nanomaterials: a stateof-the-art review. Int J Biol Macromol 2013;59:46–58.

Introduction to nanocomposites

2

Abu Tariqa, Showkat Ahmad Bhawanib, Mohd Razip Asaruddinb, and Khalid M. Alotaibic a Department of Chemistry, Aligarh Muslim University, Aligarh, Uttar Pradesh, India, b Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia, cDepartment of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia

2.1

Introduction

Dimensionality plays a critical role in determining the properties of matter. A material’s nanostructure is considered as the key aspect in the induction and development of novel properties and in the nano-level control of the structure. Nanotechnology, therefore, is a highly promising field of the current century, in which technical applications in the field of semiconductors, inorganic and organic materials, energy storage, biotechnology, and biomedicine are expected to be redesigned and totally restructured.

2.1.1 Definition of nanocomposites The term “nanotechnology” can be described as the controlled processing of materials that consist of at least one component with one dimension smaller than 100 nm in size. This technology aims to combine all core area of science; chemistry, physics, biology, and material science in order to establish and achieve new materials with peculiar properties that can be exploited to develop and produce advanced biomedical and electronic devices, high performance advanced materials, and other consumer products. It is anticipated that the commercialization of nanotechnology will bring large and widespread technological advancements that will improve the quality of life and benefit society around the world. In a comprehensive sense, the term “composite” can be defined as a material that is comprised of two or more distinct components [1]. It could also be defined as a mixture of two or more distinct components that are combined in an attempt to incorporate the best properties of all the components. Jean-Marie explained composite as a material that consists of two phases, a continuous phase called a “matrix” and a discontinuous phase known as “reinforcement or reinforcing material.” A nanocomposite is a material that consists of at least one component with one dimension at or around 10
9 m [2]. A scale comparison of materials is reported in Table 2.1. Another definition of nanocomposites states that “nanocomposites are solid materials consisting of multiple phased components as one, two or three dimension of less than 1 nm size” [3]. The peculiar properties such as mechanical, electrical, thermal, Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00012-8 Copyright © 2021 Elsevier Inc. All rights reserved.

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Table 2.1 Comparison of materials and their approximate sizes. Material

Size

Coin Carbon fiber Carbon-carbon chemical bond

10
3 m 10
6 m 10
10 m

optical, electrochemical, catalytic, and medicinal characteristics of the obtained nanocomposites will be significantly different from those of the component materials from which the nanocomposites are produced. Many exciting new materials with unique and peculiar properties are being produced within this rapidly expanding field. The obtained nanocomposites have the possibility of new properties other than the properties of the parent materials. Thus, nanocomposites can be explained as nanomaterials comprised of either one or more separate components in order to obtain nanomaterials with peculiar properties of each added component. Nanoparticles such as clay, metal, and carbon nanotubes (CNTs) serve as matrix fillers in a nanocomposite, typically a polymer matrix [4]. Nanocomposites are a future alternative, addressing and overcoming existing microcomposite and monolithic limitations and are becoming potential ingredients for a variety of products. The potential benefits of nanocomposites over parent materials and other composites are listed below: l

l

l

smaller gap between fillers and small filler size owing to high surface/volume ratio; enhanced mechanical properties, such as high ductility, retaining strength, and scratch resistance; and improved optical characteristics.

The key drawbacks of nanocomposites are related to their strength and impact efficiency associated with incorporation of nanoparticles into the composite bulk matrix. Moreover, cost-effectiveness and lack of understanding of the relationship between formulation, characteristics, and framework needed for easier exfoliation of particles and diffusion are other limitations of nanocomposites.

2.1.2 History of nanocomposites For almost five decades, nanocomposites have been the choice of researchers for various studies, but few sources reported the significance of organoclay and how it is transformed into plastics of choice. As early as 1950, nanocomposites were first reported and polyamide nanocomposites first came to light in 1976. The first reference in the literature was reported in 1949 with regard to polymer/clay nanocomposite technology and is attributed to Bower who performed DNA absorption by montmorillonite (MMT) clay [5]. In addition, other studies conducted in the 1960s showed that the surface of clay could serve as an initiator of polymerization [6, 7] and the monomers

Introduction to nanocomposites

17

could be embedded between clay mineral platelets [8]. It is also of immense importance that Greenland, in 1963, prepared nanocomposites of polyvinyl alcohol/MMT in an aqueous medium [7]. However, Toyota researchers had done an extensive and detailed study of composites consists of polymer/layered silicate clay mineral that made nanocomposites a potential contender for research in both academic and industrial laboratories. In the early 1990s, study on a Nylon-6 nanocomposite was reported by Toyota Central Research Laboratories (TCRL), Japan. In that study it was reported that a very lesser amount of nanofiller was loaded, resulting in a significant enhancement in the mechanical properties of nylon-6 [9]. The nylon-6 nanocomposite accompanied with organophilic clay nanocomposites was used by Toyota in manufacturing of timing belts in cars [10–12]. This newly produced material was found to have 4.2 wt% with an increase of 40% in rupture tension, 68% enhanced Young’s modulus, and 126% increase in flexural modulus; moreover, it was reported that heat distortion temperature was found to be increased to 152°C from 65°C when compared with the pure form of polymer [13]. Acquarulo and O’Neil [14] also reported a similar kind of study from TCRL on polymer-layered silicate-mineral composites and the clay mineral that is creating a huge wave of interest for use in nanocomposite—montmorillonite (MMT) or nanoclay, sometimes also called bentonite. Thermoplastic nanocomposites, such as polyamide and polypropylene, have since been introduced by several companies for automotive applications [15]. The gas barrier, through using polyamides or polyesters [16], is another highlighted application of nanocomposites.

2.2

Classification of nanocomposites

Nanocomposites are classified into three broad groups, based on the matrix materials of which they are composed: l

l

l

ceramic matrix nanocomposites (CMNC) polymer matrix nanocomposites (PMNC) metal matrix nanocomposites (MMNC)

Nanocomposites can also be categorized as nanocomposites, which can be classified further as: (a) nonpolymer-based nanocomposites; metal/metal nanocomposites metal/ceramic nanocomposites ceramic/ceramic nanocomposites (b) polymer-based nanocomposites polymer/ceramic nanocomposites inorganic/organic polymer nanocomposites inorganic/organic hybrid nanocomposites polymer/layered silicate nanocomposites polymer/polymer nanocomposites biocomposites l

l

l

l

l

l

l

l

l

nonpolymer

and

polymer

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Table 2.2 Types of nanocomposites and their examples. Type of nanocomposite

Examples

Ceramic matrix nanocomposite (CMNC) Polymer matrix nanocomposite (PMNC)

Al2O3/SiC, SiN/SiC, SiO2/Ni, ZnO/Co, TiO2/Fe2O3, Al2O3/ SiO2/ZrO2, Al2O3/CNT, Al2O3/TiO2, NdAlO3/Al2O3 Thermoplastic/thermoset polymer/layered silicates, polyester/TiO2, polymer/CNT, polymer/layered double hydroxides, MMT/N6/PCL/PMMA /PU/Epoxy, Clay/PCL, PLA, HDPE, PEO, PVA, PVP, PVA Fe-Cr/Al2O3, Ni/Al2O3, Co/Cr, Fe/MgO, Al/CNT, Mg/CNT, Cu-Al2O3, Al/Mo, Cu/W, Cu/Pb, Fe/MgO, W/Cu, Nb/Fe, Al-C60

Metal matrix nanocomposite (MMNC)

A brief description of three broad categories of nanocomposites is given below. Various examples of these categories of nanocomposites are given in Table 2.2.

2.2.1 Ceramic matrix nanocomposites (CMNCs) Ceramic matrix nanocomposites are those materials where different ceramic phases are deliberately inculcated into the matrix to improve properties such as wear resistance and chemical and thermal stability. However, the key drawback of ceramics is their brittleness and low hardness, preventing them from being utilized in industries for various applications. This limitation has been controlled through the formation of ceramic matrix nanocomposites (CMNCs); an example of CMNC is a matrix system where energy dispersing or dispelling components such as platelets, fibers, or particles are integrated into the ceramic matrix to minimize fragility and enhance fracture resilience [17–19]. The pioneering research of Niihara [20, 21] revealed the potential of ceramic matrix nanocomposites (CMNCs), predominantly that of an Al2O3/SiC system. The observable strengthening of the Al2O3 matrix after addition of a low volume, i.e., 10%, of SiC particles of suitable size and hot pressing of the resultant mixture has been confirmed by most studies reported so far. The mechanism, intended to harden the material, is based on the crack-bridging role of nanosize reinforcement, and has been reported by some researchers [22].

2.2.2 Polymer matrix nanocomposites (PMNC) In PMNC, nanocomponents are typically fillers known as nanofillers and are graded as: 1D-linear (e.g., carbon nanotubes (CNTs)), 2D-layered (e.g., MMT), and 3Dpowder (e.g., silver nanoparticles (AgNPs)) [23]. The effect of attraction between nanocomposites at molecular level is observed owing to the interaction between nanofiller and polymer matrix. As a result, the incorporation of a small amount of nanofiller to the matrix system with dimensions below 100 nm produces a change

Introduction to nanocomposites

19

in the properties of the composite material. Several methods including an in situ solvent method or mixing melted polymer matrix method are applied to produce nanocomposites. The obtained PMNCs have properties such as increased abrasion resistance, high thermal stability, and high barrier capacity [24]. Owning to their ease of production, lightweight and often ductile nature, polymer materials are widely used in a variety of industries. They have certain drawbacks, however, such as low modulus and strength as compared to metals and ceramics. In this context, addition of whiskers, platelets, or particles as reinforcements to the polymer matrix system is a very effective potential approach to enhance mechanical properties. In order to enhance heat and impact resistance, flame retardancy, and mechanical strengthening and to reduce electrical conductivity and gas permeability with respect to oxygen and water vapor, polymers have been incorporated with several inorganic compounds, either natural or synthetic [25]. In addition, metal and ceramic reinforcements provide striking pathways to certain peculiar electronic, magnetic, optical, or catalytic characteristics derived from inorganic nanoparticles that add to other polymeric properties such as the ability to process and form film [26]. Polymers can be strengthened using this technique while retaining their lightweight and ductile character [27–32]. Another significant aspect is that, as will be seen later, nanoscale reinforcements have an exceptional potential to generate new phenomena, leading to special properties in these materials.

2.2.3 Metal matrix nanocomposites (MMNC) Metal matrix nanocomposites (MMNC) are multiphase components that are made up from a ductile metal or alloy matrix that contains nanosized reinforcement materials as implants. The peculiar properties of MMNC comprise both metal and ceramic features such as high ductility, resilience, strength, and modulus. Therefore, MMNC are ideal for the manufacture of materials with enhanced strength in high shear/compression processes and elevated operating temperature capabilities. These materials show exceptional potential for applications in various fields such as the aerospace and automotive industries and the production of structural materials [33].

2.3

Structure and processing of nanocomposites

2.3.1 Structure of nanocomposites The nanocomposite structure typically consists of a matrix containing nanosized reinforcement components in the form of particles, whiskers, fibers, nanotubes, etc. [34]. Various nanocomposite characterization equipment and techniques have been used by investigators and researchers, including atomic force microscopy (AFM), scanning tunneling microscopy (STM), scanning and transmission electron microscopy (SEM/TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), etc. [33, 35–41].

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

2.3.2 Processing of nanocomposite The raw materials used in preparation of CMNC matrix systems include Al2O3, SiC, SiN, etc., whereas in PMNC, a wide range of polymers such as vinyl and condensation polymers, polyolefins, and specialty polymers including a whole range of biodegradable moieties are used; however, in MMNC, metal matrices such as Al, Mg, Pb, W, Sn, and Fe are employed. All the materials used as reinforcements in the nanocomposite systems are of nano-range size. Natural and synthetic crystalline reinforcements like Fe and other metal powders, clays, silica, TiO2, and other metal oxides have been used, although the most common are clays and layered silicates owing to their availability, low particle size, and well researched interaction chemistry [24, 42–46]. Known techniques are used to prepare most of these reinforcements; chemical, mechanical (e.g., ball milling), vapor deposition, etc. Most of the processing techniques of the three kinds of nanocomposites, considering their nano dimensions, remain almost the same as in the case of microcomposites. This is also valid for composites that are carbon nanotube (CNT) reinforced. Details of all such processes are described in the coming section.

2.3.2.1 Processing methods for CMNCs A wide range of methods and techniques are identified and applied for the preparation of CMNCs [21, 47–59]. New techniques mainly include single source precursor techniques focused on melt spinning of hybrid precursors followed by curing and pyrolyzing the fibers. Some of the older methodologies include traditional powder technique [60], polymer precursor pathway [61, 62], spray pyrolysis [63], vapor technique (CVD and PVD) [64], and chemical methods that comprise sol-gel technique, colloidal, and aggregation approaches, and template synthesis as well [65]. Table 2.3 represents the processes, advantages, and limitations of the techniques involved in the manufacturing of CMNCs. (a) Powder process: (i) selection of mostly small size, uniform and high purity powders as raw materials, (ii) wet ball milling method or attrition milling procedure in organic and aqueous medium is applied to mix the material, (iii) drying of obtained material by using heat through lamps and or ovens or just by application of freeze drying technique, (iv) hot pressing/gas pressure/slip casting/injection molding or pressure filtration technique is applied for compacting of the solid material [21, 51]. (b) Polymer precursor process: Si-polymeric precursor is mixed with the matrix material followed by pyrolysis of the obtained mixture using oven to generate reinforced particles [52–55, 66]. (c) Sol-gel process: The procedure includes the hydrolysis and polycondensation reactions of the organic dissolved inorganic precursors resulting in the formation of 3D polymers containing metal-oxygen bonding. The process is followed by drying to obtain solid material, which in turn can be treated thermally to compact the material [56–59].

2.3.2.2 Processing methods for PMNCs Several procedures have been studied for the preparation of polymer nanocomposites, which include layered materials and materials containing CNTs [43, 67–88]. The most

Introduction to nanocomposites

21

Table 2.3 Representation of techniques, limitations, and advantages of different CMNC matrix systems. Technique

Matrix system

Advantages

Limitations

References

Powder process

Al2O3/SiC

Simple

[38, 52]

Polymer precursor process

Al2O3/SiC, SiN/ SiC

Sol-gel process

SiO2/Ni, ZnO/ Co, TiO2/Fe2O3, La2O3/TiO2, Al2O3/SiC, TiO2/ Al2O3, Al2O3/ SiO2, Al2O3/ SiO2/ZrO2, TiO2/ Fe2TiO5, NdAlO3/Al2O3

Better reinforcement dispersion, possibility of formation of fine particles Simple, versatile, low processing temperature, high chemical homogeneity, high purity products, etc.

Low phase dispersion, rate of formation Phase segregation caused by ultrafine particles and agglomeration Greater shrinkage, low void formation, when compared to mixing process

[16, 53–56]

[57–72]

frequently used ones are: (i) intercalation of the polymer/prepolymer from solution, (ii) in situ intercalative polymerization; (iii) melt intercalation; (iv) direct mixing of polymer and particulates; (v) template synthesis; (vi) in situ polymerization; and (vii) sol-gel technology. Table 2.4 shows the different aspects of the PMNCs. (a) Intercalation/prepolymer from solution: The process is employed for layered reinforcing materials where the polymer can intercalate, often for layered silicates, with intercalation of the polymer or prepolymer from solution; usage of a solvent where the polymer or prepolymer is soluble and the silicate layers are swellable. (b) In situ intercalative polymerization: This is a technique through which polymers are formed between the intercalated sheets. During this process, swelling of layered silicates within liquid monomer is followed by the polymerization initiated through heat or radiation, or by suitable initiator diffusion, or by the use of any organic initiator [97, 98]. In the early stages, only Nylon-MMT nanocomposite was prepared, later, production of thermoplastics was observed using this technique. This technique is very suitable for the production of thermoset-clay nanocomposites [99]. Slow reaction rate, and dependence of clay exfoliation through swelling of clay/formation of oligomer due to incomplete polymerization are some limitations of this technique [100]. (c) Melt intercalation: This technique is comprised of molten layered silicate and does not require any solvent and polymer matrix. At an elevated temperature, organoclay is mechanically mixed with thermoplastic polymer through classical methods such as extrusion or

Table 2.4 Different PMNC matrix systems and their advantages and limitations. Technique

Matrix system

Advantages

Limitations

References

Intercalation/ prepolymer from solution

Clay with PCL, PLA, HDPE, PEO, PVA, PVP, PVA, etc.

Industrial use of large amounts of solvents

[24, 67–69, 89]

In situ intercalative polymerization

MMT with N6/PCL/PMMA /PU/epoxy

Preparation of homogeneous filler dispersions, preparation of intercalated nanocomposites based on polymers with low or even no polarity Simple process, based on the dispersion of the filler in the polymer precursors

[70, 71, 90, 91]

Melt intercalation

MMT with PS/PEO/PP/PVP, clayPVPH

Template synthesis

Hectorite with PVPR, HPMC, PAN, PDDA, PANI

Direct mixing In situ polymerization

PVA/Ag; PMMA/Pd polyester/TiO2 PET/CaCO3, epoxy vinyl ester/Fe3O4; epoxy vinyl ester/γ-Fe2O3; poly (acrylic acid)(PAA)/Ag, PAA/Ni and PAA/Cu AgNO3, NiSO4, and CuSO4 Polyimide/SiO2; 2-hydroxyethyl acrylate (HEA)/SiO2, polyimide/silica. PMMA/SiO2, polyethyl-acrylate/SiO2, polycarbonate/SiO2 and poly (amideimide)/TiO2

– –

Arduous control of intragallery polymerization; restricted applications Restricted applications to polyolefins that constitute the majority of used polymers Restricted applications; based mainly in aqueous polymers, side product contamination – –

Simple, versatile, low processing temperature, high chemical homogeneity, high purity products, etc.

Greater shrinkage, low void formation, when compared to mixing process

Sol-gel technology

Environmentally friendly; use of polymers not suited for other processes; compatible with industrial polymer processes Large production on plant scale; simple technique

[41, 72, 92, 93]

[80, 94–96]

– –

[56–59]

Introduction to nanocomposites

(d)

(e) (f)

(g)

23

injection molding [101]. In this step, nanocomposites are formed through exfoliation of polymer chains. This technique is also very handy in the preparation of thermoplastic nanocomposites. The limitation of this technique is limited applications of polyolefins. Template synthesis: Template synthesis is used to obtain clay minerals within the polymer matrix. It is obtained by applying an aqueous or gel solution, which comprises the silicate building blocks and the polymer. The aqueous polymer act as a template for the layer formation. During the process, nucleation occurs and the formation of inorganic host crystals is achieved. Mixing of polymer: Mixing of either polymer or monomer with reinforcing materials allows the formation of nanocomposites. In situ polymerization: The polymeric matrix, a monomer, is infused with inorganic particles dispersed all around the polymer. Furthermore, an appropriate catalyst is added to the mixture for polymerization to occur using a classical molding technique. An ultrasonicator is used for dispersion to occur in epoxy systems. Sol-gel technology: This technique involves the incorporation of organic molecules and monomers into sol-gel matrices, and introduction of organic groups through the chemical bond formation. Theoretically, the sol-gel method, without the involvement of the onium ion, will felicitate the dispersion of silicate layers in a one-step process, presenting a large number of disadvantages. Firstly, high temperatures are needed for the synthesis of clay minerals, which causes polymers to decompose. Aggregation tendency during the growth of silicates is a drawback with this technique.

