Advances in Biomedical Polymers and Composites: Materials and Applications 9780323885249

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Advances in Biomedical Polymers and Composites Materials and Applications

Advances in Biomedical Polymers and Composites Materials and Applications Edited by

Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, India

Sarika Verma Materials for Radiation Shielding and Cement Free Concrete Division, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Bhopal, India

Pallab Datta Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India

Ananya Barui Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, India

S. A. R. Hashmi Integrated Approach for Design and Product Development Devision, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Bhopal, India

Avanish Kumar Srivastava CSIR-Advanced Materials and Processes Research Institute, Bhopal, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2023 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. ISBN: 978-0-323-88524-9 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisitions Editor: Edward Payne Editorial Project Manager: Sara Greco Production Project Manager: Surya Narayanan Jayachandran Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Contents List of contributors .................................................................................................xxi

CHAPTER 1 Introduction to biomedical polymer and composites .................................................................... 1 1.1 1.2 1.3

1.4

1.5 1.6

Soham Chowdhury, Adhish Singh and Bidyut Pal Introduction ....................................................................................1 Classification of polymers and composites ...................................2 Fabrication techniques polymer composites..................................3 1.3.1 Electrospinning ................................................................... 4 1.3.2 Melt extrusion ..................................................................... 4 1.3.3 Solution mixing................................................................... 4 1.3.4 Latex technology................................................................. 6 Polymers and their composites for biomedical applications.........7 1.4.1 Natural polymers and their composites .............................. 7 1.4.2 Synthetic polymers and their composites......................... 13 1.4.3 Gas-permeable polymeric membranes ............................. 18 1.4.4 Other polymeric composites ............................................. 19 Challenges and future trends........................................................22 Conclusion ....................................................................................23 References.................................................................................... 24

CHAPTER 2 Foundation of composites........................................... 31 2.1 2.2 2.3

2.4 2.5

Umesh Kumar Dwivedi and Neelam Kumari Introduction ..................................................................................31 Classification of composites ........................................................32 History of composites ..................................................................33 2.3.1 Fiberglass in 20th century................................................. 34 2.3.2 Composite material in our daily life ................................ 35 Why composites? .........................................................................35 Advantages of composites ...........................................................35 2.5.1 Design flexibility ............................................................ 35 2.5.2 Light weight .................................................................... 36 2.5.3 High strength................................................................... 36 2.5.4 Strength related to weight............................................... 36 2.5.5 Corrosion resistance........................................................ 36 2.5.6 High-impact strength ...................................................... 36 2.5.7 Consolidation of many parts........................................... 37 2.5.8 Dimensional stability ...................................................... 37

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2.6

2.7 2.8

2.9

2.10

2.11

2.5.9 Nonconductive ................................................................ 37 2.5.10 Nonmagnetic ................................................................... 37 2.5.11 Radar transparent ............................................................ 37 2.5.12 Low thermal conductivity............................................... 38 2.5.13 Durable ............................................................................ 38 Applications of composites..........................................................38 2.6.1 Aerospace/aircrafts............................................................ 38 2.6.2 Appliances......................................................................... 38 2.6.3 Automobile and transportation ......................................... 38 2.6.4 Infrastructure ..................................................................... 38 2.6.5 Environmental ................................................................... 39 2.6.6 Applications of electricity................................................. 39 Limitation of composites .............................................................39 Biocomposites and classification .................................................40 2.8.1 Biomedical composites ..................................................... 40 2.8.2 Basic requirements and parameters for biomedical applications ....................................................................... 40 2.8.3 Biomedical polymer composites....................................... 42 Applications of biocomposites.....................................................43 2.9.1 Tissue engineering ............................................................ 45 2.9.2 Orthopedic......................................................................... 46 2.9.3 Dental ................................................................................ 47 2.9.4 External prosthetic and orthotics ...................................... 49 2.9.5 Biocompatibility on skin................................................... 51 2.9.6 Healing of fracture and wound dressing .......................... 51 Fabrication techniques of biomedical composites.......................54 2.10.1 Hand layup molding........................................................ 54 2.10.2 Open contact molding method........................................ 54 2.10.3 Liquid molding and injection molding........................... 55 2.10.4 Vacuum resin transfer molding process ......................... 55 2.10.5 Compression molding ..................................................... 56 2.10.6 Tube rolling..................................................................... 57 2.10.7 Automated fiber/tape placement process........................ 57 Conclusion ....................................................................................58 References.................................................................................... 58

CHAPTER 3 Biopolymer-based composites for drug delivery applications—a scientometric analysis .................... 61 Kunal Pal, Deepti Bharti, Preetam Sarkar and Doman Kim 3.1 Introduction ..................................................................................61

Contents

3.2 Scientometric analysis..................................................................62 3.2.1 Coauthorship analysis ....................................................... 64 3.2.2 Cooccurrence analysis ...................................................... 66 3.2.3 Analysis of the citations of the articles ............................ 75 3.3 Conclusion ....................................................................................80 References.................................................................................... 80

CHAPTER 4 Characteristics and characterization techniques of bacterial cellulose for biomedical applications—a short treatise ................................... 83 Kumar Anupam, Richa Aggrawal, Jitender Dhiman, Priti Shivhare Lal, Thallada Bhaskar and Dharm Dutt 4.1 Introduction ..................................................................................83 4.2 Biomedical applications of bacterial cellulose ............................84 4.2.1 Wound healing applications ........................................... 86 4.2.2 Diagnosis of ovarian cancer ........................................... 93 4.2.3 Shape memory material .................................................. 93 4.2.4 Preventing deterioration of salmon muscle and slowing down the lipid oxidation ................................... 94 4.2.5 Lipase immobilization .................................................... 94 4.2.6 Tissue engineering .......................................................... 94 4.2.7 Implantable devices in regenerative medicine ............... 98 4.2.8 Drug delivery .................................................................. 99 4.2.9 Bone healing ................................................................. 100 4.2.10 Wound dressing............................................................. 100 4.3 Conclusion ..................................................................................105 References.................................................................................. 106

CHAPTER 5 Engineering scaffolds for tissue engineering and regenerative medicine.............................................. 109 5.1 5.2 5.3

5.4

Ibrahim Fatih Cengiz, Rui L. Reis and Joaquim Miguel Oliveira Introduction ................................................................................109 Scaffolds properties and characterization ..................................110 Fabrication of scaffolds..............................................................114 5.3.1 Scaffold fabrication methods .......................................... 114 5.3.2 Patient-specific scaffolds ................................................ 117 Conclusion ..................................................................................120 Acknowledgments ..................................................................... 120 Declaration of conflict of interest ............................................. 121 References.................................................................................. 121

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CHAPTER 6 Recent trends in polymeric composites and blends for three-dimensional printing and bioprinting ....... 131 6.1 6.2 6.3

6.4

6.5 6.6 6.7

Sriya Yeleswarapu, K.N. Vijayasankar, Shibu Chameettachal and Falguni Pati Introduction ................................................................................131 Need of synergistic approach in polymeric materials ...............132 Blends and composites of natural and synthetic polymers .......133 6.3.1 Synthetic polymers based composites .......................... 134 6.3.2 Natural polymers based composites ............................. 135 3D printing techniques employed to print polymeric materials .....................................................................................138 6.4.1 Extrusion-based 3D printing ........................................... 139 6.4.2 Vat polymerization.......................................................... 140 6.4.3 Powder bed fusion........................................................... 141 6.4.4 Laser-assisted bioprinting ............................................... 143 Application of value-added polymers........................................143 Current challenges and possible solutions.................................145 Conclusion ..................................................................................147 References.................................................................................. 147

CHAPTER 7 Polymers for additive manufacturing and 4D-printing for tissue regenerative applications .... 159 7.1 7.2

7.3

7.4

Bhuvaneshwaran Subramanian, Pratik Das, Shreya Biswas, Arpita Roy and Piyali Basak Introduction ................................................................................159 Polymers for 4D printing ...........................................................161 7.2.1 Hydrogels ........................................................................ 162 7.2.2 Shape memory polymers ................................................ 163 7.2.3 Elastomer actuators ......................................................... 164 7.2.4 Thermoresponsive polymers ........................................... 165 Application of 4D printing technology......................................166 7.3.1 Engineered tissue constructs........................................... 166 7.3.2 Medical devices .............................................................. 169 7.3.3 Drug delivery implants ................................................... 170 Conclusion ..................................................................................178 Reference ................................................................................... 178

CHAPTER 8 Bioprinting of hydrogels for tissue engineering and drug screening applications ............................. 183 Ece O¨zmen, O¨zu¨m Yıldırım and Ahu Arslan-Yıldız 8.1 Advancements in bioprinting technology ..................................183

Contents

8.2 Bioinks........................................................................................186 8.3 Hydrogel bioinks ........................................................................188 8.4 Applications of hydrogel bioinks...............................................193 8.4.1 Bone tissue engineering .................................................. 198 8.4.2 Cartilage tissue engineering............................................ 198 8.4.3 Cardiac tissue engineering .............................................. 203 8.4.4 Skin tissue engineering ................................................... 203 8.4.5 Vascular tissue engineering ............................................ 204 8.4.6 Neural tissue engineering................................................ 205 8.4.7 Drug screening ................................................................ 206 8.5 Challenges of bioprinted hydrogels in tissue engineering and drug screening .....................................................................209 8.6 Conclusion and future perspectives ...........................................210 References.................................................................................. 211

CHAPTER 9 Smart polymers for biomedical applications........... 223 9.1 9.2 9.3 9.4 9.5 9.6 9.7

Deepti Bharti, Indranil Banerjee, Preetam Sarkar, Doman Kim and Kunal Pal Introduction ................................................................................223 Temperature-sensitive smart polymers ......................................225 Applications of temperature-sensitive smart polymers .............226 pH-sensitive smart polymers......................................................228 9.4.1 Applications .................................................................... 230 Photosensitive polymers.............................................................232 9.5.1 Applications .................................................................... 234 Enzyme-responsive polymers ....................................................237 9.6.1 Applications .................................................................... 238 Conclusion ..................................................................................240 References.................................................................................. 241

CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery...................................................................... 247 10.1 10.2 10.3 10.4 10.5

Kunal Pal, Bikash K. Pradhan, Doman Kim and Maciej Jarze˛bski Introduction ................................................................................247 Anatomy and protection mechanism of eye ..............................248 Properties of chitosan.................................................................250 Some recent applications of chitosan nanoparticles in ocular delivery............................................................................254 Conclusion ..................................................................................257 References.................................................................................. 258

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CHAPTER 11 Appraisal of conducting polymers for potential bioelectronics ........................................................... 265 11.1 11.2 11.3 11.4 11.5 11.6 11.7

Rimita Dey and Pallab Datta Introduction ................................................................................265 Sensors and actuators used on conducting polymers ................267 Energy storage from conducting polymer .................................272 Energy harvesting based on polymer.........................................279 Organic light-emitting diodes ....................................................283 Electrochromic materials and devices .......................................285 Conclusions ................................................................................289 References.................................................................................. 289

CHAPTER 12 Shape-memory polymers .......................................... 299 Deepshikha Rathore 12.1 Introduction ................................................................................299 12.2 Various shape-memory polymers ..............................................300 12.2.1 Cross-linking ................................................................. 300 12.2.2 Thermal transitions ....................................................... 301 12.2.3 Categorization of shape-memory polymers.................. 302 12.3 Mechanism of shape-memory polymers....................................302 12.4 Composites using shape-memory polymers ..............................303 12.4.1 Functionalization of shape-memory polymers by silicate ........................................................................... 304 12.4.2 Functionalization of shape-memory polymers by magnetic particles .................................................... 304 12.4.3 Functionalization of shape-memory polymers by carbon fillers ................................................................. 306 12.4.4 Functionalization of shape-memory polymers by biocompatible materials........................................... 307 12.5 Limitations of shape-memory polymers ....................................308 12.5.1 Recovery time and activation process .......................... 308 12.5.2 Recovery force and work capacity ............................... 310 12.6 Conclusion ..................................................................................312 References.................................................................................. 312

CHAPTER 13 Rapid prototyping...................................................... 315 Umesh K. Dwivedi, Shashank Mishra and Vishal Parashar 13.1 Introduction ................................................................................315 13.2 Preprocessing, the process, and postprocessing in rapid prototyping .................................................................................319 13.2.1 Preprocessing ................................................................ 319

Contents

13.3 13.4 13.5

13.6

13.2.2 The process ................................................................... 320 13.2.3 Postprocessing ............................................................... 320 Contemporary rapid prototyping systems..................................321 13.3.1 Available rapid prototyping systems ............................ 323 Applications................................................................................329 Advancements in the rapid prototyping technology..................334 13.5.1 Improvement of product quality ................................... 335 13.5.2 Improvement on versatility of rapid prototyping ......... 335 13.5.3 Multifunctional fabrication process.............................. 336 13.5.4 Printable and embeddable functions............................. 336 13.5.5 Fiber-reinforced polymer composites........................... 337 13.5.6 Functionally graded materials using rapid prototyping .................................................................... 338 13.5.7 Comparison with traditional manufacturing................. 338 Conclusion ..................................................................................339 References.................................................................................. 339

CHAPTER 14 Self-assembled polymer nanocomposites in biomedical applications ........................................... 343 14.1 14.2

14.3

14.4

Anurag Dutta, Manash Jyoti Baruah, Satyabrat Gogoi and Jayanta Kumar Sarmah Introduction ................................................................................343 Methods of preparation of self-assembled polymer nanocomposites ..........................................................................345 14.2.1 Polymer grafting on/from the modified surface of nanoparticles ................................................................. 346 14.2.2 Layer-by layer assembly technique .............................. 347 Applications of the self-assembled polymer nanocomposites in biomedical science ......................................350 14.3.1 Drug delivery ................................................................ 350 Future prospects and conclusion................................................356 References.................................................................................. 357

CHAPTER 15 Thermoresponsive polymers and polymeric composites ................................................................ 363 Mh Busra Fauzi, Samantha Lo, Maheswary Thambirajoo, Zawani Mazlan, Izzat Zulkiflee, Syafira Masri, Isma Liza Mohd Isa and Sabarul Afian Mokhtar 15.1 Introduction ................................................................................363 15.1.1 Thermoresponsive polymers ......................................... 363 15.1.2 Thermoresponsive polymeric composites .................... 364 15.2 Mechanisms ................................................................................366

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15.3

15.4

15.5 15.6

15.2.1 Protein adsorption ......................................................... 366 15.2.2 Cells adhesion and attachments.................................... 369 15.2.3 Thermoresponsive behaviors ........................................ 371 Form of thermoresponsive polymers and polymeric composites ..................................................................................374 15.3.1 Hydrogels ...................................................................... 375 15.3.2 Nanoparticles................................................................. 376 15.3.3 Micelles ......................................................................... 377 15.3.4 Films.............................................................................. 378 15.3.5 Interpenetrating networks ............................................. 379 15.3.6 Polymersomes ............................................................... 380 Applications of thermoresponsive polymers .............................381 15.4.1 Vascular applications .................................................... 381 15.4.2 Gene delivery ................................................................ 383 15.4.3 Drug delivery ................................................................ 384 15.4.4 Wound healing .............................................................. 385 Future perspectives.....................................................................388 Conclusion ..................................................................................390 References.................................................................................. 390 Further reading .......................................................................... 397

CHAPTER 16 Ceramic particle dispersed polymer composites ................................................................ 399 16.1 16.2

16.3

16.4 16.5

16.6

Bhabatosh Biswas, Gurudas Mandal, Apurba Das, Abhijit Majumdar and Arijit Sinha Introduction ................................................................................399 Matrices used in ceramic particle dispersed polymer composites ..................................................................................401 16.2.1 Biodegradable matrices................................................. 401 16.2.2 Nonbiodegradable matrices .......................................... 404 Reinforcements used in ceramic particle reinforced composites ..................................................................................405 16.3.1 Reinforcement from natural resources ......................... 405 16.3.2 Reinforcements from synthetic resources .................... 405 Fabrication of ceramic particulate dispersed composites........406 16.4.1 Methods of composite fabrication ................................ 406 Curing of the composites ...........................................................411 16.5.1 Room-temperature curing ............................................. 411 16.5.2 High-temperature curing ............................................... 411 Different types of ceramic particle dispersed composites ..................................................................................412

Contents

16.6.1 Particulate-reinforced composites................................. 412 16.6.2 Hybrid composites ........................................................ 415 16.7 Characterization..........................................................................416 16.7.1 Structural properties...................................................... 416 16.7.2 Charpy impact strength test .......................................... 418 16.7.3 Atomic force microscopy.............................................. 419 16.8 Summary.....................................................................................424 References.................................................................................. 424

CHAPTER 17 Electrospinning for biomedical applications........... 433 17.1

17.2

17.3

17.4

17.5

Srividya Hanuman, Steffi Zimran, Manasa Nune and Goutam Thakur Introduction ................................................................................433 17.1.1 Theory of electrospinning............................................. 433 17.1.2 Principle of electrospinning .......................................... 434 Parameters influencing fiber production ...................................436 17.2.1 System parameters ........................................................ 437 17.2.2 Solution parameters ...................................................... 438 17.2.3 Ambient parameters ...................................................... 440 Polymers for fabrication of electrospun fibers ..........................440 17.3.1 Synthetic polymers........................................................ 440 17.3.2 Natural polymers........................................................... 444 17.3.3 Composite and hybrid ................................................... 449 Applications of electro-spun fibers in tissue engineering applications.................................................................................450 17.4.1 Use of electro-spun polymers in neural tissue engineering .................................................................... 450 Conclusion ..................................................................................455 References.................................................................................. 455 Further reading .......................................................................... 463

CHAPTER 18 Advances in biomedical polymers and composites: Drug delivery systems ..................................................... 465 Aalok Basu and Amit Kumar Nayak 18.1 Introduction ................................................................................465 18.2 Synthesis of polymer composites ..............................................466 18.2.1 Hydrothermal method ................................................... 466 18.2.2 In situ polymerization ................................................... 467 18.2.3 Electrospinning method ................................................ 468 18.2.4 Three-dimensional printing technology........................ 469

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18.3 Characterization and drug release properties ............................470 18.3.1 X-ray diffraction ........................................................... 470 18.3.2 Fourier transform infrared spectroscopy ...................... 471 18.3.3 Thermal analysis ........................................................... 472 18.3.4 Scanning electron microscopy ...................................... 472 18.3.5 Determination of drug loading into composites........... 473 18.3.6 Estimation of drug release from composites................ 473 18.3.7 Mathematical treatment of drug release kinetics ......... 474 18.3.8 Mechanisms for controlling drug release from composites..................................................................... 476 18.4 Applications in drug delivery ....................................................477 18.4.1 Tumor-targeted drug therapy ........................................ 478 18.4.2 Ophthalmic drug delivery ............................................. 479 18.4.3 Buccal drug delivery..................................................... 479 18.4.4 Drug delivery for bone tissue regeneration .................. 483 18.5 Conclusion and future perspectives ...........................................484 References.................................................................................. 485

CHAPTER 19 Natural gums of plant and microbial origin for tissue engineering applications ......................... 497 Kunal Pal, Deepti Bharti, Goutam Thakur and Doman Kim 19.1 Introduction ................................................................................497 19.2 Scientometric analysis................................................................499 19.3 Natural gums ..............................................................................500 19.3.1 Gellan gum.................................................................... 501 19.3.2 Xanthan gum ................................................................. 504 19.3.3 Guar gum....................................................................... 506 19.4 Conclusion ..................................................................................508 References.................................................................................. 509

CHAPTER 20 Polymers and nanomaterials as gene delivery systems...................................................................... 513 Sundara Ganeasan M, Amulya Vijay, M. Kaviya, Anandan Balakrishnan and T.M. Sridhar 20.1 Introduction ................................................................................513 20.2 Types of gene delivery...............................................................514 20.2.1 Germline gene therapy.................................................. 515 20.2.2 Somatic gene therapy.................................................... 516 20.3 Methods and techniques used in gene delivery.........................517 20.3.1 Nanoparticle gene delivery systems ............................. 517 20.3.2 Liposome gene delivery systems.................................. 520

Contents

20.3.3 Microbubble gene delivery systems ............................. 521 20.3.4 Viral and nonviral gene delivery systems .................... 523 20.4 Polymers and bioceramics for gene delivery ............................526 20.4.1 Natural polymer chitosan.............................................. 527 20.4.2 Synthetic polymers........................................................ 527 20.5 Applications of gene delivery ....................................................531 20.5.1 Cancer............................................................................ 531 20.5.2 Cardiovascular............................................................... 532 20.5.3 Kidney ........................................................................... 533 20.5.4 Bone .............................................................................. 534 20.6 Conclusion ..................................................................................535 Acknowledgment ....................................................................... 536 References.................................................................................. 536

CHAPTER 21 Essential oil-loaded biopolymeric films for wound healing applications ..................................... 541 21.1 21.2 21.3

21.4

Kunal Pal, Preetam Sarkar, Goutam Thakur and Doman Kim Introduction ................................................................................541 Wound healing physiology ........................................................542 Essential oils...............................................................................546 21.3.1 Mechanisms of promoting wound healing by essential oils .................................................................. 550 21.3.2 Methods of preparation of essential oil-loaded films............................................................................... 552 21.3.3 Essential oil-loaded biopolymeric films for wound healing applications ...................................................... 554 Conclusion ..................................................................................557 References.................................................................................. 558

CHAPTER 22 Biomedical antifouling polymer nanocomposites...... 563 Javad B.M. Parambath, Mahreen Arooj and Ahmed A. Mohamed 22.1 Introduction ................................................................................563 22.2 Mechanism of antifouling ..........................................................567 22.2.1 Strategies of antifouling................................................ 567 22.2.2 Natural antifouling ........................................................ 568 22.3 Biomedical antifouling...............................................................569 22.3.1 Nanogel engineering ..................................................... 569 22.3.2 Zwitterionic nanomaterials ........................................... 573 22.3.3 Superhydrophobic surfaces and wettability.................. 576

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22.4 Computational studies ................................................................577 22.4.1 Exploring the antifouling properties of polymers using computational methods ....................................... 577 22.4.2 Effect of surface hydration on antifouling properties....................................................................... 578 22.4.3 Polyzwitterions.............................................................. 579 22.5 Conclusion ..................................................................................581 Acknowledgments ..................................................................... 582 References.................................................................................. 582

CHAPTER 23 Application of antiviral activity of polymer ............. 591 Shradha Sharma, Howa Begam and Ananya Barui 23.1 Introduction ................................................................................591 23.2 Types of antiviral polymers .......................................................592 23.2.1 Polysaccharides ............................................................. 592 23.2.2 Antiviral peptide polymer............................................. 595 23.2.3 Nucleic acid polymers .................................................. 597 23.2.4 Polymer-drug conjugates .............................................. 597 23.2.5 Metal containing polymers ........................................... 599 23.2.6 Dendrimers .................................................................... 599 23.3 Application of antiviral polymers ..............................................601 23.3.1 Drug delivery system .................................................... 601 23.3.2 Polymers in protective application ............................... 603 23.3.3 Food packaging ............................................................. 607 23.4 Concluding remarks ...................................................................609 References.................................................................................. 610

CHAPTER 24 Biosensor: fundamentals, biomolecular component, and applications ................................... 617

24.1 24.2 24.3 24.4 24.5 24.6 24.7

Manoj Kumar Tripathi, C. Nickhil, Adinath Kate, Rahul M. Srivastva, Debabandya Mohapatra, Rajpal S. Jadam, Ajay Yadav and Bharat Modhera Introduction ................................................................................617 Fundamentals of biosensor.........................................................618 24.2.1 Principle of biosensor ................................................... 618 Classification of the biosensors .................................................618 Characteristics of the biosensors................................................621 Biopolymers for the development of biosensors .......................622 24.5.1 Biopolymer composites ................................................ 623 Biomolecular component of biosensor ......................................625 Recent trends in biosensors........................................................626

Contents

24.8 Recent applications of biosensors..............................................627 24.9 Merits and limitation of biosensors ...........................................630 References.................................................................................. 630

CHAPTER 25 Polymeric materials in microbial cell encapsulation............................................................ 635 25.1 25.2

25.3

25.4 25.5

Memoona Akhtar, Muhammad Farrukh Sarfraz, Samra Fatima and Muhammad Atiq Ur Rehman Introduction ................................................................................635 Encapsulation method ................................................................637 25.2.1 Nanoprecipitation .......................................................... 638 25.2.2 Emulsification ............................................................... 638 25.2.3 Coacervation ................................................................. 639 25.2.4 Capillary encapsulation method ................................... 639 25.2.5 Electrospinning ............................................................. 640 25.2.6 Layer-by-layer self-assembly method .......................... 642 25.2.7 Spray drying .................................................................. 643 Applications................................................................................644 25.3.1 Intestinal tract health..................................................... 644 25.3.2 Bioavailability and nutrient synthesis .......................... 645 25.3.3 Probiotics’ antimicrobial potential ............................... 646 25.3.4 Cancer prevention ......................................................... 646 25.3.5 Tissue engineering ........................................................ 647 25.3.6 Methylene blue dye remediation from water ............... 647 25.3.7 In Agriculture and the food processing ........................ 647 25.3.8 Drug delivery ................................................................ 648 Conclusion ..................................................................................649 Future considerations .................................................................649 References.................................................................................. 650

CHAPTER 26 Carbon nanotubes based composites for biomedical applications ........................................... 657 Sarika Verma, Ramesh Rawat, Vaishnavi Hada, Ram Krishna Shrivastava, Kunal Pal, Sai S. Sagiri, Medha Mili, S. A. R. Hashmi and A.K. Srivastava 26.1 Introduction ................................................................................657 26.2 Carbon nanotube based composites for biomedical applications.................................................................................659 26.2.1 Carbon nanotube nanocomposites for biosensors ........ 660 26.2.2 Carbon nanotube nanocomposites for drug delivery.......................................................................... 661

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26.2.3 Carbon nanotube nanocomposites for cancer treatment........................................................................ 663 26.2.4 Carbon nanotube nanocomposites for tissue engineering .................................................................... 663 26.3 Toxicity of carbon nanotubes ....................................................665 26.4 Future prospective ......................................................................665 26.5 Conclusion ..................................................................................666 Acknowledgment ....................................................................... 667 Conflict of interest..................................................................... 667 References.................................................................................. 667

CHAPTER 27 Cryogels as smart polymers in biomedical applications............................................................... 675 27.1 27.2 27.3 27.4 27.5 27.6 27.7

27.8

O¨zlem Bic¸en U¨nlu¨er, Ru¨stem Kec¸ili, Rıdvan Say and Arzu Erso¨z Introduction ................................................................................675 What is cryogel?.........................................................................676 Cryogel preparation method.......................................................677 The precursors in cryogel preparation .......................................681 The cross-linking strategy in cryogel preparation.....................682 Characterization of cryogels ......................................................683 The biomedical applications of the cryogels.............................686 27.7.1 Cryogels in bioseparation process ................................ 687 27.7.2 Cryogels in wound dressing applications ..................... 690 27.7.3 Cryogels in tissue engineering applications ................. 692 27.7.4 Cryogels in drug release applications........................... 700 Conclusion ..................................................................................704 References.................................................................................. 704

CHAPTER 28 Naturally derived ceramics polymer composite for biomedical applications ..................................... 711 E. Shinyjoy, S. Ramya, P. Saravanakumar, P. Manoravi, L. Kavitha and D. Gopi 28.1 Introduction ................................................................................711 28.2 Preparation of biogenic-derived biocomposites ........................716 28.2.1 Materials........................................................................ 716 28.2.2 Various biocomposites from biowaste materials ......... 716 28.2.3 Zinc-substituted hydroxyapatite/cellulose nanocrystals biocomposite ............................................ 717 28.2.4 Hydroxyapatite reinforced with polyvinylpyrrolidone/aloe vera biocomposite .............. 718

Contents

28.2.5 Hydroxyapatite/carboxymethyl cellulose/sodium alginate biocomposite ................................................... 718 28.2.6 Characterization ............................................................ 718 28.3 Results and discussion................................................................721 28.3.1 Egg shell derived hydroxyapatite/cellulose nanocrystals biocomposite ............................................ 722 28.3.2 Crab shell extracted hydroxyapatite/poly (vinylpolypyrrolidone)/aloe vera biocomposite ........... 725 28.3.3 Fish bone derived hydroxyapatite/biopolymer composite ...................................................................... 730 28.4 Conclusion ..................................................................................739 Acknowledgments ..................................................................... 739 References.................................................................................. 740

CHAPTER 29 Molecularly imprinted polymers (MIPs) for biomedical applications ........................................... 745 29.1 29.2

29.3

29.4

Ru¨stem Kec¸ili, O¨zlem Bic¸en U¨nlu¨er, Arzu Erso¨z and Rıdvan Say Introduction ................................................................................745 Molecular imprinting technology ..............................................746 29.2.1 Key parameters for the preparation of molecularly imprinted polymers ....................................................... 746 29.2.2 Approaches for the preparation of molecularly imprinted polymers ....................................................... 749 Applications of molecularly imprinted polymers in biomedical science .....................................................................751 29.3.1 Drug delivery ................................................................ 751 29.3.2 Bio-imaging and cancer therapy................................... 755 29.3.3 Sensing and separation processes ................................. 758 Conclusions and future perspectives..........................................762 References.................................................................................. 763

CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery in the medical field .................................... 769 Ana Mafalda Pinto, Marisol Dias, Lorenzo M. Pastrana, Miguel A. Cerqueira and Sanna Sillankorva 30.1 Introduction ................................................................................769 30.2 Phage therapy .............................................................................769 30.2.1 Regulatory approval of phage therapy ......................... 773 30.2.2 Phage application in medicine...................................... 773

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30.3 Bacteriophage encapsulation......................................................776 30.3.1 Encapsulation of phages in natural polymers............... 777 30.3.2 Phage encapsulation for wound healing applications ................................................................... 780 30.3.3 Phage encapsulation to prevent and manage gastrointestinal diseases ................................................ 782 30.4 Conclusions and future perspectives..........................................784 Funding ...................................................................................... 786 References.................................................................................. 786 Index ......................................................................................................................795

List of contributors Richa Aggrawal Department of Chemical Engineering, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Haryana, India Memoona Akhtar Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Kumar Anupam Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India; Chemical Recovery and Biorefinery Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India Mahreen Arooj Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Ahu Arslan-Yıldız Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey Anandan Balakrishnan Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, Chennai, Tamil Nadu, India Indranil Banerjee Department of Bioscience & Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India Manash Jyoti Baruah Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India Ananya Barui Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Piyali Basak School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Aalok Basu Department of Pharmaceutics, Dr. BC Roy College of Pharmacy and Allied Health Sciences, Durgapur, West Bengal, India Howa Begam Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Deepti Bharti Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India

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Thallada Bhaskar Material Resource Efficiency Division, CSIR-Indian Institute of Petroleum, Dehradun, Uttarakhand, India; Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India O¨zlem Bic¸en U¨nlu¨er Faculty of Sciences, Chemistry Department, Eskis¸ehir Technical University, Eskis¸ehir, Turkey Bhabatosh Biswas Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, Delhi, India Shreya Biswas School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Ibrahim Fatih Cengiz 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal Miguel A. Cerqueira INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Shibu Chameettachal Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Soham Chowdhury Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Apurba Das Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Pratik Das School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Pallab Datta Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India Rimita Dey Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Jitender Dhiman Biotechnology Division, Central Pulp, and Paper Research Institute, Saharanpur, Uttar Pradesh, India

List of contributors

Marisol Dias INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Dharm Dutt Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India Anurag Dutta Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India; Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India Umesh K. Dwivedi Amity School of Applied Sciences, Amity University Jaipur, Jaipur, Rajasthan, India Umesh Kumar Dwivedi Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India Arzu Erso¨z Chemistry Department, Faculty of Science, Eskis¸ehir Technical University, Eskis¸ehir, Turkey; Bionkit Co Ltd., Eskis¸ehir, Turkey Samra Fatima Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Mh Busra Fauzi Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Sundara Ganeasan M Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India Satyabrat Gogoi Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India D. Gopi Department of Chemistry, Periyar University, Salem, Tamil Nadu, India Vaishnavi Hada Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India Srividya Hanuman Manipal Institute of Regenerative Medicine, Bangalore, Karnataka, India; Manipal Academy of Higher Education, Manipal, Karnataka, India S. A. R. Hashmi Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India

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Rajpal S. Jadam ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Maciej Jarze˛bski Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznan´ University of Life Sciences, Poznan´, Poland Adinath Kate ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India L. Kavitha Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India M. Kaviya Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India Ru¨stem Kec¸ili Department of Medical Services and Techniques, Yunus Emre Vocational School of Health Services, Anadolu University, Eskis¸ehir, Turkey Doman Kim Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea Neelam Kumari Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India Priti Shivhare Lal Physical Chemistry, Pulping and Bleaching Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India Samantha Lo Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Abhijit Majumdar Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, Delhi, India Gurudas Mandal Department of Metallurgical Engineering, Kazi Nazrul University, Asansol, West Bengal, India P. Manoravi Materials Chemistry and Metal Fuel Cycle Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamil Nadu, India Syafira Masri Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

List of contributors

Zawani Mazlan Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Medha Mili Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Shashank Mishra Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India Bharat Modhera Department of Biotechnology, MANIT, Bhopal, Madhya Pradesh, India Ahmed A. Mohamed Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Debabandya Mohapatra ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Isma Liza Mohd Isa Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Sabarul Afian Mokhtar Department of Orthopaedics and Traumatology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Amit Kumar Nayak Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Odisha, India C. Nickhil Department of Food Engineering & Technology, Tezpur University, Tezpur, Assam, India Manasa Nune Manipal Institute of Regenerative Medicine, Bangalore, Karnataka, India; Manipal Academy of Higher Education, Manipal, Karnataka, India Joaquim Miguel Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal Ece O¨zmen Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey

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Bidyut Pal Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India Javad B.M. Parambath Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Vishal Parashar Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India Lorenzo M. Pastrana INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Falguni Pati Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Ana Mafalda Pinto Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, Portugal; INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Bikash K. Pradhan Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India S. Ramya Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Deepshikha Rathore Amity School of Applied Sciences, Amity University Rajasthan, Jaipur, Rajasthan, India Ramesh Rawat Department of Chemistry, Institute for Excellence in Higher Education, Bhopal, Madhya Pradesh, India Muhammad Atiq Ur Rehman Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Rui L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal

List of contributors

Arpita Roy Polymer Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology (ISM), Dhanbad, Jharkhand, India Sai S. Sagiri Department of Food Science, Agricultural Research Organization, AgroNanotechnology and Advanced Materials Research Center, the Volcani Institute, Rishon Lezion, Israel P. Saravanakumar Department of Chemistry, Periyar University, Salem, Tamil Nadu, India Muhammad Farrukh Sarfraz Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Preetam Sarkar Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India Jayanta Kumar Sarmah Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India Rıdvan Say Department of Chemistry, Faculty of Sciences, Anadolu University, Eskis¸ehir, Turkey; Bionkit Co Ltd., Eskis¸ehir, Turkey Shradha Sharma Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India E. Shinyjoy Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Ram Krishna Shrivastava Department of Chemistry, Institute for Excellence in Higher Education, Bhopal, Madhya Pradesh, India Sanna Sillankorva INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Adhish Singh Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Arijit Sinha Department of Metallurgical Engineering, Kazi Nazrul University, Asansol, West Bengal, India T.M. Sridhar Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India

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A.K. Srivastava Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Rahul M. Srivastva Department of Biotechnology, MANIT, Bhopal, Madhya Pradesh, India Bhuvaneshwaran Subramanian School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India Goutam Thakur Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Maheswary Thambirajoo Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Manoj Kumar Tripathi ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India O¨zlem Bic¸en U¨nlu¨er Chemistry Department, Faculty of Science, Eskis¸ehir Technical University, Eskis¸ehir, Turkey Sarika Verma Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Amulya Vijay Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, Chennai, Tamil Nadu, India K.N. Vijayasankar Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Ajay Yadav ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Sriya Yeleswarapu Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India O¨zu¨m Yıldırım Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey

List of contributors

Steffi Zimran Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Izzat Zulkiflee Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

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Introduction to biomedical polymer and composites

1

Soham Chowdhury, Adhish Singh and Bidyut Pal Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India

1.1 Introduction Synthetic or natural materials that can be used in conjunction with or as a substitute for living tissues of the human body are called biomaterials (Ulery, Nair, & Laurencin, 2011). Advancements in synthetic materials and fabrication techniques, aided further by cutting-edge sterilization and surgical procedure, have established the clinical use of biomaterials in the current medical practice (Ramakrishna, Mayer, Wintermantel, & Leong, 2001). Biomaterials are extensively used to replace and/or regenerate the function of degenerate or damaged tissues and organs, thereby assisting in healing and improving the functioning and the overall quality of life of the patient (Salernitano & Migliaresi, 2003). Since the biological tolerance of the same material is host-dependent, the term “biocompatibility” was coined to characterize the suitability of materials in a clinical setting. The term “biocompatibility” suggests that when implanted in living tissue, the implant material can produce desirable biological and chemical reactions (Wintermantel, Mayer, Ruffieux, Bruinink, & Eckert, 1999). This necessitates both surface and structure compatibility. A material is surface-compatible when the chemistry, biology, and surface morphology of the implant surface are suitable to the host tissue. Structural compatibility is characterized by an optimal interaction at the implant/tissue interface when the implant material adapts to the mechanical behavior of the surrounding host tissue. Desirable interactions between the biomaterial and the host tissue are achieved when both the surface compatibility and the structural compatibility are met. Biomaterials that are being used in the form of implants (bone plates, joint replacements, sutures, bone graft, vascular grafts, dental implants, etc.) and medical devices (artificial hearts, pacemakers, biosensors, etc.) are anticipated to perform satisfactorily in the aggressive environment of the body (Camilo et al., 2017). Scaffolds developed not only have to have sufficient mechanical strength to bear the structural loads but should also possess an internal porous network that will facilitate tissue ingrowth, cell proliferation, and vascularization (Mayer, Karamuk, Akaike, & Wintermantel, 2000). The material used in the scaffold must actively interact with the surrounding tissue to prevent immune rejection. Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00005-X © 2023 Elsevier Inc. All rights reserved.

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Needless to say, the physiological environment of a patient also depends upon their conditions and activities. Recently, there has been an increased interest in the development of partially or completely resorbable biomaterials for clinical applications to replace the bio-stable materials that are currently being used. Significant research has been carried out to evaluate biodegradable polymeric materials’ efficacy for various biomedical applications (Karamuk, Mayer, Wintermantel, & Akaike, 1999). Degradable polymeric biomaterials were found to be useful for developing temporary prostheses and degradable porous scaffolds for replacing or regenerating damaged tissues. They can also be used in drug carriers for controlled release of the drug. Polymeric materials are being used extensively for applications in tissue engineering due to their availability in a wide variety of compositions, forms (fiber, gel, solid, fabrics, and films), properties and ease of fabrication into complex shapes and structures (Dhand et al., 2016). Tissues are generally categorized into hard and soft tissues. As the name suggests, the hard tissues (bone, tooth) are stiffer than soft tissues (skin, ligament, cartilage, blood vessels). Thus metals and ceramics are generally used for hard tissue applications, and polymers are used for the latter. However, as the load carried by a material is directly related to its stiffness, metals and ceramics pose several complications in orthopedic applications such as stress-shielding, implant loosening, periprosthetic fracture, etc., due to the stiffness mismatch with the neighboring bone tissue. It has been reported that matching the stiffness of the implant material to the neighboring host tissue can significantly reduce the stress-shielding as well as promote favorable bone remodeling around the implant (Apostu, Lucaciu, Berce, Lucaciu, & Cosma, 2017). Although polymers seem like an interesting choice due to their low modulus of elasticity, they cannot be used as they are not capable of sustaining the mechanical loads observed at the implantation site on account of their low strength (Park, Oh, & Lee, 2018). To overcome the shortcomings of using homogeneous materials in isolation, composite biomaterials are being developed. Human tissues are composite materials exhibiting anisotropic properties, which depend on the orientation and structure of the components (Ramakrishna et al., 2001). Polymer composite materials, that is, fiber-reinforced polymers, are characterized by low elastic modulus along with high strength, making them suitable for orthopedic applications. The functionality and performance of a composite implant can be made to optimally adapt to the mechanical and physiological properties of the neighboring host tissue by controlling the composition and orientation of the reinforcement phase. This chapter presents a brief overview of the characteristics, fabrication techniques, and biomedical applications of polymeric materials and polymer composites.

1.2 Classification of polymers and composites Polymeric biomaterials can be broadly grouped into natural polymers and synthetic polymers (Choudhary, Saraswat, & Venkatraman, 2019). The natural polymers can be further grouped into proteins, polysaccharides, and polynucleotides. Among them, proteins and polysaccharides are most commonly used for applications in tissue engineering.

1.3 Fabrication techniques polymer composites

FIGURE 1.1 Classification of composite materials based on the nature and dispersion of the reinforcement phase. Adopted from Wang, M., & Zhao, Q. (2019). Biomedical composites. In: Encyclopedia of biomedical engineering (pp. 34 52). Elsevier, with permission from Elsevier.

Quite a few composites have been evaluated for applications in the medical industry. A broad classification of composite materials is shown in Fig. 1.1. According to the type of the matrix phase, composites can be classified into metal composites, ceramic composites, and polymer composites (Wang & Zhao, 2019). Generally, the characteristics of the composite are determined by the type of matrix. For example, polymer matrix composites are usually ductile in nature. Alternatively, based on the type of reinforcement, composites can be categorized into fiber-reinforced, particle-reinforced, and structural composites. Particlereinforced composites are further categorized as large-particle and dispersionstrengthened materials, depending on the size of the particulate reinforcement. The fiber-reinforced composites can be further subcategorized based on the length of the fiber (long fiber and short fiber). The long fibers are dispersed in a continuous and aligned manner within the composite, whereas in the case of the short fibers, the dispersion can either be regular or randomly oriented. Fiber-reinforced composites are favored due to the enhancement in strength and controllability of the mechanical properties by adjusting the composition, length, orientation, and concentration of the fibers. The structural composites are composed of multiple homogeneous composite layers and can be further grouped into laminates and sandwich panels. Laminates are constructed by a sequential stacking and cementing of multiple anisotropic layers. Due to their unique fabrication, laminates possess excellent in-plane stiffness. Sandwich panels are fabricated by sandwiching a low-density layer with two stiff face sheets. Sandwich panels are generally lightweight and have excellent resistance to bending.

1.3 Fabrication techniques polymer composites Polymer composites are fabricated using various processes. The processes are designed to fabricate polymer composites suitable for various applications, including biomedical applications. Selected techniques are discussed in the following sections.

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1.3.1 Electrospinning The electrospinning technique utilizes high-strength electric fields to process ultrafine fibers up to 2 nm in diameter. The fabrication of nanofibers with a large surface area is performed using this technique in comparison with generally used spinning techniques. A coaxial electrospinning system is illustrated in Fig. 1.2. The basic components of an electrospinning system are spinneret, high-voltage power supply, and grounded collector (Ponnamma, Sadasivuni, Cabibihan, & AlMaadeed, 2017; Zagho & Elzatahry, 2016). Electrospinning can be divided into two types, namely, vertical and horizontal sets (Ponnamma et al., 2017; Zagho & Elzatahry, 2016). When a high voltage is applied to the polymeric solution, a charged liquid jet is formed from the Taylor cone tip. As the solvent evaporates, the liquid jet dries, resulting in the formation of fibers.

1.3.2 Melt extrusion For the production of polymeric composites, the most frequently utilized technique is melting extrusion. Polymer and fillers are mixed together with the help of a twin-screw extruder, which is used for a specific period at a specific temperature (Ponnamma et al., 2017). Compression molding is used to give shape to the polymer composites after the extrusion to get the final product. The amount of filler present determines the mechanical and thermal properties of the polymer composite produced by this technique. Nanocomposites were prepared by Majeed, Al Ali AlMaadeed, and Zagho (2018) having 3 wt.% nanotubes and 97 wt.% low-density polyethylene (LDPE) mixed at 180 C for 7 min. The preparation of titania nanotubes from commercially available TiO2 powder is illustrated in Fig. 1.3. LDPE galleries and the nanotubes are found to be incompatible; therefore maleic anhydride polyethylene can be utilized as compatible material. The mixture was then compression molded for 5 min at 180 C and 200 MPa, to produce thin films (Majeed et al., 2018).

1.3.3 Solution mixing The solution mixing technique fabricates polymer composites by dissolving polymers into a certain solvent at a fixed temperature (Ponnamma et al., 2017). This is followed by fillers being homogeneously distributed in the polymer solution. The mixture is constantly stirred at a fixed temperature. Then a mold is used to dry the resulting mixture at a specified temperature (Zepp et al., 2020). Al-Marri, Masoud, Nassar, Zagho, and Khader (2015) synthesized poly(vinyl alcohol) (PVA)/Cloisite 20A polymeric composites by utilizing PVA polymer solution and suspending Cloisite 20A filler at 70 C for 30 min. A square aluminum mold was used to dry the mixture at 25 C. The PVA polymer solution was prepared by mixing PVA pellets in chloroform and constant stirring at 60 C for 2 h (Al-Marri et al., 2015).

1.3 Fabrication techniques polymer composites

FIGURE 1.2 (A) Schematic illustration of a coaxial electrospinning system, (B) overview of the fabrication process of alginate hydrogel composites reinforced with polycaprolactone nanofibers, and (C) optical images of the fabricated composites with 0.0, 0.023, 0.085, and 0.122 volume fraction of nanofibers, respectively (Jang, Lee, Seol, Jeong, & Cho, 2013). Adopted from Jang, J., Lee, J., Seol, Y.-J., Jeong, Y. H., & Cho, D.-W. (2013). Improving mechanical properties of alginate hydrogel by reinforcement with ethanol treated polycaprolactone nanofibers. Composites Part B: Engineering, 45, 1216 1221, with permission from Elsevier.

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FIGURE 1.3 (A) Scanning electron micrographs of commercially available TiO2 powder; (B) scanning electron micrograph, (C) transmission electron micrograph, and (D) X-ray diffraction analysis (XRD) diffractograms of hydrothermally prepared titania nanotubes. Adopted from Majeed, K., Al Ali AlMaadeed, M., & Zagho, M. M. (2018). Comparison of the effect of carbon, halloysite and titania nanotubes on the mechanical and thermal properties of LDPE based nanocomposite films. Chinese Journal of Chemical Engineering, 26, 428 435, with permission from Elsevier.

1.3.4 Latex technology Latex technology is used to fabricate conductive polymer composites. This is achieved by incorporating conductive fillers into the polymer network. Process upscaling and easy processing make this technique advantageous. The polymer network shows homogeneously distributed fillers (Grossiord, Hermant, & Tkalya, 2012). Also, the highly viscous polymer network can take the nanofillers when using the technique. The three steps involved in this process are as follows: disperse nanofillers into a colloidal mixture, the addition of polymer latex, and drying

1.4 Polymers and their composites for biomedical applications

of colloidal mixture (Grossiord et al., 2012). Carbon nanotubes (Rode, Sharma, & Mishra, 2018) and graphene polymer composites (Pei, Ai, & Qu, 2015), which have various biomedical applications, can be fabricated using this technique.

1.4 Polymers and their composites for biomedical applications 1.4.1 Natural polymers and their composites 1.4.1.1 Collagen Collagen (Col), a naturally occurring protein, is the major constituent of both the soft tissue (skin, cartilage, blood vessels) and the hard tissue (teeth, bone). It provides strength and structurally supports the tissues (Lee, Singla, & Lee, 2001). There are 29 different types of Col, out of which Ⅰ, Ⅱ, Ⅲ, Ⅴ, and ⅩⅠ types are widely studied for purposes of tissue repairing. Since Type Ⅰ Col does not elicit an allergic response, it has found multiple applications in tissue engineering (Parenteau-Bareil, Gauvin, & Berthod, 2010). The biocompatibility, flexibility, and biodegradability make Col a suitable polymer for biomedical applications. Studies have found that Col sponges used as scaffolds assist in the adhesion and growth of cells and tissues (Freyman, Yannas, & Gibson, 2001; O’Brien, Harley, Yannas, & Gibson, 2005). Moreover, it promotes osteoblast proliferation and induces osteoblast differentiation, thereby enhancing bone formation (Seol et al., 2004). A study was conducted to evaluate the performance of Col gel infused with mesenchymal cells in the repair of osteochondral defects. At the implant site, the formation of hyaline cartilage and bone, having mechanical stability significantly lower than the surrounding host tissue, was observed (Wakitani et al., 1994). Studies indicate that the biological properties of Col depend on the orientation of the Col fibers. A study was reported comparing the activity of random Col fiber scaffolds and aligned Col fiber scaffolds fabricated using the electrospinning technique. It was observed that rabbit conjunctival fibroblast cells proliferated faster on the aligned fibrous scaffolds. The rapid degradation rate of Col protein results in poor mechanical properties (Zhong et al., 2006). To overcome this shortcoming, Col composites are made using natural (glycosaminoglycans) and synthetic polymers (polyglycerol methacrylate), characterized by good mechanical strength and osteoconductivity, for promoting the repair of bone tissue (Daamen, 2003; Woerly, Marchand, & Lavalle´e, 1991).

1.4.1.2 Silk Silk fibroin is a natural polymeric protein processed from silkworms (Bombyx mori) and insects. The biocompatibility characteristics associated with silk, such as high strength, biodegradability, flexibility, and permeability to water and oxygen make it a promising biomaterial for tissue engineering. The application of

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Gauze

silk is hindered majorly due to sericin protein, a common contaminant that elicits adverse reactions at the site of application (Puppi, Chiellini, Piras, & Chiellini, 2010). Studies indicated that silk has the potential to enhance the proliferation and differentiation of osteoblastic cells and simulate chondrogenesis and osteogenesis from mesenchymal cells extracted from bone marrow (Meinel et al., 2004; Vepari & Kaplan, 2007). The efficacy of silk sericin in the preparation of wound dressing gauze is illustrated in Fig. 1.4. A study investigated the biological activity of silk fibroin hydrogel postapplication for treating cancellous defects in rabbits. The results indicated an increased healing rate of the defective bone and enhanced quality of the newly formed bone tissue when silk fibroin hydrogel was present, compared to the control material, commercial synthetic poly(D,L-lactide glycolide) copolymer (Fini et al., 2005). Semi-IPN nanocomposite hydrogel H2N H HO

(B)

O HO

HSP00

OH O

OH NH2

HSP20

O

O H2N

OH

D3

D6

D9

D13

(C)

(A)

HSP00

Gauze

D0

HSP20

8

FIGURE 1.4 (A) Sericin/poly(NIPAm/LMSH) nanocomposite hydrogel dressings were applied at wound sites in rats, (B) wound healing after treating with gauze, HSP00, and HSP20 nanocomposite hydrogel dressings, and (C) histological evaluation done on the skin at the 13th day by hematoxylin and eosin (H&E) staining, after treatment with gauze, HSP00, and HSP20 nanocomposite hydrogel dressings (Yang et al., 2017). Adopted from Yang, C., Xue, R., Zhang, Q., Yang, S., Liu, P., Chen, L., . . . Wei, Y. (2017). Nanoclay crosslinked semi-IPN silk sericin/poly(NIPAm/LMSH) nanocomposite hydrogel: An outstanding antibacterial wound dressing. Materials Science and Engineering: C, 81, 303 313, with permission from Elsevier.