2.3.2.3 Processing methods for MMNCs The raw materials used for the preparation of MMNCs are Al, Mg, Pb, Sn, Fe, and W, with similar reinforcements as used in CMNCs and PMNCs. The frequently used techniques for preparation of MMNCs include spray pyrolysis [102, 103], rapid solidification process (RSP) [104], liquid metal infiltration [105], vapor techniques (CVD/ PVD) [106], electrodeposition [105], and several chemical process such as colloidal [104] and sol-gel technique [106]. Metal falling-drop quenching methods [107] and one-pot synthesis of the quantum dots (CDs) and gold nanoparticles (AuNPs) [108] are some novel methods for the preparation of MMNCs. Table 2.5 represents the various parameters of MMNCs. (a) Spray pyrolysis: This process includes dissolution of the starting materials in a suitable solvent, forming a liquid source that generates mist on usage of ultrasonic atomizer. Carrier gas is applied to carry the mist into a preheated chamber where vaporization of the droplets occurs and they are trapped in filters, in turn promoting the decomposition of these droplets to produce respective oxides. Formation of oxide is followed by selective reduction of these metal oxides to produce required metallic materials. (b) Liquid infiltration: The matrix metal material is mixed with fine particles of reinforcements. Thermal treatment of the obtained material is done during which the metal melts and surrounds the reinforcements by liquid infiltration. Thermal treatment is further continued at below melting point of matrix, in turn internal porosity is eliminated and consolidation is observed. (c) Rapid solidification process (RSP): During this process, melting of metal components is performed together; when the melt is kept above the critical line of miscibility gap of

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Table 2.5 Various techniques, advantages, and limitations of MMNC matrix systems. Technique

Matrix system

Spray pyrolysis

Fe/MgO, W/Cu

Liquid infiltration

Pb/Cu, Pb/ Fe, W/Cu/ Nb/Cu, Nb/ Fe, Al-C60

Rapid solidification process (RSP)

Al/Pb, Al/X/ Zr (X ¼ Si, Cu, Ni), Fe alloy

RSP with ultrasonics

Al/SiC

High energy ball milling

Cu-Al2O3

CVD/PVD

Al/Mo, Cu/ W, Cu/Pb

Advantages

Limitations

References

Efficacious production of ultrafine, spherical, and homogeneous powders in multicomponent systems, reproductive size and quality Contact time matrix and reinforcement is less; fast solidification, production on both lab and plant scale Simple; efficacious method

Expensive in producing large quantities of uniform, nanosized particles

[109]

Use of high temperature; precipitation of reinforcements; formation of byproducts during processing

[109–113]

Only metal-metal nanocomposites; induced agglomeration and nonhomogeneous distribution of fine particles –

[114–118]

[119]

–

[120]

Cost; optimization of many parameters; relative complexity

[121–124]

Excellent dispersion without agglomeration, even with fine particles Homogeneous mixing and uniform distribution Capacity to produce pure and high dense materials; uniform thickness films; adhesion at high deposition rates; good reproducibility

Introduction to nanocomposites

25

Table 2.5 Continued Technique

Matrix system

Sol-gel/ colloidal processes

Ag/Au, Fe/ SiO2, Au/ Fe/Au

(d) (e) (f)

(g)

Advantages

Limitations

References

Simple; low processing temperature; versatile; high chemical homogeneity; rigorous stoichiometry control; high purity products

Weak bonding, low wear-resistance, high permeability, and difficult control of porosity

[57, 125– 128]

different components, homogeneity is obtained. To solidify rapidly, melt spinning or like processes are applied to the melt. RSP with ultrasonics: In this technique, ultrasonics are applied to mix and improve wettability between the matrix and the reinforcements. High energy ball milling: During this process, nanocomposites are obtained through milling of powders together until the required nanosized alloy is achieved. CVD/PVD: PVD: Production of vapor-phase by evaporating/sputtering different components followed by supersaturation of the vapor-phase in an inert environment to prepare metal nanoparticles through condensation. The nanocomposites are condensed by thermal treatment under inert environment. CVD: Chemical reactions are applied to obtain vapors of materials, followed by condensation. Chemical processes: Colloidal method: Metal particles are prepared by the reduction of inorganic salts chemically in a solution, followed by condensation, and thermal treatment and drying of the obtained solid materials under reducing environment such as H2, in order to promote selective oxide reduction and produced metal components. Sol-gel process: Mesoporous silica containing 0.1 M HAuCl4 (aq.) and 0.6 M NaBH4 (aq.) is used to produce two micelle solutions. Mixing of these two solutions under ultraviolet light until complete reduction of gold is performed.

2.4

Benefits and new trends in nanocomposites

Nanocomposite systems have been extensively studied since the 1990s including the very powerful CNTs. Nanocomposites have proved to be highly potent and provide a wide range of benefits including increased properties in materials, reduces solid waste (lower gage thickness films), lower reinforcement usage, and also show enhanced manufacturing capacities, especially in the area of packaging. As can be witnessed, a wide range of applications of nanocomposite systems are available, which include both the production of new advanced materials and the enhancement of properties of various known devices like fuel cells, sensors, and coatings [129]. The massive switch

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

of nanocomposite production from laboratory scale to plant scale is expected to be extensive in the next few years. It is observed that ceramic and metal-based nanocomposites have a large impact over a wide range of industries, including aerospace, the automotive sector, electronics, and the military [65]. Some of the notable applications of CMNCs include acid fuchsin removal [130], photocurrent [131], and biomedical applications. Those ceramics which are used in biomedical applications are known as bioceramics. In biomedical applications, nanocrystalline ceramics offers various advantages such as higher resistance, durability, decreased elasticity, durability, and reduced rejection risk. The enhanced properties like resistance, strength, stability, hardness, and toughness of nanocomposites can be achieved by the combination of bioactive and mechanical properties to create a new range of advanced materials for medical devices, prosthetics, and implants. There are a variety of CMNCs available that include alumina/silicon carbide (Al2O3/SiC) nanocomposites, ceramic/CNTs composites, alumina/zirconia (Al2O3/ ZrO2) nanocomposites, etc. Zirconia and alumina are well studied materials that have potential for orthopedic implants owing to properties such as low wear rates, hardness, and excellent biocompatibility. Zirconia is well suited for dental applications where color and translucency are essential requirements [132]. Gamal-Eldeen and coworkers showed that ferromagnetic glass ceramic (CaO-ZnO-Fe2O3-SiO2) nanocomposites are promising agents for the treatment of bone cancer Saos-2 cells [133]. The enhancement in properties of polymer-based nanocomposites enable a variety of industrial applications. More common applications are packaging, power tool housing, fuel and solar cells, fuel tanks, impellers and blades of vacuum cleaners, power hoods, covers of devices, and plastic containers. Some other applications of PMNCs are discussed below: (a) Gas barriers for packaging and sports goods: Various PMNCs, such as butyl rubber and styrene butadiene to ethylene propylene diene monomer rubber, have been commercially used in barrier applications. The most general polymer barriers are applied for isolation of CO2, O2, N2, HCl, HNO3, and H2SO4. Surgical gloves and chemical protectives production for chemical warfare agents and contamination of medicine are another important application of PMNCs [134]. (b) Energy storage systems: The efficacy of fuel cells increases immensely on infusion of nanomaterials to cells. The proton exchange membrane plays a very important role in transport from anode to cathode [135]. The incorporation of clay thermoset polymer in fuel cells enhances the proton conductivity, increases ion exchange capacity, and enhances the rate of conductivity even at high humidity and increased mechanical properties [136, 137]. (c) Optical glass and membranes: The clay incorporated polymers, when applied to coat transparent materials, enhance the toughness together with hardness of material without hampering the light transmission [138]. (d) Electronics and automobile sectors: The clay nanocomposites are efficiently applied in the field of electronics and automobiles. Specialty elastomers and polyimides, when incorporated with nanoclay, have the ability to reduce solvent transmission. (e) Coatings: It is an important process to modify surface properties. The infusion of thermosetting polymer nanocoatings increases durability and wettability. Weather, chemical, and abrasion resistance are other properties that are enhanced due to nanopolymers.

Introduction to nanocomposites

27

A wide range of PMNCs, including insulating, semiconducting, or metallic NPs, are in the forefront of usage and have been produced to prepare specific types of required products. The enhanced mechanical properties of PMNCs are the main reason for this extensive usage. CNT-ceramic composites are other potential nanocomposites that are successfully applied in the area of aerospace, sports goods, composite mirrors, and automotive spares. Moreover, it can be used in flat panel displays, gas storage devices, toxic gas sensors, and conducting paints [139]. Al2O3-CNT composites, apart from toughness and hardness, also show enhanced contact damage resistance. Engineering and biomedical applications are another important area where CNT nanocomposites are effectively applied [140]. The automotive industry has clearly edged out other industries for applications of PMNCs, as these are applied in tires, fuel systems, gas separation membranes of fuel cells, seat textiles, mirror housing, door handles, engine covers, timing-belt covers, and intake manifolds [141, 142]. Other promising applications are manufacturing of pollution filters that have been made up of porous polymer nanocomposites [25] and air bag sensors where nano-optical platelets are used to transmit signals at high speeds to bring down possible impact injuries [143]. There are polymer/inorganic nanocomposites with enhanced permeability, interfacial resistance, water management, and conductivity that are considered as promising replacements for traditional Nafion PEM fuel cells and are currently under research and trials [144]. Another important application is the networks obtained from nanofiber donor cells and tissue growth [145]. Nanosized silver (Ag), AgO, or other silver salts comprised of nanocomposites are used to achieve antibacterial effects for dendrimer-based drugs [146]. Prostheses are prepared from gelatin incorporated with hydroxyapatite particles or other nanocomposites obtained from this filler [147]. Promising applications of nanocomposite systems are shown in Table 2.6. A wide range of biomedical applications are reported in various research articles, Table 2.7 represents the various applications. Genetic disorders such as Parkinson’s disease, cystic fibrosis, and cancer is another area where use of nanocomposites is reported. Polyethyleneglycol (PEG), graphene oxide (GO), reduced graphene oxide (rGO), and other nanocomposite systems are widely used for the treatment of various diseases through gene delivery and tissue engineering. Controlled release and targeted delivery of drugs are another potential area of research and applicability in which nanocomposites will play a role in the future.

2.5

Future perspective and conclusion

Nanocomposites are a new area of science under consideration for a variety of its applications in various industries and laboratories. This field is interdisciplinary in nature, and encompasses chemistry, physics, biology, and material science with the infusion of engineering. The diversity of this field lies in the merger of various fields of science to produce novel and unique materials, which in turn are used for applications in the areas of aerospace, biomedical and medicine, environmental protection, transportation, catalysis, etc. With every passing day and improvement in quality of

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Table 2.6 Promising applications of nanocomposite systems. Nanocomposite systems

Applications

Ceramic matrix nanocomposites (CMNCs) SiO2/Fe ZnO/Co BaTiO3/SiC, PZT/Ag SiO2/Co, SiO2/Ni, metal oxides/metal Al2O3/SiC, Si3N4/SiC PbTiO3/PbZrO3 Al2O3/NdAlO3 and Al2O3/LnAlO3 TiO2/Fe2O3 Al2O3/Ni

High performance catalysts, data storage technology Field effect transistor for the optical femtosecond study of inter particle interactions Electronic industry, high performance ferroelectric devices Optical fibers, chemical sensors, opto-electronic sensor devices, catalysts Structural materials Microelectronic and micro-electromechanical systems Solid-state laser media, phosphors, and optical amplifiers High-density magnetic recording media, ferrofluids, and catalysts Engineering parts

Metal matrix nanocomposites (MMNCs) Fe/MgO Ni/PZT Ni/TiO2 Al/SiC Cu/Al2O3 Ni/TiN, Ni/ZrN, Cu/ZrN Al/Al2O3, Au/Ag

Catalysts, magnetic devices Wear resistant coatings and thermally graded coatings Photo-electrochemical applications Aerospace, naval, and automotive structures Electronic packaging High-speed machinery, tooling, optical, and magnetic storage materials Microelectronic industry, optical devices, light energy conversion

Polymer matrix nanocomposites (PMNCs) Epoxy/MMT Thermoplastic olefin/ clay Polycaprolactone/SiO2 Polyimide/SiO2 Polycarbonate/SiO2 SPEEK/laponite Nylon-6/LS Shape memory polymers (SMP)/SiC PET/clay

Polyimide/clay

Materials for electronics Beverage container applications Bone-bioerodible for skeletal tissue repair Microelectronics Abrasion resistant coating Direct methanol fuel cells Automotive timing-belt—TOYOTA Medical devices for gripping or releasing therapeutics within blood vessels Food packaging applications that include packing for processed meats, cheese, confectionery, cereals and boil-inthe-bag foods, fruit juice and dairy products, beer and carbonated drinks bottles Automotive step assists—GM Safari and Astra Vans

Introduction to nanocomposites

29

Table 2.7 Various applications of nanocomposites in gene delivery and tissue engineering. Nanocomposite systems

Applications

References

Alginate nanoTiO2 needle Bacterial cellulose/silk fibroin sponge Chitin-chitosan/nano TiO2, chitosanhalloysite nanotubes Chitosan/gelatin/nSiO2 Graphene-bioactive glass Starch/cellulose nanofibers Keratin-bacterial cellulose (modified) Polyethylenimine (PEI)-graphene oxide (GO) complex Polyethyleneglycol (PEG)-BPEI-rGO Gellan gum-PEI nanocomposites Silica-collagen nanocomposites Graphene-based nano-carrier

Tissue engineering Tissue engineering Freeze drying, solution mixing Bone tissue engineering Bone tissue engineering Tissue engineering Skin tissue engineering Gene delivery

[148] [149] [150]

Gene Gene Gene Gene

[157] [158] [159] [160]

delivery delivery delivery delivery

[151] [152] [153] [154] [155, 156]

life, the future of nanocomposites seems exciting and they have proved to be suitable materials with peculiar properties that meet the emerging demands of our progress. There are different routes to prepare desired nanocomposites, such as CMNC, MMNC, and PMNC, but limitations and drawbacks lead to new avenues. Today, applications of these nanocomposite systems are prevalent with many unexplored areas to pursue, thus creating a new highly advanced and peculiar material era. In the near future, it is expected to see revolutions in the fields of power production, storage devices for hydrogen storage, and as fuel cells, supercapacitors, and batteries [143]. Other futuristic projects in automotive industries include: (i) enhanced fire retardancy of nanocomposites that are widely used in interiors; (ii) increased weather resistance for exterior parts applications; and (iii) nanocarbons used to produce bipolarity that is applied to prepare fuel cells. In the case of CMNCs, coating and internal structure of combustion engines have received highlighted attention. In case of these three types of nanocomposites, various processing parameters get much anticipated attention without getting into their high commercialization values. The problems such as de-agglomeration and compatibility can be overcome through the modification of surfaces via homogeneous dispersion of reinforcements. The other area under consideration for future research is the replacement of expensive reinforcements, such as CNTs, with cheap and easily available natural origin polymeric reinforcements for wide range of applications. From the above discussions, a conclusion has been drawn that for every new technology, unique properties in materials are required and are considered as bench mark and the prerequisite. In this context, nanocomposite systems have emerged as suitable materials to meet the high expectations of scientific and technological advancements and concurrent demands. A large number of applications have already been explored, however, a wide range of potential applications of these materials are possible that

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

open the window for new vistas in the future. Thus, all the three types of nanocomposite systems provide opportunities and create worldwide interest among researchers and scientists for their enhanced and peculiar properties.

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Chitosan and its derivatives-based dimensional frameworks as carrier for gene delivery

3

Mohammad Ashfaqa and Asrar Ahmadb a BS Abdur Rahman Crescent Institute of Science and Technology, Chennai, India, bCenter for Sickle Cell Diseases, College of Medicine, Howard University, Washington, DC, United States

3.1

Introduction

Genes are the specific sequence of nucleotides in DNA and RNA that contains specific instructions to code for their respective proteins. These are located on chromosomes, known as a functional unit of inheritance. These codes are a vital resource in order to understand life and its components. Recently, gene therapy has drawn much more attention to the biomedical and pharmaceutical fields because of its huge therapeutic effects in the treatment of various diseases [1–4]. In this technique, a genetic disorder is treated at its genetic roots by inoculating a gene into the cells, rather than by drugs or surgery. This technique involves a series of practices that may help in the delivery of genetic material into cells using vectors to facilitate the expression or the suppression of specific proteins. The accomplishment of gene therapy mostly depends on the ability of the vector or vehicle to transmit a gene with minimal or no side effects to the target site. In gene therapy, finding an effective vector that is safe and productive is the most significant obstacle. Several adverse effects related to vectors and their suitability in gene delivery have been documented over the past two decades, focusing much emphasis on related hazards rather than therapeutic benefits [5–7]. Even though gene therapy is an auspicious treatment option for several diseases such as cancer, inherited genetic disorders, and certain viral infections, the technique remains dicey due to its serious adverse effects and is still being examined to make it safe before it becomes successful. Viruses are very successful in cell transduction, but because of their extreme adverse effects and restrictions, they still have major issues in gene therapy [8, 9]. Therefore, there is an enormous growth in the development of nonviral gene delivery systems. Several studies have recently centered on polymeric nanoparticles because they can enhance the therapeutic effectiveness of drugs by monitoring their efficient delivery time and circulation time [10, 11]. The advantage of nonviral vectors is that they have a favorable safety profile with low immunogenicity, but low transfection efficiency is also associated with them [12]. Due to their possible drug delivery Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00014-1 Copyright © 2021 Elsevier Inc. All rights reserved.

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

mechanism, recently a huge interest in chitosan-based nanocomposites has been established due to their high biocompatibility, low cytotoxicity, and favorable properties as gene carriers [13, 14]. Chitosan-based nanocomposites will be evaluated in this chapter and summarized as a gene delivery method, along with their physicochemical properties, chemical modification range, and gene delivery mechanism. Recently, researchers have investigated many chitosan-based gene delivery systems. Finally, along with more approaches and future insights, a thorough overview of recent applications of gene delivery systems will be provided in this chapter.

3.1.1

Gene delivery system

The gene delivery mechanism is typically consisting of an intracellular gene delivery into particular cells in order to achieve therapeutic effectiveness by regulating the abnormality or function of the cells. The crucial role of the gene delivery system is one-time administration of genes that efficiently restores a gene by replacing flawed or omitted genes. Nowadays, typically two major forms of gene therapy, germline and somatic gene therapy (limited to somatic cell alterations), and two forms of gene delivery, viral and nonviral, are used in gene delivery systems. Nonetheless, each delivery system has certain benefits and drawbacks [15].

3.1.2

Germline gene therapy

Usually, in this process, DNA is transferred into the cells, which produce reproductive cells/sperm/eggs. Germline gene therapy is as simple in those genetic defects that can be treated precisely by using germline cells directly rather than by targeting them. This process has almost never been tested on humans, but multiple transgenic techniques have been used on different species. This method includes gene transport to nuclei (somatic cells are taken at metaphase stage), ex vivo amendment of egg cells, in vitro culture alteration of mouse cells (embryonic stem cells), pronuclear microinjection, and transgenic delivery within the sperm cells using direct/indirect injection to genital parts [16, 17].

3.1.3

Somatic gene therapy

In this process, genes are inserted into the diploid cells, where genetic materials are not transferred into offspring. Somatic gene therapy is a relatively safe process because it affects only targeted cells. However, somatic gene therapy has a short life due to tissues dying and being replaced with newer cells. Moreover, the transfer of genes to the cells also has some issues. In numerous disorders, somatic gene therapy is appropriate and necessary, even considering other complexities. There are typically three types of somatic gene therapy:

Chitosan and its derivatives-based dimensional frameworks

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(1) Ex vivo delivery—the genetic materials are isolated from the target tissue in this process, cultured and operate in vitro, then transferred to the targeted cells/tissue. The advantage of these processes is that there are mainly no immunological issues. (2) In situ delivery—in this process, genetic material is directly transferred into the targeted tissue. This process is effective due to the proper administration of the genetic material. (3) In vivo delivery—in this process, genetic materials are transferred by using an appropriate vector, either viral or nonviral. Insufficient targeting is one main issue associated with this delivery system [18–20]. In this context, improvement in targeting and development of vectors might resolve such issues.