1.4 Polymers and their composites for biomedical applications

1.4.1.3 Hyaluronic acid Hyaluronic acid (HA), also known as hyaluronan, is a biodegradable polysaccharide that naturally occurs in the extracellular matrix of connective tissues. It assists in structural support and plays an important role in maintaining water balance and lubrication between articulating cartilage surfaces. HA is mainly extracted from vitreous humor, synovial fluid, and umbilical cord (Malafaya, Silva, & Reis, 2007). The viscoelasticity, swelling capability, and poor immunogenic response of HA make it a potential biomaterial for applications in the encapsulation of cells and drug delivery systems (Kang et al., 2009; Wieland, Houchin-Ray, & Shea, 2007). Moreover, the extensive availability, biodegradability, biocompatibility, and amenability to chemical modification have made HA a potential polymer for tissue engineering (Allison & Grande-Allen, 2006). Studies indicated that the polyanionic and hydrophilic surface of HA does not favor attachment of anionic cell surfaces, thereby impeding cell growth and tissue remodeling (Shu, Liu, Palumbo, & Prestwich, 2003). To overcome this limitation, Shu et al. (2003) coated the surface of HA with extracellular matrix proteins, resulting in a significant improvement in cellular attachment and tissue formation. Further expansion of its applications has been achieved by modification of its chemical characteristics by photo linking and covalent crosslinking (Allison & Grande-Allen, 2006). In a recent study, the in vitro performance of HA-based Hyaft-ll biodegradable polymer infused with human vascular endothelial cells was evaluated. Formation of the subendothelial matrix was observed within 24 h (Fig. 1.5). The study indicates Hyaft-ll-based biopolymers can be used for making scaffolds that facilitate endothelialization in vascular grafts (Turner, Kielty, Walker, & Canfield, 2004).

1.4.1.4 Chitosan Naturally occurring Chitosan is a biodegradable and biocompatible polysaccharide. It has wide applications in pharmaceutics, cosmetics, and the food industry (Perinelli et al., 2018). Chitosan is produced by partial deacetylation chitin, obtained mainly from the cuticles of various crustaceans through chemical hydrolysis (Chandy & Sharma, 1990). The extensive availability, antibacterial activity, hydrophilicity, and nonimmunogenic properties indicate why Chitosan is preferred for tissue engineering applications (Nair & Laurencin, 2007). Researchers have recently developed an antibacterial and biocompatible derivative of chitosan, 1,3-diethyl-2-thiobarbituric acid (CS-DETBA). CS-DETBA showed enhanced inhibition to the growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus bacteria and was observed to have no cytotoxic effects on the human gastric adenocarcinoma (AGS) cells (Rizwan et al., 2017). A study was carried out to determine the proliferation of mesenchymal stem cells produced from bone marrow in a chitosan/tripolyphosphate scaffold (Fig. 1.6). From the results, it was concluded that the developed scaffold has the potential to be used in bone regenerative medicine (Xu et al., 2018). Under in vivo and in vitro

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FIGURE 1.5 Confocal micrographs illustrating attachment of rat BM-MSCs on chitosan scaffolds after 24 h of culture in (A) 1%(w/w) TPP, (B) 3 M NaCl 1 1% (w/w) TPP, (C) 6 M NaCl 1 1% (w/w) TPP, (D) 3 M NaCl 1 0.25% (w/w) TPP, (E) 3 M NaCl 1 2%(w/w) TPP, and (F) 3 M NaCl 1 6% (w/w) TPP. Adopted from Turner, N. J., Kielty, C. M., Walker, M. G., & Canfield, A. E. (2004). A novel hyaluronan-based biomaterial (Hyaff-11®) as a scaffold for endothelial cells in tissue engineered vascular grafts. Biomaterials, 25, 5955 5964, with permission from Elsevier.

conditions, chitosan stimulates bone formation due to its osteoconductive ability. However, its poor mechanical stability restricts the maintenance of precise shape, thereby reducing its application.

1.4.1.5 Cellulose Cellulose is a naturally occurring polysaccharide found in the cell walls of plants. Cellulose is involved in the provision of structural support. It also occurs in other microorganisms like fungi, algae, and bacteria (Puppi et al., 2010). The hydrophilicity and biocompatible characteristics of cellulose make it a potential biomaterial for application in tissue engineering and drug delivery (Klemm, Heublein, Fink, & Bohn, 2005). Cellulose hydrogel membranes are prepared by casting cellulose/1-butyl-3methylimidazolium chloride into assembly molds, followed by coagulation in

1.4 Polymers and their composites for biomedical applications

FIGURE 1.6 Confocal micrographs illustrating attachment of rat bone marrow mesenchymal stem cells on Chitosan scaffolds after 24 h of culture in (A) 1%(w/w) tripolyphosphate (TPP), (B) 3 M NaCl 1 1% (w/w) TPP, (C) 6 M NaCl 1 1% (w/w) TPP, (D) 3 M NaCl 1 0.25% (w/w) TPP, (E) 3 M NaCl 1 2%(w/w) TPP, and (F) 3 M NaCl 1 6% (w/w) TPP. Red represents the actin network with rhodamine-phalloidin and nuclei, and chitosan shown in blue and green, respectively. Adopted from Xu, Y., Han, J., Chai, Y., Yuan, S., Lin, H., & Zhang, X. (2018). Development of porous chitosan/tripolyphosphate scaffolds with tunable uncross-linking primary amine content for bone tissue engineering. Materials Science and Engineering: C, 85, 182 190, with permission from Elsevier.

water. Depending on the techniques adopted, porous cellulose hydrogels possessing suitable morphological features and mechanical properties can be prepared for applications in the delivery of pharmaceutical agents as a drug carrier, contact lenses, or wound healing material (Peng, Wang, Xu, & Dai, 2017). In a study, it was observed that cellulose enhances the proliferation and growth of human chondrocytes, as illustrated in Fig. 1.7, and thus can be utilized in cartilage tissue engineering (Svensson et al., 2005). In a recent study, a cellulose-based composite reinforced with silver and chitosan nanoparticles was fabricated. The composite was found to possess an excellent antibacterial inhibition to the growth of E. coli and S. aureus, and an increased proliferation rate of fibroblastic cells was observed within an incubation period of 3 days (Haider et al., 2018). The findings indicate this composite can be used for preparing scaffolds for wound dressing. An effective polysaccharide capsule has been developed for orally administering a hydrophobics drug (Ibuprofen) by physical crosslinking of hydroxyethyl

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FIGURE 1.7 Scanning electron microscopic (SEM) micrographs of bovine chondrocyte cell attachment on unmodified bacterial cellulose (BC) and modified BC (BC-P1, BC-P2, BC-S) surfaces. Adopted from Svensson, A., Nicklasson, E., Harrah, T., Panilaitis, B., Kaplan, D. L., Brittberg, M., & Gatenholm, P. (2005). Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials, 26, 419 431, Adopted with permission from Elsevier.

cellulose (HEC) and carboxymethyl cellulose (CMC). It was observed that the drug was completely released from HEC into the intestinal fluid within 8 h. However, upon mixing of HEC and CMC resulted in a prolonged (24 h) and sustained release of the drug from the carrier (Chen, Wang, & Yan, 2017). The performance of gelatin-based chitosan and hydroxyethyl cellulose was evaluated for applications in tissue engineering. A reduction in stiffness, enhanced flexibility, and mechanical strength comparable to soft tissues were observed for the gelatin poly(ethylene glycol) (PEG)/hydroxyethyl cellulose (G/PEG/HEC) hydrogel. A biological evaluation of the hydrogel was reported on rat myoblasts and human fibroblasts cell lines. The results showed good cell adhesion and enhanced proliferation, indicating its potential for biomedical applications (Dey et al., 2018).

1.4 Polymers and their composites for biomedical applications

1.4.2 Synthetic polymers and their composites 1.4.2.1 Polycaprolactone Polycaprolactone (PCL) is a polymer made up of semipolar ester groups and nonpolar methylene groups. It finds its use in tissue engineering as it is biocompatible and has high elasticity. The Food and Drug Administration (FDA) gave approval to PCL for its use in various biomedical applications, which include drug delivery systems, tissue repair as sutures and scaffolds, etc. (Woodruff & Hutmacher, 2010). The slower degradation rate of PCL makes it a better material for implants and a drug carrier (Cipitria, Skelton, Dargaville, Dalton, & Hutmacher, 2011). When used as a bulk material, PCL shows slow adhesion and cell proliferation. In order to increase the bioactivity of PCL, its polymeric composites are being attempted. Apart from that, surface functionalization is also utilized for the same. A blend of PCL, chitosan-1,3-diethyl-2-thiobarbituric acid-PCL (CS-DETBAPCL), is prepared for its use in tissue engineering. The cytotoxicity response on AGS cells of this blend is negligible, and the growth of bacterial strains (S. aureus, E. coli, P. aeruginosa) was significantly hampered (Xu et al., 2018). Recently, with the help of the electrospinning method, a PCL/chitosan/magnesium oxide nanofiber was manufactured. Mechanical stability of PCL/MgO (25 MPa) is better than that of PCL/ chitosan (3 MPa). A cellular study suggests the noncytotoxic nature of the composites due to the attachment of 3T3 cells on its surface. Also, this study endorses PCL as a suitable polymer for bone regeneration and wound healing (Rijal et al., 2018). With or without bioactive ceramics, PCL can be utilized either way in hard tissue regeneration as a scaffold material. Interaction of bone marrow mesenchymal stem cells (BMSCs) with pure PCL indicates an insignificant influence on its functioning by the degradation by-products of PCL (Sukanya & Mohanan, 2018). PCL/forsterite scaffold was fabricated, which, as compared to pure PCL, showed improvement in bioactivity and cytotoxicity of scaffolds that were used for bone regeneration. The content of forsterite in the composites affects the cellular behavior of the composites (Diba, Tapia, Boccaccini, & Strobel, 2012). To repair the orbital fractures of the white rabbit, a thin membrane patch of PCL/β-tricalcium phosphate was three-dimensional (3-D) printed. It was observed that a 40% reduction of fracture volume took place after 2 months. Also, within 4 months, the mesh implant showed growth of new bone. This study pointed out that the use of the 3-D printed membrane patch for filling defective spaces in the bone. This could be an encouraging methodology as an inflammatory response was prevented at the site of application (Ho Han et al., 2018).

1.4.2.2 Poly(L-lactic acid) Poly(L-lactic acid) (PLLA) can be manufactured from the polymerization of L-lactide or derived from natural sources. It is a degradable polymer. The use of PLLA as scaffolds for tissue regeneration, drug delivery carrier, and pin in fixing implants in bone and sutures has been heavily investigated. FDA approved Sculptra for commercial use in facial atrophy treatment in which PLLA is used as

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an injectable material (Stratton, Shelke, Hoshino, Rudraiah, & Kumbar, 2016). However, PLLA showed the inflammatory response at the site of application as it undergoes rapid degeneration due to its high crystalline nature (Lasprilla, Martinez, Lunelli, Jardini, & Filho, 2012). When fabricated with the other polymers as a composite, this issue can be resolved. Cui et al. (2013) prepared novel PLLA/Rg3 scaffolds that help reduce inflammation and also studied the response of composite to skin regeneration. The proliferation of fibroblast cells was hindered due to uniform surface morphology and interconnected pores of the scaffolds. This points out the ability of fabricated composites to restore damaged skin due to severe burns. With the help of the electrospinning technique, a defect-free fiber of PLLA/polyglycerol sebacate (PGS) was manufactured (Cui et al., 2013). Superhydrophilicity was achieved due to the use of PGS in fibers. The elastic modulus was observed to drop sharply from 35.9 to 7.4 MPa when there was an increase in 25% concentration of PGS in the fibers. Also, the stretching ability was improved by twofolds. A study found PLLA/PGS to be a promising biomaterial for nerve regeneration as it showed adhesion and proliferation of A59 nerve cells, as illustrated in Fig. 1.8

FIGURE 1.8 Scanning electron micrographs illustrating the distribution of A59 cells on the membrane surfaces for 1-day cell culture on (A) poly(L-lactic acid) (PLLA) membrane, and (B) PLLA: polyglycerol sebacate (PGS) (25 wt.% PGS) membrane and 5-day cell culture on (C) PLLA membrane, and (D) PLLA:PGS (25 wt.% PGS) membrane. Adopted from Yan, Y., Sencadas, V., Jin, T., Huang, X., Chen, J., Wei, D., & Jiang, Z. (2017). Tailoring the wettability and mechanical properties of electrospun poly(l-lactic acid)-poly(glycerol sebacate) core-shell membranes for biomedical applications. Journal of Colloid and Interface Science, 508, 87 94, with permission from Elsevier.

1.4 Polymers and their composites for biomedical applications

(Yan et al., 2017). A study was conducted by dual coating the Mg alloy with hydroxyapatite and PLLA in order to improve its biomedical application (Diez, Kang, Kim, Kim, & Song, 2015). The dual-coated alloys enhanced the biological response and mechanical stability in comparison to single-coated or noncoated samples.

1.4.2.3 Poly(methyl methacrylate) Poly(methyl methacrylate) (PMMA) is a synthetic polymer with superior mechanical properties and self-hardening ability, which helps to fix an artificial joint to the bone (Lee & Rhee, 2009). Due to its inert nature, PMMA is a suitable material to provide immediate structural support to metallic implants in bone. However, in between an implant and bone, the PMMA is found to be a weak link (Renterı´a-Zamarro´n, Corte´s-Herna´ndez, Bretado-Arago´n, & Ortega-Lara, 2009). Repeated interfacial movements lead to osteolysis and loosening of the implants (Goodman, 2005). With the help of bioactive ceramic as filler in between the polymer matrix, the osteoconductivity and mechanical stability of the composite can be improved (Shinzato et al., 2000). Hydroxyapatite reinforced with PMMA provides improved anchorage of human osteoblast cells, increased activity of alkaline phosphatase, and cell proliferation (Dalby, Di Silvio, Harper, & Bonfield, 1999). In a study, Renterı´aZamarro´n et al. (2009) found good apatite forming ability and compressive strength in PMMA containing 39% wollastonite. Another study showed the use of PMMA/SiO2 CaO nanocomposites in dental composites as a filler material and bone cement (Lee & Rhee, 2009).

1.4.2.4 Poly(lactic-co-glycolic) acid Poly(lactic-co-glycolic) acid (PLGA) is a biodegradable polyester. It is formed by combining PLLA and poly(glycolic acid) (PGA). PLGA has been the center of attention of researchers due to its biocompatibility and modifiable nature of its surface properties which encourage its interaction with biological materials and make it a right fit for tissue engineering applications (Gentile, Chiono, Carmagnola, & Hatton, 2014). FDA approved Osteofoam as a PLGA scaffold for hard tissue regeneration (Shen, Hu, Bei, & Wang, 2008). To treat craniosynostosis, a uniform mix of PLGA and polyisoprene was studied. The scaffolds were made with porous structures with pores of a size that promotes the growth of C2C12 cell lines and forms an extracellular matrix. Marques, dos Santos, O’Brien, Cartmell, and Gough (2016) proposed to use these scaffolds for soft tissue engineering as their strength was similar to that of natural soft tissue. For tendon regeneration, PLGA/silk scaffolds exhibited good mechanical stability and also the tendency to simulate mesenchymal progenitor cells to undergo adhesion and differentiation (Fig. 1.9) (Sahoo, Toh, & Goh, 2010). For its use in hard tissue engineering, PLGA/silk composites supported by hydroxyapatite were manufactured (Sheikh et al., 2015). It was observed that over the surface of

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FIGURE 1.9 (A) Unseeded and bone marrow mesenchymal stem cell seeded scaffold specimens at mechanical testing, (B) load displacement curves for the biohybrid scaffolds obtained after mechanical testing, (C) loads at which the representative scaffolds fail, and (D) stiffness obtained for the scaffolds. Adopted from Sahoo, S., Toh, S. L., & Goh, J. C. H. (2010). A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials, 31, 2990 2998, with permission from Elsevier.

microspheres of chitosan/PLGA, there was a proliferation of MC3T3-E1 (Jiang, Abdel-Fattah, & Laurencin, 2006). PLGA finds limited use in drug delivery applications due to the acidic nature of its by-products of degradation. Research is being done to resolve this issue by changing the concentration of PGA. To slow the degradation rate, the ratio of PGA to that of PLLA was increased, producing less acidic by-products (Houchin & Topp, 2008).

1.4.2.5 Polyvinylidene fluoride Polyvinylidene fluoride (PVDF) is one of the most common fluorinated polymers. It has a semicrystalline structure and is a nonreactive polymer. Fluoride polymers have exceptional biocompatibility, thermal stability, chemical resistance, and stimulus-response that make them biomaterials with many biomedical applications (Cardoso, Correia, Ribeiro, Fernandes, & Lanceros-Me´ndez, 2018). For hard tissue regeneration, the ability of piezoelectric PVDF was examined by implanting PVDF films in Wistar rats. After a period of 28 days, the implanted

1.4 Polymers and their composites for biomedical applications

films led to remarkable bone regeneration without any inflammatory response (Ribeiro et al., 2017). PVDF finds its use in a variety of pharmaceutical applications and hygienic products. Due to these uses of PVDF, it is exposed to microorganisms that can form biofilms. To examine its antibacterial properties against P. aeruginosa, different PVDF composites were prepared with several nanofillers. Nanofillers used in the composites were graphene nanoplatelets (GNPs), zinc oxide nanorods (ZnO-NRs), and ZnO-NR-decorated GNPs (ZNGs) (Bregnocchi et al., 2016). The antimicrobial activity observed in the composites with GNPs and ZNGs nanofillers was much better than in composite with ZnO-NRs. The nanostructure formed by GNPs and ZNGs provided a greater interacting surface with bacteria resulting in good antibacterial activity. PVDF/HAP film was prepared using the solvent casting method to investigate its cytotoxicity for potential application in repairing bone defects (Braga et al., 2007). The cell viability revealed that the composites are noncytotoxic. The mechanical stability of composites was observed to be reduced by the use of hydroxyapatite (HAP) in the PVDF matrix. These studies established the biocompatibility of the samples, and thus they can have applications in bone and dental restoration. Fluorinated membranes have hydrophobic nature, which impedes the attachment of cells and consequently cell proliferation. A composite of PVDF was fabricated with reduced graphene oxide (RGO) in an attempt to enhance its cytocompatibility (Pei et al., 2015). Human umbilical vein endothelial cells were cultured on membranes of RGO/PVDF composite in order to study cellular proliferation response and adhesion. The composites were observed to be better than the pure PVDF. Furthermore, the incorporation of RGO assisted the conversion of alpha-phase PVDF to beta-phase PVDF, which assisted endothelial cells to secret prostacyclin, having antithrombotic functions.

1.4.2.6 Poly(ethylene glycol) PEG is a polyester that can exist in various molecular weights. It is soluble in several organic solvents and water. PEG has a unique capability to maintain solubility and chemical reactivity after surface functionalization and chemical modifications, demonstrating variable biomedical uses. Also, PEG has the capability to not affect the activity of active proteins of cells while interacting with cell membranes (Harris, 1992). A nanosystem was fabricated with PEG supported by carbon nanotubes in the form of nano-cocoons. To study its efficacy as a drug delivery carrier, it was loaded with curcumin. These nanosystems show no toxicity to blood and also promote the growth of L929 fibroblast cell lines. In a saline medium, curcuminloaded nanosystems dispersed effectively and interacted with C6 glioma brain cancer cells. On the other hand, curcumin alone was not able to penetrate brain cancer cells (Simon, Flahaut, & Golzio, 2019). For application as a wound dressing material, polyethylene oxide and poly(ethylene glycol) dimethacrylate was

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used to fabricate hydrogel film (Haryanto & Mahardian, 2018). When poly(ethylene glycol) dimethacrylate was added to hydrogel, it increased the vapor transmission, mechanical strength, and percentage elongation. The tensile strength improved from 5% to 20%. The water vapor transmission rate was observed to be close to the ideal value for favorable conditions for the healing of wounds. PEG/cellulose scaffolds that were environment friendly, nontoxic, and biocompatible were manufactured. A network of close-grained sheets was noticed when PEG was added to regenerated cellulose. This alteration in scaffolds increased compressive strength by 33 times that of regenerated cellulose (Teng et al., 2018).

1.4.3 Gas-permeable polymeric membranes Polymeric membranes have extensive application as oxygenators or for hemodialysis that are used in cardiac surgery in infants, treating underdeveloped lungs and chronic problems. Reduced gas efficiency is found for gas-permeable membranes because blood components (proteins, platelets) have a tendency to get deposited over their surface (Kolobow, Borelli, & Spatola, 1986). 2-Methacryloyloxyethyl phosphorylcholine (MPC) copolymer was prepared to resolve the aforementioned issue by surface modification of conventional polymers. Substrate polymer was coated with MPC copolymers and alkyl methacrylate, which resulted in inhibition of cell adhesion. Also, when blood lacking anticoagulants was put in direct contact with the polymer, the protein adsorption decreased (Ishihara et al., 1992). An oxygenator membrane made up of poly(MPC-co-dodecyl methacrylate) (PMD) skin film adhered to polyethylene (PE) was fabricated (Iwasaki, Uchiyama, Kurita, Morimoto, & Nakabayashi, 2002). This showed that the gas permeability improved with the presence of MPC in the polymer film. PMD/PE membrane under oxygen gas permeation analysis showed similar results in comparison to PE membranes besides the fact that MPC in PMD was more than 0.2 unit mole fraction. It was also observed that there was a significant decrease in protein adsorption on the surface of PMD in comparison to the PE surface. The membrane surface decreases the protein adsorption as the surface restricts the hydrogen bonding with water (Lu, Lee, & Park, 1992). With the help of plasma-induced surface modification, polysulfone (PSF) membranes were fabricated to investigate its application as an artificial lung (Wang et al., 2016). The steric hindrance and surface hydrophilicity of the PSFPEG-Heparin (Hep) membrane was improved, which led to the decrease of adsorption rate of fibrinogen and bovine serum albumin in contrast to pure PSF. However, pure PSF showed better adhesion of platelets. On the other hand, the PSF-PEG-Hep membrane exhibited a steep decline in platelet adhesion with the increase of the molecular weight of PEG. Therefore good platelet adhesion resistance was observed in PSF-PEG10,000-Hep and PSF-PEG6000-Hep. Furthermore, exceptional gas exchange performance was shown by the PSF-PEGHep membrane in the presence of porcine blood. To examine its use as membrane

1.4 Polymers and their composites for biomedical applications

oxygenators, the PSF-PEG-Hep membrane was synthesized utilizing PSF chloromethylation, PEGylation, and heparin immobilization process (Zheng et al., 2016). The blood oxygenation results for the same were at par with the commercially available membrane oxygenators indicating its potential application as an oxygenator in treating various lung diseases. For use as an extracorporeal membrane oxygenator, PSF membranes were fabricated through surface modification with low-temperature plasma treatment. Surface modification of PSF was performed with three additives, Acrylic acid (AA) with heparin (Hep), MPC, and Col. (Zheng, Wang, Huang, Fan, & Li, 2017). PSF-AA-Hep showed the least protein adsorption with an increasing trend, followed by PSF-MPC, PSF-Col, and pure PSF. The same trend was followed for platelet adhesion. It is expected that charged groups from heparin exhibited steric hindrance, which restricted the protein adsorption. Similarly, biomimetic structures from Col or MPC, and hydrophilic groups from AA were the reason behind the trend followed by these membranes. Gas permeation activity of surfacemodified PSF membrane was low as compared to pure PSF, as its surface was covered with grafted molecules. Thus the study showed that a modified PSF membrane could be potentially applied as a membrane oxygenator or in artificial respiratory devices (Zheng et al., 2017).