Plasmid DNA is normally inserted into cells and the genetic information is converted into proteins. With the help of the gene delivery system, various obstacles have been resolved such as targeted delivery to the target cells, transportation from the cellular membrane, degradation of endolysosomes, and intracellular transportation of DNA to the nucleus. Viral gene delivery vectors have higher transfection efficiency over multiple targeted cell ranges. However, the inflammatory response and cryogenic affects are among the main disadvantages. In this context, certain problems related to the gene delivery system could be overcome by designing a newer gene delivery system with the aid of polymers. Numerous cationic polymers such as poly (D,L-lactide-co-glycolide; PLGA), i-poly (ethylene-imine; PEI), poly (L-lysine), and tetra amino-fullerene, etc. have been extensively used for the delivery of drug as well as genes. Moreover, natural polymers, mainly chitosan, hyaluronic acid, gelatin, and collagen, have been commonly used in various biomedical application including gene delivery. These polymeric composites are advantageous because of their targeting ability, high stability, and efficient uptake by endocytosis [21–25]. To enhance transfection efficiency, the cationic polymers interact with DNA to form complexes that are used to deliver genes in vitro and in vivo. Moreover, higher biocompatibility, low immunogenicity, and minimal toxicity make a suitable alternative candidate for viral or lipid-mediated transfection [26].

3.2

Chitosan

Chitosan is a polysaccharide cationic polymer synthesized from partial deacetylation of chitin of insect shells that consist of repeating units of glucosamine and N-acetylglucosamine. At acidic pH, it is positively charged and soluble, while at neutral pH, it is insoluble. Chitosan’s pKa value is about 6.5. The characteristics of chitosan such as hydrophobicity, solubility, and interaction ability with polyanions might be changed by changing the number of the amine group. Furthermore, the solubility of chitosan (neutral and basic) might be enhanced by surface functionalization to produce trimethyl chitosan derivatives. Chitosan has higher solubility and higher degradation due to its low molecular weight and lower deacetylation frequency. Chitosan is extensively used in the gene delivery system due to its several advantages such as being biodegradable, biocompatible, and inexpensive, having low immunogenicity, high positive charge, and a nontoxic nature, and easily forming polyelectrolyte

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

complexes with DNA [27]. The positively charged chitosan easily binds with the cellular membranes, increases the permeability of cells, and decreases the transepithelial electrical resistance of the cells. Moreover, trans-and-para cellular permeability increases with increasing the dose, molecular weight, degree of deacetylation, and the pH of the chitosan solution, thereby making it efficient for use as a carrier for gene delivery [21, 28–34]. However, there is still concern about low water solubility, changes in surface charges at physiological pH, and relatively poor targeting capability. Therefore, there is a need to develop a newer/modified chitosan-based gene delivery system that resolves such issues and makes an effective gene delivery system. In this context, researchers are constantly working on the design criteria and gene delivery specifications using the chitosan-based gene delivery system [27, 33, 35].

3.2.1

Derivatives of chitosan

For specific improved applications, researchers always try a change in the structural properties of chitosan, which can easily be achieved by chemical modification. Fortunately, due to its hydroxyl, acetamido, and amine functional groups, chitosan is suitable for chemical modification. Chemical modifications will therefore not alter the basic chitosan skeleton and retain the original physicochemical and biochemical properties while introducing new or enhanced properties [36]. Numerous derivatives of chitosan are made such as triethyl, trimethyl, and dimethyl ethyl chitosan by alkylation of the amino group of chitosan. These derivatives are derived with the help of an appropriate aldehyde in the presence of a reducing agent and then applied in various applications. Moreover, by alteration of the hydroxyl functional group of chitosan, thiolated, hydrophobic, quaternized, and chemically grafted chitosan derivatives have been synthesized with newer properties to chitosan [37–39]. Therefore, the derivatives of chitosan have improved numerous properties including increased drug permeation efficiency, protection of acid-sensitive biomolecules or drugs, the enhanced release of drugs, easy solubility at neutral pH, and achieved targets [40,41].

3.2.2

Chitosan-based frameworks for gene delivery

The superlative polymeric or chitosan-based delivery system requires some characteristics such as the formation of stable complexes with genetic materials, targeting the preferred cells, protecting genes against nuclease degradation, and facilitating cellular transportation. Typically, after the release of genes in the cytoplasm, the chitosanbased delivery mechanism escapes from endosomes and lysosomes, thereby interacting with the target elements with insignificant toxicity [35,42]. Moreover, due to the versatile characteristics of chitosan, various factors must be considered while choosing chitosan as effective carriers for gene delivery, mainly molecular weight, deacetylation degree, and chitosan derivatives [35,43]. Usually, two types of polymeric nanoparticles (NPs)-based delivery systems are utilized: nucleic acid (DNA or RNA) entrapping and surface binding systems.

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The chitosan polymer and anionic DNA or RNA interaction might be utilized by the surface binding system. The entrapping system (DNA or RNA) is protected by nuclease degradation. Moreover, low immunogenicity, exceptional biocompatibility, and insignificant toxicity make their use safer in numerous end applications; mainly drug delivery, plants, tissue engineering, and gene delivery. Hence, DNA or RNAchitosan NPs or nanosphere (NS) might be efficiently used for the delivery of genes. Recently, numerous chitosan-based gene delivery systems have been used, mainly self-assembling polymeric, chitosan/DNA complexes, DNA-chitosan nanospheres, and siRNA-chitosan NPs. In the spontaneous development of nanomaterials or chitosan-based gene delivery systems, the positively charged chitosan interacts with the negatively charged genetic materials. After the cellular uptake by endocytosis, genetic materials escape from endosomes and release siRNA and miRNA (Fig. 3.1). Usually, there are two types of nanocapsules based on the nature of encapsulated liquid: with and without chitosan. The first, PLGA, makes it possible to entrap siRNA within the polymeric matrix, and the second, PLGA, is disbanded in the organic phase, and the aqueous solution of PVA and chitosan contains siRNA. Fig. 3.2 shows a schematic representation of the formation of nanocapsules with and without chitosan.

3.2.3

Chitosan-DNA NPs for gene delivery

The cationic polymers or polysaccharides, mainly chitosan, are extensively used in biomedical and pharmaceutical industries due to their capacity to regulate the release of drugs, DNA, proteins, vaccines, and genes. In addition, chitosan has also been studied for the delivery of a gene as a nonviral DNA carrier. Numerous studies focus on the chitosan-DNA-based composite for the delivery of genes. For example, Erbacher et al. synthesized the chitosan-vector/DNA complex for the delivery of genes. The data suggests that the prepared chitosan-vector/DNA complex was stable, and easily transfected HeLa cells. The expression of genes increased with increasing the time. Moreover, chitosan was considered more proficient as compared to the PEI [44]. Roy et al. synthesized chitosan-DNA NPs containing the Lac Z gene to produce chitosan-DNA-Lac Z-based NPS for the delivery of peanut allergen gene (pCMVArah2). The prepared chitosan-DNA-Arah2-based NPs were orally administered into mice to produce IgA and IgG2a. The data suggested that the chitosan-DNA-Arah2 based NPs-based delivery system effectively modulate anaphylactic response that shows prophylactic utility in food allergy [45]. Lu et al. synthesized chitosan-DNA (CS-DNA), PEI-chitosan-DNA (CP-DNA), PEI-DNA, and Lipofectamine, which acted as nonviral genes for osteoarthritis gene therapy. The data suggested that CP-DNA NPs show high transfection efficiency with low cytotoxicity against both chondrocytes and synoviocytes [46]. Fig. 3.3 shows microscopic images of chondrocytes, synoviocytes transfected with CP/DNA, plasmid-DNA, CS-DNA, PEI-DNA, and Lipofectamine. The transfection efficiency of CP-DNA was equivalent to Lipofectamine and significantly greater than CS-DNA, PEI-DNA, and p-DNA, as shown in the figure. Cifani et al. synthesized a chitosan-plasmid DNA-based complex for effective gene delivery. The prepared chitosan-plasmid DNA-based complex was incorporated with

Endosome

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Fig. 3.1 Synthesis of the chitosan-based gene delivery system and its mechanism. From Cao Y, Tan YF, Wong YS, Liew MWJ, Venkatraman S. Recent advances in chitosan-based carriers for gene delivery. Mar Drugs 2019; 17(6):381 with permission. Copyright @ 2019 Cao et al. Creative Commons Attribution (CC BY) license.

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Fig. 3.2 A schematic representation of the formation of nano capsules with and without chitosan. From Vauthier C, Zandanel C, Ramon AL. Chitosan-based nanoparticles for in vivo delivery of interfering agents including siRNA. Curr Opin Colloid Interface Sci 2013;18(5):406–18 with permission.

Fig. 3.3 Microscopic images of chondrocytes, synoviocytes transfected with CP/DNA, plasmid-DNA, CS-DNA, PEI-DNA, and Lipofectamine; (A) chondrocytes, and (B) synoviocytes. From Lu H, Dai Y, Lv L, Zhao H. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis. PLOS One 2014;9(1):e84703 with permission. Copyright @ 2014 Lu et al. Creative Commons Attribution License.

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GFP reporter and tested against HEK293 cells. The data showed that the complex based on chitosan-plasmid DNA had a high transfection efficiency of around 150% compared to the HEK293 cells. Moreover, the highest transfection efficiency was observed at 5 days of exposure [47]. These studies suggested that the chitosan is an extraordinary polysaccharide that efficiently delivered the genes to treat numerous diseases because of its positively charged surface, high biocompatibility against numerous cell lines, and biodegradability. However, poor water solubility, poor targeting ability, and changes in surface charge at physiological pH remain concerns; therefore, chitosan as a gene delivery vehicle is still unacceptable in clinical practice.

3.2.4

Chitosan-RNA NPs for gene delivery

The chitosan-RNA-based complex, or NPs, polyplexes, or nanocapsules have been used for the delivery of various genes to treat numerous diseases [48]. For example, Howard et al. synthesized chitosan-siRNA-based NPs for gene therapy and tested against NIH 3T3 and H1299 cells. The data suggested that the prepared chitosansiRNA shows RNA interference in both cell lines [49]. Katas et al. synthesized chitosan NPs by two processes, namely simple complexation and ion gelation. The prepared chitosan NPs were incorporated with siRNA for the delivery of siRNA to treat various diseases and tested against CHO K1 and HEK 293. The data suggested that the efficiency of the transfection depends on the siRNA interaction and the ion gelation process and has better performance as compared to the simple complex [50]. Dehousse et al. synthesized chitosan-siRNA and trimethyl-chitosan (TC)-siRNA for the delivery of siRNA and tested against HEK293 cell lines. The data suggested that the TC-siRNA NPs were stable with high transfection efficiency. Moreover, both NPs are favorable for siRNA delivery [51]. Santos-Carballal et al. synthesized chitosan-miRNA and tested against MCF-7 cell lines. The data suggests that the chitosan-miRNA effectively downregulates miRNA expression in MCF-7 cell lines. Moreover, no cellular toxic effect was observed against MCF-7 cell lines [52]. For miRNA delivery to treat breast cancer, Kaban et al. synthesized chitosan-miR140 and chitosan-miR200c nanoplexes. The data suggested that both the chitosan-miR140 and chitosan-miR200c nanoplexes efficiently transfect in breast cancer cell lines [53]. Chitosan-miR200c nanoplexes were synthesized by another researcher in the same community and tested against different stages of breast cancer cell lines. The data suggests that the chitosan-miR200c nanoplexes downregulated various breast cancer cell lines. Moreover, nanoplexes decrease the angiogenesis, metastasis in cells, and increase the level of apoptosis [54]. These studies suggest that chitosans are highly desirable polymers that deliver nucleic acid and stimulate a transgenic response, and consequently upregulation of expression (like plasmid DNA and mRNA) or downregulation of expression (like siRNA and miRNA). The upregulation/downregulation of expression explains multistep processes such as nucleic acid degradation, stability at physiological condition, release from endolysosomes (proton-sponge effects), cellular internalization, and siRNA unpacking and enabling RNA interference silencing complex. However, it is still unclear which types of chitosan are suitable to deliver numerous genes, such

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as plasmid-DNA, siRNA, and miRNA, in terms of the shape and size of the complexes (nanoparticles, nanocapsules, polyplexes, and nanocomplexes). Another aspect regarding the chitosan-based gene delivery system—comparatively low transfection efficiency against various cell lines—is one of the greatest challenges. In this framework, researchers continue focusing on the different formulation, incorporation of various surface functional groups, size, and shape-based gene delivery to improve transfection efficiency.

3.3

Strategies for improving gene transfer efficiency

There is growing interest in chitosan-based gene delivery, due to high biocompatibility as well as constructive physicochemical properties, which make chitosan an extraordinary candidate for the delivery of genes. However, less transfection efficacy and poor cell specificity exhibit a limitation on their use in clinical applications. In this context, numerous strategies such as changing the molecular weight, deacetylation degree, and surface functionalization have been used to enhance transfection efficiency, as well as cell specificity. For example, K€oping-H€ogga˚rd et al. synthesized chitosan-plasmid DNA and PEI-plasmid DNA polyplexes for nonviral gene delivery. The prepared chitosan-plasmid DNA and PEI-plasmid DNA polyplexes were tested in vitro and in vivo (intratracheal administration to mice). The data suggested that the chitosan-plasmid DNA formed more stable polyplexes due to the primary amino group. The prepared chitosan-plasmid DNA polyplexes were effectively an expression of the gene in epithelial cells. Moreover, rapid gene expression was observed in PEI-plasmid DNA polyplexes. The chitosan-plasmid DNA polyplexes have comparable gene expression with other cationic lipids [55]. Lavertu et al. synthesized chitosan-DNA NPs with different molecular weight (MW) of chitosan (10, 40, 80, and 150 kDa), degree of deacetylation (DD) (72%, 80%, 92%, and 98%), depolymerized different nitrous acid to phosphate (N/P) ratio (5:1, and 10:1) and tested against HEK 293 cells (with different media pH 6.5 and 7.1). The data suggested that the two types of formulation of chitosan-DNA NPs, (1) DA-MW-N/ P (92-10-5), and (2) DA-MW-N/P (80-10-10) show higher transfection efficiency than that of the positive control. The MW and DA is one of the determining factors to enhance the expression of the gene [56]. Lee et al. synthesized chitosan-plasmid DNA complex and thiolated chitosanplasmid DNA complex for the improved and persistent delivery of genes. Initially, thiolated chitosan was synthesized with thioglycolic acid and then plasmid DNA incorporated fluorescent protein (GFP) to produce a nanocomplex. The data suggested that the prepared thiolated chitosan-plasmid DNA complex was stable and protect against DNAse I digestion. The thiolated chitosan-plasmid DNA complex easily transfected HEK293, MDCK, and Hep-2 cell lines. The GFP expression was higher in all cell lines compared with that of the chitosan-plasmid DNA complex. In addition, gene expression increased with an increase in exposure time. Due to the cross-linking of chitosan with the thiol group, the higher gene transfer and sustained GFP expression suggested the potential capacity for gene delivery [57]. Luo et al. synthesized

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guanidinylated chitosan-DNA for siRNA delivery to the lung. The data suggests that the guanidinylation of chitosan decreases the cellular toxicity as well as facilitating the internalization of siRNA, thereby increasing gene silencing ability compared to the pristine chitosan. The in vitro results were comparable to in vivo transgenic mice [58]. Oliveira et al. synthesized chitosan incorporated HA-DNA (chitosan-HA-DNA) polyplexes and transfection efficiency tested against HEK293 and epithelial cells. The data suggested that the prepared chitosan-HA-DNA polyplexes have four- to sixfold high transfection efficiency compared with that of chitosan only [59]. Yue et al. synthesized PEI grafted chitosan-BMP2 gene (PEI-chitosan-BMP2) for the delivery of BMP2 gene to mesenchymal stem cells. The data advocates that around 17.2% transfection efficiency was observed with around 80% cell viability. The expression also increases the BMP2 gene to improve differentiation of osteogenic cells [60]. Using Alizarin red, Oil-Red-O, and Toluidine blue, respectively, osteogenic, adipogenic, and chondrogenic differentiations were observed (Fig. 3.4). The pIRES2-ZsGreen1-hBMP2 plasmid was administered to bone marrow stem cells using PEI-chitosan, PEI, and chitosan. The GFP expression indicates the BMP2 gene

Fig. 3.4 PEI-chitosan-BMP2 based gene delivery system. (A) Bone marrow stem cells differentiate by Alizarin red, Oil-Red-O, Toluidine blue; (B) ZsGreen1 GFP expression in bone marrow stem cells quantified by flow cytometry. From Yue J, Wu J, Liu D, Zhao X, Lu WW. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector. Nanoscale Res Lett 2015;10(1):203 with permission. Copyright @ 2015 Yue et al. Creative Commons Attribution License.

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expression. As observed from Fig. 3.4, chitosan shows the lowest expression of BMP2 gene, whereas PEI shows the higher expression of BMP2 gene in comparison to chitosan. Upon incorporation of both chitosan and PEI (PEI-chitosan) shows the highest gene expression. The extraordinary gene expression of PEI-chitosan was observed due to the proton-sponge effects. Another research focused on the synthesis of PEI-chitosan-plasmid-DNA (PCPD) polyplexes for an effective gene delivery system. The data indicates that the PCPD polyplexes efficiently transfer genes in Hep2 cells with levels approximately 44 and 38 times higher than chitosan and PEI, respectively. Moreover, the cellular toxicity of the PCPD polyplexes is also lower than chitosan and PEI [61]. Wang et al. synthesized astaxanthin-chitosan-DNA (ACD) nanoparticles for the delivery of astaxanthin. The data showed that the prepared ACD nanoparticles enhance cellular uptake as well as improving antioxidant activity [62]. Xu et al. synthesized an arginine-chitosan-plasmid DNA (ACD)-based carrier as a gene delivery system. The ACD was incorporated with the BMP2 gene and tested against osteoblastic progenitor cells. The data suggested that the ACD-BMP2 efficiently transfect osteoblastic progenitor cells and promote the differentiation of osteogenic cells. Therefore, ACD-BMP2 is efficiently able to promote gene transfection ability within a controlled manner [63]. Another study focuses on the synthesis of glycosylated chitosanpolyethylene glycol (GCPEG) nanoparticles for gene delivery. The GCPEG was incorporated with plasmid DNA (GCPEG-DNA) and tested against L929 and Bel7402 cells. The data shows that the prepared GCPEG-DNA efficiently transfect with no cellular toxic effects on L929 cells. However, some inhibitory effects were also observed on Bel-7402 cells [64]. Raik et al. synthesized different materials, methyl glycol-chitosan (MC), and diethylaminoethyl-chitosan (DEAE-C) for the delivery of genes in Calu-3 cells. The data suggested that the DNA transfection efficiency was higher in MC compared with that of the DEAE-C. Moreover, both the delivery systems show insignificant toxicity [65]. Mohammadzadeh et al. synthesized chitosan-folic acid-plasmid DNAbased nanoparticles with different ranges of MW (50–190 and 310–375), and DA (75%–85%) using ions gelation process. The data advocates that the encapsulation of plasmid-DNA increased with increasing the MW. Moreover, high MW-based chitosan-folic acid-plasmid DNA-based nanoparticles show slow release, whereas low MW have faster release of plasmid-DNA [66]. Another study focused on the surface functionalization of chitosan using polyethylene glycol (PEG) to produce PEG-chitosan-siRNA nanoplex for gene delivery. The data suggested that a small concentration of PEGylation has no effect on the stability of a nanoplex, whereas a higher concentration of PEGylation reduces size, the surface charge of nanoplexes, and cellular uptake that knocks down siRNA efficiency [67]. Jadidi-Niaragh et al. synthesized chitosan-lactate-siRNA-based NPs to suppress the expression of CD73 on 4T1 tumor cells. The data recommends that the chitosanlactate-siRNA-based NPs efficiently suppress CD73, and recommended chitosanlactate-siRNA-based NPs for use as an effective therapeutic tool for cancer therapy [68]. Wang et al. synthesized chitosan NPs and crosslinked with tripolyphosphate (TPP-C) for the delivery of siRNA. The prepared TPP-C NPs efficiently delivered

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siRNA to the cells. Moreover, TPP-C NPs showed high biocompatibility and insignificant cellular toxicity [69]. Veilleux et al. synthesized a chitosan-siRNA polyplex for treating various diseases using gene silencing. The physicochemical properties of the chitosan-siRNA polyplex were determined by various buffers. The data suggested that the prepared chitosan-siRNA polyplex is nontoxic and has gene silencing ability, thereby effectively treating various diseases [70]. Tekie et al. synthesized thiolate-dextran-chitosan-miR-145 NPs for the treatment of cancer. The data advocates that the dextran-chitosan-miR-145 NPs were efficiently expressed in cancer cell lines. The over-expression of miR-145 diminished cancer migration, invasion, and adhesion of cells [71]. In general, changing the functional group, size, and shape of the various chitosanbased gene delivery systems has the potential to enhance the upregulation or downregulation of the expression of genes. Moreover, surface functionalization also aids advantages that reduce cellular toxicity against different cell lines. Therefore, the modified chitosan-based gene delivery system might be an alternative and safer tool for the treating of various diseases.

3.4

Challenges and limitations associated with chitosan-based nanocomposites for gene delivery

Viral vectors have been demonstrated very successfully in gene delivery systems, but they have some associated safety risks and unforeseen adverse effects. The effectiveness of a gene delivery system depends mainly on the delivery vectors and their capability to protect the genetic material from degradation with the sustained release of a sufficient quantity of nucleotides for the target cells to achieve a therapeutic effect [72]. For the advancement of gene delivery systems and their effectiveness, physical and biological barriers must be overcome with consideration of the physicochemical properties of the vector [73]. In gene delivery systems, the established serum nucleases lead to their rapid degradation after the release of the genetic material. Thus, the therapeutic genes within the endolysosomes must withstand enzymatic degradation. The introduction of chitosan as a gene delivery vector should provide vector-cargo complexes with stability, protect them from extracellular and intracellular degradation, deliver the cargo into specific cells, and allow complexes to be dissociated to facilitate transfection [74]. The degree of deacetylation (DD), the length of the polymer, and the ratio of chitosan nitrogen per gene phosphate (N/P ratio) plays an important role in the physicochemical and biological efficiency of the polymer [75]. Since they meet most of the required characteristics, chitosan-based nanocomposites have high therapeutic potential. However, some drawbacks such as low solubility at physiological pH, aggregation, poor complex stability, and untimely release within the cytoplasm exist without structural and chemical modification [76,77]. To address these limitations, many methods have been used, such as the alteration of the structure of chitosan, conjugation, grafting, copolymerization, or encapsulation into nanoparticles [74]. Chemical alteration, for instance, has been used to increase the solubility of chitosan. However, excessive modification can decrease

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the positive charge density of chitosan and affect its ability to bind to genes. The durability of nanocomposites can also increase chemical modifications to siRNA, but it also makes siRNA ineffective. Another method to stabilize the chitosan nanocomposites is the addition of negatively charged components. The hydrophobic modification to chitosan also extends many advantages, such as improved stability, easy binding to cells, better cellular absorption and defense from extracellular and intracellular degradation, and promoting DNA dissociation within cells from chitosan.

3.5

Conclusion and future assessments

The chitosan-based gene delivery system has several characteristics such as insignificant cellular toxicity, high gene delivery ability, targeting of particular cells, protection of DNA/RNA or gene cargo, slow release of the DNA/RNA, higher transfection ability, and easy escape from lysosomes. However, relatively poor solubility, changes of surface charge at physiological pH, poor stability of complex (nanoplexes, polyplexes, nanocapsules, and nanoparticles), and sometimes inability to reach targeted cells remain concerns. To overcome such drawbacks, various strategies such as chemical modification (like thiol group, and PEGylation) and different formulation of chitosan have been used to achieve high therapeutic efficacy. The chemical modifications of chitosan enhance the stability of chitosan-DNA/RNA complexes (nanoplexes, polyplexes, nanocapsules, and nanoparticles). Moreover, the chemical modification also improves the biocompatibility of the chitosan-DNA/RNA complexes against different cell lines. However, higher chemical modification might reduce the gene delivery efficiency. With the help of several strategies incorporated with chitosan-based gene delivery, the various limitations associated with chitosanbased gene delivery might be overcome. Moreover, higher encapsulation of DNA/ RNA and its controlled release has proved advantageous over the existing gene delivery system. Extensive research on new strategies to improved chitosan-based gene delivery is still needed to improve transfection efficiency.

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[62] Wang Q, Zhao Y, Guan L, Zhang Y, Dang Q, Dong P, et al. Preparation of astaxanthinloaded DNA/chitosan nanoparticles for improved cellular uptake and antioxidation capability. Food Chem 2017;227:9–15. [63] Xu X, Qiu S, Zhang Y, Yin J, Min S. PELA microspheres with encapsulated arginine– chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation. Artif Cells Nanomed Biotechnol 2017;45(2):330–9. [64] Jin J, Fu W, Liao M, Han B, Chang J, Yang Y. Construction and characterization of Galchitosan graft methoxy poly (ethylene glycol) (Gal-CS-mPEG) nanoparticles as efficient gene carrier. J Ocean Univ China 2017;16(5):873–81. [65] Raik VS, Andranovitsˇ S, Petrova AV, Xu Y, Lam KJ, Morris AG, et al. Comparative study of diethylaminoethyl-chitosan and methylglycol-chitosan as potential non-viral vectors for gene therapy. Polymers 2018;10(4). [66] Mohammadzadeh R, Shahim P, Akbari A. Formulation of a pH-sensitive cancer celltargeted gene delivery system based on folate-chitosan conjugated nanoparticles. Biotechnol Appl Biochem 2021;68(1):114–21. https://doi.org/10.1002/bab.1900. [67] Gut¸oaia A, Schuster L, Margutti S, Laufer S, Schlosshauer B, Krastev R, et al. Fine-tuned PEGylation of chitosan to maintain optimal siRNA-nanoplex bioactivity. Carbohydr Polym 2016;143:25–34. [68] Jadidi-Niaragh F, Atyabi F, Rastegari A, Mollarazi E, Kiani M, Razavi A, et al. Downregulation of CD73 in 4T1 breast cancer cells through siRNA-loaded chitosan-lactate nanoparticles. Tumor Biol 2016;37(6):8403–12. [69] Wang L, Zhang W, Zhou Q, Liu S, Xu W, Sun T, et al. Establishing gene delivery systems based on small-sized chitosan nanoparticles. J Ocean Univ China 2018;17(5):1253–60. [70] Veilleux D, Gopalakrishna Panicker RK, Chevrier A, Biniecki K, Lavertu M, Buschmann MD. Lyophilisation and concentration of chitosan/siRNA polyplexes: influence of buffer composition, oligonucleotide sequence, and hyaluronic acid coating. J Colloid Interface Sci 2018;512:335–45. [71] Tekie FSM, Soleimani M, Zakerian A, Dinarvand M, Amini M, Dinarvand R, et al. Glutathione responsive chitosan-thiolated dextran conjugated miR-145 nanoparticles targeted with AS1411 aptamer for cancer treatment. Carbohydr Polym 2018;201:131–40. [72] Ibraheem D, Elaissari A, Fessi H. Gene therapy and DNA delivery systems. Int J Pharm 2014;459:70–83. [73] Naldini L. Gene therapy returns to centre stage. Nature 2015;526:351–60. [74] Whinnery C, Kirsch WM. Recent advances in chitosan-based gene carrier application and design. JSM Biochem Mol Biol 2016;3(2):1018. [75] Buschmann MD, et al. Chitosans for delivery of nucleic acids. Adv Drug Deliv Rev 2013;65(9):1234–70. https://doi.org/10.1016/j.addr.2013.07.005. [76] Ji J, et al. Chemical modifications of chitosan and its applications. Polym Plast Technol Eng 2014;53:1494–505. [77] Jiang HL, et al. Chemical modification of chitosan for efficient gene therapy. Adv Food Nutr Res 2014;73:83–101. https://doi.org/10.1016/B978-0-12-800268-1.00006-8.

Alginate-based hydrogels for tissue engineering

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Abhishek Kumara,b, Anant Narayan Bhattb, Laishram Rajendra Kumar Singhc, Zoheb Karimd, and Mohd Saquib Ansaria a Department of Biomedical Sciences, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, New Delhi, India, bInstitute of Nuclear Medicine and Allied Sciences, Defence Research and Development Organization, New Delhi, India, cDr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India, dMoRe Research AB, Sweden

4.1

Introduction

The scaffolds of biocompatible biopolymers mimic the extracellular matrix without being toxic to the body and helps the damaged tissue to regenerate by providing suitable microenvironment to the cells, which enhances cell-attachment, adhesion, and migration. This improves the cell-cell cross-talk, which in turn promotes processes like cell differentiation and reepithelialization. This property of biomaterials has been utilized extensively in the field of tissue repair and engineering as this is a growing field, and many advancements are expected to generate tissues and organs quickly that are better adapted to their biological functions. Various naturally occurring biomaterials have been suggested as a scaffolding material, as they have the biocompatibility, degradability, desired mechanical strength, and biological activity in vivo. Alginate is approved by the United States Food and Drugs Administration; it is a highly abundant naturally occurring biopolymer and one of the most preferred scaffolding materials as it is nontoxic with no evidence of immunogenicity; and it shows excellent biocompatibility, chemical versatility, and biological activity [1–3]. Alginates have long-term in vivo stability and degrade slowly with efficient renal clearance [4]. Modified alginate hydrogels have immense potential for application in tissue repair and regeneration, and these modified alginate hydrogels are improving expeditiously to better imitate the physical and chemical environment of tissues.

4.2

Structure and composition of alginate

Alginates are salts of algin, which is a polysaccharide derived from the cell wall of brown algae. Ascophyllum nodosum, Macrocystis pyrifera, Laminaria hyperborean, Laminaria digitata, Laminaria japonica, and Saccharina japonica are some of the most common algae utilized for extraction of alginate. Ascophyllum nodosum contains 22%–30%, and Laminaria digitata has 25%–44% alginate of their total biomass [1, 5]. Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00002-5 Copyright © 2021 Elsevier Inc. All rights reserved.

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Alginate is extracted by initially treating the algae with aqueous NaOH [6]; furthermore, the extracts are filtered, followed by the addition of calcium chloride to precipitate the alginate. A final treatment of dilute HCl leads to the formation of alginic acid. The resulting compound is further purified, which ultimately results in a hydrophilic powder known as sodium alginate [7]. The molecular weight of seaweed-derived, commercially produced alginate may vary from 32 KDa to 400 KDa. Alginates are anionic, and their structure consists of two monomers: α-L-guluronic acid (G) joined together by α(1,4)glycosidic bonds and β-D mannuronic acid (M), which are connected by β(1,4) glycosidic bonds [19] (Fig. 4.1). The biopolymer is formed by blocks of M-M, G-G, and G-M polymeric sequences. Divalent cations like Ca2+, Ba2+, Zn2+, or Mg2+ associate with the G monomer block with very high affinity and lead to the formation of interconnected ionic bridges causing cross-linking between multiple alginate chains (Egg-box model), which helps alginate to attain a hydrogel form. Commercially available alginates are usually characterized by their G:M ratio, where high G content corresponds to strong cross-links, resulting in a highly viscous hydrogel. In biomedical applications, alginate with G content >50% is preferred as it does not elicit an immune response with no reported induction of cytokine production, nor does it cause any reactive oxygen species (ROS) generation [2, 3, 20, 21].

4.3

Hydrogels

Hydrogels are formed of a network of water-insoluble polymeric chains. They are a type of colloidal gel having water as its dispersion medium. The gels can contain over 99% water of its total volume; regardless of the high water content they also retain their stiffness. The high water content and the desired stiffness renders the gel an ECM-like property, which makes them suitable to provide mechanical support for an injured tissue, as well as serving as a platform for delivering biomolecules to the injured tissue for its repair and regeneration (Fig. 4.2).

4.3.1 Alginate hydrogel biological interaction Alginate is Food and Drug Administration (FDA) certified and is considered safe for human administration. It has high in vivo compatibility, with no evident toxicity, involves no thrombogenic complications, and does not generate any immune response. Alginate hydrogels have a dense, negatively charged surface and high water content that makes protein adsorption and cell attachment difficult. The cell surface receptors are also unable to recognize the molecule [22], making it safe for multiple medical implementations such as a material for dental impressions, wound management bandages, thickener in pharmaceuticals, tablet disintegrator, and dispersion stabilizer [2]. In normal physiological conditions, the alginate chains are stable, but the crosslinked hydrogels disintegrate with time mostly because of calcium ions being

Fig. 4.1 (A) Structure of monomers of alginate. (B) Structural representation of different blocks of monomers in a single alginate polypeptide.

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Fig. 4.2 Schematic representation of peptide-loaded modified alginate hydrogels acting as extracellular matrix providing adhesion and attachment to cell for proliferation and differentiation.

exchanged by much readily available sodium ions present in higher concentration in the physiological environment. This should be considered when designing an alginate hydrogel biomaterial for in vivo use as only those alginate chains that have a molecular weight equal to or less than 50 kDa will be efficiently cleared by the renal system of the body [4].

4.3.2 Alginate hydrogel cell-crosslinking Alginate exhibits good biocompatibility, but for it to be able to provide anchorage to cells, it should provide specific receptor-ligand interactions. Alginate being completely formed of inert monomers, it lacks the necessary ligands required to provide anchorage to cells. In case, alginate hydrogels are used in combination with cells; the hydrogels are modified to make them compatible for interaction with the cells. An increased cell-hydrogel matrix interaction is observed upon covalently conjugating the hydrogel with heparin-binding peptides and related peptide sequences. Multiple techniques have been extensively implemented to cross-link such alginate-based hydrogels with required adhesion ligands to make the cell-surface receptors latch onto these ligands. These strategies involve immobilizing arginine, glycine, and aspartic acid (RGD peptide) onto alginate using aqueous carbodiimide chemistry [23–25]. These modifications provide excellent cellular adaptability toward the hydrogel matrix and have the ability to initiate biological interactions between the peptide-coupled hydrogel and cells.

4.4

Alginate hydrogels in tissue engineering

Sodium alginate hydrogels have a lot of potential in cell and tissue engineering applications and have been studied extensively in the field of regenerative medicine. The modulation of the physico-chemical characteristics of these alginate-based hydrogels is usually achieved by selecting the appropriate divalent cation employed in the gel

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cross-linking. The use of calcium ion for cross-linking sodium alginate hydrogels have shown to significantly increase proliferation and differentiation of osteoblasts. Using barium and strontium as a divalent cation in place of calcium leads to more rigid hydrogels [26–28] (Table 4.1).

4.4.1 Skin Skin is the most important organ involved in the maintenance of homeostasis and protects the internal organs from the external environment. In the process of healing of an injured site, the epithelial cells migrate inwards for the repairment of the wound. It is a highly regulated physiological process of tissue restoration involving coordinated action of endothelial cells, keratinocytes, fibroblasts, and immune cells [29, 30]. The repair process includes covering up of wound area by the production of the extracellular matrix, which assists healing of a wound by providing conditions for cell proliferation and blood vessel formation to deliver nutrients at the wound site [31]. It supports the inward movement of keratinocytes and helps in recruiting fibroblasts at the wound area along with providing the appropriate microenvironment for their differentiation into myofibroblasts, ultimately causing wound closure (Fig. 4.3). Drug incorporated alginate hydrogels can be ideally modified to act as an extracellular matrix and accelerate the healing process. Wound healing properties of sodium alginate films have been well established with findings that suggest rapid and efficient healing with faster wound closure [29].

4.4.2 Cartilage The cartilage is a rubber-like tissue that covers the joints at the end of long bones. Cartilage provides lubrication and has the property to bend and resist stretching, assisting in load-bearing activities and exercises along with helping in various Table 4.1 Different protein and peptide-based alginate hydrogels for tissue engineering. Alginate hydrogel uses Skin regeneration Cartilage repair

Bone restructuring and repair Cardiac tissue regeneration

Type of modifications

References

Basic fibroblast growth factor loaded alginate, alkaline poly(ethylene glycol)-based hydrogels TGFβ1/BMP-4-affinity-bound bilayered alginate hydrogel, chondrocyte-seeded alginate hydrogels, dental-derived MSCs incorporated hydrogels Calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste, TGFβ3/BMP-2-loaded alginate hydrogel Fullerenol-alginate hydrogel, IGF-1, and HGF alginate hydrogels

[8, 9] [10–12]

[13–15]

[16–18]

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Fig. 4.3 Schematic representation of wound healing by the application of alginate hydrogels on the site of injury showing the recruitment of keratinocytes and fibroblasts followed by fibroblast differentiation into myofibroblasts, ultimately sealing the wound.

physical movements. Cartilage’s extracellular matrix is made up of a collagen mesh containing slowly dividing chondrocytes and negatively charged proteoglycans [32]. Any injury and trauma to the cartilage lead to defects, which does not heal properly. The insufficient cartilage regeneration causes severe pain, rendering the person disable, ultimately requiring a total joint replacement to treat the condition [10]. Alginate-polyacrylamide-based hybrid hydrogel with silica nanoparticles has shown immense potential as a substitute for cartilage [33]. This alginate-polyacrylamidesilica hydrogel consists of short chains of alginate that dissipate the strain energy via ionic cross-linking, providing the structure healing properties by reforming the ionic cross-links. The long polyacrylamide chains offer structural integrity, and the silica embedded in the hydrogel encourages cell proliferation. RGD-enriched alginate hydrogels when mixed with hyaluronic acid shows cartilage healing potential by specifically increasing chondrogenesis of the encapsulated chondrocytes [34–36].

4.4.3 Bone Bone is a rigid organ constituted of nanocomposites of organic collagen nanofibers and inorganic materials such as hydroxyapatite (HAP). Alginate has a great scaffold forming ability, which deems this biomaterial a good choice for engineering and restructuring bone tissue. The requirements for a material to be bone healing are much complex. The material needs to be biocompatible, along with the support of cell adhesion, migration, and proliferation. The biomaterial structure needs to be rigid and porous, with the porosity of more than 100 μm [27]. The porous scaffolding material allows the movement of osteogenic cells into the scaffolds, providing ample

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Fig. 4.4 Diagrammatic representation of bone tissue repair with alginate hydrogel scaffold showing assisted movement and attachment of osteoprogenitor cells into the porous scaffold and their simultaneous differentiation into osteoblasts followed by mineral deposition.

conditions for their division and differentiation into cells that enhance mineral deposition for bone formation (Fig. 4.4). The scaffold-forming ability of alginate hydrogels has been implemented in the production of bone healing biomaterials. Calcium silicate can cross-link silk fibroin and sodium alginate, forming an interpenetrating network of the two compounds. The increased concentration of calcium silicate inside the hydrogel increases the porosity of the structure, enhancing its hydrophilicity, compression resistance, and increasing the bioactivity, which in turns promotes osteogenic differentiation [37]. Macroporous alginate foams have been employed with an evident role in the promotion of osteogenic differentiation [38].