1.4.4 Other polymeric composites Polymers and ceramics are the most widely used materials in biomedical applications (Holzapfel et al., 2013). But they individually do not meet all the basic requirements like biocompatibility, mechanical load bearing capability, corrosion, and wear resistance. So researchers are developing composites with polymer matrix reinforced with ceramic to introduce bioactive characteristics of the polymer coupled with mechanical strength and corrosion resistance of ceramics (Dziadek, Stodolak-Zych, & Cholewa-Kowalska, 2017). A study was conducted by Gil-Albarova et al. (2012) to evaluate the in vivo performance of cross-linked glutaraldehyde, gelatin-coated nanocrystalline hydroxyapatite binding peptide (HABP) scaffolds. HABP foam cylinders were implanted at the site of artificially created bone defects in the femurs of New Zealand rabbits. The foam was found distended after being filled with bodily fluids, thereby ensuring proper fixation without cementation. In the histological and radiological studies conducted after 4 months, it was found that the implantation treated the critical-sized bone defect with osseointegration and osteoconduction at the surface of the foam, illustrated in Fig. 1.10. The results indicate that a gelatin-coated hydroxyapatite scaffold has the potential to be used for clinical conditions in orthopedics and dentistry (GilAlbarova et al., 2012). 3-D printed biomimetic Col/hydroxyapatite (CHA) composite material scaffolds were prepared using a low-temperature additive manufacturing technique (Lin et al., 2016). The CHA scaffolds facilitated the proliferation of bone marrow stromal cells and enhanced the osteogenic response in vitro. The scaffolds were

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FIGURE 1.10 (A) Macroscopic image of the femoral bone specimen of the rabbit after necropsy, (B) histological staining image illustrating the integration of bone tissue (indicated with black arrows) in the HABP foam cylinders; Masson’s trichrome staining images illustrating: (C) formation of new bone tissue in the form of trabeculae (indicated with a white star) around the foreign material (shown with black arrow) and bone marrow (shown with black asterisk), and (D) membranous ossification process (indicated with black wavy arrow) with newly formed bone tissue (shown with white star) around the foreign material (black arrow). Adopted from Gil-Albarova, J., Vila, M., Badiola-Vargas, J., Sa´nchez-Salcedo, S., Herrera, A., Vallet-Regi, M. (2012). In vivo osteointegration of three-dimensional crosslinked gelatin-coated hydroxyapatite foams. Acta Biomaterialia, 8, 3777 3783, with permission from Elsevier.

implanted in defective femoral condyles of rabbits to investigate the in vivo performance of the scaffolds. The scaffold constructed with 600 μm diameter rods was found to have optimal mechanical stability and, prior to degradation, promoted cell penetration and mineralization along with improved healing of the bone defects. The results indicate that 3-D printed CHA scaffolds are suitable for applications in tissue engineering and regenerative medicine. In a recent study conducted by Alonso-Sierra et al. (2017), biomimetic inorganic/organic composites were fabricated, and the porosity analysis of the same was carried out using X-ray microtomography. Hydroxyapatite was prepared by gel casting method and was molded into the 3-D hierarchical interconnected porous structures by adding PMMA microspheres. The organic matrix was burned off during sintering to achieve a controllable porosity of the hydroxyapatite

1.4 Polymers and their composites for biomedical applications

scaffolds. Finally, two organic phases, gelatin, and Col were used to generate an organic inorganic composite material, after extraction from the bovine tail. The compressive strengths of the gelatin and Col-based composites were found to be roughly three times that of the natural bone tissue. The achieved pore size distribution will facilitate cell proliferation, ingrowth of intermediate tissue, ensure vascular incursion, and nutrient supply. The study showed that this composite is a promising material to substitute bone tissue and can be used in the manufacturing of prostheses. Recently, a study evaluated the cytotoxicity in vivo and antibacterial behavior of a PCL/hydroxyapatite/gelatin scaffold loaded with doxycycline. The antibacterial study showed that the codelivery of hydroxyapatite nanoparticles along with doxycycline inhibited the bacterial growth of Gram-positive S. aureus and Gramnegative Porphyromonas gingivalis bacteria. In vitro doxycycline release profile in the phosphate buffer medium was characterized by two steps. Initial burst release of the drug from the scaffold was found to be 60% within an hour, followed by a continual release of the remaining drug for 55 h. The anticancer activity of the scaffold was studied by testing the sensibilities of three cancer cells (A-431, 4T1, and CACO-2) to the codelivery system. It was found that the A-431 exhibited the most synergistic effect compared to 4T1 and CACO-2 cells. Based on the anticancer and antibacterial results obtained in this study, the developed doxycycline-loaded PCL/hydroxyapatite/gelatin composites were found suitable as a drug delivery system (Ramı´rez-Agudelo et al., 2018). A three-component poly(lactic acid) (PLA)/PCL/wollastonite material system was fabricated to evaluate their suitability as tissue engineering scaffolds (Goswami, Bhatnagar, Mohanty, & Ghosh, 2013). The porous foams prepared from the composites were tested in vitro for biocompatibility by seeding in osteoblast cells. The prepared foams exhibited enhanced proliferation and fashion of osteoblast cells over the 7-day incubation period. The materials were also tested under compressive loads in dry and wet conditions for simulating their performance in physiological conditions. Increasing the wollastonite content led to an improvement in hydrophilicity of the polymers, and that facilitated better implant tissue adhesion. It was found that composite (PLCLW8) containing the highest amount of CaSiO3, facilitated cell adhesion and proliferation at a faster rate compared to the pure polymer (PLCL15). In a study conducted by Bheemaneni, Saravana, and Kandaswamy (2018), cytotoxicity and mechanical properties of poly(butylene adipate-co-terephthalate)/ wollastonite biocomposites were evaluated. The composite exhibited good formation of hydroxyapatite layer after being soaked in simulated body fluid within 5 days. Increasing the wollastonite content led to enhancement of the tensile strength of the composite. The composite facilitated enhanced MG63 cell proliferation within a short incubation period. It was found that adding the bioactive silicate filler (wollastonite) helped in biomineralization as well as cell proliferation compared to the pure polymer [poly(butylene adipate-co-terephthalate)] (Bheemaneni et al., 2018).

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Recently, hydroxyapatite/PLLA electrospun membranes were fabricated to study their potential applicability as bone substitutes (Santos et al., 2017). The biocompatibility of the hybrid composite membranes was studied. Results of in vitro MG63 osteoblast cell culture suggest that the membranes promote better cell proliferation compared to PLLA membranes, and processing methods do not induce any cytotoxic effect in the cells. The results of the metabolic activity indicate a faster rate of cell growth on the surface of the hydroxyapatite/PLLA when compared to PLLA and the control membranes. The simple and scalable processing method used makes the fabricated polymer microparticles fiber membranes a potential bone substitute. In a recent study, thin PLA composite films reinforced with coralline hydroxyapatite were fabricated. The coralline hydroxyapatite content of the composite promoted human adipose-derived stem cell attachment and proliferation, contrary to no cellular activity seen on the surface of pure PLA. The bioactivity and osteoconductivity of the ceramic, in addition to the flexibility and biodegradability of the polymer matrix, make this composite a potential biomaterial for designing scaffolds (Macha et al., 2017). Hydroxyapatite/ultra-high molecular weight PE composites with different ratios of hydroxyapatite were prepared for evaluating the role of the ceramic filler in biocompatibility (Mirsalehi, Sattari, Khavandi, Mirdamadi, & Naimi-Jamal, 2015). All the samples facilitated enhanced adhesion and proliferation of the MG63 cells compared to the control material. The results obtained indicate that the composite with higher hydroxyapatite content exhibited better bioactivity due to enhanced differentiation and proliferation of osteoblast cells. It was concluded that composites having polymer matrix reinforced with bioactive ceramic fillers have potential applicability as nontoxic biomaterials that can initiate bone ingrowth on their surface.

1.5 Challenges and future trends The use of polymers and polymeric composites for fabricating new biomaterials and improving the existing biomaterials is gaining a lot of traction in the biomedical industry. In a nutshell, this can be attributed to the broad spectrum of mechanical and biological properties exhibited by the polymer and polymer composite biomaterials. The composite biomaterials can be tailored for designing implants with an overall structure and interface properties that can elicit favorable responses from the surrounding tissues. Despite this, the present areas of application of these biomaterials in medicine are significantly lower than what was extrapolated a few years back. In many cases, although the biomaterials showed a lot of potential in the research and development stage, they were not included in the production and subsequent commercial distribution of medical devices in any capacity (Salernitano & Migliaresi, 2003). The major critical issues hindering the use of these materials are summarized here.

1.6 Conclusion

The experimental and clinical data supporting the long-term applications of composite materials are remarkably less compared to the same for homogeneous materials. One positive aspect of composites is the controllability of material properties by varying the constituents and the type and orientation of the reinforcement phase. This makes composite suitable for designing implants with optimized adaptability to the mechanical properties of the host tissue (Chung, Im, Kim, Park, & Jung, 2020). However, the additional design parameters make the fabrication process more complicated. An ideal biomedical scaffold should possess excellent biocompatibility, must be fully resorbable, have mechanical stability, can be easily molded into the required shape tailored to the implantation site, and have an internal geometry that facilitates tissue ingrowth and ensure proper vascularization (Palmieri, Sciandra, Bozzi, De Spirito, & Papi, 2020). The lack of satisfactory standardized tests for evaluating the biocompatibility of composites makes it difficult to comment on the suitability of a composite biomaterial scaffold in a particular clinical setting. Fatigue testing of a material is important for determining its use in an implant as in vivo loads are predominantly repetitive or cyclic in nature. There is no adequate standardized testing available for assessing the performance of composite materials under fatigue loads. Moreover, compared to the monolithic materials, determining the fatigue behavior of composite is much more complicated. Overcoming the challenges mentioned here is integral before polymer composites can be extensively used in the biomedical industry. However, the future prospects for the widespread commercial use of composite biomaterials in medicine are very promising. 3-D printing and advanced manufacturing techniques have to facilitate the fabrication of scaffolds with the desired porous network without the use of any cutting tools, molds, or dies. Hence, this can be used to construct patient-specific scaffolds that are compatible with the mechanical properties of the neighboring tissue at the implantation site (Nakayama, Shayan, & Huang, 2019). Composite materials might also find applications in other clinical areas where materials with new properties are needed for developing more efficient alternative devices and surgical techniques.

1.6 Conclusion In this chapter, a detailed review of the recent developments in fabrication techniques and biomedical applications of polymers and polymeric composites has been reported. From the recent trends, it can be observed that the composite approach for developing new biomedical scaffolds and modifying existing materials is becoming more and more important in the field of biomaterials. This can be attributed to the tailorable manufacturing of composite materials and properties comparable to the host tissue. The successful development of novel polymers and polymeric composites targeting particular medical applications depends upon the

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firm grasp of the fabrication technique, material properties of the component materials, and a thorough understanding of biology and medical science. Based on such a foundation, new and innovative polymer and polymeric composites can be tailored and fabricated for a variety of applications in biomedical engineering. However, for the application of composite materials in a clinical setting, medical professionals need to be convinced about their long-term durability and reliability. Compared to monolithic materials, the experimental and clinical data supporting polymer and polymeric composite biomaterials are relatively small. Thus further research into polymers and polymeric composites can elucidate their long-term durability under physiological conditions. The successful development and applications of polymer and polymeric composites demand harmonious cooperation between the healthcare and engineering industries.

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CHAPTER

Foundation of composites

2

Umesh Kumar Dwivedi and Neelam Kumari Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India

2.1 Introduction For several years composites have been used to solve technical problems. Polymer-based composites were introduced in the 1960s and more industries began to take notice. Since then, composites have become a standard engineering material. The increase use is due to the product performance and their light weight components (Pal, Jit, Tyagi, & Sidhu, 2011). Composites are composed of two or more distinct materials called constituent materials. These constituents are not chemically bonded together and have different properties. It is the combination of different constituents that result in superior physical properties. The constituent phase that is continuous throughout the composite is termed the matrix. The particles/fibers which are dispersed in the matrix making it stronger are called the reinforcements. Processing of composites is affected by both the reinforcements and the matrix. The main function of the matrix is to carry load, maintain the reinforcement fibers in proper orientation, and bind the reinforcements together. It distributes the load throughout the reinforcements through interface and protects them from environmental damage. The reinforcing phase provides the toughness and strength. The matrix material may be metallic, polymeric, or ceramic, and reinforcement may be fiber or a particulate. Particulates in a composite should be evenly distributed. The reinforced particulate composites are less expensive, less rigid, and much weaker as comparable to fiber-reinforced composites. The particulate composites have disadvantages such as brittleness and processing difficulties when the particulate/matrix volume percentage is more than 30% 40%. Fibrous composites are formed by stacking single sheets of continuous fibers into laminate to obtain the desired rigidity and strength and can include 60% 70% of fibers. Due to small diameter of fibers, composites are very strong. Examples of fibers include carbon, glass, and Kevlar. Final properties of composites depend on the type of matrix and reinforcement. Continuous and discontinuous fibers have different properties. For instance, continuous fibers have greater strength and rigidity, whereas discontinuous fibers are random in orientation. This random orientation tends to decreases the strength and rigidity of the final Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00015-2 © 2023 Elsevier Inc. All rights reserved.

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product. Continuous fibrous composites are used where high strength and rigidity are required. Discontinuous fibrous composites are used in low strength and rigidity applications.

2.2 Classification of composites On the basis of the matrix phase, composites are divided into polymer, ceramic, and metal (Malhotra, Goda, & Sreekala, 2012). Three main types of polymer used as matrices are thermoset, thermoplastic, and elastomers as shown in Fig. 2.1. Once cured, thermosets cannot be melted. Thermosets have good affinity to heterogeneous materials, for example, epoxy, polyester, etc. The most common resins used in thermoset composites are epoxy, phenolic, and polyester. Thermosets have the advantage over thermoplastic at high temperature and creep resistance. On the other hand, thermoplastic materials are ductile and tougher than thermoset materials. Thermoplastic are reversible in nature, that is, they can be reshaped by the application of pressure and heat. Thermoplastic molecules are flexible and reformable into previous shape due to the cross-link behavior. Some thermoplastic resins are polypropylene, polythene, etc. The term “elastomer” stands for elastic polymer and is frequently interchangeable with the term “rubber.” Elastomers have both viscous and elastic properties, generally having low longitudinal stress-to-strain ratio and high yield point as compared to other materials. Polymers are made up of monomers formed mostly from the elements of carbon, hydrogen, silicon, and oxygen. Examples of polymers are polyvinyl chloride, polyethylene, etc. Elastomers exhibit no definite form above 370 C. Natural rubber, polybutadiene, butyl rubber, etc. are some examples of elastomers. Two types of matrices are mainly used for fabrication process, that is, thermoset and thermoplastics. An inflexible solid will be formed by processing above melting temperature and curing the thermoset resin. Since the resin is intractable, it cannot be melted and remolded into a different shape, whereas thermoplastic can be remolded to new shape by heating the previously cured shape. These polymer composite matrices are favored over other materials due to their undemanding fabrication, greater strength-to-density ratio, and corrosion

FIGURE 2.1 Types of polymer.

2.3 History of composites

FIGURE 2.2 Classification of composites.

resistance. They can be classified on the basis of different reinforcements as shown in Fig. 2.2. Composites with a polymer matrix can be used where the addition coatings such as oil and grease cannot be tolerated. Different polymer possesses different properties such as biocompatibility, biodegradibilty, and high mechnical strength which define their uses in appropriate applications. These matrices are easily processed, are low in cost, and have low relative density. On the other hand, low rigidity, low strength, and low transition temperature limits their use. These requirements can be attained by combining nonmetal reinforcements in the polymer matrix. Using metal reinforcements, natural or synthetic fibers, and ceramic particles are often employed in a polymer matrix. Metallic fibers have limited use due to their high density and reaction affinity with the matrix. Due to excellent properties of polymer composites, these are extensively useful in medical fields such as tissue engineering, dentistry, implantation, prosthesis, etc. The biomedical materials are basically a combination of natural fibers and polymer matrix. These composites have excellent biocompatibility and match the morphology of a living being. The classification of biocomposites, fabrication techniques, and its biomedical applications are discussed later in this chapter.

2.3 History of composites In ancient times builders and artisans reinforced mud with straw making bricks. Around 1500 BCE strong and sturdy buildings made up of these bricks were used

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by Egyptians and Mesopotamian settlers (Scala, 1996). Pottery and boats were reinforced in this manner, as well. The Mesopotamians were first credited for use of composites in the form of plywood, created from glued woods. Later, composite bows were invented by the Mongols in 1200 CE. These bows were made of the combination of wood, bone, bamboo, and horns bonded with animal glue. These bows were extremely deadly weapons on the Earth during that time. Mongolian bows provided the emperor with military dominance until the invention of gunpowder in the 14th century. In Indian history, women contributed to the field of composite by reinforcing cow dung with hay to make cow dung cakes. These cow dung cakes are still being used today to cook food in villages. The fashionable era of composites did not fully begin until scientists developed plastics. In 1869 a chemical revolution changed the composite revolution. People started to experiment with resin. These resins completely transformed from liquid to solid state. Glue and binders were some natural resins obtained from plants and animals. Within the early 1900s plastics such as vinyl, polystyrene, phenolic, and polyester were created. These plastic materials were far better than natural resin. However for better strength and rigidity, reinforcements were required. Fiberglass was introduced by Owens Corning in 1935. An incredibly strong, light weight structure was created by combining fiberglass with plastic polymer. This is the start of the fiber-reinforced polymer (FRP) industry as we all know it today. World War II brought the need for light weight, strong, and weather-resistant composite materials and the production of FRP began. Before WWII, aircraft wings consisted of wood and plastic resin. Aircrafts used during WWII were light weight and corrosion resistance as they consisted of fiberglass. By 1947 an automobile of fully composite body was tested, which led to the manufacture of 1953 Corvette. Fiberglass was employed in pipes on a commercial scale and later adopted by the refining industries. In addition to light weight and strength of fiberglass, researchers learned that fiberglass was transparent to radio frequency (RF) bands. Fiberglass was quickly adaptable to electronic radar equipment during the war. By WWII, military wanted fiberglass instead of FRP composites.

2.3.1 Fiberglass in 20th century In the 20th century the world of automobiles and boats fully depended on fiberglass composites. After 1950s the outer body of boats were reinforced with fiberglass materials. After this, fiberglass wakeboards were introduced (Sharma, Bhanot, Singh, Undal, & Sharma, 2013). During this period, classic cars were manufactured by the automobile industry using fiberglass impregnated with resin and molded with fine-shaped dies. In 1953 a new composite Chevrolet Corvette was manufactured. These composites are carbon fiber-derived composites and proved to be slug- and knife-resistant, upgrading safety for the military and police forces.

2.5 Advantages of composites

2.3.2 Composite material in our daily life In the present, composites are employed in the technology of our daily life, for medical supplies, and to the department of defense. Nowadays automotive and aerospace fields are fully dependent on composite technology. Military and police forces utilize these new composites in gunpowder, firearms, automobiles, and aircrafts. Some composites are very light weight and this opens diverse applications in the medical field and equipment technology, etc. National parks are using composites to rebuild foot and vehicle paths and bridges without the need to demolition natural surroundings or use heavy machinery. As technology in composite manufacturing grows, this draws our attention to sustainability and the composites effect on the environment.

2.4 Why composites? The most essential reason to use composite materials is the adaptability of their properties. Composites are chosen due to their improved rigidity and strength while being light weight and to increase acceleration or range in transportation. Composites can be used as fire and blast protection as they are thermal insulators and are also used in parts of machinery. Other reasons to use composites in various applications are durability, strength, rigidity, stiffness, and corrosion-resistant properties. Nowadays, composite materials are used in large number of engineering fields such as aviation, automobile, and robotics. Composite materials have certain strengths and weaknesses which should be taken in consideration. However, the major driving force that is responsible for development of new composite materials is the many combinations of reinforcements and matrices possible to meet the requirements of applications. Composites can be engineered and produced to be sturdy and hardy in a particular alignment, whereas metals are of same strength in all directions. The sheer repertoire of the composites is mind boggling.

2.5 Advantages of composites 2.5.1 Design flexibility Designers can experience unlimited flexibility while designing different shapes from thermoset polymer-based composites. They can be formed into the most intricate shaped components and be made in a large range of densities and chemical formulations to precise performance specifications. Molding of composite in any shape is easier than other materials. Fiberglass composites are materials which can easily form any complex shape. Most recreational boats are built from fiberglass composite material, lowering the cost of boats and improving the boat

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design. The surface of the piece can be finished with a varity of textures, smooth to pebbly.

2.5.2 Light weight Composites are light weight, collated to various metals and woods. The light weight of composites are vital in automobile and aircraft industries. For example, light weight materials result in better fuel efficiency. While designing airplanes, the weight of the craft is always considered by the engineers; reducing the aircraft’s weight reduces the amount of fuel it needs as it acquires more speed. More composites are employed to build modern aircrafts than metal. The new Boeing 787 Dreamliner is made up of composites (Giurgiutiu, 2015).

2.5.3 High strength Aluminum and steel are weaker in strength in comparison to composites. Metals have equal stiffness and strength in all directions, whereas composites have strength in a specific direction in which it required.

2.5.4 Strength related to weight The strength-to-weight ratio can be used when deciding which composite to use for a specific application. Steel itself is a very rigid, heavy material. Other material may be strong and light weight, like bamboo poles. Composites are often styled to be both firm and light weight. Because of these features, composites are used to manufacture airplanes which need a highly compact material on the underneath side. Composite materials exhibit excellent properties in one direction due to unidirectional alignment of fibers in matrix. For example, composites made with metal fibers that must be in an alignment, require more precise processing. But the metal used is often heavy, which adds weight to the finished composite. Composites have strength without being substantially heavy. Composites that have higher strength-to-weight ratios are in structures today.

2.5.5 Corrosion resistance The matrix in composites protect the reinforcement materials from seasonal damage and harmful chemicals that can damage other materials. Composites are better choice for protecting chemicals in stores and outdoors. Composites can stand up in bad weather conditions and during temperature changes.

2.5.6 High-impact strength Many composites can absorb impacts, blast from an explosion or sudden force of bullet. Because of this unique property, bullet-proof vests and panels are made by

2.5 Advantages of composites

using composites. Airplane, buildings, and military vehicles can be shielded from explosions by composites.

2.5.7 Consolidation of many parts Whole assembly of metal parts can be replaced by a single composite materials. Time can be saved by using less number of parts or components in machinery. Less number of parts used means less maintenance needed for a machine.

2.5.8 Dimensional stability Composites are free from temperature effect. Size and shape of composites can remain the same in any weather condition. As the weather changes to hot or cool, wet or dry, swelling and shrinking of wood takes place. For example doors of houses that are made of wood, swell and shrink during weather changes. In rainy seasons wood doors swell and more force is required to close the doors (Selzer & Friedrich, 1997). A situation where tight fit is required, composites can be a better choice. Composites are used in the wings of aircrafts, as the plane gains altitude, size and shape of wings do not change.

2.5.9 Nonconductive Many composites are nonconductive, they do not conduct electricity and are insulators. Electrical utility poles and the electric circuit boards in electronics are made with composites. However, conposites can be made conductive by using conductive filler reinforcement in matrix.

2.5.10 Nonmagnetic Composites are nonmagnetic as they contain no metals in matrix. Any metal reinforcements are shielded by the matrix. They can be used around delicate equipment. The lack of magnetic intervention from the composites used in the housing allows bulky magnets utilized in magnetic resonance imaging machine to perform better. Furniture is also made up to composite material. Floors and concrete walls of hospitals are made from reinforced concrete. In a whole, nonmagnetic nature of composites helps in development of infrastructure of houses and buildings.

2.5.11 Radar transparent Composites are radar transparent materials, which means that radar signals to pass easily through the material. This property of composites makes it a suitable material to use anywhere, whether the radar is on ground or in the atmosphere. The US Air Force’s B-2 spy plane is made up of composite, which is nearly invisible to radar. Radar signals cannot capture the stealth aircraft.

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2.5.12 Low thermal conductivity Composites do not conduct heat or cold easily as they are good insulators. Door, panels, and windows of buildings that are made from composites work as extra protection in severe weather.

2.5.13 Durable Structures made with composites are long-lasting, very durable, and need little maintenance. Many composites have been in use for 50 years.

2.6 Applications of composites Today, thermosetting composites are employed in a vast range of industrial applications that have been described in the following sections:

2.6.1 Aerospace/aircrafts Wings, fuselages, and bulkheads are some applications in aerospace which are made of thermoset composites for commercial, military, and civilian use.

2.6.2 Appliances Thermoset composites have wide range of application in the appliance industries, such as washing machine, hair dryers, refrigerators, microwave ovens, freezer panels, electric controls panels, handles of cookware, power tools, side trims, and vent trims.

2.6.3 Automobile and transportation Composites are widely employed in automobile industry and in transportation. Composite materials are light weight and strong. Due to these properties, they are used for the internal components of cars, buses, and trains and also the outer body parts.

2.6.4 Infrastructure Composites are being used in infrastructure applications such as in buildings, bridges, and roads. Strength and durability of composite material are the major properties designers want in the material that we use in our daily life. Thermoset composites are used in construction, replacing many traditional materials. In homes, doors, wall panels, fixtures, roofing, sinks, shower stalls, etc. are architectural components that can be replaced by thermoset polymer composite with

2.7 Limitation of composites

some additional reinforcement. These composites are having far better result in comparison to traditional materials—aluminium and steel.

2.6.5 Environmental Composites are often seen in applications in corrosive environments. These materials are said to be ideal for many types of application, such as natural resource refineries, chemical processing plants, water purification and treatment facilities, etc. Some of the ordinary things that are made of thermoset materials are tanks, hoods, pumps, pipes, fans, and cabinets.

2.6.6 Applications of electricity Composite materials have many applications in electrical industries due to their high dielectric permittivity, good arc resistance, and scratch resistance etc. Examples of components made of these composites are microwave antennas, wiring boards, substation equipment, motor controller, standoff insulators, control system components, circuit breakers, arc chutes, terminal boards, metering devices, gear switch, bus supports, and lighting components.

2.7 Limitation of composites Some of resins used in composite materials can withstand a temperature of 150 C or less, which means the resin will weaken at 140 C 150 C. This may increase the risk of fires in aircrafts or vehicles. Fires within composite materials can spread toxic gases and microparticles in atmosphere, causing health hazards. Temperatures above 300 C can result in structural breakage. Some composites are very brittle in nature and due to this, these materials can be easily broken (Lau, Ho, Au-Yeung, & Cheung, 2010). Composites involve such assembly where out-of-plane direction of reinforcement leads to poor strength of composites. The primary load is carried out by matrix which impacts mechanical susceptibility and requires more careful repairing as compared to structures made of metals. Due to brittle characteristic of some composites, they easily get damaged as compared to wrought metals. Cast metals also have some brittle character. Metal reinforced composites have low shelf lives because of oxidation of metal with time which introduces corrosion, abrasion, etc. and require refrigerated transport and storage. Some special equipment is required to hot curing. Curing process takes time for heating and cold. Pressure and tooling is required to repair at original cure temperature. Cleaning of composite should be done before repairing to make them free from all contaminates present. Some fibers and matrices absorb moisture that leads to the need of the composite to be dried at a particular temperature before repair.

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2.8 Biocomposites and classification 2.8.1 Biomedical composites It is worth addressing that the most noteworthy advancements in the field of composites are biomedical ones. Biomedical composites are used to supplement or replace tissue in the human body. Several uses of biomedical materials were noted from primeval civilization. Artificial nose and eyes were found on Egyptian mummies. For the repairing and reconstructing the defective parts of body, People of India generally use biomaterials such as wax, glue, etc. Some of these biomaterials are useful today for the purpose of cleaning and shinning household furniture. Over the centuries, advancement in many composite materials has promoted bioresearch in many ways such as medical practices these days. Medical practice these days utilizes biomedical composites in several applications, such as limb replacements, joint replacements, and dental implants (Pye, Lockhart, Dawson, Murray, & Smith, 2009). Medical devices, such as biosensor pacemaker, artificial hearts, blood vessels, or organs, are widely used to supplement and bring back the function of traumatized and degraded tissues to help in healing and improving function, thus increasing the life span of patients. Biomedical materials and biocomposites are fabricated to operate with the body.

2.8.2 Basic requirements and parameters for biomedical applications 2.8.2.1 Biocompatibility Biocompatibility is the ability of graft material to function without damaging the systematic response of the body. Prior to use, the biomaterials first undergo toxicity and compatibility testing to determine the safety. Some materials such as nickel and chromium can cause allergic reactions to skin or tissue.

2.8.2.2 Corrosion Corrosion is oxidation deterioration of metals. When the metal corrodes that means it will weaken the mechanical property of metals and this will weaken the chance for complete bone healing. Several types of corrosion could take place during orthopedic surgery to repair broken bones. Most common corrosion is galvanic corrosion. In galvanic corrosion metals which are in electrical contact with one another undergo corrosion. The metals are immersed into an electrically conductive medium, that is, in human body. The corrosion not only harms the surface of the orthopedic grafts but also acts as stresser which damages other soft tissue attached to it.

2.8.2.3 Mechanical properties Mechanical properties must be kept in mind while designing a bone fixation device or an implant. Design of implanting device required attention to size,

2.8 Biocomposites and classification

structure, long time stability, and mechanical properties. Mechanical properties depend on a number of factors, such as the wear resistance properties under stress and strain from use, how it shows fatigue, it’s viscoelasticity and isotropic nature. For example, in dental prostheses, silicon carbide/carbon fibers composite have low elastic modulus with appropriate strength with that of natural teeth which reduces the chance for stress, giving replaced teeth a better lifetime (Ramakrishna, Mayer, Wintermantel, & Leong, 2001).

2.8.2.4 Pores Pores are defined by several measurments, such as degree, size shape, and interconnectivity, and are a essential part of the biomedical application. Low-cost titanium-based composites are porous in nature to provide interconnectivity with the cells it is placed with. Titanium rods do not harm the soft tissues of body.

2.8.2.5 Eye glasses Composites have replaced the glass used in spectacles, as well as being used for the frames. The plastic glasses are stronger and lightweight compared to normal glass. The raw material used for plastic glasses is cellulose acetate which is strong and flexible. The embedment of certain particulate such as nickel and titanium makes the glass mechanically more stronger. These glasses can be coated with a scratch resistant film.

2.8.2.6 Biodegradability and bioabsorbable polymer Biodegradable polymer are used for various applications in medical fields such as surgical sutures, drug delivery system, bone fixation, aid in cell adhesion, drug carrier, vascular grafts, artificial skin, etc. These polymers are biologically decomposable and do not harm the cells in body.

2.8.2.7 High cell adhesion and less inflammation In many biological applications controlling surface to cell adhesion is very important parameter. Cell to surface adhesion is a crucial factor studied in tissue growth research which indicates whether cell culture could develop into functional tissue. Biomedical composites must have high cell adhesion properties. In medical implantation, adhesion between implanting device and peripheral tissues is of enormous value to make implants successful. It must be kept in mind that implanting a medical device should create less inflammation to the surrounding tissues.

2.8.2.8 Wear resistance In biomedical composite applications such as in dentistry, bone graft, and bone cement, the wear resistance behavior of composites is very important. Wear causes less stability and lifetime of joint or dental implant. High wear resistance properties of some polymers such as polyurethane, calcium phosphate, polyethylene terephthalate, etc. enhances the long-term stability of planted device. On the

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other hand, proper alignment of reinforcement fillers in the polymer matrix improves the mechanical properties and wear resistance of the replaced part. Wear debris is minimized by incorporating fillers in composites.

2.8.3 Biomedical polymer composites Biomedical polymers are macromolecular compounds obtained from natural origin. Biomedical composites are flexible and are resistant to chemical attack. These composites have good compatibility with body. There are a wide variety of biomedical composites available with different physical and chemical properties. These materials are easily moldable in any shape. Biomedical composites are classified into two types, depending on their origins: • •

Natural biomedical polymer Synthetic biomedical polymer

2.8.3.1 Natural biomedical composites Natural biopolymers are derived from living organisms and easily replace injured tissue. Biocomposites have stimulated, or prompted researchers as it is the best eco-friendly alternative on the market. Fiber reinforced polymer is made of a polymer matrix reinforced with fibers (Bharath & Basavarajappa, 2016). Natural fibers are usually extracted from plants, such as pineapple, hemp, etc. Several varieties of plants are currently in use and more are being researched. Plant fibers have proven their position in the field because their properties are absolutely remarkable compared to other sources of fibers. These fibers have low density, are renewable, biodegradable, highly porous, and available in large quantities. Their mechanical and physical properties make them better than other types of composites available on the market. Some fibers are obtained from living plants, however some fibers can be extracted from the waste of the crop. Fiber properties vary from one plant to plant, but the properties also vary depending on the harvesting methods, crop management, and the treatments the plant undergoes. Biocomposites are eco-friendly, however, many different industries are researching to find out whether they are completely biodegradable. An epoxy matrix, that is not biodegradable, with fibers from the kenaf plant produces a partially biodegradable biocomposite. When designing a biodegradable biocomposite, it is important to select a biodegradable matrix. Polylactic acid (PLA) is a biopolymer and widely available in the market however, it is appropriate and is one option present in the market; however, it is appropriate only for those applications that do not require high mechanical performance at high temperatures or do not need long-term durability. Other options available in the market are polyglycolic acid, poly-b-hydroxyalkanoates, and polycaprolactone. Natural fibers are sensitive to temperature and must be kept at temperatures lower than 200 C at all times. To satisfy this requirement, matrices having low melting point are needed. Due to

2.9 Applications of biocomposites

its lower mechanical strength, these polymers are rarely used. Thus these biocomposites have limited applications.

2.8.3.2 Synthetic biomedical composites Advancements in production techniques and material science work together in the production of synthetic biocomposites that play a crucial part in tissue engineering. Composites can be designed or fabricated by using materials such as man-made resin and inorganic materials. The objective of this amalgamation is to maximize the advantages and minimize the drawbacks. Required characteristics can be attained by adding reinforcing inorganic fiber to methacrylate or urethane dimethacrylate synthetic polymers. Poly(methyl methacrylate) (PMMA) is the most suitable polymer for restoration of dentin and enamel. Biocomposites made up of ceramic and glasses are also used in dental tissue regeneration. The embedded inorganic particles are used to overcome inherent weakness of the polymer. Over the past few decades, scientists have made great advancements toward bone and dental tissue engineering by developing porous resin with desired mechanical properties. Some of the synthetic materials or polymers used as biodegradable polymers are: 1. 2. 3. 4.

polyesters polylactides polypeptides polyglutamic acid polyanhydrides

2.9 Applications of biocomposites Applications of biocomposites are many in the medical and dental fields, such as tissue regeneration, wound healing, and even cancer treatments, (Fig. 2.3) and in various parts of human body (Fig. 2.4). Biomedical composites can be used alone and as a complement to standard materials. Biocomposites can be used for improving health and safety in the production of these polymers as they are lighter in weight and environment friendly. In designing biocomposites and anticipating their performance, many issues must be taken into account related to their biological reaction. As the number of scaffold material increases, therefore the host material can be variable. If the reinforcement is biocompatible, the host material, that is, matrix with reinforcement will itself be biocompatible to each other. The biocomposites can be styled in such a way that there will be no interaction of filler particles with the host tissue, but this is demanding because it means eliminating all spaces at the fiber or particle matrix interface throughout fabrication. Bulk form of composite structures directly affects the host material reaction with body part and may lead to serious infection as compared to tiny/particulate form of composite which will not cause severe effects. For instance, ultrahigh-molecular-weight polyethylene is a highly cross-linked, biocompatible

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FIGURE 2.3 Commercial applications of biomedical polymer.

FIGURE 2.4 Various applications of biocomposites in human body.

2.9 Applications of biocomposites

polymer that has recently become a prominent material in hip replacement as an acetabular cup, whereas fibrous forms of acetabular cup are generally biocompatible but cause more adverse reactions (Ge, Kang, & Zhao, 2011). While nanofillers, whiskers, microspheres are small in size and cause less irritant to human body, the immune cells could envelop the composite and transport it to other parts of the body. This may result in the release of enzymes that could influence the functioning of composites, such as by changing the speed at which degradation is expected. In addition, in dental and orthopedic moving parts, friction increases. New voids at the interface could occur and could cause abrasions to the host. Exposure of the filler particle to the host is also a concern. Therefore the interlinkage of filler material to the host boundary is important for composite performance. The tissue response may affect the material in different ways, such as puffiness of facial spaces with body liquid and fibrous tissue. It should be noted that thermoset polymers are uncommon in tissure implants in human body because they contain nonreacted monomers and cross-linking agents. The size of glass fibers is another important issue to kept in mind during reinforcing in thermoset and thermoplastic polymer. Some glass pieces may also percolate from the matrix if they are not fully removed during processing, but this is not a concern if the application is on the outer part of body. Following are a few examples to illustrate the use of composites in the medical and dental fields.

2.9.1 Tissue engineering The main purpose of biomedical composites in tissue engineering is to regenerate the damaged tissue. The biocomposites act as a template or scaffold to support the growth of new cells and are cell- and tissue-specific materials that help the tissue integration (Seal, Otero, & Panitch, 2001). Cells attached through biodegradable polymer composites have smaller dimension as compared to actual cells available in body. Repairing the blood vessels and healing the wound at the same time is a major requirement in the medical science. Bone of human body is considered composites of protein having a different morphology such as a variety of shape, structure, and size. These composites consist of hydroxyapatite (HA) nanocrystals that are deposited along the collagen fibers in bone. Collagen fibers have low elastic modulus. These fibers are oriented in the direction of stress. About 70% of dry bone weight can be covered by HA. According to Wolff’s law, bone can remodel and adapt itself according to applied mechanical surroundings. Depending on the location in the body, there are different kinds of fracture. To attain proper fixation of bone, it is required to follow the proper procedure of implantation with control development of extra tissue and fracture point trauma. After the fracture is healed, all implants can be eliminated from patient body. Biodegradable polymers used for bone scaffold are propylene fumarate and chitosan, with a copolymer such as lactide glycolide. Khan et al. (Ambrosio, Sahota, Khan, & Laurencin, 2001) prepared calcium phosphate scaffold. This material

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was inoculated for 8 weeks in simulated body fluid. Simulated body fluid pH and scaffold mass decreased firstand than increased. The composite scaffolds increased reprecipitation of calcium phosphate simulated body fluid for the implant. Researchers are paying more attention to the bioactive ceramics because of their excellent bone tissue binding nature. Reproduction of the osteoblasts occurs due to contact between blood plasma and HA layer coating on the surface of embedded composite. In most of studies HA particles are reinforced in highdensity polyethylene (HDPE) matrix as a bone replacement material (Bonfield, Wang, & Tanner, 1998; Di Silvio, Dalby, & Bonfield, 2002; Ladizesky, Pirhonen, Appleyard, Ward, & Bonfield, 1998; Salernitano & Migliaresi, 2003; Wang & Bonfield, 2001; Wang, Deb, & Bonfield, 2000; Wang, Joseph, & Bonfield, 1998). The successful marketing of HA-based composite was started in 1995 by a company name HAPEX. The bone substitutes manufactured and traded by HAPEX suffered from drawbacks of strain failure, low stiffness and strength as compared with cortical bone. This is why these substitutes cannot be used in load-bearing applications.

2.9.2 Orthopedic As summarized by Evans and Gregson (1998) composite materials are widely used in orthopedic applications, especially in bone fixation plates, hip prosthesis, bone cements, and bone grafts. The most common materials for hip joint supplement of femoral stem are 3161 stainless steel and some alloy of cobalt, chromium and titanium. These alloys are 10 times than the bones in body they are replacing. Cortical bone in the body has stiffness of 16 GPa and tensile strength of 92 MPa (Katz, 1966), whereas titanium has tensile strength and stiffness of approximately 800 MPa and 110 GPa, respectively, which is clearly more as compared to the bone it replaces. This causes unpleasant bone reshaping and stress shielding, resulting in a loss of bone mass and implant displacement over time, particularly in the proximal part. Fiber reinforcements can be used to match tensile strength of bone. Carbon fibers reinforced in polysulfone matrix have stiffness and tensile strength of about 170 GPa and 900 MPa, respectively. Carbon fiber polysulfone composites are difficult to produce and have low durability but they continue to be fabricated for the inherent advantages of flexibility and radiolucency. Carbon debris from the composites can be mitigated by polishing with titanium alloy and HA (Baˇca´kova´, Stary´, Kofroˇnova´, & Lisa´, 2001). A resorption-rich bone plate is required to eliminate the need for another implant after complete fracture fixation healing. The rate of degradation must be controlled and degradation by-products must be nontoxic that aids to balance the mechanical parameters such as stiffness loss in implantation and increases healing strength. Composite bone plates of carbon fiber with PLA and calcium phosphate fiber with PLA are both fully resorbable (Iftekhar, 2004). These composites however do not have suitable mechanical properties and degrade rapidly. Bone cement is used to fill the voids and enhance adhesion between host bone tissues

2.9 Applications of biocomposites

and implant bone which has been used to reinforce with fiber matrix to prevent loosening and enhance strength. PMMA powder mixed with methacrylate-type monomer is used as typical bone cement during fixation. Creep deformation and fatigue life of PMMA matrices can be enhanced by adding a low volume fixation of graphite, carbon, and Kelvar fibers (Kelly, Cahn, & Bever, 1994).

2.9.3 Dental Composites have so far been the most successful in the dental field. There are rigorous design requirements, which are hard or difficult to obtain from materials such as ceramic alloys and metal alloys. Whether it is to fabricate crowns, restore fillings, or fill in missing teeth, the composite must matched the color and diaphanous with adjacent teeth and keep its shine (Furtos, Silaghi-Dumitrescu, Lewandowska, Sionkowska, & Pascuta, 2016). The antiwear stress and fracture must be matched with the strength of above tooth. The tooth must be isotropically stable inside the mouth and withstand the varying temperatures of foods. Organic and inorganic inclusions added to polymer resin matrix are used in repairing fillings as shown in (Fig. 2.5). Ceramics, calcium silicate, calcium fluoride, and crystalline quartz are some of the particles used to increase strength and wear resistance. Fatigue fractures and water absorption can be reduced by using silica particles as reinforments and treating the composites with a silane coating. These treatments may improve retention of matrix (Beatty, Swartz, Moore, Phillips, & Roberts, 1998). Normally, the size of the filler particles are between 20 nm and 50 nm in size. Filler volume of about 80% can be used to fabricate composites. Fused silica particles of between 20 and 50 nm in size ared used in microfilled dental resin to a volume of 42%. These composites are colorless and can be coated with an opaque gloss however, the mechanical strength of these composites is not enough for posterior teeth and hard to handle the coating due to low viscosity of gloss (Furtos et al., 2016). Particle size from 0.1 to 10 μm are used in hybrid dental resin. These dental resins provide easy handling due to higher filler volume around 80% particle content and higher viscosity. Hybrid resins have low water absorption compared with microfilled resin. Commercial dental resin could change shape due to polymerization shrinkage of 1.2% 2.9% and up to 1.5% water absorption (Beatty et al., 1998). These composites have difficulty adhering to dentine, causing fitting and leakage problems. Using all ceramic dental composites can enhance stress bearing on crowns and bridges as shown in (Fig. 2.5). Decreasing fractures in is one of the very important challenges for dental composites. A common type of composite is In-Ceram made from alumina glass, in which there are slip casting molds, sintering of a skeleton of alumna particle, followed by melt seepage of glass into the porious areas (Wolf, Vaidya, & Francis, 1996). Fracture toughness was not affected by thermal expansion mismatch between alumina and glass. It is necessary to match the hardness of composites with the enamel of opposing teeth to reduce the wear stress. Hardness of teeth can be enhanced by coating composites with alumina silicate calcium phosphate.

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CHAPTER 2 Foundation of composites

FIGURE 2.5 (A) Natural tooth crown with screw at lower part along with natural tooth and (B) broken tooth image before tooth filling and (C) after the filling of tooth with composite.

Posterior and anterior teeth can be replaced by composite materials. The broken teeth replaced by the oral prostheses done by PMMA provides excellent mechanical properties to the posterior teeth. The composites in the dentistry field has several crucial requirements, such as long-term durability, dimensional stability, wear resistance, minimal shrinkage in polymerization, and higher strength. Filling microcavities that may form are responsible for the excellent mechanical and wear resistance properties. The adhesion between the filler and microcavities results from lack of material involvement (Ensaff, O’Doherty, & Jacobsen, 2001).

2.9 Applications of biocomposites

Orthodontic archwires are another application of dental composites. Fiber volume fraction ranges from 30% to 75%, depending on the glass fiber yarns used the strength and the modulus of titanium wires. Hyoxyapatite ceramic particles reinforced in polyethylene matrix produce orthodontic brackets, which have good isotropic properties and excellent adhesion to enamel.

2.9.4 External prosthetic and orthotics Skeletal bone is an important part of human body that supports and protects the essential organs. Red blood cells and white blood cells are produced by the bones. Bone grafting is an invasive procedure that transplants bone into the injured bone in order to heal a fracture that is tremendously complex. Bone grafts serve as a structural framework for limb formation, maturation, and remolding. Several materials are used as bone repairing materials as shown in Fig. 2.6. Wood, aluminum, and leather are traditional orthotics and prosthetics materials and these are being widely substituted with extraordinary composites and thermoplastics. Fiberreinforced composites are light weight, can be very small in size, and are safe to use; that is why they have become a very attractive option in this area. These traditional composites are designed and manufactured using a specific thermoset to be very strong and exterior to the body. Composites have been used to interface transtibial (TT) and transfemoral prostheses (TP) with the residual limb as well as to spread out the load over the surface (Paul, 1999). Only 1 2 kg weight can be targeted by TT and TP prosthesis, which makes carbon-reinforced composites ideal. Socket frame is made up of tape of carbon fiber and resin. Stiffness of bone socket and grafts can be tailored using methyl methacrylate, which is a blend of rigid and flexible polymeric matrix.

FIGURE 2.6 Different approaches for bone repair.

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CHAPTER 2 Foundation of composites

Stainless steel in artificial arms has been replaced by carbon fiber-reinforced (CFR) tubes. Carbon fiber/nylon hybrid composites have exhibited excellent contact resistance as compared to single-carbon nylon composites. Injection molding technique is used to fabricate CFR/nylon 6, 6 hybrid composites. These fabricated composites have excellent vibration damping and shock absorption properties. For the prosthetic foot unit (shown in Fig. 2.6), various composites have the property to store flexural energy and reuse it during motion of body. According to individual patient needs and their characteristics, modification of the foot unit and prosthesis have been done to preserve the flexural energy of the implant. For this requirement the flex foot unit contains long CFR epoxy composites rod that preserves the unbroken length of prosthesis (Fu et al., 2015). In the carbon-copy foot, posterior Kevlar-reinforced nylon block and anterior CFR plastic leaf springs were the materials that are used to produce keel and provide dual stage support to flexion (Mir et al., 2018). At last, nylon-reinforced silicon elastomers have been used to construct a tough plaster to make a bendable foam that is wrapped over the simulated body part. The orthotics were originally designed to support tissue injury. These days their application can be found in splinting material for cast formation, replacing the old cotton and plaster of Paris. Besides strength, they have the advantages of better X-ray transmission and lower water adsorption. Fig. 2.7 depicts different types of implanting devices in human body.

FIGURE 2.7 Different types of prosthesis and replacement in body.

2.9 Applications of biocomposites

2.9.5 Biocompatibility on skin Skin is considered as the first organ of the immune system as it fights against the harmful antigens and pathogens. Skin can be damaged in several ways, such as burns, cuts, injuries, infections, etc., but cannot heal itself. A critical requirement in the field of tissue engineering, is to develop biocompatible and biodegradable material for skin healing and repairing (Jeong, Park, & Lee, 2017; Zhao et al., 2019). The structure of the grafts should match the structure of natural skin extracellular matrix. Several natural and synthetic hydrogels are being fabricated and developed for skin regeneration such as collagen, hyaluronic acid, polyviny alcohol, and polyethylene glycol (Bahadoran, Shamloo, & Nokoorani, 2020). Thin films made of polyvinyl alcohol and sodium alginate were synthesized for skin burns (Kamel, Abd El-messieh, & Saleh, 2017). The wound healing properties of chitosan can be more efficient by reinforcing it with banana peel powder. Kamel and his colleagues synthesized a nanocomposite membrane from banana peel powder. Banana peel powder acts as an ionic cross-linker (Ghiasi, Chen, Rodriguez, Vaziri, & Nazarian, 2019). Nanocomposite membrane has high surface-to-volume ratio which enhances cell proliferation and cell migration. Another compound for wound dressing is spider web and silk. Spider’s web can stop the bleeding and heal the wound rapidly.

2.9.6 Healing of fracture and wound dressing A complex process that involves unique and highly integrated series of events is used to heal fractures in the body. The process of repairing a endochrondal fracture begins with pro-inflammatory and ends with remolding phase (Neumann & Epple, 2006). In initial stage a hematoma is formed from the blood vessels ruptured by the injury. Inflammatory cells invade hematoma and remove the necrotic debris. A combination of degranulating macrophages, leukocytes, platelets, and mast cells penetrates the fractured area and amplifies the initial inflammatory responses by activating additional pro-inflammatory cytokines and peptide signaling molecules that trigger and promote growth and repair, as well as begin to clean up necrotic debris. In a typical trauma, the fracture undergoes several types of tissue malfunction. Naturally using bone grafts can make the situation even worse, therefore there is high demand for bone cement as a substitute. Unfortunately medical application of xenograft is generally carriers’ viral infection. Xenograft is generally use for transplantation of tissue, organ of one other species to human body which unfortunately causes viral infection. Other disadvantage of xenograft is it is easy resorption and low osteogenicity as compare to autogenous bone in body. Calcium phosphate is widely used as a bone substitute material due to their chemical similarity to the mineral component of mammalian bone and teeth (Gazdag, Lane, Glaser, & Forster, 1995). Some commonly used calcium phosphate cements are listed in Table 2.1. Most importantly, it is nontoxic and biocompatible.

51

Table 2.1 Existing calcium phosphate and its properties. Molar ratio of Ca/P 0.5 1.0 1.33 1.5

Material name

Chemical formula

Solubility at 25 C, -log(Ks)

Solubility at 25 C, (g/L)

Range of pH stability in aqueous solutions at 25 C

Mono-calcium phosphate monohydrate (MCPM) Dicalcium phosphate dihydrate (DCPD), Octacalcium phosphate (OCP) β-Tricalcium phosphate (β-TCP)

Ca(H2PO4)0.2H2O

1.15

Approx. 18

Between 0.0 and 2.0

CaHPO4
2H2O

6.60

Approx. 0.088

Between 2.0 and 6.0

Ca8(HPO4)2(PO4)4
5H2O

96.5

Approx. 0.0081

Between 5.5 and 7.0

β-Ca3(PO4)2

28.9

Approx. 0.0005

Stable at temp 100 C

2.9 Applications of biocomposites

Calcium phosphate cement has exhibited bioactive behavior and ability to integrate into living tissue by the same process active in remodeling healthy bone. Calcium phosphate cement is used to fill the defect after the injury or breakage of bone (Fig. 2.8). The major drawback of calcium phosphate cement is its poor mechanical properties resulting in poor load-bearing capacity. For a better result calcium phosphate cement is reinforced with a different material of same or more biocompatibility and having more strength. Many patients suffer from blood loss and infection during and after a personal injury. Slow wound healing generates microbial infections due to its molecular and cellular behavior that can be controlled by growth of secreting cells. The fundamental wound healing process is fabrication of collagen fibers due to transportation of fibroblast to the wound. Wound and its nearby location is safeguard by wound dressing. Some of wound dressing materials are polymers, cotton, hydrofibers, films, foams, and alginates. Some important examples of naturally extracted wound dressing materials are silk sericin and spiders’ web derived from silkworm and spiders respectively, due to their antibacterial, moisture absorption, and oxidation resistance properties. These materials immediately stop the blood loss and healing of the wound can begin. Some synthetic wound dressing materials are kaolin/polyurethane, silver-cobaltdoped bioactive glass nanoparticles, cross-linked polymer network films, etc. (Zhang, 2002).

FIGURE 2.8 Radiographs demonstrating the use of bio-net materials. (A) A posterior image of the both leg shows the use of calcium phosphate bone cement and additional Fixation device made of u-HA reinforced polymer (arrows) to fill the defect after bone brake. Bone cement can be used as an adjunct to increase the purchase of screws during fracture fixation (arrow). (B) Image (1) showing before treatment and image (2) showing after treatment, a biodegradable polymer impart.