4.4.4 Cardiac tissue In cardiac tissue, myocardial infarction causes massive damage to the ECM that supports maintenance and tissue repair. This causes detrimental effects on the systolic and diastolic functions [39, 40]. Biomaterials can be ideally modified to mimic these properties of the ECM to provide cell attachment and interaction while eliminating the issues of biocompatibility that may occur in the case of animal-derived ECM. Alginate being an inert biopolymer is an ideal candidate for the addition of RGD and HBP peptides, which in turn provides excellent cell-receptor interaction for cell adhesion, migration, and cell differentiation. In the case of cardiac ECM injury caused by a myocardial infarction (MI), RGD/HBP-modified alginate hydrogels have shown promise in supporting the cardiac tissue repair process by providing temporary tissue support and preserving cardiac function [41, 42]. Utilization of cell-embedded hydrogels on the infarcted area has a positive role in maintaining the wall stress by providing support to the cardiac wall while also providing the cells required for regeneration.

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Limitations of using alginate hydrogels

Alginate hydrogels have seen a notable level of success in tissue repair and engineering and have been employed in multiple forms and modifications for the purpose. Still, some aspects need to be looked upon before considering it to be the biomaterial of choice for the same. The major drawback to be considered before using alginates in tissue engineering is the inherent inert nature of alginate in a human physiological environment, which makes it resistant to enzymatic degradation due to lack of production of any alginate degrading enzymes in the body. Alginate hydrogels are also unable to provide the required microvasculature to the seeded cells making the resulting microenvironment nutrient-deprived, which tends to hinder the viability and proper functioning of the cells. Proper adherence of the repair cells require modifications involving incorporation of peptides onto the hydrogel polymers to support cellular adhesion and attachment further supplemented with factors to assist differentiation and proliferation of the migrating repair cells. Engineering and repairing tissues of single-cell types is relatively easy but as the complexity of the tissue increases, the difficulties associated with providing growth support to multiple cells having unique ECM composition requirement also increases, which is still a challenge that needs to be addressed.

4.6

Conclusion

Alginate has been a significant biopolymer in the biomaterial-based construction, repair, and regeneration of tissues. Modification of alginate biopolymers have been derived extensively to assist the physical structure of the tissue for maintaining tissue integrity to chemically introduce ligands onto hydrogels for increased cellular interaction and also to achieve a modified microenvironment for cells to grow, differentiate, and proliferate. Incorporation of ligands, drugs, and cells can easily be achieved in the matrix scaffolds of alginate, which renders it suitable for tissue-targeted regeneration therapy. Even though most of the tissue engineering work has reaped positive results, but much is still needed to be improved to attain better tissue compatibility and synergy. Studies need to be carried out for developing a versatile and tissuespecific alginate-based hydrogel composite for improved tissue restructuring and engineering.

References [1] Rowe RC, Sheskey P, Quinn M. Handbook of pharmaceutical excipients. Libros DigitalesPharmaceutical Press; 2009. [2] Ueno M, Oda T. Chapter six—biological activities of alginate. In: Kim S-K, editor. Advances in Food and Nutrition Research, vol. 72. Academic Press; 2014. p. 95–112. https://doi.org/10.1016/B978-0-12-800269-8.00006-3.

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[3] Andersen T, Strand BL, Formo K, Alsberg E, Christensen BE. Chapter 9, Alginates as biomaterials in tissue engineering. vol. 37. The Royal Society of Chemistry; 2012. p. 227–58. https://doi.org/10.1039/9781849732765-00227. [4] Al-Shamkhani A, Duncan R. Radioiodination of alginate via covalently-bound tyrosinamide allows monitoring of its fate in vivo. J Bioact Compat Polym 1995;10: 4–13. https://doi.org/10.1177/088391159501000102. [5] Qin Y. Alginate fibres: an overview of the production processes and applications in wound management. Polym Int 2008;57:171–80. https://doi.org/10.1002/pi.2296. [6] Clark DE, Green HC, Kelco Co. Alginic acid and process of making same. US Patent 2,036,922, April 7 1936. [7] Rinaudo M. Main properties and current applications of some polysaccharides as biomaterials. Polym Int 2008;57:397–430. https://doi.org/10.1002/pi.2378. [8] Liu Q, Huang Y, Lan Y, Zuo Q, Li C, Zhang Y, et al. Acceleration of skin regeneration in full-thickness burns by incorporation of bFGF-loaded alginate microspheres into a CMCS–PVA hydrogel. J Tissue Eng Regen Med 2017;11:1562–73. [9] Lee W, Park J, Kim K, Kim S, Park D, Chae M, et al. The biological effects of topical alginate treatment in an animal model of skin wound healing. Wound Repair Regen 2009;17:505–10. [10] Ruvinov E, Re’em TT, Witte F, Cohen S. Articular cartilage regeneration using acellular bioactive affinity-binding alginate hydrogel: a 6-month study in a mini-pig model of osteochondral defects. J Orthop Translat 2019;16:40–52. [11] Lee CSD, Gleghorn JP, Choi NW, Cabodi M, Stroock AD, Bonassar LJ. Integration of layered chondrocyte-seeded alginate hydrogel scaffolds. Biomaterials 2007;28:2987–93. [12] Moshaverinia A, Xu X, Chen C, Akiyama K, Snead ML, Shi S. Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration. Acta Biomater 2013;9:9343–50. [13] Zhao L, Weir MD, Xu HHK. An injectable calcium phosphate-alginate hydrogelumbilical cord mesenchymal stem cell paste for bone tissue engineering. Biomaterials 2010;31:6502–10. [14] Moshaverinia A, Ansari S, Chen C, Xu X, Akiyama K, Snead ML, et al. Co-encapsulation of anti-BMP2 monoclonal antibody and mesenchymal stem cells in alginate microspheres for bone tissue engineering. Biomaterials 2013;34:6572–9. [15] Gonzalez-Fernandez T, Tierney EG, Cunniffe GM, O’Brien FJ, Kelly DJ. Gene delivery of TGF-β3 and BMP2 in an MSC-laden alginate hydrogel for articular cartilage and endochondral bone tissue engineering. Tissue Eng Part A 2016;22:776–87. [16] Hao T, Li J, Yao F, Dong D, Wang Y, Yang B, et al. Injectable fullerenol/alginate hydrogel for suppression of oxidative stress damage in brown adipose-derived stem cells and cardiac repair. ACS Nano 2017;11:5474–88. [17] Ruvinov E, Leor J, Cohen S. The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction. Biomaterials 2011;32:565–78. [18] Ruvinov E, Leor J, Cohen S. The effects of controlled HGF delivery from an affinitybinding alginate biomaterial on angiogenesis and blood perfusion in a hindlimb ischemia model. Biomaterials 2010;31:4573–82. [19] Ehrenfreund-Kleinman T, Golenser J, Domb AJ. Polysaccharide scaffolds for tissue engineering. Scaffolding tissue engineering, CRC Press; 2005. p. 37–54. [20] Laurienzo P. Marine polysaccharides in pharmaceutical applications: an overview. Mar Drugs 2010;8:2435–65. https://doi.org/10.3390/md8092435.

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[21] Senni K, Pereira J, Gueniche F, Delbarre-Ladrat C, Sinquin C, Ratiskol J, et al. Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering. Mar Drugs 2011;9:1664–81. https://doi.org/10.3390/md9091664. [22] Gandhi JK, Opara EC, Brey EM. Alginate-based strategies for therapeutic vascularization. Ther Deliv 2013;4:327–41. https://doi.org/10.4155/tde.12.163. [23] Lee KY, Kong HJ, Larson RG, Mooney DJ. Hydrogel formation via cell crosslinking. Adv Mater 2003;15:1828–32. https://doi.org/10.1002/adma.200305406. [24] Lehenkari PP, Horton MA. Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. Biochem Biophys Res Commun 1999;259: 645–50. https://doi.org/10.1006/bbrc.1999.0827. [25] Koo LY, Irvine DJ, Mayes AM, Lauffenburger DA, Griffith LG. Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. J Cell Sci 2002;115:1423. [26] Saul JM, Williams DF. Hydrogels in regenerative medicine. Handbook of polymer applications in medicine and medical devices. William Andrew Publishing; 2011. p. 279–302. [27] Venkatesan J, Bhatnagar I, Manivasagan P, Kang K-H, Kim S-K. Alginate composites for bone tissue engineering: a review. Int J Biol Macromol 2015;72:269–81. [28] Wang Y. Programmable hydrogels. Biomaterials 2018;178:663–80. [29] Summa M, Russo D, Penna I, Margaroli N, Bayer IS, Bandiera T, et al. A biocompatible sodium alginate/povidone iodine film enhances wound healing. Eur J Pharm Biopharm 2018;122:17–24. [30] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature 2008;453:314–21. [31] Sinno H, Prakash S. Complements and the wound healing cascade: an updated review. Plast Surg Int 2013;2013:1–7. [32] Huang H, Tan Y, Ayers DC, Song J. Anionic and zwitterionic residues modulate stiffness of photo-cross-linked hydrogels and cellular behavior of encapsulated chondrocytes. ACS Biomater Sci Eng 2018;4:1843–51. [33] Arjmandi M, Ramezani M. Mechanical and tribological assessment of silica nanoparticlealginate-polyacrylamide nanocomposite hydrogels as a cartilage replacement. J Mech Behav Biomed Mater 2019;95:196–204. [34] Seo Y, Lee H, Lee JW, Lee KY. Hyaluronate-alginate hybrid hydrogels prepared with various linkers for chondrocyte encapsulation. Carbohydr Polym 2019;218:1–7. [35] Park H, Lee HJ, An H, Lee KY. Alginate hydrogels modified with low molecular weight hyaluronate for cartilage regeneration. Carbohydr Polym 2017;162:100–7. [36] An H, Lee JW, Lee HJ, Seo Y, Park H, Lee KY. Hyaluronate-alginate hybrid hydrogels modified with biomimetic peptides for controlling the chondrocyte phenotype. Carbohydr Polym 2018;197:422–30. [37] Zheng A, Cao L, Liu Y, Wu J, Zeng D, Hu L, et al. Biocompatible silk/calcium silicate/ sodium alginate composite scaffolds for bone tissue engineering. Carbohydr Polym 2018;199:244–55. [38] Catanzano O, Soriente A, La Gatta A, Cammarota M, Ricci G, Fasolino I, et al. Macroporous alginate foams crosslinked with strontium for bone tissue engineering. Carbohydr Polym 2018;202:72–83. [39] Akhyari P, Kamiya H, Haverich A, Karck M, Lichtenberg A. Myocardial tissue engineering: the extracellular matrix. Eur J Cardiothorac Surg 2008;34:229–41. [40] Dobaczewski M, Gonzalez-Quesada C, Frangogiannis NG. The extracellular matrix as a modulator of the inflammatory and reparative response following myocardial infarction. J Mol Cell Cardiol 2010;48:504–11.

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[41] Ruvinov E, Sapir Y, Cohen S. Cardiac tissue engineering: principles, materials, and applications. Synth Lect Tissue Eng 2012;4:1–200. [42] Ruvinov E, Cohen S. Alginate biomaterial for the treatment of myocardial infarction: progress, translational strategies, and clinical outlook: from ocean algae to patient bedside. Adv Drug Deliv Rev 2016;96:54–76.

Hyaluronic acid-based hydrogel for tissue engineering

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Maya Asyikin Mohamad Arif Faculty of Resource Science and Technology, University Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia

5.1

Introduction to tissue engineering

The past 30 years have seen increasingly rapid advances in the field of tissue engineering, an interdisciplinary field that involves biomaterials science, cell biology, cellmaterial interaction, as well as surface characterization. Tissue engineering plays an important role in the restoration, preservation of damaged tissues or whole organs, as well as in the construction of new tissues to replace the lost tissues. Restoration or creation of new tissues normally involves four components, which are progenitor or stem cells, biomaterial scaffold, signaling proteins, and bioreactors. To develop a tissue, usually, the stem cells are first isolated from the tissue of interest, normally obtained from patients’ small tissue biopsy. The isolated cells are then cultured and harvested in vitro. The isolated cells are then loaded into a three-dimensional biomaterial scaffold that has similar properties with the normal extracellular matrices (ECMs) of the selected tissues. Subsequently, the cell-implanted scaffolds are injected into the patient either through a needle or other minimally invasive delivery procedure. The fabricated tissue can also be transplanted into a patient’s body through surgery. Of all the key components, the design of biomaterial scaffold with optimum characteristics is very crucial to ensure success in tissue engineering. Over the years, the role of hydrogels as a biomaterial scaffold in tissue engineering has received increased attention thanks to their desirable framework for cell growth and survival, on top of their unique properties and resemblances with the natural extracellular matrices (ECMs).

5.2

Overview of hydrogel

A hydrogel is referred to as three-dimensional (3D) cross-linked polymer scaffolds that form a macromolecular network capable of maintaining high water content. The hydrogel can be prepared from natural polymers such as collagen, gelatine, alginate, hyaluronic acid, and chitosan [1], as well as from synthetic materials such as polyethylene glycol (PEG) [2, 3], polyacrylamide (PAA) [4–6], polydimethylsiloxane (PDMS). Hydrogels can either be formed through physical or chemical cross-linking methods. To mimic the ECM and regenerate new tissue, the design of hydrogel must adhere to several criteria. For instance, a hydrogel scaffold should contain 3D Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00006-2 Copyright © 2021 Elsevier Inc. All rights reserved.

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Fig. 5.1 Structure of hyaluronic acid.

architecture of a hydrophilic polymer network that allows the diffusion of glucose and other nutrients, which promotes cell growth. The designed hydrogel also should be biodegradable, biocompatible, as well as demonstrating enhanced vascularization. For tissue engineering purposes, the biomaterial used to construct the hydrogel must also be accessible and economically feasible. In comparison with synthetic polymers, natural polymers such as polysaccharide and polypeptides [7, 8] have been widely used to prepare hydrogels owing to their inherent biocompatibility, biodegradability, and biological activity [9]. Of particular interest is hyaluronic acid (HA) (Fig. 5.1), a natural linear polysaccharide having high molecular weight composed of repeating disaccharide units of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine. In the human body, HA performs essential biochemical and physiological roles, including maintaining the mechanical integrity, homeostasis, viscoelasticity, and lubrication of the tissue in most tissues’ extracellular matrix due to its high molecular weight and its capacity to absorb a high volume of water [10]. Natural HA shows unique properties such as having similar water content as human tissue, and shows excellent biocompatibility, biodegradability, and highly reproducible. Hence, they are often used as a building block for new biomaterial scaffolds in the field of tissue engineering and regenerative medicine. However, pure HA also has limitation for application in tissue engineering due to their rapid degradation and poor biomechanical properties. Therefore, the following section discusses a few approaches that have been performed to modify the structure of hyaluronic acid to enhance their degradation and biomechanical properties.

5.3

Chemical modification of hyaluronic acid

In 2014, Ning Cui group has reported a novel facile method to synthesize functional hyaluronic acid (HA) hydrogels containing hydrazone and disulfide bonds in their crossbridges [11]. This light yellow-colored hydrogel was obtained through the reaction of 2,5-hexanedione with 3,30 -dithiodipropionate-hydrazide-modified HA (DTPH-HA) in the absence of a chemical catalyst. The hydrogel consists of a highly porous surface with pore size in the range of 50–400 μm. Besides, this hydrogel is also a good candidate for biomaterial scaffold in cell delivery and tissue regeneration owing to their good elastic properties, good thermostability, and rapid enzyme degradation rate [12, 13]. In another work done by Tavsanli and Okay [14], mechanically strong HA hydrogels with tunable viscoelastic and mechanical properties were

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prepared via free-radical copolymerization of methacrylated hyaluronic acid (GMHA) and N,N-dimethylacrylamide (DMA) in aqueous solutions. During the copolymerization reaction with DMA, the use of GMHA as multifunctional cross-linker induces the formation of interpenetrated and entangled polymer networks. As DMA was replaced by metacrylic acid monomer, the mechanical properties of the hydrogel were enhanced due to the stronger extent of noncovalent interactions. In this work, strong hydrogels with a Young’s modulus of around 200 kPa that can sustain up to 20 MPa stresses at 96% compression could be prepared by varying the synthesis parameters. To sum up, structural modification of hyaluronic acid is crucial to improve their properties, particularly for tissue engineering application. The next section discusses examples of hyaluronic acid-based hydrogel used in drug delivery, hemorrhage control, and articular cartilage regeneration.

5.4

Application of hyaluronic acid-based hydrogel in tissue engineering

5.4.1 Hyaluronic acid-based hydrogel for hemorrhage control Hemorrhage or uncontrolled bleeding can cause severe complications, including hypotension, organ collapse, and even death. Over the years, several attempts have been made to control excessive bleeding by encouraging hemostasis in a range of forms, including the use of a hemostatic agent. However, the challenge in the design of an ideal hemostatic agent is to ensure that the agent can exhibit effective hemostasis and good biocompatibility. Hence, homeostasis agents should be made of natural and safe components, with nonprotein-based main components and biomolecules that facilitate blood coagulation. For instance, an albumin-based compound, Bioglue, may cause cytotoxic effects due to the unreacted glutaraldehyde impurities leading to minimal tissue growth, nerve loss, and mutagenesis [15]. One recent example of in situ injectable hydrogels reported by Luo et al. was used as tissue sealants for hemorrhage control. These hydrogels were constructed through self-crosslinking reactions of gelatin (sc-G) and hyaluronic acid/gelatin (HA/G) [16]. These injectable hydrogels were prepared through crosslinking reactions of gelatin and hyaluronic acid/gelatin with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), respectively. HA/G hydrogel synthesized had short gelation time (50% for anhydrous composite) and resulted in good dispersion of fibers in the hydrogel. To date, there are a limited number of studies on 3D printing of cellulose-based inks for biomedical applications, and Table 11.6 summarizes some recent research on 3D printed cellulosic biomedical materials.