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2.10 Fabrication techniques of biomedical composites There are several approaches for fabrication of the constituents of biomedical composites Several of the approaches are taken from a different industry, but some methods are developed to satisfy a particular design or fabrication challenge. Selection of the fabrication technique depends up the requirements of the particular part, such as the design, application and the material to be used. Molds are often used to provide the required during composite fabrication process. Specialized tools are required to ensure the correct shape during the molding and the curing process.

2.10.1 Hand layup molding Hand layup is the most common fabrication approach for polymer-based thermoset composites. Hand layup technique comprises arranging plies (sheets of fiber) by hand onto an implement use for making coated stack. The stack is placed onto the mold, if being used and they system placed under a vacuum. Resin is then infused into the plies of fiber. There are a number of curing techniques but the most basic is simply to permit the composite to cure at a particular temperature. The whole process of curing is performed in vacuum. Application of heat can accelerate curing. High heat and constant pressure are required for curing high-performance thermoset polymer-based composites. Autoclaves can also be used for curing, but are expensive to purchase and to operate. Many parts can be cured simultaneously by manufacturers having autoclaves. Autoclave temperature, vacuum, pressure, and inert atmosphere are controlled by the computer systems. The computer system also provides remote supervision of curing process. Thin laminates are cured by methods using an electron beam (E-beam). This is an effective method. In Ebeam curing, the amalgamated layup is exposed to a stream of electrons that delivers radiation causing polymerization and cross-linking in sensitive resins. An analogous manner is curing through X-ray radiation or microwave. In thermoset resin a photoinitiator has been added, and this initiator is activated by ultraviolet (UV) radiation. Activating the photoinitiator in the resin sets off a cross-linking reaction. UV radiation curing is termed a fourth alternative for curing. Only light permeable resin and reinforcement can be cured using this method.

2.10.2 Open contact molding method One-sided molds can be inexpensiveand the basic method for creating fiberglass composites in one-side molds is the open contact molding method. This is lowcost and effective method to fabricate composites. Open molding technique is typically used for cabs of truck, hulls of boat, decks, bathtubs, shower stalls, and other large simple shapes. In this process a rigid one-sided mold is used to provide the surface finish to one side of a component. The thickness of composite depends on how much of composite piles are placed in the mold. The plies can

2.10 Fabrication techniques of biomedical composites

be placed in the mold either by hand layup or by spray up. To prepare mold surface, a gel coating is applied on the inside of the mold to help with the release step. The sprayed gel is then cured and mold is ready. In the spray up process, reinforcement fibers are sprayed into a mold using a chopper gun, which slices the fibers into short lengths. These chopped fibers are then sprayed directly onto a stream of resin in order to make the resin and fibers adhere to each other perfectly. In this process the volatile organic compounds reduced by activated piston that further activates the spray guns and nonfluid impingement spray heads distribute gel coating on fiber and after gel coating the resin is cured in low pressure along with fiber/particulate. Workers compressed the laminate with a roller in next step of the method. Other core material such as wood, foam, etc. may then be added and the core may then be sprayed with another layer. The molded portion is then cooled, cured, and taken out from mold. Hand layup or spray up technique require fewer number of labors. Faster production rate of resin infusion process makes the industries to switch to hand layup technique as an alternative process for fabrication and encourage the fabricators to process this method wherever possible.

2.10.3 Liquid molding and injection molding Resin transfer molding (RTM) is a common alternative for fabrication and is also known as liquid molding. The RTM process has very impressive results and benefits. Generally, the materials used in RTM are less costly and can be stored at room temperature as compared to pre-impregnated (prepreg) materials. This method generally produces a very thick shape and eliminates most postfabrication work. All exposed surface gets smooth finish and complex parts are dimensionally accurate having good surface details with this method. Before closing the mold, it is possible to put inserts inside the preform. The RTM process allows for integration of other hardware and additional core materials. RTM process requires less cycle time to complete and could be automated for greater effieiency. It reduces a cycle time from several days to just hours or perhaps minutes. In RTM process before injection into mold, the resin and catalyst are mixed, where in reaction injection molding (RIM) two separate streams, one of resin and one of the catalyst, are injected into the mold. Therefore all the chemical reactions take place inside mold rather than in the dispensing head. In the automobile industry, RIM and other quick reaction approaches are combining together fabricate structural parts but without A-class finish. The fiber and resin mixture can be sprayed up onto a vacuum mold by means of programmable robots. Robotic spray up can be directed to regulate fiber alignment.

2.10.4 Vacuum resin transfer molding process Vacuum resin assisted transfer molding (VARTM) is a fast emerging molding technique. The difference between resin transfer molding process and vacuum-

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assisted process is the vacuum. VARTM can operate at moderate heat and does not require heavy, expensive equipment. VARTM can produce large complex parts inexpensively at one time. As the whole process in VARTM is carried out in presence of vacuum tight seal; therefore a canopy/plastic film is placed at the highest and it is a two-sided mold, where resin is injected from one side and reinforcement injected from another side. The resin enters the mold through various ports and feed lines, termed manifold ports. These are drawn by the vacuum that facilitates the wetting of fibers in matrix. There are many applications of structures made from theis method, such as bus, car, bikes, marine transportation, and construction work on ground, many infrastructural structures in building, medical field, etc. This method is first used by Boeing cooperation (Chicago III) and by NASA. The first firm that used this process was small and provided laminate pieces for aerospace without the use of an autoclave.

2.10.5 Compression molding Compression molding is a high-volume and high-pressure molding process appropriate for producing strong pieces. This method employs durable metal dies which are very expensive. Thermoplastic composite can be molded with fibers, fabrics, optical strands, etc. When production quantity requirements exceeds 15,000 parts, use of the compression molding technique is very beneficial. Compression molding technique produces very simple structures, has lower tooling costs and less waste. as compared to other techniques In this method the molding material is preheated in an oven before being added to the molding cavity. A large hydraulic press is required to compress the top of the mold onto the material inside the mold to form the desired shape. The material is then cured by means of pressure. By using sheet molding compound (SCM), large parts can be fabricated in forged mold dies kept in preheated oven. SCM is a sheet of composite fabricated by inserting fiberglass or other fiber strands in chopped form and using glue or resin paste to combine two or more sheets. Low-pressure SCM offers a low capital investment free from volatile organic compounds emission and potentially a supreme quality surface finish to the structure part. Automotive industry suppliers use carbon fiber-reinforced sheet molding material, to take advantage of the benefits of carbon, its high strength and stiffness. During the oven cure, the discharge gases of inserted material are trapped within the microcracks of cured compound. These microcracks can be prevented using SMC technique. Composite producers in industrial markets are formulating their own resins and compounding SMC inhouse to satisfy need for customized material fabrication, in applications that require UV radiation protection, impact and moisture resistance, or have high surface-quality demands. Injection molding can be a fast, high-volume, lowpressure, and closed process using, most typically, reinforced thermoplastics, such as nylon reinforced with chopped glass fiber. In last 20 years automated injection molding process has seized some market operated by thermoplastic and metal casting manufactures. Transfer molding is a closed mold process where a nozzle

2.10 Fabrication techniques of biomedical composites

forces the material into the molder cavity where the material is settled under the application of temperature and pressure. Filament binding is a repeatable method that has low costing of material. Structural part yields by filament method have high strength. Golf shafts are made by using filament winding method. Pressure vessels, rods, pipe, tubes, etc. are used in several other businesses. Glass fibers and resins have been used for many years such as RTM in pultrusion process, but in last 10 years this method has set roots in advance composite applications. During this low-cost, simple, and continuous process, a forged resin bath is required to heat the resin so that reinforced fibers can pull through it to make specific shapes by passing through many forming bushings. The shape and curing of fabric takes when the fabric moves through the heated die filled with resin. After cooling, desired length of the fabric and resin mixture is formed. Requirement of post processing is negligible to provide smooth finish to the structures by converting the fibers and resin into thermoplastic polymer. Pultrusion of good range of continuous, solid or hollow profiles takes place and therefore the process is often custom-tailored to fit specific applications.

2.10.6 Tube rolling Tube rolling may be a long-standing composite manufacturing process which will produce finite-length tubes and rods. It is mainly applicable to small-diameter cylinder having diameter of approximately 6 in. or 152 mm that will be rolled properly. Typically an adhesive prepreg fabric is employed depending on the part. The fabric is precut in shapes that are styled to attain the essential ply schedule for the applications. A cylindrical tube is rolled over the surface under certain pressure to compress the other material pieces and to remove the part of the bulk from the flat surface. Only primary rows of fibers fall on verity 0-degree axis by rolling mandrel.

2.10.7 Automated fiber/tape placement process Fibers can be continuously placed under a mandrel at high speeds using a programmable robotic head to cut and then clamp the tows. More than 32 tows can be clamped and cut simultaneously. The mandrel cuts the fibers into a shortest possible length. The automated machine attached to dual mandrel tubes or station produces fiber more efficiently and increases production. A 5-axis overhead bridge-like structure supports the fiber placement head and is fitted to a winder of filament to deliver custom system. Automated fiber placement (AFP) has certain advantages such as less processing speed, material scrap, wastage, labor cost, and improved part uniformity. Often the method is employed to provide complex shapes having bulky parts. Automated tape laying (ATL) is a similar, faster automated process within which prepreg tape, instead of single tows, is used to make structure. It is used for fragments having complicated angles. Tape layup is flexible, allowing breaks within the process and straightforward alignment changes,

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CHAPTER 2 Foundation of composites

and it may be adapted for both thermoset and thermoplastic materials. The top of the machine contains a coil of tacky winder and a tape cutter. In either case, the pinnacle could also be set at the tip of a multiple axis robot that rotates around a cylindrical tube. ATL is quicker than AFP and might place more material over wider distances; AFP is well matched to shorter courses and places the material over the counter surfaces.

2.11 Conclusion The composite materials have superior properties for application in medical field, which makes them a promising material in research and development. Biomedical composites are cost effective, provide better strength and stiffness to the parts they are replacing, composed of natural, biodegradable, and biocompatible matrices with certain reinforcements. Open molding, injection molding, and compression molding are some highly reported techniques of fabrication of composites. Polymer composites have received a great deal of attention from researcher and medical professional due to their great effectiveness in dental technology, bone cement technology, bone fracture healing, tissue engineering, prosthesis, cancer treatment, etc. Polymer-based bone scaffolds show great strength, biodegradability, biocompatibility, and high cell adhesion, and low inflammatory reaction upon implantation. However, composites have certain limitations, such as having poor cell attachments and releasing acidic by-products. These drawbacks could be assuaged in future by integrating nanobioreinforcements, with more biocompatibility, having bioactive molecules and nontoxic nature.

References Ambrosio, A. M., Sahota, J. S., Khan, Y., & Laurencin, C. T. (2001). A novel amorphous calcium phosphate polymer ceramic for bone repair: I. Synthesis and characterization. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 58(3), 295 301. Bahadoran, M., Shamloo, A., & Nokoorani, Y. D. (2020). Development of a polyvinyl alcohol/sodium alginate hydrogel-based scaffold incorporating bFGF-encapsulated microspheres for accelerated wound healing. Scientific Reports, 10(1), 1 18. Baˇca´kova´, L., Stary´, V., Kofroˇnova´, O., & Lisa´, V. (2001). Polishing and coating carbon fiber-reinforced carbon composites with a carbon-titanium layer enhances adhesion and growth of osteoblast-like MG63 cells and vascular smooth muscle cells in vitro. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 54(4), 567 578. Beatty, M. W., Swartz, M. L., Moore, B. K., Phillips, R. W., & Roberts, T. A. (1998). Effect of micro filler fraction and silane treatment on resin composite properties.

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Malhotra, S. K., Goda, K., & Sreekala, M. S. (2012). Part one introduction to polymer composites. Polymer Composites, 1, 1 2. Mir, M., Ali, M. N., Barakullah, A., Gulzar, A., Arshad, M., Fatima, S., . . . Asad, M. (2018). Synthetic polymeric biomaterials for wound healing: A review. Progress in Biomaterials, 7(1), 1 21. Neumann, M., & Epple, M. (2006). Composites of calcium phosphate and polymers as bone substitution materials. European Journal of Trauma, 32(2), 125 131. Pal, H., Jit, N., Tyagi, A. K., & Sidhu, S. (2011). Metal casting A general review. Advances in Applied Science Research, 2(5), 360 371. Paul, J. P. (1999). Strength requirements for internal and external prostheses. Journal of Biomechanics, 32(4), 381 393. Pye, A. D., Lockhart, D. E. A., Dawson, M. P., Murray, C. A., & Smith, A. J. (2009). A review of dental implants and infection. Journal of Hospital Infection, 72(2), 104 110. Ramakrishna, S., Mayer, J., Wintermantel, E., & Leong, K. W. (2001). Biomedical applications of polymer-composite materials: A review. Composites Science and Technology, 61(9), 1189 1224. Salernitano, E., & Migliaresi, C. (2003). Composite materials for biomedical applications: A review. Journal of Applied Biomaterials and Biomechanics, 1(1), 3 18. Scala, E. P. (1996). A brief history of composites in the US-The dream and the success. JOM, 48(2), 45 48. Seal, B. L., Otero, T. C., & Panitch, A. (2001). Polymeric biomaterials for tissue and organ regeneration. Materials Science and Engineering: R: Reports, 34(4 5), 147 230. Selzer, R., & Friedrich, K. (1997). Mechanical properties and failure behaviour of carbon fibre-reinforced polymer composites under the influence of moisture. Composites Part A: Applied Science and Manufacturing, 28(6), 595 604. Sharma, P., Bhanot, V. K., Singh, D., Undal, H. S., & Sharma, M. (2013). Research work on fiber glass wool reinforced and epoxy matrix composite material. International Journal of Mechanical Engineering and Robotics Research, 2, 106 124. Wang, M., & Bonfield, W. (2001). Chemically coupled hydroxyapatite polyethylene composites: structure and properties. Biomaterials, 22(11), 1311 1320. Wang, M., Deb, S., & Bonfield, W. (2000). Chemically coupled hydroxyapatitepolyethylene composites: processing and characterisation. Materials Letters, 44(2), 119 124. Wang, M., Joseph, R., & Bonfield, W. (1998). Hydroxyapatite-polyethylene composites for bone substitution: effects of ceramic particle size and morphology. Biomaterials, 19 (24), 2357 2366. Wolf, W. D., Vaidya, K. J., & Francis, L. F. (1996). Mechanical properties and failure analysis of alumina-glass dental composites. Journal of the American Ceramic Society, 79(7), 1769 1776. Zhang, Y. Q. (2002). Applications of natural silk protein sericin in biomaterials. Biotechnology Advances, 20(2), 91 100. Zhao, Y., Li, Z., Song, S., Yang, K., Liu, H., Yang, Z., . . . Lin, Q. (2019). Skin-inspired antibacterial conductive hydrogels for epidermal sensors and diabetic foot wound dressings. Advanced Functional Materials, 29(31), 1901474.

CHAPTER

Biopolymer-based composites for drug delivery applications—a scientometric analysis

3

Kunal Pal1, Deepti Bharti1, Preetam Sarkar2 and Doman Kim3 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 3 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

3.1 Introduction Drug delivery vehicles are regarded as the formulations or devices that are responsible for delivering drugs. Controlled drug delivery systems are a specific type of formulations and devices that allow controlling the rate of release of the drugs to maintain their level at the site of action. Such systems can also be designed to deliver drugs at a specific time or as per the demand. These systems form an interface between the drug and the patient. Biopolymers (e.g., chitosan, alginate, hyaluronic acid, etc.) have been explored for designing controlled drug delivery systems. These are the polymers that are available in nature in abundance. Fig. 3.1 (Balart, GarciaGarcia, Fombuena, Quiles-Carrillo, & Arrieta, 2021) summarizes the classification and sources of the biopolymers. Due to this reason, they are inexpensive. The biopolymers are inherently biocompatible and nonimmunogenic (Gheorghita, AnchidinNorocel, Filip, Dimian, & Covasa, 2021). They are available in a wide range of chemistries, which allow researchers to design delivery systems with desirable properties. It is possible to deliver drugs at a specific site. For example, it is possible to protect some medications from the harsh gastric pH and deliver the same to the duodenal or intestinal region (Zhao, Maniglio, Chen, Chen, & Migliaresi, 2016). On the contrary, some drugs are to be released in the gastric region of the gastrointestinal tract. Similarly, some of the biopolymeric formulations can cross the blood brain barrier to deliver drugs to the central nervous system. Further, the chemical modifications (e.g., carboxylation, thiolation, acetylation, and conjugation) of the biopolymers can be carried out (Dmour & Taha, 2018). The modifications can expand the functionality and applications of the biopolymers. For example, PEGylation of the Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00024-3 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 3.1 Classification and sources of biopolymers. Reproduced from Balart, R., Garcia-Garcia, D., Fombuena, V., Quiles-Carrillo, L., & Arrieta, M. P. (2021). Biopolymers from natural resources. Polymers, 13(15). https://doi.org/10.3390/polym13152532. under Creative Commons License.

biopolymeric nanoparticles allows the researchers to develop stealth delivery systems that can have an increased residence time in the blood (Suk, Xu, Kim, Hanes, & Ensign, 2016). This, in turn, improves the bioavailability of the drugs and their efficiency. The properties of the biopolymer-based systems may further be improved by developing their composites. These types of formulations may provide improved therapeutic benefits over the conventional delivery systems. In this chapter, we hereby perform the scientometric analysis to analyze how the use of biopolymeric composites in drug delivery systems has evolved over the years. The collection of data was carried out from the Web of Science (WoS) database. Analysis of the data was carried out using the VoSviewer software, a freeware. After analyzing the data, we identify the commonly used biopolymers for developing composite-based drug delivery systems. Subsequently, we discuss the properties of these biopolymers. Finally, we recognize the most-cited literature in the said field and briefly discuss the research finding in that literature.

3.2 Scientometric analysis The articles for the scientometric analysis were searched using the keywords: biopolymer (All Fields) and composite (All Fields), and “drug delivery”

3.2 Scientometric analysis

(All Fields). The search was carried out in the WoS database on October 10, 2021. The search returned with 451 publications. Among the publications, there were 313 articles, 137 review articles, seven proceeding publications, six early access publications, one book chapter, and one meeting abstract. Further 450 publications were in the English language, while one publication was in the Chinese language. Subsequently, the publications in the English language and categorized as articles were selected for further analysis. The application of these filters returned with 312 publications. It was observed from the results that the first publication on the proposed topic was during the year 2002. The analysis of the data deciphered that 74 articles were published as Open Access articles. Fig. 3.2 deciphers the growth of the publications over the years till the current day. It is important to note that the biopolymeric composite based drug delivery systems have evolved considerably in the last decade. Since the year 2016, there have been more than 30 publications each year. The highest number of publications was achieved in the year 2020. Fig. 3.3 shows the TreeMap chart of the top five publishers who have published the articles. It could be seen that the highest number of publications were published by Elsevier (134 articles) followed by Wiley (49 articles), Springer Nature (22 articles), MDPI (22 articles), and Royal Society of Chemistry (17 articles), respectively. The top five journals that published the publications were Journal of Applied Polymer Science (23 articles), International Journal of Biological Macromolecules (21 articles), Carbohydrate Polymers (14 articles), Materials Science Engineering C: Materials for Biological Applications

FIGURE 3.2 Histogram of publications on a time scale.

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FIGURE 3.3 Most number of articles published by the publishers.

(13 articles), and Journal of Drug Delivery Science and Technology (7 articles), respectively. It was interesting to note that all the articles were from the Elsevier Publishing group. Most articles were published from the countries People’s Republic of China (52 articles), India (48 articles), Iran (37 articles), the United States (37 articles), and Spain (22 articles), respectively. Thereafter the bibliometric records of the articles were downloaded as the Tab delimited file. The data so obtained were then analyzed using Vosviewer software. The Vosviewer software is a free software that several researchers are using to analyze bibliometric data.

3.2.1 Coauthorship analysis Initially, coauthorship analysis was carried out. The details of the coauthorship analysis when the analysis was carried out using the relationship of the authors, who had at least three articles, are tabulated in Table 3.1. It can be observed that the total link strength, which provides information regarding the collaborative research amongst the authors, was highest for Giuseppe Cavallaro, Giuseppe Lazzara, Lorenzo Lisuzzo, and Stefana Milioto. These authors also shared an equal number of documents and citations, which indicated that these authors were collaborating. Fig. 3.4 summarizes the coauthorship analysis among the authors. The analysis of Fig. 3.4 suggests that the authors Giuseppe Cavallaro, Giuseppe Lazzara, Lorenzo Lisuzzo, and Stefana Milioto are collaborators. This group formed the largest network. It can also be observed from Fig. 3.4 that the authors Hriday Bera and Aldo R. Boccaccini are also collaborators. However, the number of documents and the citations of these authors were different (Table 3.1). This is suggestive of the fact that the author Hriday Bera, who has four articles against

3.2 Scientometric analysis

Table 3.1 Coauthorship analysis at the individual level of the authors. #

Author

Documents

Citations

Total link strength

1 2 3 4 5 6 7 8 9 10

Cavallaro, Giuseppe Lazzara, Giuseppe Lisuzzo, Lorenzo Milioto, Stefana Abbasi, Yasir Faraz Bera, Hriday Boccaccini, Aldo R. Castro, Guillermo R. Holban, Alina Maria Tan, Huaping

3 3 3 3 3 4 3 3 3 3

100 100 100 100 12 44 150 101 17 15

9 9 9 9 3 3 0 0 0 0

FIGURE 3.4 Author network analysis.

his name, has also worked in the aforesaid field wherein Aldo R. Boccaccini was not part of the research. The coauthor relationship of authors from different organizations was analyzed from Table 3.2. Table 3.2 enlists the organizations from where at least five articles were published. It is evident that the authors of the University of Tehran and the Tehran University of Medical Sciences collaborated. Similarly, the authors

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Table 3.2 Coauthorship analysis based on the author’s organization. #

Organization

Country

Documents

Citations

Total link strength

1 2

University of Tehran Tehran University of Medical Sciences University of the Basque Country Politehnica University of Bucharest Indian Institute of Technology University of Minho

Iran Iran

7 5

127 215

2 2

Spain

5

310

1

Romania

6

34

1

India

8

97

0

Portugal

5

295

0

3 4 5 6

from the University of the Basque Country and the Politehnica University of Bucharest collaborated. The number of articles from the University of Tehran and the Politehnica University of Bucharest was higher than the Tehran University of Medical Sciences and the University of the Basque Country, respectively. This is suggestive of the absence of collaboration among the authors of the paired organizations in some cases. Subsequently, the coauthorship analysis based on the country of affiliation of the authors was carried out. The summary of the analysis is tabulated in Table 3.3. During the analysis, countries with at least 15 articles were taken into consideration. It can be seen that the collaboration of the authors from the United States was the highest with the authors from other countries. A careful observation suggests that the number of articles from the authors of the United States was comparatively lower than or equal to those from the authors from countries like the Peoples’ Republic of China, India, and Iran, respectively. But the collaboration of the authors was in the order of Peoples’ Republic of China, Iran, and India, respectively. The software segregated the countries into two groups (Fig. 3.5). In the first group, there were five countries, namely the United States, Peoples’ Republic of China, Iran, India, and Malaysia. The other group consisted of the countries Spain and Brazil.

3.2.2 Cooccurrence analysis The analysis of the cooccurrence of the keywords was carried out. The software detected 1858 keywords from the downloaded bibliometric data. For the analysis, the keywords that appeared at least five times were selected. This narrowed down the number of keywords to 134. Henceforth, the names of the biopolymers were chosen manually. It was found that there were 11 biopolymers that have been explored for the development of composites for drug delivery applications. Postidentification of the keywords, the bibliometric data were used to obtain the

3.2 Scientometric analysis

Table 3.3 Coauthorship analysis based on the author’s country. #

Country

Documents

Citations

Total link strength

1 2 3 4 5 6 7

United States Peoples’ Republic of China Iran India Malaysia Spain Brazil

37 52 37 48 15 22 18

1464 870 961 821 246 916 392

20 15 12 11 10 10 6

FIGURE 3.5 Coauthorship analysis based on the author’s country.

Density Visualization plot (Fig. 3.6). The visual analysis of the Density Visualization plot suggested that biopolymers like chitosan, alginate, cellulose, and hyaluronic acid are closely related to drug delivery applications. Accordingly, the properties of the above-mentioned polymers will be discussed briefly in this section.

3.2.2.1 Chitosan Chitosan is synthesized by the partial deacetylation of chitin, the second-most abundant naturally occurring polymer. In other words, chitosan is not available in nature. It is derived by biological or chemical modification of chitin. Herein, it is noteworthy to mention that cellulose is the most abundant natural polymer. Chitin is extracted from various natural sources, including the cell walls of fungi, shrimps, crabs, and insects (Fig. 3.7; da Silva Alves, Healy, Pinto, Cadaval, & Breslin, 2021). However, the main sources of chitosan are shrimps and crabs. The chemical structures of chitin and chitosan have been provided in Fig. 3.8 (Younes & Rinaudo, 2015). Both chitin and chitosan are linear polysaccharides. Chitosan carries a positive charge when solubilized in acidic solutions (Matica, Aachmann,

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FIGURE 3.6 Cooccurrence analysis of the keywords from the bibliometric data.

FIGURE 3.7 (A) Sources of chitin and (B) methods of preparation of chitosan. Reproduced from da Silva Alves, D. C., Healy, B., Pinto, L. A. D. A., Cadaval, T. R. S. A., & Breslin, C. B. (2021). Recent developments in chitosan-based adsorbents for the removal of pollutants from aqueous environments. Molecules (Basel, Switzerland), 26(3). https://doi.org/10.3390/molecules26030594. under Creative Commons License.

3.2 Scientometric analysis

FIGURE 3.8 Chemical structure of chitin and chitosan. Reproduced from Younes, I., & Rinaudo, M. (2015). Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs, 13(3), 1133 1174. https://www.mdpi.com/16603397/13/3/1133. under Creative Commons License.

Tøndervik, Sletta, & Ostafe, 2019). Accordingly, chitosan is regarded as a polycationic polymer. Chemically, chitosan is composed of two subunits, namely, β-(1 4)-linked D-glucosamine and N-acetyl-D-glucosamine (Cheung, Ng, Wong, & Chan, 2015). These subunits are randomly arranged throughout the polymer skeleton. The polymer has found applications in various fields of study like food packaging, tissue engineering, drug delivery, wound healing, and bioremediation. The main advantage of chitosan is its biodegradability. It is also nonimmunogenic and hence does not elicit immunological reactions. The biodegradable products of chitosan do not trigger an inflammatory reaction (Zhao et al., 2018). The matrices of chitosan promote mammalian cell attachment, migration, and differentiation (Huang et al., 2015). This suggests that chitosan is not only a biocompatible polymer but also can promote cell proliferation. Further, due to the polycationic nature of chitosan, it has inherent antimicrobial properties. The antimicrobial properties of chitosan are against not only the Gram-positive and Gram-negative bacteria but also fungi (Kong, Chen, Xing, & Park, 2010). The mechanisms of antimicrobial activities of chitosan are summarized in Fig. 3.9 (Ke, Deng, Chuang, & Lin, 2021).

3.2.2.2 Alginate Like chitosan, alginates are also linear polysaccharides. However, unlike polycationic chitosan, alginates are polyanionic. Alginates can be readily solubilized in water. Hence, the polymer matrices of alginates swell when immersed in aqueous mediums. It is mainly obtained from the cell walls of brown algae (Rabille´ et al., 2019). Extraction of alginates from some bacterial strains (e.g., Pseudomonas and Azotobacter) has also been reported. There are two subunits that are arranged in block-like patterns that are organized either homogeneously or heterogeneously. The subunits of the alginates include 1,4-linked β-D-mannuronic acid (M) and 1,4

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FIGURE 3.9 Antimicrobial activity of chitosan: (A) Gram-positive bacteria, (B) Gram-negative bacteria, and (C) fungi. Reproduced from Ke, C.-L., Deng, F.-S., Chuang, C.-Y., & Lin, C.-H. (2021). Antimicrobial actions and applications of chitosan. Polymers, 13(6). https://doi.org/10.3390/polym13060904. under Creative Commons License.

α-L-guluronic acid (G) monomers (Fig. 3.10). If the M and G subunits are present heterogeneously, then the blocks are regarded as MG blocks. On the contrary, poly-M and poly-G blocks are formed in the alginates if the subunits are homogenously present (Barbu et al., 2021). These subunits may be arranged in three possible ways, viz., consecutive G subunits, consecutive M subunits, and alternating MG or GM subunits (Sun & Tan, 2013). Since the polymer is polyanionic, it can be ionically crosslinked with positive ions like calcium ions and different metal ions (e.g., cobalt ions, manganese ions, etc.) (Fig. 3.10; Dodero et al., 2019). The polymer is inherently biocompatible and biodegradable. Some of the properties of alginates are summarized in Fig. 3.11 (Gheorghita Puscaselu, Lobiuc, Dimian, & Covasa (2020). The properties of the alginate matrices can be enhanced multifolds through physical and chemical methods. The affinity of the mammalian

3.2 Scientometric analysis

FIGURE 3.10 (A) Chemical structure of alginates and (B) mechanism of ionic crosslinking using calcium ions (herein, M stands for calcium ions). Reproduced from Dodero, A., Pianella, L., Vicini, S., Alloisio, M., Ottonelli, M., & Castellano, M. (2019). Alginate-based hydrogels prepared via ionic gelation: An experimental design approach to predict the crosslinking degree. European Polymer Journal, 118, 586 594 under Creative Commons License.

FIGURE 3.11 Properties of alginates. Reproduced from Gheorghita Puscaselu, R., Lobiuc, A., Dimian, M., & Covasa, M. (2020). Alginate: From food industry to biomedical applications and management of metabolic disorders. Polymers, 12(10). https:// doi.org/10.3390/polym12102417. under Creative Commons License.

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cells toward the alginate matrices can be tuned by affixing various ligands like peptide and sugar molecules as pendant groups. Functionalization of the alginates can also allow us to tune the biological properties of the alginate matrices (Dalheim et al., 2016). One such method of functionalization of alginates is shown in Fig. 3.12 (Huamani-Palomino, Co´rdova, Pichilingue L, Venaˆncio, & Valderrama, 2021). Further, the blending of alginates with other polymers has been proposed to improve the properties of the alginate matrices.

3.2.2.3 Cellulose Cellulose is the most abundant polysaccharide in nature and is an organic compound. It is inherently biocompatible and biodegradable. The polysaccharide can be easily found in the cell walls of green plants (Mihranyan, 2011). It can also be extracted from various algae. Chemically, it is a polymer of D-glucose molecules. The process of polymerization is attained by the formation of β(1-4)-glycosidic bonds among the glucose molecules (Fig. 3.13; Tayeb, Amini, Ghasemi, & Tajvidi, 2018). The polymerization of the glucose molecules occurs during the replication of the plant cells. The formed polysaccharide, that is, cellulose, is a linear-chain polysaccharide like chitosan and alginates. In nature, cellulose exists

FIGURE 3.12 Functionalization of sodium alginate by oxidation and reductive amination. Reproduced from Huamani-Palomino, R. G., Co´rdova, B. M., Pichilingue L, E. R., Venaˆncio, T., & Valderrama, A. C. (2021). Functionalization of an alginate-based material by oxidation and reductive amination. Polymers, 13(2). https://doi.org/10.3390/polym13020255. under Creative Commons License.

3.2 Scientometric analysis

FIGURE 3.13 Chemical structure of cellulose. Reproduced from Tayeb, A. H., Amini, E., Ghasemi, S., & Tajvidi, M. (2018). Cellulose nanomaterials— Binding properties and applications: A review. Molecules (Basel, Switzerland), 23(10). https://doi.org/ 10.3390/molecules23102684. under Creative Commons License.

FIGURE 3.14 Mechanism of interconversion of the cellulose polymorphs. Reproduced from Naomi, R., Bt Hj Idrus, R., & Fauzi, M. B. (2020). Plant- vs. bacterial-derived cellulose for wound healing: A review. International Journal of Environmental Research and Public Health, 17(18). https:// doi.org/10.3390/ijerph17186803. under Creative Commons License.

only as type-I and type-II polymorphs, even though other polymorphs have also been proposed (Bian, Yang, & Tu, 2021). Fig. 3.14 depicts the mechanism of interconversion of the polymorphs. In recent years, cellulose of bacterial origin has also been reported by many researchers. In recent years, cellulose of bacterial origin has also been reported by many researchers. Bacterial cellulose is synthesized by the bacteria of the genera Gluconacetobacter, Agrobacterium, and Sarcina (Naomi, Bt Hj Idrus, & Fauzi, 2020). The mechanism of cellulose synthesis by the bacteria is summarized in Fig. 3.15. Since cellulose has many hydrophilic hydroxyl groups, cellulose is innately hydrophilic. A single unit of the glucose molecule in the cellulose backbone consists of three hydroxyl groups. However, as cellulose is a macromolecule, it is not soluble in water or aqueous solutions. Additionally, it is also not soluble in many organic solvents. It is easy to chemically modify the cellulose structure that has been associated with the chemistry of cellulose. The modification can be achieved without compromising the natural property of the polymer. The cellulose chain length, which plays a significant role in governing the cellulose matrices’ physical properties, depends on the cellulose source (Costa et al., 2019). The cellulose that is obtained from wood

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FIGURE 3.15 Mechanism of synthesis of bacterial cellulose. Reproduced from Naomi, R., Bt Hj Idrus, R., & Fauzi, M. B. (2020). Plant- vs. bacterial-derived cellulose for wound healing: A review. International Journal of Environmental Research and Public Health, 17(18). https:// doi.org/10.3390/ijerph17186803. under Creative Commons License.

pulp may contain 300 1700 units of glucose molecules. On the other hand, bacterial cellulose may consist of 800 10,000 units of glucose molecules.

3.2.2.4 Hyaluronic acid Hyaluronic acid is a glycosaminoglycans-based natural polymer. It is an unbranched heteropolysaccharide. The presence of the polysaccharide can be widely found in nature. Hyaluronic acid is not only found in humans and animals but also in algae, yeasts, and bacteria (Fallacara, Baldini, Manfredini, & Vertuani, 2018). The polysaccharide is composed of two subunits, namely N-acetyl-D-glucosamine and D-glucuronic acid, which are connected through ß-1,3-glycosidic linkages. Fig. 3.16 depicts the chemical structure of hyaluronic acid. The figure also depicts the hydrophilic and hydrophobic groups that are present in the hyaluronic acid chain. Additionally, the formation of hydrogen bonds by the polysaccharide in an aqueous solution has been shown. These hydrogen bonds stabilize the secondary structure of hyaluronic acid. A combination of the hydrophobic interactions and the hydrogen bonds causes the formation and stabilization of ßsheet tertiary structure in the aqueous solutions of hyaluronic acid. From the chemical structure of hyaluronic acid, it is clear that hyaluronic acid is a polyelectrolyte. Accordingly, the physical properties of the hyaluronic acid matrices are governed by the pH and ionic strength. The chemical modification of hyaluronic

3.2 Scientometric analysis

FIGURE 3.16 (A) Schematics of the chemical structure of hyaluronic acid, and (B) demarcation of hydrophilic groups, hydrophobic groups, and hydrogen bonds within the hyaluronic acid polymer chain. Reproduced from Fallacara, A., Baldini, E., Manfredini, S., & Vertuani, S. (2018). Hyaluronic acid in the third millennium. Polymers, 10(7). https://doi.org/10.3390/polym10070701. under Creative Commons License.

acid is straightforward and allows to tailor of the physical and chemical properties of the hyaluronic acid as per the requirement. Fig. 3.17 summarizes the possible ways for chemical modifications of hyaluronic acid and the polysaccharide types used in the pharmaceutical industries.

3.2.3 Analysis of the citations of the articles The articles were then analyzed for their citations. The articles that have received at least 100 citations were selected. The list of such articles is tabulated in Table 3.4. In the present section, the summary of the research works in the selected articles will be discussed.

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FIGURE 3.17 (A) Schematic representation of the ways to modify the chemical structure of hyaluronic acid, and (B) types of hyaluronic acid used in pharmaceutical industries. Reproduced from Fallacara, A., Baldini, E., Manfredini, S., & Vertuani, S. (2018). Hyaluronic acid in the third millennium. Polymers, 10(7). https://doi.org/10.3390/polym10070701 under Creative Commons License.

Leach and Schmidt (2005) have reported the synthesis of photocrosslinkable hyaluronic acid polyethylene glycol hydrogel as tissue engineering scaffolds. The developed scaffolds were used as delivery matrices for the proteins. The hyaluronic acid biopolymer was chosen in the study because of its inherent biocompatibility and nonimmunogenicity. Chemically, the biopolymer is a glycosaminoglycan, which is a nonadhesive polysaccharide. Additionally, biopolymer also plays an important role in various biological processes. Some of the notable biological processes include its ability to modulate angiogenesis and the inflammatory response. The authors reported that they have previously developed a photocrosslinkable derivative of hyaluronic acid, namely photopolymerizable glycidyl methacrylatehyaluronic acid. The derivative was found to be biocompatible. The derivatization of the biopolymer allowed the authors to control the degradation rates of the polymeric constructs of the derivatized biopolymer. Most importantly, the properties of

3.2 Scientometric analysis

Table 3.4 List of documents that have received at least 100 citations. #

Document

Citations

Reference

1

Characterization of protein release from photocrosslinkable hyaluronic acidpolyethylene glycol hydrogel tissue engineering scaffolds Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury Biodegradable and thermo-sensitive chitosan-g-poly(N-vinylcaprolactam) nanoparticles as a 5-fluorouracil carrier Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications Pectin/carboxymethyl cellulose/ microfibrillated cellulose composite scaffolds for tissue engineering Electrophoretic deposition of gentamicinloaded bioactive glass/chitosan composite coatings for orthopedic implants Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan nanoparticles

324

Leach and Schmidt (2005)

113

Baumann, Kang, Tator, and Shoichet (2010)

123

121

Rejinold, Chennazhi, Nair, Tamura, and Jayakumar (2011) Coimbra et al. (2011)

143

Ninan et al. (2013)

107

Pishbin et al. (2014)

172

Hosseini, Rezaei, Zandi, and Farahmandghavi (2015)

2

3

4

5

6

7

the derivatized biopolymer could be tailored using peptide moieties. The authors reported that the hydrogels of pristine derivatized biopolymer and derivatized biopolymer/polyethylene glycol were suitable for the release of the protein molecules. In the study, bovine serum albumin was used as the model protein. The authors reported that the hydrogel developed with 1% of the derivatized biopolymer could quickly release the model protein. It was observed that .60% of the model protein was released within 6 h. Interestingly, an increase in the concentration of the derivatized biopolymer or polyethylene glycol delayed the release of the model protein. The analysis of the released protein molecules confirmed that the developed hydrogels did not alter the native monomeric form of the model protein. Lastly, the authors reported that the release of the model protein was prolonged to several weeks when the model protein was incorporated within the poly(lactic-co-glycolic acid) microspheres, which were incorporated afterward within the hydrogels. The authors concluded in their study that the novel photopolymerizable composites were suitable for the delivery of proteins in their active form and could be explored successfully for tissue engineering applications. Traumatic spinal cord injury (SCI) is a severe type of injury that primarily results in the life-long disability of the patients. A typical SCI may induce lasting

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paralysis lower than the position of injury. To date, there is no cure for SCI. However, various researchers have reported that molecular medicine based therapy has shown great success in animal models. The primary aim of the treatment of SCI is to prevent degeneration of the functional tissue and rejuvenate the functionality of the already degenerated tissues to the extent possible. In this regard, Baumann et al. (2010) have reported the local delivery of the bioactive agents directly to the site of action. The use of such a delivery system allows circumventing the blood spinal cord barrier. This delivery system is essential because most bioactive agents cannot permeate through the blood spinal cord barrier and cannot elicit therapeutic activity. The matrix of the delivery system was developed using a biopolymeric combination of hyaluronan and methylcellulose. The developed matrix was an injectable hydrogel that could be quickly injected within the intrathecal space. The incorporation of the poly(lactic-co-glycolic acid) nanoparticles altered the rheological properties of the biopolymeric hydrogels. This observation was explained by the increased hydrophobic interactions between the methyl groups of methylcellulose and the nanoparticles. Such an increase in the rheological properties was related to the better stability of the composite hydrogels. Further, it was found that the developed hydrogels were well tolerated within the intrathecal space of the spinal cord of rats. The microglial activation was also quite limited in the presence of the hydrogels. The incorporation of the composite hydrogel within the healthy rats did not affect their locomotor function. The use of thermo-sensitive nanoparticles has been used for cancer treatment. The anticancer drugs are encapsulated within the thermo-sensitive nanoparticles, which are then directly injected into the bloodstream. Such drug delivery systems are expected to deliver the drugs directly to the tumor site. Rejinold et al. (2011) have reported the synthesis of nanoparticles for the treatment of cancer. Composite nanoparticles were synthesized using chitosan-g-poly(N-vinylcaprolactam). The synthesis of the nanocomposite was achieved by the ionic crosslinking method. 5-Fluorouracil, a well-established anticancer drug, was incorporated within the nanocarrier. The drug molecules formed intermolecular hydrogen bonding with thermo-responsive chitosan-g-poly(N-vinyl caprolactam). The in vitro drug release studies divulged an increased amount of drug release above 38 C, which was the lower critical solution temperature for the nanocomposite matrix. It was observed that the cancer cells were capable of uptaking the 5fluorouracil containing nanoparticles, thereby eliciting anticancer activity. The authors concluded that the developed composites were suitable for exploring as a delivery vehicle in cancer treatment. Composite scaffolds have found applications in tissue engineering. Highly porous scaffolds are used considerably in tissue engineering applications. The scaffolds help in the three-dimensional regeneration of the cells to develop targeted tissues and are capable of delivering bioactive agents like growth factors and drugs. One such application is bone tissue engineering. Coimbra et al. (2011) have developed a polyelectrolyte complex scaffold using the polysaccharides

3.2 Scientometric analysis

pectin and chitosan for possible bone tissue engineering. The polyelectrolyte complex is formed when polycationic and polyanionic polymers are mixed. Such polyelectrolyte complex systems have also been explored as drug delivery systems. Herein, pectin is a polyanionic polymer, and chitosan is a polycationic polymer. In the study, pectin derived from citrus fruits was used, while the chitosan used in the study was derived from crab shells. The aqueous solutions of the polysaccharides were used to develop the polyelectrolyte complex. During the preparation of the polyelectrolyte complex, the pH was maintained at 4.5. Thereafter the hydrogels were converted to scaffolds by the freeze-drying method. The elemental analysis of the scaffolds suggested the presence of both chitosan and pectin. This suggests that both the polymers retained their chemical identity in the scaffolds. The scaffolds were highly porous, but the pores were irregular in shape and size. Further, it was observed that the mass of the developed scaffolds was reduced to half when immersed in phosphate buffer solution (pH 7.4) for 1 month. Human osteoblast cells were able to adhere and proliferate over the scaffolds suggesting the biocompatibility and noncytotoxic nature of the developed scaffolds. The authors concluded that the scaffolds could be incorporated with bioactive inorganic materials (e.g., hydroxyapatite) to develop composite scaffolds. Ninan et al. (2013) have reported the synthesis of composite scaffolds of pectin, carboxymethyl cellulose, and microfibrillated cellulose for tissue engineering applications. The scaffolds were also prepared by the freeze-drying (lyophilization) method. The crosslinking of the composite matrix was carried out by the ionic crosslinking method. For crosslinking, a calcium chloride solution was used. It was observed that the porosity of the scaffolds was dependent on the composition of the scaffold matrix. The pore sizes of the developed scaffolds were in the range of 10 250 μm. Such pore sizes have been reported to be sufficient enough for tissue engineering applications. The crystalline lattice planes of microfibrillated cellulose could be deciphered within the composite scaffolds by the X-ray diffraction (XRD) study. The incorporation of microfibrillated cellulose within the scaffolds improved the thermal stability and reduced the degradation rate of the composite scaffolds. The composite scaffolds were also found to improve the cell viability of the NIH3T3 cells, which are acquired from mouse embryonic fibroblasts. The metallic implants are nonbioactive. Such implants may be made bioactive by coating the implants with bioactive materials. These implants have been proposed as a long-term solution for treating bone defects that are larger than a critical size. In this regard, Pishbin et al. (2014) has proposed electrophoretic deposition of glass and chitosan-based multifunctional composite coatings. The coatings were also loaded with gentamicin, an antibiotic drug. The electrophoretic deposition technique allowed the researchers to deposit glass, chitosan, and gentamicin onto a stainless steel substrate in a single step. This helped the researchers to reduce the complexity of the coating process. The coating allowed the deposition of hydroxyapatite over the coated stainless steel substrate, indicating its bioactivity. Also, the proliferation of the MG-63 cells, osteoblast-like cells, was promoted. Gentamicin was released in a sustained manner from the coatings. In

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the first 5 days, 40% of the gentamicin was released from the coated substrate. The gentamicin-loaded coatings showed antimicrobial activity against Staphylococcus aureus. The authors concluded that the developed method would help the implant manufacturers to coat the metallic implants efficiently. Hosseini et al. (2015) have reported the synthesis of chitosan nanoparticles by the ionotropic gelation method. Sodium tripolyphosphate was used as the ionic crosslinker for the chitosan molecules. The prepared nanoparticles were spherical and had sizes in the range of 40 80 nm. The zeta potential of the synthesized nanoparticles was 110 mV. Subsequently, the nanoparticles were used to develop nanocomposites wherein fish gelatin was used as the polymer matrices. The distribution of the nanoparticles within the gelatin matrix was homogenous when the filler content was low. However, at higher filler content, agglomeration of the filler nanoparticles was observed. Infrared spectroscopy suggested that hydrogen bonding was prevalent among the chitosan nanoparticles and the gelatin matrices. The inclusion of the nanofillers improved the mechanical stability of the films but consequently reduced the elongation at break. The composite films could significantly improve the ultraviolet barrier and the water vapor permeability properties. The results suggested that the nanocomposite films were superior to the pristine gelatin film.

3.3 Conclusion There has been continuous development in designing composite biopolymers over the last two decades, specifically in the field of drug delivery. Nontoxic, biodegradable, and biocompatible nature of natural polymers are a few attributes that support their constant development. The natural abundance of biopolymers allows its maximum utilization for controlled delivery systems. The biocomposite polymeric system can prevent drugs/bioactive agents from the harsh microenvironment until it reaches the targeted site. The current chapter made an effort to perform a qualitative and quantitative evolution of biocomposites for drug delivery applications over time through scientometric study. Analysis of the data was carried out using freeware, VoSviewer software. The data obtained from the density visualization plot of the cooccurrence keywords suggested chitosan, alginate, cellulose, and hyaluronic acid are closely associated composite-based drug delivery systems. The chapter covered a brief description of these biopolymers and the possibility of their structural modification to serve efficiently as drug delivery systems. In the final section of the chapter, we listed a few of the most-cited literature in the field and tried to summarize those research works.

References Balart, R., Garcia-Garcia, D., Fombuena, V., Quiles-Carrillo, L., & Arrieta, M. P. (2021). Biopolymers from natural resources. Polymers, 13(15). Available from https://doi.org/ 10.3390/polym13152532.

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Barbu, A., Neamtu, B., Z˘ahan, M., Iancu, G. M., Bacila, C., & Mire¸san, V. (2021). Current trends in advanced alginate-based wound dressings for chronic wounds. Journal of Personalized Medicine, 11(9). Available from https://doi.org/10.3390/jpm11090890. Baumann, M. D., Kang, C. E., Tator, C. H., & Shoichet, M. S. (2010). Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury. Biomaterials, 31(30), 7631 7639. Available from https://doi.org/10.1016/j.biomaterials.2010.07.004. Bian, H., Yang, Y., & Tu, P. (2021). Crystalline structure analysis of all-cellulose nanocomposite films based on corn and wheat straws. BioResources, 16(4), 8353 8365. Cheung, R. C., Ng, T. B., Wong, J. H., & Chan, W. Y. (2015). Chitosan: An update on potential biomedical and pharmaceutical applications. Marine Drugs, 13(8). Available from https://doi.org/10.3390/md13085156. Coimbra, P., Ferreira, P., de Sousa, H. C., Batista, P., Rodrigues, M. A., Correia, I. J., . . . Gil, M. H. (2011). Preparation and chemical and biological characterization of a pectin/ chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications. International Journal of Biological Macromolecules, 48(1), 112 118. Available from https://doi.org/10.1016/j.ijbiomac.2010.10.006. Costa, C., Medronho, B., Filipe, A., Mira, I., Lindman, B., Edlund, H., . . . Norgren, M. (2019). Emulsion formation and stabilization by biomolecules: The leading role of cellulose. Polymers, 11(10), 1570. Dalheim, M. Ø., Vanacker, J., Najmi, M. A., Aachmann, F. L., Strand, B. L., & Christensen, B. E. (2016). Efficient functionalization of alginate biomaterials. Biomaterials, 80, 146 156. da Silva Alves, D. C., Healy, B., Pinto, L. A. D. A., Cadaval, T. R. S. A., & Breslin, C. B. (2021). Recent developments in chitosan-based adsorbents for the removal of pollutants from aqueous environments. Molecules (Basel, Switzerland), 26(3). Available from https://doi.org/10.3390/molecules26030594. Dmour, I., & Taha, M. O. (2018). Natural and semisynthetic polymers in pharmaceutical nanotechnology. Organic materials as smart nanocarriers for drug delivery (pp. 35 100). Elsevier. Dodero, A., Pianella, L., Vicini, S., Alloisio, M., Ottonelli, M., & Castellano, M. (2019). Alginate-based hydrogels prepared via ionic gelation: An experimental design approach to predict the crosslinking degree. European Polymer Journal, 118, 586 594. Fallacara, A., Baldini, E., Manfredini, S., & Vertuani, S. (2018). Hyaluronic acid in the third millennium. Polymers, 10(7). Available from https://doi.org/10.3390/polym10070701. Gheorghita, R., Anchidin-Norocel, L., Filip, R., Dimian, M., & Covasa, M. (2021). Applications of biopolymers for drugs and probiotics delivery. Polymers, 13(16), 2729. Gheorghita Puscaselu, R., Lobiuc, A., Dimian, M., & Covasa, M. (2020). Alginate: From food industry to biomedical applications and management of metabolic disorders. Polymers, 12(10). Available from https://doi.org/10.3390/polym12102417. Hosseini, S. F., Rezaei, M., Zandi, M., & Farahmandghavi, F. (2015). Fabrication of bionanocomposite films based on fish gelatin reinforced with chitosan nanoparticles. Food Hydrocolloids, 44, 172 182. Available from https://doi.org/10.1016/j.foodhyd.2014.09.004. Huamani-Palomino, R. G., Co´rdova, B. M., Pichilingue L, E. R., Venaˆncio, T., & Valderrama, A. C. (2021). Functionalization of an alginate-based material by oxidation and reductive amination. Polymers, 13(2). Available from https://doi.org/10.3390/polym13020255. Huang, R., Li, W., Lv, X., Lei, Z., Bian, Y., Deng, H., . . . Li, X. (2015). Biomimetic LBL structured nanofibrous matrices assembled by chitosan/collagen for promoting wound healing. Biomaterials, 53, 58 75.