11.2.4 Chitosan Chitosan is a deacetylated form of chitin (N-acetyl glycosamine) produced by the hydrothermal process and is a well-known polysaccharide thanks to its high biocompatibility and interesting biological properties, such as antibacterial and mucoadhesive effect. Unfortunately, due to its strong cationic property and insolubility in nonacidic solutions, it cannot be used readily in bioprinting formulations. In recent years, several chemical modifications on chitosan have been performed to make this polysaccharide printable via extrusion or light cure-based systems (Fig. 11.5). When used in the native form, an alkali treatment is required to convert chitosan to neutral pH to start gelation after the bioprinting process. Chitosan (6% w/v, in acetic acid) was added to an acetone solution of poly(ε-caprolactone)-diacrylate/poly(ethylene glycol)-diacrylate baseline resin and used in digital light processing (DLP) projection in which visible light illumination (13.43 mW/cm at a wavelength of 400–460 nm) is used [77]. The blend was printed in DLP at 100 mm line-width at chitosan concentration up to 15% (w/v). In addition, with increased chitosan content in the resin, a significantly high number of cells (L929 fibroblast) was counted compared to baseline polymer without chitosan due to biocompatibility of chitosan. However, chitosan (10%)-containing bioink could decrease tensile strength 0.53 from 0.81 MPa compare to chitosan-free baseline resin. In a similar study, DLP printing technology was applied to a photocurable methacryl-chitosan solution (CHI-MA), which can be cured as hydrogels by UV irradiation (10 mW/cm2, 10 s) [78]. Using the CHI-MA with a high degree of methacrylate substitution (33.6%), a 150 μm thick hydrogel layer can be printed within a reasonably short time period, at high fidelity and resolutions (up to 50 μm). A cytotoxicity test with seeded human

Table 11.6 Examples of studies on 3D bioprinting or printing of cellulose-based materials. Materials

Cells

3D printing method

Main aim of the research

Main outcomes

CNC (0%–4% w/v), alginate (2%–6% w/v), water (94%–98% w/v) [65]

Fibroblasts or human hepatoma cells in bioink (106 cells/mL)

Pneumatic extrusion 3D bioprinting

To determine the printability of the bioink composition and to 3D print a honeycomb structure containing fibroblast and hepatoma cells for liver tissue engineering.

l

l

A commercial bioink (Cellink, Sweden), which contains 2% (w/w) plantderived CNF and 0.5% (w/w) sodium alginate and polycaprolactone (PCL) as a separate support [66]

Primary human nasal chondrocytes (hNC) in bioink (20
106cells/mL)

Pneumatic microvalve contact dispensing method for 3D bioprinting of bioink and thermoplastic extrusion method for 3D printing of support structure

To determine the functionality of nanofibrillated cellulose and alginate for auricular cartilage tissue engineering.

l

l

Only the bioink consisting of 2% alginate, 4% CNC and 1% (w/v) CaCl2 for gelation exhibited good shear-thinning property, extrudability, and high shape consistency after deposition. Cell viabilities of fibroblast and hepatoma cells decreased to 58.91% and 49.51%, respectively, after 3 days in bioprinted structures due to the lack of cellbinding sites in the hydrogel. Cell-laden cellulosealginate auricle constructs exhibit good printability and stability after 3D bioprinting. The constructs enabled the synthesis of new cartilage, indicated by a significant increase in the amount of cartilagespecific extracellular

matrix components such as collagen and markers such as aggrecan (ACAN) and cartilage oligomeric protein (COMP). CNF (1.5%–2.25% w/v), alginate (0.25%–1% w/v), water (97.50% w/v) [67]

Human nasoseptal chondrocytes (hNC) in bioink and L929 fibroblasts for cytotoxicity assays (15
106 cells/mL)

Pneumatic microvalve 3D bioprinting based on electromagnetic jetting

To 3D bioprint human chondrocytes in CNF/ alginate bioink and to evaluate its printability

l

l

CNF was used to improve the shape fidelity of alginate and the bioinks were cross-linked with 90 mM CaCl2. The bioink, consisting of 2% (w/v) CNF and 0.50% (w/v) alginate, was considered as optimum composition for 3D bioprinting according to the results of rheology, compression, and shape deformation tests. The viability of hNC decreased from 95.3  0.1% to 69.9  13.3% after embedding in the bioink and cross-linking the constructs. However, there were no significant viability changes between nonprinted and printed cells; therefore, it was Continued

Table 11.6 Continued Materials

Cells

3D printing method

Main aim of the research

Main outcomes concluded that the preparation and mixing were what caused a decrease in viability, not the bioprinting process.

CNF/alginate (60/40 ratio: 1.8% CNF + 1.2% alginate and 80/20 ratio: 2.4% CNF + 0.6% alginate) and another bioink of CNF/ hyaluronic acid (95% vol% CNF and 5 vol% hyaluronic acid) [68]

Human-induced pluripotent stem cells (iPSCs) iPSCs and/or irradiated chondrocytes in bioink (20
106 cells/mL)

Pneumatic inkjet 3D bioprinting

To test the given 3D bioprinted hydrogel combinations for cartilage regeneration with iPSCs.

l

l

The pluripotency of iPSCs in 3D printed CNF/alginate (60/40, dry weight % ratio) hydrogels, which were cross-linked with 100 mM CaCl2, was maintained, hyaline-like cartilaginous tissue with collagen type II was observed after 5 weeks. The proliferation of iPSCs in 3D printed CNF/hyaluronic acid gels, which were crosslinked with 0.001% (v/v) H2O2, was low and no pluripotency was seen. Therefore, CNF/alginate was concluded to be suitable for bioprinting iPSCs coculture with irradiated chondrocytes to support cartilage.

Alginate/methylcellulose (3/1, 3/3, and 3/9, dry weight % ratio) [69]

Mouse fibroblast L929 cells in bioink (3
106 cells/mL)

Pneumatic extrusion 3D bioprinting

To optimize the 3D bioprintability of alginate/ methylcellulose blend hydrogel with improved adhesion of overlapping layers.

l

l

BC blended with 10 wt% PCL solution at a weight ratio of 5:95 [70]

Mouse embryo fibroblast cells (NIH/3T3) were used to evaluate cytocompatibility.

A filament-type fused deposition modeling (FDM) 3D printer was modified to connect a syringe pump and a high voltage power supply.

To obtain 3D structures with a better resolution by applying a high voltage to polymer solution via an electrohydrodynamic 3D printing method.

l

l

Treatment of the 3D printed structures in trisodium citrate (TSC) solution and crosslinking in a solution of CaCl2 after 3D printing highly enhanced the strength of adhesion between the layers in a concentration-dependent manner. Immediately after 3D printing, good cell viability of >95% was achieved in the alginate/ methylcellulose structure. The average spacing between the 3D printed filaments of the BC/PCL (5:95 wt ratio) composite scaffold was 100 μm and filament depth dimensions were 4.7  1.03 μm. BC was used to adjust the hydrophilicity of PCL, which strongly influences cell proliferation. BC/PCL exhibited a better cell viability (>155%) after Continued

Table 11.6 Continued Materials

Cells

3D printing method

Main aim of the research

Main outcomes 72 h in cell culture compared to pure PCL scaffold.

Nanocellulose prepared with TEMPO mediated oxidation used as a substrate, and nanocellulose prepared with a combination of carboxymethylation and periodate oxidation used as 3D printing inks [71]

No cells were used.

Pneumatic extrusion 3D printing following freezedrying to remove water content.

To investigate the potential use of nanocellulose as a wound dressing material and its ability to inhibit bacterial growth.

l

l

Cellulose (5% w/v) in N-methylmorpholineN-oxide (NMMO, in 50 wt % H2O) [72]

No cells were used in inks or tested.

Pneumatic extrusion 3D printing following freezedrying to remove water content.

To determine the printability and the mechanical properties of 3D printed cylindrical cellulose scaffolds.

l

Nanocellulose produced with carboxymethylation and periodate oxidation procedure yielded homogeneous and thin ( chitosan lactate > glycol chitosan > chitosan glutamate > chitosan HCl [39–43]. In vitro studies have evaluated the biologically favorable methylpyrrolidinone and HCl on fibroblast compartments, showing that methylpyrrolidinone chitosan produced a 35% decrease in cell feasibility, while chitosan-HCl produced a 70%–80% decrease in cell feasibility [44]. It is known that the chitosan DD is directly proportional, due to its biocompatibility. With increase in DD, a rise in chitosan’s feasibility was observed due to the occurrence of free amino sites, which permit the contact between chitosan and compartments. The solvent effect on chitosan with respect to biocompatibility was also studied by formulating chitosan flakes in acidic solvents like lactic acid and acetic acid. It was

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observed that chitosan in acetic acid produced skin irritation, whereas chitosan produced in lactic acid exhibited toxicity toward skin. However, in chitosan biocompatibility as well as biodegradability, the capacity of chitosan to interrupt monomers has been widely studied in vivo and in vitro [34, 45–47]. Biodegradability is an important property of chitosan because it helps to control metabolic rate; it must therefore be examined thoroughly. The biodegradation rate of chitosan is frequently measured, based on the envisioned application. For known examples of tissue engineering, the rate of biodegradation of chitosan must be slow to sustain its structural and motorized veracity until the tissue is produced, as well as in drug deliveries applications. Studies revealed that the rate of biodegradation of chitosan is not proportional to DD. It was noticed that higher DD of chitosan shows a lower rate of degradation, which encourages insignificant provocative response, whereas lower DD chitosan shows a minimal rate of degradation, which induces a severe inflammatory response due to the growth of amino saccharides creating a provocative effect and becoming poisonous [48]. The rate of biodegradation of chitosan depends on the MW. It was observed that MW of chitosan fluctuated from 30,000 to 40,000 Da and it was removed through renal approval [49]. However, above this MW, chitosan was initially degraded through enzymatic action, such as microflora or lysozyme, or chemically modified degradation, such as degradation via acid catalyzed in the abdomen before renal approval. Lysozyme is the main enzyme accountable for the breakdown of chitosan [50]. Enzyme degradation is introduced through growth of proteins, biofouling, and cells on the substance, which leads to immune system recognition and consequent exclusion. Recently, scientific studies observed that chitosan showed antiinflammatory activities. The antiinflammatory is a resistant response of external bodies which can be protected by physical injury, toxic chemicals, as well as pathogens [51].

12.4

Significance of chitosan nanocomposites

Chitosan nanocomposite resources have a new area of interest, based on the introduction of supporting fillers with magnitudes in nanometric scale, such as nanofillers with at least one dimension within 100 nm [52]. The nature and superficial functionalities of nanocomposites as nanofillers could show alterations in their activities like enhanced barrier and mechanical properties, as well a high transparency. These kinds of property improvements depend on their nano-scale dispersal, even at a lower scale of nanofiller introduction, which has a higher characteristic proportion and large surface area. The efficiency in terms of reinforcement of nanocomposites can compete for the safe composites with 40%–50% of packing along with usual fillers [18, 53–55]. Chitosan nano-range composite, with its matrix and small-size benefits, results in tougher materials. In these cases, the proportion of supporting stage is usually lower, for instance, 5–10 wt%, to keep the chitosan percentage comparatively high. This results in materials that look exactly like the matrix in terms of bioactivities

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and biocompatibility [56]. The introduction of nano-supports in a chitosan matrix is an influential approach to address the traditional disadvantages of chitosan biopolymer. The nanocomposites are actively functionalizing in some areas, including biomedicine, cosmetics, nanotechnology, the nutrition industry, cultivation, ecological protection, various industries, fabrics, etc. Bio-nanocomposite with chitosan is very popular, for example, in chitosan/clay. The chitosan can make a nanocomposite in the presence of montmorillonite (MMT) clay, and there is a great deal of information about the drug discharge performance of chitosan/MMT nanocomposite flake hydrogels and scaffolds. Recent studies have explained that chitosan/MMT nanocomposites signify an advanced and hopeful period of development of sorbent resources [57–59]. The application of chitosan nanocomposite in term of films, composites, and coatings in packing industries have become an interesting subject due to the need to increase the shelf-life of various food goods. The mechanical aspects of nanocomposite chitosan scaffolds can be enhanced through the accumulation of bioactive glass ceramics and nano-hydroxyapatite, which are extensively utilized in tissue engineering. A large number of studies show the applications of chitosan nanocomposite scaffolds and films comprising Au and Ag nanoparticles to treat patients with profound injuries and wounds by utilizing their antibacterial properties. In previous studies, the performance of chitosan nanomaterials as biosensors has been meaningfully increased through the incorporation of nanomaterials in the detecting layers, e.g., carbon nanotubes (CNTs), nanoparticles, and nanowires. However, for the enhancement of electrodes and biosensing capacities of chitosan nanomaterials, CNTs are stated to advance the physicochemical and mechanical aspects and electric conductivities of synthesized chitosan/CNT nanocomposites [60]. The unique reinforcing behavior of graphene sheets was also studied as a feature in chitosan biopolymers. Recent reports show that the introduction of graphene oxide enhanced the tensile power and Young’s modulus of chitosan/graphene oxide nanocomposites by 122% and 64%, respectively. Modern research on biocomposites of chitosan/metal oxide nanocomposites has focused on AgO, ZnO, and TiO2, since they have first-rate photocatalytic efficiency and are constant in both basic and acidic solvents [61–64]. Chitosan/magnetic nanomaterial, such as CoFe2O4 and Fe3O4, was employed in bio-based applications for protein immobilization, enzymes, peptides, drug delivery, bio-adsorbents, biosensors, and gene delivery. The development of nanotechnology has provided new prospects to chitosan nanomaterials, where nano-sized particles have been used as steadying agents to advance their film forming, biocompatibility, nontoxicity, and higher mechanical activity. The nano-ranged particles offer individually diverse physical, chemical, optical, and magnetic properties as associated to bulk stage [65, 66]. However, Zuo et al. [67] studied the chemical functionalized chitosan/graphene oxide nanocomposites as shown in Fig. 12.3 through amide bonding, which formed graphene oxide/chitosan nanocomposites. This nanocomposite also exhibited an enhanced ductile strength of 2.5-fold and showed high glass transitional temperature and heat stability, as associated with clean chitosan.

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Fig. 12.3 Graphic design defines the GO/chitosan nanocomposite. Adapted from reference Zuo PP, Feng HF, Xu ZZ, Zhang LF, Zhang YL, Xia W, Zhang WQ. Fabrication of biocompatible and mechanically reinforced graphene oxide-chitosan nanocomposite films. Chem Cent J 2013;7(1):39.

12.5

Applications of chitosan nanocomposites

Composite materials are produced by mixing more than one element with diverse physicochemical properties to produce a special properties-based material. The chitosan derivatives are first-rate resources for medicinal-based applications, which are safe for human beings. Biologists have industrialized several categories of chitosan-derived nanocomposites to provide superior functions in biomedicine-based fields [68]. Numerous applications of chitosan have been summarized for medical purposes, as presented in Fig. 12.4. Cisplatin, which serves as an antineoplastic medicine that demonstrates therapeutic activities against tumors, is one good example of a nanocomposite. It has high tumor attraction with adjacent effects, e.g., neurotoxicity, ototoxicity, and nephrotoxicity. Efforts have been made to lower the adjacent effects through various biological approaches. Cisplatin multifaceted with alginate (Pt-based drug) may be accumulated in kidneys, which lowers the nephrotoxicity, but retains the antitumor activities.

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Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering

Fig. 12.4 Various applications of chitosan nanocomposites.

The effective release of the drug by the nanomaterials was obtained through electrostatic interaction between chitosan and alginate. These two polymers can be used in drug deliveries and protein transfer, respectively. The oral management of cisplatin was verified on 180 mice with the growth of sarcoma; the subjects did not exhibit weight loss, and immunogenicity was encouraged through the medication with modification of the tumor activities [69]. The L-leucine conjugation along with chitosan was effective in making a different nanocomposite in terms of enhanced solubility. The in vitro assessment exhibited the inflammatory and toxicity effects of chitosan nanocomposite in contrast to the respiratory epithelial cell line, BEAS-2B. The nanomaterials prepared between 10 and 30 nm had less toxicity effect and have been convenient for respiratory medication deliveries applications. However, in angiogenesis therapy, antiangiogenic agents have been measured as an active treatment for tumors [70]. New efforts have been made for regenerating tumor suppressor activities of p53 subsequent from its ability of initiating, via transcription, an extensive diversity of genes that control cell sequence capture, apoptosis, and destruction of angiogenesis p53 effects by utilizing the gene delivery approach. A multifunctional anticancerous polymer associated with chitosan-graft, containing low molecular weight-based chitosan, has been prepared as a nanovector for the difficult codelivery of medicine and genes in cancer therapy [71].

12.6

Application in gene delivery

Gene treatment is a method usually employed as genomic material for remediation purposes of several diseases. It is presently used in treatment of diseases such as autoimmune, cancer, cardiovascular, and cystic fibrosis. The gene distribution includes the DNA inside the multitude of cells to inhibit the growth of cancer cell. Enormous resources have been devoted to the problems of gene distribution. For this,

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well-organized nanoparticles are prepared from bio-materials with respect to biomimetic properties and higher surface area to volume aspects. The resistant power toward the rabies virus glycoprotein (G protein) can be formed through repetition of humanoid adenovirus form 5 (Ad5) [72]. The duplication of substandard concept was significantly active for the protection from rabies virus. Nonviral approaches to gene delivery have been projected in order to address the drawbacks. The oral gene distribution system is judged to be the best approach due to its ease of management, suitability, and and persistent obedience. The gene programming chloramphenicol acetyl transformation is reported to be effectively delivered orally via chitosan. Oral delivery of gene therapy is becoming common practice, but is encountering a number of problems due to the nature of the gastrointestinal tract. The high acidic pH and presence of degradative enzymes in the stomach cause destruction of material that is orally delivered [36]. Furthermore, pancreatic secretions are also a source of destruction of the transporter and material to be distributed. In addition, a gene to be carried orally has to overcome the obstacles of the intestinal area to influence the specified target, which is a challenge for the transporter to achieve via paracellular way. These problems lead to lower oral bioavailability for several peptide and protein drugs. The drug-based applications of nucleic acids are judged to be an appropriate mechanism for inoculations or in gene treatment. The formulation of the gene in nanomaterial is intended to address the bodily and degradative obstacles. Several functional nanomaterials have been employed for problematic gene delivery, as shown in Fig. 12.5. The captured gene may be mutated by using such nanomaterials and can recover cellular uptake via endocytosis.

Fig. 12.5 Multifunctional nanomaterial for gene delivery. Adapted from reference Sanvicens N, Marco MP. Multifunctional nanoparticles—properties and prospects for their use in human medicine. Trend Biotech 2008; 26(8):425–33.

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The biodegradable polymer that received much attention in the earlier period as a constituent of the oral gene delivery mechanism was chitosan, although a diversity of polymers and several types of lipids have been used to produce gene-loaded nanomaterials [73]. The influences affecting chitosan deliveries of nucleic acids include chitosan’s molecular weight and the control ratio between the luciferase plasmid and chitosan. The pH of pure cultured medium assists the in vitro transfection performance. A vector for tumor cell gene treatment was successfully offered by Tat tagged and folate improved N succinyl-chitosan (Tat-Suc-FA) self-assembly nanomaterials. Chitosan grafted with polyethyleneimine-candesartan was employed for the synthesis of several functional copolymer-anticancer conjugates as an embattled delivery nano-gene for possible cancer treatment. Nanomaterials of polyethylene attached calcium phosphate and carboxymethyl produced an anionic-charged mixture for siRNA deliveries via single-step self-assembly development in an aqueous medium. Low-density lipoprotein separated from anthropoid plasma and then overloaded with siRNA was effective in suppressing the multi-drug resistant gene of tumors [74]. Cholesterol siRNA/LDL combined with N-succinyl chitosan nanomaterials which created problem for doxorubicin. Tumor-based folic acid-polyethylene/siRNA glycol chitosan oligosaccharide lactate nanomaterials play a dynamic role in gene delivery to cancerous sites through an ionic gelation system and are active in ovarian cancer gene therapy. Poly-siRNA-tGC nanomaterials with a usual diameter of 370 nm and the accessibility of psi-tGC-NPs, which targets definite cytokines, result from poly-siRNA self-polymerization. This occurs via thiol groups for siRNA and results in thiolated glycol-chitosan biopolymers employed for rheumatoid arthritis treatment [75]. Multiwalled carbon nanotubesbased materials of various sizes have been functionalized in the presence of chitosan-folic acid nanomaterials through ionotropic gelation, developed as gene delivery materials. The instantaneous gene delivery to tumor cells can be addressed through a chitosan/magnetic graphene nanomaterials stage. The respirational syncytial virus gene encapsulation concept can be achieved through employing a specialist nanomaterial, which transfers poly (2-hydroxyethyl methacrylate) nanospheres enclosed in a cationic chitosan shell [76].

12.7

Future perspective and concluding remarks

Several research magazines and articles on chitosan derivatives and their nanocomposites reflect the significant value of chitosan. Many important technological developments have been used to increase the abilities and advance the consistency of chitosan-based nanocomposites for diverse applications, particularly in gene delivery and the medical field. Chitosan is basically a biocompatible, natural, biodegradable, toxin-free, hydrophilic polymer, which has ideal properties for biomedicine applications [5]. In addition, chitosan itself and chitosan nanocomposites are astonishing resources that have an extensive range of useful applications. Chitosan has some drawbacks that limit its application, such as basic and insoluble nature in neutral media. Several nanoparticles and other polymers, such as carbon nanotubes, have served as supportive candidates for the emerging and enormously varied applications of chitosan nanocomposites. Furthermore, chitosan can be improved with

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various functional groups to regulate the hydrophobic, anionic, and cationic parameters. This is why chitosan is comparatively unique among biopolymers. The most unusual properties of chitosan arise from primary amines groups with their backbone. These types of structures communicate to the polysaccharides, which have highly appreciated physical and chemical properties, but also have specific contacts with proteins, cells, and living creatures. In this chapter, the importance of chitosan bio-nanocomposites and basic properties of chitosan have also been studied. Making an accurately biodegradable product provides both environmental and humanoid protection. At the least, production must be nontoxic. Other eco-friendly qualities are use of sustainable ingredients, produced in ways that will not damage the bionetwork [77]. Obviously, chitosan polysaccharide displays a capacity for gentle applications in healthcare products, which are not toxic and are safe materials. However, care is necessary to ensure that the material remains pure, as metal, or many supplementary impurities, could possibly produce harmful properties, individually or in derivative combinations and in various quantities. The cross-linking or derivatization and unreacted substances must be carefully removed to avoid unintended consequences, as many substances are toxic. It was shown that, for superior applications such as biomedical gene delivery and food packing, the thermal and mechanical aspects of pure chitosan are not adequate. Additionally, several studies focus on schemes with chitosan nanocomposites [78]. The addition of a small amount of clay nanomaterials improved thermal or mechanical properties, overcoming the restrictions of chitosan alone being functionalized in fields which need a definite thermal and strength stability. The chitosan nanocomposites and polymeric system can offer a good result in terms of gene delivery, as well as drug discharge enzyme immobilization, high biocompatibility, and toxin-free properties offering a broad diversity of applications such as medical, material engineering, and pharmaceuticals in the future.

Acknowledgments The authors gratefully acknowledge Universiti Sains Malaysia, 11800 Penang Malaysia under the Research University Grant 1001/PKIMIA/8011070.

Conflicts of interest There are no conflicts to declare.

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Abu Tariqa, Showkat Ahmad Bhawanib, Mehvish Nisarb, Mohd Razip Asaruddinb, and Khalid M. Alotaibic a Department of Chemistry, Aligarh Muslim University, Aligarh, Uttar Pradesh, India, b Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Kota Samarahan, Sarawak, Malaysia, cDepartment of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia

13.1

Introduction

Biomaterials have received considerable attention over past few decades as a potential ingredient in the field of biotechnology, biomedicine, tissue engineering, and drug and gene delivery, all due to enhanced consciousness for green and sustainable environment. Natural biopolymers are readily available, inexpensive, degradable, and are environmentally friendly. Starch is such a naturally occurring polymeric carbohydrate, which is abundant, inexpensive, renewable, nontoxic, and biodegradable polymer, and has great potential in the biological applications as excipients. Starch is a storage polysaccharide present in plants and exists as granules in chloroplast of leaves and as amyloplast of seeds, pulses, and tubers [1]. Starch is a second most abundant naturally occurring biomaterial found in plant roots, stalks, tubers, seeds, fruits, and in crops such as rice, corn, wheat, and potato [2, 3]. Native starch is comprised of two homopolymers or glucans, known as amylose; a linear polymer and amylopectin; a highly branched polymer. Starch consists of repeating monomeric units of α-glucose joined together with a D-(1–4) and/or D-(1–6) linkages. In molecular starch structure, the linear amylose is composed of anhydrous glucose units mainly linked with α-(1–4)-D-glucosidic bonds; however, amylopectin is branched with α-(1–6)-D-glycosidic linkages [4]. Fig. 13.1 presents the complete molecular structure of amylose and amylopectin. Most of the granular mass of starch, about 70%, is amorphous and consists of amylose and small portion of amylopectin and rest 30% is crystalline in nature and is made up of amylopectin [1]. However, the ratios between the two glucans, i.e., amylose and amylopectin, vary mainly depending on the source and geographical origin of the starch (Table 13.1). The molecular order and crystallinity solely depend on the nature of source of starch such as corn, potato, and wheat [5]. The crystallinity of starch is of three types based on types of source of starch: type A is from cereal starches such as maize, rice, and wheat; type B is from cassava, sago, and potato, whereas type C consists of both type A and type B crystallinity patterns typically found in bean and other root starches.

Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00007-4 Copyright © 2021 Elsevier Inc. All rights reserved.

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Fig. 13.1 Structure of amylose and amylopectin.

Table 13.1 Percentage of amylose and amylopectin in starch from different sources. Source of starch

Amylopectin (%)

Amylose (%)

Potato Corn Rice Wheat Cassava Sorghum Banana

76–83 75–83 65–85 75–80 28–81 75 76–83

17–24 17–25 15–35 20–25 19–22 25 17–24

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It is evident that a large numbers of hydroxyl groups are present on starch molecular chains and thus starch is pronounced as hydrophilic in nature. These hydroxyl groups have potential to get oxidized and reduced and may actively contribute to the formation of ethers, esters, and hydrogen bonds [6]. It is then expected from the starch cluster to provide a large number of binding sites for the effective DNA condensation. Moreover, enzymatic biodegradability of starch is a well-known aspect; therefore, it is observed that some enzymes native to cells may release loaded materials on the effective admittance of gene delivery vectors into the cells. A few recent studies focused on this aspect of starch showing potential results, thus establishing starch nanocomposites as a cost-effective and readily modifiable gene delivery system. Furthermore, the starch nanocomposites are modified as cations with positively charged amino groups [7, 8], oligoamine residues [9], and polymeric amine [10] for starch-based gene delivery vector systems. It is evident from various inferences that the native starch is not as effective as the modified starch due to some limitations which come hand in with the starch molecules. In order to pacify these limitations, various modification processes of starch are performed via physical and chemical routes [11]. One possible and potent way to broaden the applications for starch biopolymers is to prepare starch-based nanocomposites. Nanotechnology is a branch of material science which deals in the preparation and applications of nanoscale materials of size ranging from 1 to 100 nm [12]. In early days, nanomaterials were composed of only hard materials; however, a significant growth is observed over the last decade in nanomaterials, which are composed of soft materials or both hard and soft materials in combination [13]. Composites are composed of two types of components: matrix and fillers. Matrix is to support and protect the filler materials, transmitting and distributing load among the fillers [14–17], whereas fillers in the form of fibers, sheets, or particles embedded in matrix phase are stronger components of composites and are to provide reinforcement to matrix. These reinforcing materials or fillers are of nanoscale size in nanocomposites. Nanoparticles or fillers enhance the properties of nanocomposites. The mechanical properties such as tensile strength and physical properties viz. barrier properties, permeability, or fire retardancy, etc., as well as optical properties are found to be increased when compared to the native or traditional composites [18]. Nanocomposites are therefore those composite materials which are comprised of these nanoscale materials, either as single or multiple components system or as incorporated materials into bulk. The properties of nanocomposite depend on matrix material, size and shape of nanoscale material, loading, degree of dispersion, etc. Nanocomposites based on starch and cellulose are new-generation bio-nanomaterials that are considered potential agents in the field of pharmacology, biomedicine, tissue engineering, gene and drug delivery, biosensors, etc. [19]. High specific area, controlled drug release, biodegradability, biocompatibility, non-toxicity, and excellent chemical reactivity are various unique properties which keep nanocomposites at par with other typical nanomaterials [20, 21]. Nanotechnology including nanoparticles (NPs)/nanocomposites (NCs) and biopolymers are the center of attraction since many years and have been a promising

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bet in the biomedical field such as gene delivery, controlled drug delivery [22, 23], tissue engineering [24, 25], and imaging in DNA [26–28]. In biomedical sciences, gene therapy is considered a potential therapeutic process to address and treat various serious diseases which are incurable. Gene therapy is to alter defective and missing gene sequences to treat inherited/and or acquired diseases which include acquired immunodeficiency syndrome (AIDS), cancer, cardiovascular diseases, and other genetic disorders. The like of cancer and other genetic disorders are treated by delivering therapeutic genes to specific site cells where they help in producing desired proteins and inhibit unwanted gene expressions [29, 30]. A gene carrier agent and the genetic material comprise a successful gene formulation. The role of carrier agent is to protect the genetic material and to introduce targeted gene delivery properties with controlled release kinetics. The vector or carrier of genetic material is designed efficiently which overcomes the challenges incurred during production of nontoxic, nonimmunogenic, and noncarcinogenic vectors. There are two main types of carriers or vectors for gene delivery: viral and nonviral. A large number of nanoscale materials are used to produce a number of potential gene carriers in the gene delivery applications. These materials include lipids, graphene, biopolymers, carbon nanotubes (CNTs), and other types of inorganic NPs. It has been observed that the functionalized NPs including nanocomposites have produced potential gene delivery platforms owing to several unique properties such as small size and superior stability. Among different available polymeric gene delivery vectors, polysaccharide-based polymers such as chitosan, dextran, and hyaluronic acid have an edge over synthetic polymers, for instance, polyethyleneimine (PEI), etc., owing to the unique characteristics like biodegradability, cytotoxicity, biocompatibility, etc. [31–36].

13.2

Starch-based nanocomposites

Starch a biopolymer widely used in various biomedical applications has been considered an important agent in the production of composite bionanomaterials and is an excellent alternative to synthetic materials [37, 38]. Several researchers have reported the preparation and applications of starch-based nanocomposites. Kalambar, Rizvi and coworkers have successfully produced starch-based nanocomposites by reactive extrusion processing from starch polycaprolactone (PCL) blends in the presence of montmorillonite (MMT) nanoclay. The obtained nanocomposites of starch-PCL blends in nanoclay show significant enhancement in mechanical properties such as strength, modulus, or strain [39]. Fama et al. synthesized biodegradable starch-based nanocomposites by reinforcing nanocomposite materials based on starch matrix with multiwalled carbon nanotubes (MWCNTs). They reported that wrapping of CNTs with starch-iodine complex allows to prepare starch-based nanocomposite with carbon nanotubes used as reinforcement in a very small quantity; moreover, they reported an increase in the high storage modulus and low water vapor permeability of obtained biodegradable starch-based nanocomposite owing to carbon nanotubes [40].

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13.2.1 Starch as matrix There are large a number of reported nanocomposites where thermoplastics and elastomeric polymers are used as matrices [14, 16, 41, 42]. Based on types of applications, compatibility between materials, cost and processing; matrices are chosen. However, recent developments in the field of biopolymeric materials with properties like biodegradability and environmental sustainability pave way for starch, ploy (lactic acid), polyhydroxybutyrate, etc. to be considered a potential option for matrices in nanocomposites [18, 43–46]. These nanocomposites are comprised of starch either as matrix or as reinforcement. Starch has been consistently used as a matrix to provide a greater support to the nanoreinforcements incorporated. The obtained nanocomposites usually show great improvement in mechanical and physical properties such as yield strength, modulus, moisture resistance, thermal stability, barrier property, and biodegradation, to name a few. The enhanced properties are the result of homogeneous dispersion of the nanofillers and the strong interface adhesion, which is the cause of rigid nanocomposite network. Furthermore, organomodifiers are used to increase hydrophobicity and reduced compatibility with the hydrophilic starch matrix [47]. There are several factors that result in the improvement of matrix characteristics [47], as mentioned below: (a) Addition of plasticizer(s) or additive(s) during the production of starch-based nanocomposites. (b) Source of starch as matrix. (c) Modification of native starch chemically. (d) Surface area/nanoreinforcement aspect ratio and other mechanical properties. (e) Annealing conditions and processing of nanocomposites during preparation. (f) Presence of other polymers in nanocomposite framework.

Significant efforts have been made to study starch as matrix in starch-based nanocomposites, keeping in view changes in mechanical properties of nanocomposites owing to the botanical origin of starch, addition of additives like plasticizers, and the choice of fillers used. Lu et al. reported the role played by strong interactions between the glycerol plasticized starch matrix and the cottonseed linter fillers in reinforcing properties [48]. Angles and Dufresne studied transcrystallization of plasticized starch and cellulose whiskers, and observed that there is a strong loss of performance owing to the importance of the filler or filler interactions [49]. Konwarh et al. studied and reported the differential templating attributes of starch under aging and sonication for biomimetically generated silver nanoparticles. The starch in this study was used as a macromolecular matrix [50]. Chitin/chitosan nanoparticles were uniformly decorated on the starch matrix by Chang and coworkers [51, 52]. The loading was performed both on low and high levels; on low level, addition of fillers enhancement in properties was observed, whereas when reinforcement was on higher side, aggregation occurred. The starch-clay nanocomposite was developed by Chung et al. by using starch solution as matrix. They observed a considerable increase in the strength and elastic modulus of the matrix, up to 30% for former and 35% for latter property when compared to native starch solution. Moreover, they

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observed that dilution of starch solution results in the decrease of elastic modulus for both native and nanocomposite starch. The addition of clay in the nanocomposite increases the stability of starch-based products during storage and transportation [53]. A self-assembled starch-based nanocomposite was prepared through bioinspired bottom-up technique. The obtained nanocomposite was a result of starch and bacterial cellulose (BC) nanofibrills. The BC obtained from Gluconacetobacter bacteria is a self-assembled material [54].

13.2.2 Starch as reinforcement Reinforcement is usually provided by the nanoscale materials or NPs such as carbon nanotubes, nanofibrils, whiskers, and nanocrystals. Starch nanocrystals are used as reinforcing material or fillers in starch-based nanocomposites. These nanocrystals of starch could be produced via different routes. Crystalline or amorphous starch NPs are obtained through production route [55]. Hydrolysis of starch results in the production of nanocrystals (crystalline form nanoparticles), whereas regeneration and mechanical treatment of starch give rise to both crystalline and amorphous nanoparticles. The production route used for processing of nanocomposite determines the size and other important properties such as tensile strength and elongation at break [55]. There are several studies witnessing the change in properties of nanocomposite when starch NPs were added to the different matrices. It is observed that the addition of starch NPs to the thermoplastic matrix increases values of several important properties such as elastic modulus, strength at break, and glass transition temperature (Tg) of the obtained nanocomposite [55–60]. Angellier and coworkers prepared a latex starch-based nanocomposite using natural rubber matrix and aqueous suspension of waxy maize starch nanocrystals as reinforcement [61]. Starch nanocrystals were obtained via sulfuric acid hydrolysis of waxy maize starch granules. They studied the properties of resultant nanocomposite and found that the swelling behavior is increased; however, a decrease in the water uptake of the nanocomposite film was observed. Although starch-based nanocomposites have remarkable properties, yet few disadvantages were reported and to overcome those disadvantages, different forms of nanofillers were used in the formation of nanocomposites. Several characteristics such as flammability, conductivity, shrinkage, processability, weight, or visual appearance were observed when addition of these nanofillers is performed. These nanofillers are inexpensive, affordable, and easily available that give rise to high-scale enhancement in physical, mechanical, and barrier properties of the nanocomposites [62–64]. These nanofillers are organic or inorganic particulate materials which include plates, spheres, flakes, fibers, whiskers, fibrils, and sheets. Nanofillers can be classified as isodimensional or zero-dimensional particles, microcrystalline cellulose, or nanowhiskers, nanofibrils, nanocrystals cellulose, and lamina or sheet particles. The commonly used inorganic fillers are carbon nanotubes, carbon black, clay, and fumed silica, whereas cellulose-based fibrils are most common organic fillers. Xie and coworkers [65] have studied the starch-based nanocomposites and showed their

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potential in both fundamental and industrial research. A large number of fillers have been used such as inorganic like MWCNT’s [66] and organic nanofillers (cellulose nonofibrils) [67, 68] in starch-based nanocomposites. Nanocomposites based on starch blends, i.e., starch blended with other polymers such as poly (lactic acid)/polylactide (PLA) [69–74], polycaprolactone (PCL) [39, 75–81] and poly(vinylalcohol) (PVA) [82–84] have also been reported.

13.3

Applications of starch-based nanocomposites

Native starch possesses several useful properties such as biodegradability, biocompatibility, non-toxicity, and other mechanical properties, which proved handy in the development of bionanocomposites for various applications in biomedical sciences [85, 86]. It is observed that various nanocrystals, nanoparticles, and nanocomposites of starch have been prepared in the past few years and their applications in the field of biomedicine is reported as well. These resultant materials are considered safe materials; however, in a few sensitive human recipients, some potential allergic reactions are expected and observed [87]. Moreover, starch-based nanocomposites have been largely applied in biomedical applications such as bone regeneration, drug delivery, tissue engineering, gene delivery, and other pharmaceutical products [88–99].

13.3.1 Gene delivery Gene therapy gained a remarkable ground in the past few decades and is considered a potential therapeutic strategy for treatment of various incurable diseases [29, 30]. Moreover, starch is the most abundant biopolymer widely used as a pharmaceutical excipient and in packaging industry owing to properties such as biodegradability, biocompatibility, and nontoxicity [100–103]. Starch molecules are modified through cationic amino groups such as quaternary ammonium groups to make it potential gene carrier [7, 8]. Moreover, oligoamine residues and polymeric amine are other materials which have been used to typically engineer vectors based on starch for successful gene delivery [9, 10]. Few examples of gene delivery by starch-based nanocomposites are given in Table 13.2. Studies have reported the formation of polyplex-based gene delivery system comprised of cationic polymer/DNA polyplexes [109–114]. The complex is internalized by cells of cell line such as HepG2 via clathrin-dependent endocytosis (CME) pathway, which initiates the formation of clathrin-coated vesicles and subsequent production of early and late endosomes, and later fuses with lysosomes. Moreover, cationic starch should bind with DNA to form stable polyplex complex and is used to avoid degradation of DNA in lysosomal complexes. Thus, keeping in mind the importance of starch in composite, Eliyahu and coworkers [104] developed a spermine-modified starch-based (SMS) nanocomposite, which is capable of enhancing the proton buffering capacity of starch, and increases the possibility of release of endosomal escape of the polyplexes.

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Table 13.2 Gene delivery applications of starch based nanocomposites. Composition

Applications

References

SMS nanocomposites

Enhancing the proton buffering capacity, release of endosomal escape of the polyplexes Structure Transfection activities with gene carriers Gene delivery and Drug delivery Drug delivery

[104]

[106, 107] [108]

Gene delivery

[7, 8]

Plasmid pAcGFP1-C1 (pDNA), SMS Fe3O4 NPs Fe3O4 /starch-g-polyethylene phthalate hydrogel nanocomposite Starch, quaternary ammonium groups

[105]

High transfection efficacies of spermine-modified starch-based nanocomposites were reported by Huang and coworkers [105]. They have chosen plasmid pAcGFP1-C1 (pDNA) as the model gene to form nanocomplexes with SMS and the transfection efficiencies were studied. Furthermore, they have studied the structure-transfection activity relationships of SMS gene carrier. Modification of Fe3O4 NPs by using biopolymers such as starch, chitosan, dextran or synthetic polymers like polyethylene glycol has been achieved to reduce the toxicity, oxidation, and were applied for various treatments [106, 107]. Such nanocomposites are effective gene and drug delivery vectors and are promising agents to deliver therapeutic molecules to targeted sites. Hamidian et al. prepared Fe3O4/starch-g-polyethylene phthalate hydrogel nanocomposite via graft copolymerization technique where starch is used as biopolymer backbone, and found that the nanocomposite is effective in release of drug [108].

13.3.2 Miscellaneous applications Apart from gene delivery, a numerous other applications are reported by various workers. Fama and his coworkers produced a starch-based nanocomposite which is comprised of a very small amount of multiwalled carbon nanotubes (MWCNT), which is observed to be a potent agent in preparation of tissue scaffolds; moreover, it could be used for bone-regenerating treatments as well [90]. A team of researchers have shown that the presence of nanotubes exhibits strong antimicrobial effects, as it severely damages cell walls of Escherichia coli [89]. When wrapped with starch-iodine matrix, MNCWTs comprised of nanocomposite exhibit highly enhanced tensile and impact properties. Tissue engineering is another field where the starch-based nanocomposites have proved their worth. Many fibrous structures analogous to natural extracellular matrices are found to be potential scaffolds for tissue engineering applications. A Starchbased scaffold is developed by Martins and his team using SPCL micro-motifs and

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polycaprolactone nanomotifs. Analysis of the resultant scaffold is done with SEM and microcomputed tomography showing multilayered scaffold fabrication with predominant cell spreading and attachments on nanofiber meshes. The inference made with these results showed that the integration of nanoscale fibers into scaffolds enhances biological performances in the bone tissue engineering. Over the past few decades, a drastic improvement has been achieved in the field of drug delivery systems owing to the growth and development of polymers and biopolymeric materials. A targeted and controlled drug delivery systems are the need of the hour to overcome the drawbacks caused by classical administration of drugs. More recently, a drug delivery system based on starch-based nanocomposites was introduced, especially that of starch-based biodegradable plastics which are used to enhance drug delivery and to eliminate the need to retrieve surgical materials after administration of drugs [91]. In case of high dosage requirement, such a method is promising [92]. Hydrogels are another form of materials which are highly effective in tissue engineering applications due to promising antimicrobial characteristics. Eid [93] prepared a starch-based hydrogel via gamma radiation polymerization process and reported the formation of silver nanoparticles within the hydrogel from the silver nitrate present in it. Another study reported by Abdel-Halim and Al-Deyab showed the formation and biomedical applications of nanocomposites from silver NPs, starch, and polyacrylamide [94]. A bionanocomposite based on hylloysite, a clay mineral nanotube and plasticized starch matrix, was prepared by Schmitt and coworkers [97]. They have reported the increase in the thermal stability and tensile strength of starch without any loss of its ductility. Bacterial effects and electrical conductivity are few other properties exhibited by nanocomposites prepared by Voladkar et al. using waxy starch matrix [98, 99]. It is evident that these biodegradable nanocomposites are a promising alternative to classical materials for various applications in the field of packaging and biomedical sciences; however, it is observed that a little understanding of their interactions with humans and the environment is known.

13.4

Final remark and future perspective

The advent of new and advanced technologies and the enhanced consciousness of sustainable environment have been the key aspects for the production and development of bionanocomposites from natural biomaterials. With new technologies available for fabrication and characterization of nanoscale materials, the production of starch-based nanocomposites has become possible. The resultant starch-based nanocomposites possess enhanced mechanical and physical properties which further can be easily tailored by the addition of nanofillers or reinforcements such as clays, carbon nanotubes, and cellulose nanofibrils. There is a great interest in the studies related to gene delivery by starch-based nanocomposites for the treatment of fatal diseases such as AIDS, cancers, and cardiac diseases. Furthermore, delivery of drugs, tissue engineering, packaging, etc. have been promising areas of research which various researchers have

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explored and reported. Moreover, considerable efforts have been put to increase the effectiveness of these nanocomposites. However, despite all these efforts for the betterment of starch-based nanocomposites, a lot of challenges are yet to be explored to make them commercially available on a large scale for biomedical applications, especially gene delivery. The future of starch-based nanocomposites is bright and promising as a large number of new nanofibrils are yet to be incorporated and tested as reinforcements. Furthermore, future studies may focus on phase separation and heterogeneous dispersion of nanofillers apart from emphasis drawn over the processing techniques.

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Vannessa Lawai and Zainab Ngaini Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia

14.1

Introduction: Gene therapy

Gene therapy is a treatment or prevention of serious disease by repairing, replacing, or regulating the malfunctioning gene using a therapeutic gene by transferring it into a specifically targeted cell nucleus [1, 2]. Approaches in gene therapy include replacing a mutated gene with a healthy gene, inactivating a malfunctioning gene, and introducing a new gene to assist the treatment of the disease [3]. Nowadays, gene therapy is studied and applied in the treatment of genetic diseases, cancers, acquired immune deficiency syndrome (AIDS), cardiovascular disease [4], and heredopathia [5]. In general, the gene therapy process includes the identification of a mutated or defective gene, followed by cloning of an identical healthy gene called a transgene. The next step is loading the therapeutic gene into a vector that delivers the therapeutic gene into the nucleus. The delivered therapeutic gene is integrated into deoxyribonucleic acid (DNA) followed by the correction of the defective or mutated gene [6].

14.1.1 Gene delivery systems There are three main gene delivery systems: viral vector (e.g., adenovirus, herpes simplex virus, retrovirus, lentivirus, and smallpox virus), nonviral vector (e.g., cationic liposome, a cationic polymer, and nanoparticles), and electroporation [7]. The viral vector method is known to have a high transfection efficiency of 80%–90% due to the ability to enter the cells naturally and express their own protein [3]. There are some limiting factors in using viruses as vectors such as the risk of toxicity, safety, acute inflammatory response, cellular immune response, and integration of nucleic acid sequence into the host genome, which leads to inadequate expression of the gene. Other limitations are the number of genes carried by the virus, high cost of production, unsuitability for large-scale production, insertional mutagenesis, and oncogenic effects in in vivo application [8]. Gene delivery systems via nonviral vectors have low transfection efficiency of 20%–30%. However, nonviral vectors have good cell viability of 80%–90%, indicating safe transfer of larger DNA molecules [3]. Gene delivery via nonviral vectors Polysaccharide-Based Nanocomposites for Gene Delivery and Tissue Engineering https://doi.org/10.1016/B978-0-12-821230-1.00016-5 Copyright © 2021 Elsevier Inc. All rights reserved.

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offers many advantages, such as biosafety, low cost, and easy production [6]. The nonviral vectors are categorized as cationic lipids and cationic polymers [9]. The cationic charged characteristic of nonviral vectors interacts with a negatively charged DNA via electrostatic interaction, thus forming complexes (lipoplexes or polyplexes) that can condensate and deliver DNA both in vitro and in vivo [10, 11]. Lipoplexes can be easily prepared and have low immunogenicity. Nevertheless, the toxicity effect and instability of lipoplexes have limited the usage in both in vitro and in vivo conditions [12]. Commercially available cationic lipids include DOTAP (N-1 (-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium sulfate), DOTMA (N-(1-(2, 3-dioleoyloxy) propyl) dN,N,N-trimethylammonium chloride) [10], lipofectamine, effectene, and tranfectam [13]. Polyplexes are more stable than lipoplexes, although efficiency is still low [11]. Cationic polymers are divided into two groups: synthetic polymer and natural polymer. The most popular synthetic polymers used as nonviral vectors are polyethylene imine (PEI), polyethylene glycol (PEG) conjugates, and polyamidoamine (PAMAM) dendrimers. Meanwhile, poly(L-lysine) (PLL), dextran, collagen, gelatine, and chitosan are the natural cationic polymers used in gene delivery [14]. Cationic polymers have low toxicity, low immune response, and ease in preparation and handling [15]. The third gene delivery system is via electroporation, which is considered a nonviral vector delivering technique. The process uses an electrical pulse to the cells to increase the cell permeability for DNA strands uptake, resulting in a slightly higher transfection efficiency than nonviral vectors, with 50%–70% efficiency [16]. However, the drawback of this method is that more than half of the recipient cells do not survive during the electronic simulation [3]. Due to this disadvantage, the delivery of gene using nonviral vectors, especially cationic polymers, is being intensively studied.

14.1.2 Chitosan as a cationic polymer in gene delivery Categorized as a nonviral vector, cationic polymers have been extensively studied due to their ability to condense large DNA molecules and further reduce the size into small structures [17]. The cationic polymers usually bear proton amines that will interact with the negatively charged phosphate in the DNA backbone via electrostatic interactions, thus forming complexes (polyplexes) that occur spontaneously and entropically [18]. Although the cationic molecules are easily manufactured and have low immunogenicity, there are some drawbacks such as toxicity, lack of biodegradability, and low transfection efficiency [10]. The toxicity effect can be reduced, and greater stability can be achieved by the incorporation of polysaccharides into the nonviral vectors [19]. A biodegradable polysaccharide is sought to become a gene carrier, as they are known to have low toxicity and high biocompatibility [16]. In this context, chitosan is a polysaccharide compound that is also a cationic polymer in the gene delivery system. Chitosan is used as a gene carrier due to its biodegradable natural polymer property, low immunogenicity, ability to form a complex with DNA, and ability to control release and targeting studies of therapeutic molecules [20].

Chitosan magnetic nanocomposites for gene delivery

14.2

281

Chitosan

Chitosan is one of the most abundant naturally occurring amino polysaccharides produced by alkaline deacetylation of chitin. Chitosan is a linear polymer composed of N-acetyl-D-glucosamine and D-glucosamine linked by β(1–4) glycosidic bond, as shown in Fig. 14.1. It is a very basic polysaccharide compound found in the exoskeleton and internal organs of invertebrates—primarily sourced from crustacean and insect shells [21]. Commercial chitosan is produced in many countries such as India, Poland, Japan, the USA, Norway, and Australia. The production process of chitosan from chitin involves several steps, which are deproteinization, demineralization, decoloration, and deacetylation [22].

14.2.1 Properties of chitosan Chitosan has excellent chemical and biological properties due to the presence of reactive functional groups in the molecular network, in the form of amino and hydroxyl groups (Table 14.1). The biocompatibility of chitosan means that it has the advantage of not causing an adverse reaction to human cells and is easily degraded by universal enzymes in the human body [23]. The mucoadhesive property of chitosan can increase the cellular permeability and improve the bioavailability of orally administered protein drugs. Owing to the two reactive sites present in the polymer backbone, chitosan

Fig. 14.1 General structure of chitosan.

Table 14.1 Chemical and biological properties of chitosan [24, 72]. Chemical properties l

l

l

l

Biological properties

Able to chelate transitional ions

l

Biocompatible

l

Polyelectrolyte behavior Soluble in most acid solutions Able to form films

l

Biodegradable Nontoxic Antifungal, antitumor, anticholesteremic, antimicrobial Central nervous system depressant Accelerates bone formation Mucoadhesion Able to transfect cells Permeation enhancement Low immunogenicity Analgesic and hemostatic

l

l

l

l

l

l

l

l

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is easily formed into nanoparticles and modified into various derivatives. The active amino groups give chitosan the ability to form a cationic character and protonate at low pH, thus increasing its solubility properties [24].

14.2.2 Chitosan nanoparticles for gene delivery Several studies have been reported on chitosan nanoparticles as gene delivery systems. The delivery of small interfering ribonucleic acid (siRNA) (G213) using chitosan nanoparticles has been prepared via simple complexation, followed by siRNA entrapment using ionic gelation, and adsorption of siRNA onto the chitosan surface to produce particle sizes of less than 500 nm. The effect of gene silencing is higher on Chinese hamster ovary cells (CHO K1) and lower on human kidney cells (Hek 293) [15]. Other chitosan gene carriers have been developed from PEGylation of chitosan nanoparticles in polyethylene glycol (PEG). Chitosan-DNA complexes (CS-DNA) were prepared via the polymeric dispersion method, while PEG was grafted on to the complex via complex coacervation, producing CS-DNA-PEG with a mean diameter of 92.18 and 93.86 nm, respectively. Gene transfections were performed in HepG2 cells that indicated CS-DNA-PEG transferred a higher dose of DNA compared to CS-DNA complexes [25]. A chitosan-graft-PEI copolymer was prepared by an imine reaction producing complexes with particle size less than 250 nm. The cell transfection was performed using 293 T, HeLa, and HepG2 cell lines, which exhibited good gene transfection efficiency compared to control (PEI 25 K and lipofectamine) [26]. Chitosan cross-linked with α-casein was prepared via electrostatic interaction with several formulations to produce the best-optimized formulation that exhibits homogenous particle size of around 335 nm. However, the transfection efficiency on COS-7 cells was low compared to the positive control [27]. The delivery of nucleic acid using chitosan is affected by several factors such as the molecular weight of chitosan, charge ratio between luciferase plasmid and chitosan, and the pH of the culture medium [28].

14.2.3 Properties of ideal vectors and factors affecting gene transfection Generally, an ideal gene delivery carrier should have low toxicity and immune response, and be biocompatible and biodegradable. It must be efficient in cell penetration, have high stability in protecting the DNA and its target, and possess the appropriate size and surface charge [29, 30]. The size capacity of a vector to deliver the genetic material should have no limit. An easy, sustained, and high production rate is also an ideal property of vectors for commercial purposes [31]. Moreover, a vector should be able to be modified to provide targeted gene delivery and cellular uptake [32]. Particle size is another important factor in gene delivery systems due to the need for circulation in the blood and accumulation in tumor sites. Particle size and shape are important criteria in the transfection process for fluent cellular uptake and particle

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distribution in the body. In gene therapy, the nanoparticle size needs to be smaller than 200 nm, with the minimum diameter for the first uptake in the liver being 10 nm and the maximum size not more than 200 nm [33]. Thus, the suitable nanoparticle size range is between 10 nm and 200 nm. The particle sizes are normally larger than 10 nm to avoid renal filtration and rapid permeability. Meanwhile, nanoparticles smaller than 200 nm are suitable to eliminate the reticular endothelial system phagocytosis of the spleen and liver [33–35].

14.3

Magnetic nanocomposite

Magnetic nanocomposites are comprised of magnetic nanosized particles embedded in a magnetic or nonmagnetic matrix, and are commonly applied in the proximity of a magnetic field. There are four types of magnetic nanocomposites: core-shell inorganic nanocomposite, self-assembled nanocomposite, silica-based nanocomposite, and organic-inorganic nanocomposite. The latter nanocomposite is the most studied magnetic nanocomposite due to the vital properties of both organic and inorganic components being present in one compound [36]. The significant properties of both organic and inorganic material in the nanocomposites show their importance in biomedical applications. The embedding of nonmagnetic matrices such as polymers, surfactants, inorganic material, and/or biological materials via grafting or coating (Fig. 14.2) is to avoid aggregation of the magnetic nanoparticles, to stabilize the exposed magnetic nanoparticles, and improve the functionalization of the whole compound. The magnetic nanoparticles can be absorbed by the vascular endothelial system with both body proteins and phagocytosed when entering the human body, thus making them inapplicable in biomedicine [37]. Therefore, coating the active material with functional groups such as hydroxyl and carboxyl allows the nanoparticles to bind with DNA fragments, drugs, and proteins, which is essential in targeted therapy and mediated transportation. The reactive surface of magnetic nanoparticles also enables them to be coated with biocompatible polymers and therapeutic agents, which is widely applied in biomedical applications [38].

Fig. 14.2 Grafting and coating structures of magnetic nanoparticles: (A) end-grafted polymer coating; (B) full polymer encapsulation; (C) liposome encapsulation; (D) core-shell; (E) heterodimer. Reproduced with © permission from Elsevier.

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14.3.1 Types of magnetic nanoparticles Magnetic nanoparticles can be classified into metal oxide nanoparticles, metallic nanoparticles, and bimetallic nanoparticles. Metal compounds such as iron (Fe), nickel (Ni), and cobalt (Co) exhibit ferromagnetism at low temperature and room temperature. The metal oxides are also known to have magnetic behavior, of which the iron oxides are the most important due to their wide application in biomedical and industrial fields [36]. Iron oxides have three crystalline polymorphs: namely hematite (α-Fe2O3), magnetite (ε- Fe2O3, Fe3O4), and maghemite (γFe2O3, β-Fe2O3) (Fig. 14.3) [39]. These ferrites, especially magnetite and maghemite, are widely applied in various fields of biomedical application due to their good chemical stability and low toxicity [40, 41]. In biomedical applications, the most utilized magnetic nanoparticles are iron oxides (Fe2O3 and Fe3O4) due to their excellent biocompatibility, biodegradability, low cytotoxicity, ability to bind with multiple targeted ligands or antibodies, ease of preparation, and ability to enter cells through endocytosis [34]. The most exploited metal oxide nanoparticles used in biomedicine are superparamagnetic iron oxide nanoparticles (SPIONs) due to their being a highly biocompatible compound that can be prepared in various sizes [42]. SPIONs are colloidal nanoparticles, composed of a small crystal of iron oxide (magnetite or maghemite) with the modification of their surface by capping with organic acids or coating with polymer [43]. SPIONs are divided into three categories: oral SPION (300 nm–3.5 μm), standard SPION (50–150 nm), and ultrasmall SPION (less than 50 nm). They are categorized based on the hydrodynamic size of the nanoparticles [44]. The major advantage of SPIONs is their superparamagnetic behavior, where the magnetization will disappear with the removal of the external magnetic field, thus preventing agglomeration, and maintaining colloidal stability [45, 46]. Metallic nanoparticles are made of Fe, Ni, and Co, usually coated with gold or silica to form a core-shell structure and ready formed oxides in the presence of water and oxygen. However, these nanoparticles are chemically unstable and therefore they are less used in biological applications [47]. Bimetallic or metal alloys also exhibit superparamagnetic properties, but are rarely used as core material unless they have a protective inert coating applied, such as platinum (Pt) [48].

14.3.2 Preparation of magnetic nanoparticles General preparation of magnetic particles can be achieved via mechanical attrition and chemical synthesis. The commonly used approach is via chemical synthesis, which is categorized as coprecipitation, thermal decomposition and/or reduction, hydrothermal/solvothermal synthesis, and microemulsion [49]. The chemical synthesis approach is better than mechanical attrition due to the production of nanoparticles with uniform composition and size (Figs. 14.4). The coprecipitation method is a convenient and widely used method to synthesize magnetic nanoparticles due to ease of production, low cost, and less usage of dangerous materials. The synthesizing method involves the addition of a base into aqueous salt solutions (Fe2+/Fe3+) under an inert atmosphere at room temperature or high

Fig. 14.3 Crystal structure and crystallographic data of: (A) hematite; (B) magnetite; (C) maghemite (black is Fe2+, green is Fe3+, and red is O2
). Reproduced with © permission from IOP Publishing.

Fig. 14.4 Fe3O4 nanoparticles images via TEM, prepared by: (A, B) coprecipitation method; (C–F) thermal decomposition method; and (G, H) solvothermal method. Reproduced with © permission from MDPI.

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temperature, which forms Fe3O4 by nucleation and growth mechanism. The chemical reaction formation of iron oxides nanoparticles in an aqueous medium is shown in Eq. (14.1) [50]. The size, shape, and composition of the magnetic nanoparticles is affected by the type of salt used (e.g., chlorides, sulfates, nitrates), ferric and ferrous ion ratio, reaction temperature, pH value, and media ionic strength [51]. The advantages of this method are the ability to produce fine, high-purity, stoichiometric particles, and multicomponents of metal oxides [52]. Fe2 + + 2Fe3 + + 8OH ! FeðOHÞ2 + 2FeðOHÞ3 ! Fe2 O4 + 4H2 O

(14.1)

The thermal decomposition method can produce a highly monodispersed Fe3O4 nanoparticle of smaller size (below 30 nm) characterized by high crystallinity and very narrow size distributions. The process involves heating the organometallic precursor in organic solvents and surfactants to a given temperature until the nanoparticles start to cluster and grow [53]. The monodispersed distributions of nanoparticles can be achieved by controlling the nucleation and growth steps with the presence of surfactant [54]. In this process, the precursors used are acetylacetonate (M(acac)n, where M ¼ Fe, Mn, Co, Ni, Cr; n ¼ 2 or 3, acac ¼ acetylacetonate), iron oleate, and carbonyl iron, while the surfactants are fatty acids (decanoic acid, oleic acid, stearic acid, lauric acid), and the solvents are octadecene and tetracosane [55]. The hydrothermal or solvothermal method is a process to synthesize inorganic nanocrystal from aqueous solution at relatively high temperature and high pressure in a sealed pressure vessel. The process involves dissolving and recrystallizing poorly soluble or insoluble substance at ordinary temperature and pressure (