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Ke, C.-L., Deng, F.-S., Chuang, C.-Y., & Lin, C.-H. (2021). Antimicrobial actions and applications of chitosan. Polymers, 13(6). Available from https://doi.org/10.3390/polym13060904. Kong, M., Chen, X. G., Xing, K., & Park, H. J. (2010). Antimicrobial properties of chitosan and mode of action: A state of the art review. International Journal of Food Microbiology, 144(1), 51 63. Leach, J. B., & Schmidt, C. E. (2005). Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene glycol hydrogel tissue engineering scaffolds. Biomaterials, 26(2), 125 135. Available from https://doi.org/10.1016/j.biomaterials.2004.02.018. Matica, M. A., Aachmann, F. L., Tøndervik, A., Sletta, H., & Ostafe, V. (2019). Chitosan as a wound dressing starting material: Antimicrobial properties and mode of action. International Journal of Molecular Sciences, 20(23), 5889. Mihranyan, A. (2011). Cellulose from cladophorales green algae: From environmental problem to high-tech composite materials. Journal of Applied Polymer Science, 119(4), 2449 2460. Naomi, R., Bt Hj Idrus, R., & Fauzi, M. B. (2020). Plant- vs. bacterial-derived cellulose for wound healing: A review. International Journal of Environmental Research and Public Health, 17(18). Available from https://doi.org/10.3390/ijerph17186803. Ninan, N., Muthiah, M., Park, I.-K., Elain, A., Thomas, S., & Grohens, Y. (2013). Pectin/ carboxymethyl cellulose/microfibrillated cellulose composite scaffolds for tissue engineering. Carbohydrate Polymers, 98(1), 877 885. Available from https://doi.org/10. 1016/j.carbpol.2013.06.067. Pishbin, F., Mourin˜o, V., Flor, S., Kreppel, S., Salih, V., Ryan, M. P., . . . Boccaccini, A. R. (2014). Electrophoretic deposition of gentamicin-loaded bioactive glass/chitosan composite coatings for orthopaedic implants. ACS Applied Materials & Interfaces, 6 (11), 8796 8806. Available from https://doi.org/10.1021/am5014166. Rabille´, H., Torode, T. A., Tesson, B., Le Bail, A., Billoud, B., Rolland, E., . . . Charrier, B. (2019). Alginates along the filament of the brown alga Ectocarpus help cells cope with stress. Scientific Reports, 9(1), 1 17. Rejinold, N. S., Chennazhi, K. P., Nair, S. V., Tamura, H., & Jayakumar, R. (2011). Biodegradable and thermo-sensitive chitosan-g-poly(N-vinylcaprolactam) nanoparticles as a 5-fluorouracil carrier. Carbohydrate Polymers, 83(2), 776 786. Available from https://doi.org/10.1016/j.carbpol.2010.08.052. Suk, J. S., Xu, Q., Kim, N., Hanes, J., & Ensign, L. M. (2016). PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Advanced Drug Delivery Reviews, 99, 28 51. Sun, J., & Tan, H. (2013). Alginate-based biomaterials for regenerative medicine applications. Materials, 6(4). Available from https://doi.org/10.3390/ma6041285. Tayeb, A. H., Amini, E., Ghasemi, S., & Tajvidi, M. (2018). Cellulose nanomaterials— Binding properties and applications: A review. Molecules (Basel, Switzerland), 23(10). Available from https://doi.org/10.3390/molecules23102684. Younes, I., & Rinaudo, M. (2015). Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs, 13(3), 1133 1174. Available from https://www.mdpi.com/1660-3397/13/3/1133. Zhao, D., Yu, S., Sun, B., Gao, S., Guo, S., & Zhao, K. (2018). Biomedical applications of chitosan and its derivative nanoparticles. Polymers, 10(4), 462. Zhao, T., Maniglio, D., Chen, J., Chen, B., & Migliaresi, C. (2016). Development of pHsensitive self-nanoemulsifying drug delivery systems for acid-labile lipophilic drugs. Chemistry and Physics of Lipids, 196, 81 88.

CHAPTER

Characteristics and characterization techniques of bacterial cellulose for biomedical applications—a short treatise

4

Kumar Anupam1,2, Richa Aggrawal3, Jitender Dhiman4, Priti Shivhare Lal5, Thallada Bhaskar6,7 and Dharm Dutt1 1

Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India 2 Chemical Recovery and Biorefinery Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India 3 Department of Chemical Engineering, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Haryana, India 4 Biotechnology Division, Central Pulp, and Paper Research Institute, Saharanpur, Uttar Pradesh, India 5 Physical Chemistry, Pulping and Bleaching Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India 6 Material Resource Efficiency Division, CSIR-Indian Institute of Petroleum, Dehradun, Uttarakhand, India 7 Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India

4.1 Introduction With the advancement in medical science there emerges a great demand of biomaterials. Biomaterials possess great importance in the field of surgery. About 2800 years ago, before the evolution of modern medical science, great Indian surgeon Sushruta innovated medical surgery and utilized sutures made up of cellulose extracted from hemp and cotton. With modern techniques cellulose was extracted in 1838 from the cell walls of plants (Bodin, Ba¨ckdahl, Petersen, & Gatenholm, 2011; Hestrin & Schramm, 1954). Cellulose is a polymer which is made up of β-(1, 4) glucose and responsible for structural integrity of various fungi, plants, and some algae. Cellulose obtained using bacteria is called as bacterial cellulose (BC) and is in its pure form; whereas cellulose obtained from plants

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00021-8 © 2023 Elsevier Inc. All rights reserved.

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also accommodate both lignin and hemicellulose in it. Physical and chemical properties of BC and plant based cellulose are same as both own same chemical structure. Bacterial species belonging to the Acetobacter, Rhizobium, Agrobacterium, and Sarcina genus are known to produce BC (Abol-Fotouh et al., 2020; Slapˇsak, Cleenwerck, De Vos, & Trˇcek, 2013; Trˇcek & Barja, 2015; Vigentini et al., 2019). In a discovery by Brown in 1886, it was found that bacterium Acetobacter xylinum produce cellulose in the form of film. At that time these bacterium was called as “the vinegar plant” because these were the chief source of acetic acid. In the middle of 20th century, research community became curious about BC. Hestrin and Schramm (in 1954) produced microbial cellulose using A. xylinum bacterium in glucose-plentiful medium. Because of the purity and excellent physicochemical characteristics BC can be utilized in the food and medical field. BC exhibit properties which are congruent in combining surface and macro-molecular characteristics that are important in biomedical field. During the synthesis of BC large quantity of water was trapped in its molecular structure, leading to the emergence of hydrogel possessing about 90% of water. BC was utilized as a noble wound dressing material due to inestimable in vivo biocompatibility, flexibility, higher water-holding capacity, and gas exchange (Czaja, Krystynowicz, Bielecki, & Brown, 2006; Sokolnicki, Fisher, Harrah, & Kaplan, 2006). Besides this, BC maintains a physical barrier that inhibits bacteria inflow in wound and only allows drug transportation in wounded region. Water present in the BC structure assists in wound healing as it provides necessary and consistent moisture supply to dry wound and inhibit necrosis. Characterization of necrosis in wound is carried out by blackness in wound. Such property is due to the dehydration and presence of death cells. Generally, healing of wounds is promoted by removing these dead cells from it; however, in crucial time when surgery is not possible, hydrogel dressing reinforced to serve the purpose (Abdelrahman & Newton, 2011; Kavitha et al., 2014). From the above discussion it is clear that BC holds great importance in biomedical filed. Therefore this chapter aims to reiterate and consider the applications of BC in biomedical field with major emphasis on physical, chemical, and biological properties of BC along with instrumental characterization techniques in light of their different morphologies and varieties.

4.2 Biomedical applications of bacterial cellulose This section discusses different types of characteristics and characterization techniques of BC as applied in various biomedical applications depicted in Fig. 4.1. An attempt has also been made to collate these characteristics and characterization techniques according to different morphologies and varieties of BCs reported in literature and shown in Fig. 4.2. Different characteristics and characterization techniques generally used for BCs are listed in Table 4.1.

4.2 Biomedical applications of bacterial cellulose

FIGURE 4.1 Bacterial cellulose implemented in various biomedical applications.

FIGURE 4.2 Morphologies and varieties of bacterial celluloses reported in literature.

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Table 4.1 Characteristics and characterization techniques of BCs. Physical properties

Biological properties

Instrumental techniques

Tensile strength Hydrophobicity Water contact angle Surface tension Pore size/microporosity Electrical conductivity Thermal stability Optical properties Surface area Roughness Morphology Crystallinity Formability

Antimicrobial activity Cell viability Biocompatibility Biodegradability Cytotoxicity Cytocompatibility

FTIR TGA DSC SEM/FESEM BET Raman spectroscopy XRD XPS XRF 13 C CP/MAS NMR TEM AFM BET

AFM, atomic force microscopy; BC, bacterial cellulose; BET, Brunauer Emmett Teller theory; CP/MAS NMR, cross-polarization magic angle spinning nuclear magnetic resonance; DSC, differential scanning calorimetry; FTIR, Fourier transform infra-red; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TGA, thermo gravimetric analysis; XPS, X-photoelectron spectroscopy; XRD, X-ray diffraction; XRF, X-ray fluorescence.

4.2.1 Wound healing applications Bacterial cellulose-montmorillonite (BC-MMT) composites were used by UlIslam, Khan, and Park (2012a) to study its applications in biomedical field. Morphological study was performed on the composites using FESEM analysis. It revealed a 3D arrangement of microfibrils randomly arranged in BC matrix. MMT particles successfully penetrated deep inside BC sheets through pores thus confirming effective agglomeration of MMT particles on BC. FTIR spectrum of BC-MMT composites comprised a combination of FTIR spectra of PBC (pure bacterial cellulose) and pure MMT. The details of peaks are shown in Table 4.2. XRD patterns of pure BC resulted in three peaks at 2θ
values of 14.2, 16.6, and 22.4 degrees indicating crystallographic planes of 110 ; (110) and (200), respectively. The pure MMT exhibited XRD peaks at 2θ values of 8.5, 17.74, 26.52, and 45.52 degrees. The XRD peaks of PBC and pure MMT altogether make up the XRD peaks for BC-MMT composites. TGA analysis revealed that the composites had a 5% weight loss over the 80 C 120 C temperature range because of moisture loss and coordinated water molecules between the layers. BC-MMT prepared with 1%, 2%, and 4% MMT suspensions exhibited that incorporation of MMT into BC improved its mechanical properties such as tensile strength and Young’s Modulus while strain and the water-holding capacity decreased. The variations in the values of these properties of pure BC with incorporation of MMT are shown in

4.2 Biomedical applications of bacterial cellulose

Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Material

FTIR spectrum peaks

References

PBC

O-H at 3444/cm, C-H at 2896/cm, and C-O-C at 1000/cm C-H at 1424/cm O-H at 3612/cm and Si-O at 1087/ cm H-O-H at 1600/cm and Si-O at 526/cm Hydrogen bonding at 3452/cm 3150/cm and 3280/cm for N-H; Peaks at 2924/cm for υas(CH2) and 2853/cm for υs(CH2) 1528/cm and 1697/cm υ (C 5 N), 3150/cm and 3280/cm N-H stretching vibrations. 2924/cm and 2853/cm υas(CH2) and υs (CH2), 3160, 3266, 2920, 2853, and 1618/cm for N-H, CH2 and C 5 N 3300/cm for OH, 2890/cm for CH and 1060/ cm for C-O-C, 1453/cm for CH and 1678/cm for H-O-H CH at 2895/cm, CH at 1428/cm and H-O-H at 1651 cm, 3342/cm for OH group CH at 2895/cm

Ul-Islam et al. (2012a)

3361/cm & 3235/cm for O-H, 2914/cm & 2850/cm for C-H, 1064/cm, 1116/cm, and 1236/cm for C-O-C and O-H 2897/cm for CH, 1032/cm and a carboxyl group band at 1595/cm 1646/cm for glucose carbonyl of cellulose, 1900 1500/cm for carbonyl and carboxyl groups, carboxyl group of BCA sponges of 70%, 50%, and 30% alginate were shifted from 1591/cm to 1608, 1610, and 1640/cm, respectively. O-H at 3300/cm, C-H at 2820/cm and C-O-C at 1080/cm and hydrogen bonded carbonyl group at 1730/cm, CH2 at 1427/cm and O-H at 684/cm

Tsai, Yang, Ho, Tsai, and Mi (2018)

Pure MMT

BC/PHMG-Cl

Pure BC

BC/ZnO-NPs

NR/BCW nanocomposite films SMN-Zein/ BC nanocomposite films Alginate/ BC hydrogel beads BCA sponge

PBC

Kukharenko et al. (2014)

Khalid et al. (2017)

Wahid et al. (2019)

Yin et al. (2018)

Kim et al. (2017) Chiaoprakobkij, Sanchavanakit, Subbalekha, Pavasant, and Phisalaphong (2011)

Zhijiang, Chengwei, and Guang (2012)

(Continued)

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Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Continued Material

FTIR spectrum peaks

Pure P(3HB-co4HB)

C-O at 1283/cm, C-H at 2741/cm, 2889/cm & 2973/cm and C 5 O at 1726/cm, hydroxyl groups at 3433/cm PBC

Oliveira Barud et al. (2015)

Pure SF

PBC

3348/cm for O-H, 2940/cm & 2893/ cm for C-H and 1040/cm for C-O-C 1613/cm, 1550/cm and 1377/cm for amide I, II, and III groups Peaks of C-O at 1163/cm and C-O (asymmetric bridge) at 1061/cm, 1061/cm for C-O asymmetric bridge stretching & C-O-C for pyranose ring skeletal vibrations, intramolecular hydrogen bonding at 3200 3500/cm Bands of S 5 O (asymmetrical) at 1233/cm, phenyl connected to NH2 group at 1552/cm and NH2 at 3344/cm & 3391/cm both 1669/cm for C 5 O (amide I), 1634/ cm for C 5 O (A-ring), 1581/cm for C 5 O (C-ring), 1535/cm for NH2 deformation (amide II) and 1456/cm for C 5 C (aromatic ring) 2888/cm for C-H and 1159/cm for C-O (asymmetric bridge) Peaks at 3345/cm for hydroxyl groups and 1055/cm for C-O-C (pyranose skeletal ring vibration)

BC-Chi Pure BC

Pure AgSD

BC-TCH

PBC

References

1040/cm for C-O (symmetric), 1168/cm for C-O-C (asymmetric), 2900/cm for C-H and 3500/cm for O-H, 400 700/cm for O-H, 1400/cm for CH2 and 1650/cm for H-O-H, 1340/cm for C-H deformation 1500 1600/cm for amide I and 1600 1700/cm for amide II, 1500 1600/cm and 1600 1700/cm for amide I and amide II, 1610 1630/cm for amide I and 1510 1520/ cm for amide II, 1640 1660/ cm and 1535 1542/cm represented silk I. Lin, Lien, Yeh, Yu, and Hsu (2013)

Shao, Liu, Wu, et al. (2016)

Shao, Liu, Wang, et al. (2016)

Zhang et al. (2020)

(Continued)

4.2 Biomedical applications of bacterial cellulose

Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Continued Material

FTIR spectrum peaks

Pure TA

1710/cm for C 5 O and 1612/cm for C 5 C (aromatic), 1025/cm for benzene ring vibrations O-H at 3445/cm, C-H at 2920/cm & 1317/cm and C-O-C at 1157/cm, β-1,4 glycosidic bond at 1052/cm amide I at 1656/cm, amide II at 1540/cm, and amide III at 1245/cm amide I at 1653/cm and amide II at 1563/cm

PBC

Pure SS Pure HA

References

Wang, Tang, Huang, and Hui (2020)

BCW, bacterial cellulose whiskers; HA, hyaluronic acid; MMT, montmorillonite; NR, natural rubber; PHMG, polyhexamethylene guanidine hydrochloride; SMN, silymarin; SS, silk sericin; TA, tannic acid; TCH, tetracycline hydrochloride.

FIGURE 4.3 Effect of MMT incorporation on pure bacterial cellulose.

Fig. 4.3. The final inference derived from various characterizations suggested that BC-MMT composites can serve as excellent wound healing materials when used like a moist paste on wounds or skin. Kukharenko et al. (2014) combined BC with polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl) to prepare a material that can be utilized in wound healing. The material was characterized using FTIR, AFM,

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antimicrobial assay, etc. The peaks identified through FTIR spectroscopy are mentioned in Table 4.2. AFM images displayed an interconnected network of microfibrils twisted bundles. Estimated diameter of these bundles was found to be 90 200 nm. Antimicrobial activity of BC/PHMG-Cl films revealed that PHMGCl was capable of inhibiting growth of all tested microorganisms. Inhibition zone of 20 mm was observed for gram-negative bacteria (Pseudomonas syringae and Klebsiella pneumonia) as well as gram-positive bacteria (S. aureus). Overall results concluded that BC/PHMG-Cl films are excellent and safe choice for wound healing. Bacterial cellulose-zinc oxide (BC-ZnO) nanocomposite films could be utilized extensively for wound healing applications. Khalid, Khan, Ul-Islam, Khan, and Wahid (2017) studied its effectiveness by subjecting the nanocomposite films to various characterizations. Morphological analysis of the nanocomposite films was carried out using FESEM analysis. SEM images suggested that ZnO nanoparticles were strongly attached to the surface of BC. Nanoparticles were found to be homogenously distributed and deeply penetrated throughout the BC matrix. It suggested the presence of enhanced thermal, mechanical, and biological properties. XRD patterns were obtained to determine the structural characteristics of the films. It was observed that XRD patterns of nanocomposite films totally comprised of peaks obtained from XRD patterns of BC and ZnO separately. Relative crystallinity of 52.4% was observed for nanocomposite films. The details of FTIR spectra of BC are provided in Table 4.2. FTIR spectra of BC-ZnO nanocomposite films contained all the peaks of FTIR spectra of BC and additionally had more peaks at 642 and 480/cm. Nanocomposite films displayed antibacterial activity against gram-negative bacteria such as Escherichia coli (90%), Citrobacter freundii (90.9%), and Pseudomonas aeruginosa (87.4%) and Gram-positive bacteria such as Staphylococcus aureus (94.3%). This suggested that ZnO nanoparticles were successfully incorporated on the surface of BC matrix and induced antimicrobial properties into BC making it potential for application in wound healing. Wound healing activity of nanoparticles resulted that wound size tended to shrink with passage of time. It was found to be 289 6 0 mm2 (day 0) to 98.3 6 7.6 mm2 (day 15). Another observation was made that 7%, 33%, and 66% healing took place on day 5, 10, and 15, respectively. Thus nanocomposite films displayed excellent healing capacities. It was concluded that BC-ZnO nanocomposite films have an excellent potential of application in burn wound healing. Khan, Ul-Islam, Ikram, et al. (2018) discovered the effective application of 3dimensional microporous regenerated bacterial cellulose/gelatin (3DMP rBC/G) scaffolds in wound healing and skin regeneration. Materials that are considered as ideal healing materials should be able to prevent deterioration of healing, prevent foreign body reactions, and be easily removed without damaging newly formed tissue. Hence different characterizations were applied to the scaffolds for analyzing their performance. Pores with high interconnectivity were found in the surface morphology of scaffolds. The cross-sectional morphology also displayed high content of gelatin entrapment in the scaffolds which led to enhanced cell

4.2 Biomedical applications of bacterial cellulose

interactions due to surface modification. Twenty four percent gelatin introduced into the scaffold was found to be used for strong hydrogen bonds between the OH and NH groups. The presence of gelatin improved the biocompatibility of 3-D scaffolds. Elemental analysis showed that pure BC was composed of C, H, and O, and gelatin was composed of C, H, O, and N. In NDBC samples, 51.31% oxygen, 42.51% carbon, 6.17% hydrogen, and 0% nitrogen were found. It was also suggested that introduction of gelatin into the scaffolds led to cellulose surface modification and enhanced biocompatibility. SEM images revealed good quality of cell adhesion and 3-D microporous structure which supported cell growth and proliferation because of availability of free space. High biocompatibility of 3-D scaffolds was supported because of the fact that cells penetrated deep inside the extracellular matrix and proliferated well with available free space. Gelatin was also found to be contributing to the biocompatibility of the scaffolds. Cell viability tests revealed that no difference was observed for the first 3 days while increased proliferation was observed after 5 7 days of incubation. Wound healing capabilities of 3-D scaffolds were found excellent after 2 weeks of application as the wound was almost completely closed. It was also observed that with time, new skin tissues grew in place of the scaffolds which led to successful skin regeneration. For 3DMP rBC/G scaffolds, wound healing rate was found to be 66.66 6 2.35% after 1 week which increased to 93.34 6 4.45% after 2 weeks. Histopathology analysis was performed to determine the healing progress and tissue regeneration phases. H&E-stained images resulted that successful reepithelialization takes place for 3-D scaffolds. H&E staining also proved increase in healing recovery. It was concluded that 3DMP rBC/G scaffolds have a great potential in wound healing applications. Performance of BC/ZnO nanocomposite films were well investigated by Wahid et al. (2019) for its applications in wound healing. FTIR, SEM, XRD, TGA were some of the characterizations applied to BC/ZnO-NP films. Characteristics of BC/ZnO-NP films were determined with the help of content and stability analysis, antibacterial activity, etc. XRD results provided with typical diffraction peaks at 14.5, 16.7, and 22.8 degrees attributed to (100), (010), and (110) crystallographic planes of cellulose. Moreover, peaks at 31.8, 34.4, 36.2, 48.5, 56.5, 63.2, and 68.4 degrees represented (100), (002), (101), (102), (110), (103), and (112) crystal planes of ZnO having hexagonal wurtzite structure. It was also observed that no peaks for impurities were found, therefore, indicating formation of BC/ZnO-NP films with no impurities. FESEM analysis suggested uniform distribution of ZnO-NPs on the surface of cellulose. FTIR results were found in correspondence with the results obtained from XRD and FESEM analysis. FTIR spectra gave various characteristic peaks as shown in Table 4.2. The peak at 438/cm was attributed to ZnO-NPs. The TGA results show that the nanocomposite film showed significant weight loss between 250 C and 330 C because of dehydration of absorbed water and thermal decomposition of BC. It was also suggested that BC/ZnO nanocomposite films possessed better thermal stability at elevated temperatures. The UV-visible spectra displayed a peak at 362 nm,

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confirming existence of ZnO-NPs in the nanocomposite films. Photocatalytic activity revealed that the intensity of the peak at 464 nm reduced on enhancing the UV exposure time. It was estimated that almost 91% of MO was degraded by nanocomposite films. The antibacterial activity of nanocomposites was tested against Gram-positive as well as Gram-negative bacteria. The results discovered a sturdier consequence on Gram-positive bacteria (Bacillus subtilis) than Gramnegative bacteria (P. aeruginosa). It was concluded that introduction of ZnO-NPs onto BC surface successfully induced antibacterial properties into BC films. Bacterial cellulose/Bacillus subtilis (BC/BS) biocomposite was utilized by Savitskaya, Shokatayeva, Kistaubayeva, Ignatova, and Digel (2019) for its potential applications in wound healing. There are three major steps involved in destruction of bacteria through development of pores inside the BM (bacterial membrane). These are (1) binding to BM, (2) accumulation within BM, and (3) creation of channels. Various characterizations were applied to the biocomposite to determine its wound healing capability. SEM images revealed porous and microfibrillar structure of BC films which was highly suitable for impregnation of BS cells. Hence, SEM micrographs established effective agglomeration of BS P-2 cells to the surface of BC films. Antagonistic activity was first determined using agar diffusion test. The test confirmed antibacterial efficacy of this biocomposite as the inhibition zone was found to be in the range of 13 22 mm around fiber mats. Time kill test was also used to determine the antagonistic activity of biocomposite by revealing its bactericidal effect. Time kill test resulted in a time-dependent and concentration-dependent antimicrobial effect. It was observed that there was 100% reduction in bacterial growth in case of gram-negative bacteria in 24 h, while the same reduction was observed in just 10 h for gram-positive bacteria. Killing pattern of Staphylococcus epidermidis and S. aureus were found to be similar while good bactericidal effect was observed against E. coli and P. aeruginosa. The biocomposite was also studied for its proteolytic activity. It was suggested that BS agglomerated on BC matrix successfully produced proteases which were capable of digesting milk-casein. Average diameter of the zones was found to be 16 mm. It was suggested that the proteolytic enzymes produced by BS cells led to faster wound healing. Effective wound dressing is one that promotes epithelium regeneration. Dead tissues in contact of wound also promoted bacterial growth. Hence, it becomes mandatory for the wound healing material to be capable not only of inhibiting bacterial growth but also killing any further microbial growth. It was suggested that proteases produced with the help of BC/BS biocomposite supported healing of burned skin wounds, pressure sores, and leg ulcers. Study of epithelialization time and wound contraction were conducted on the biocomposite films. A very high wound healing activity was observed through BC/BS biocomposite films. Tests conducted on a group of rats displayed complete healing within a period of 7 days. It was found that epithelium effectively covered the wounds in moist environment. Hundred percentage of wound closure was achieved for animals treated with BC/BS biocomposite on seventh day. In conclusion, it was suggested that BS P-2 has advantageous and positive effect on wound healing.

4.2 Biomedical applications of bacterial cellulose

4.2.2 Diagnosis of ovarian cancer BC possesses extraordinary physicochemical as well as mechanical properties, however, it lacks anticancer activity. On the other hand, chitosan displays unique anticancer activity but has weak mechanical properties. Hence, Ul-Islam et al. (2019) employed 3-D scaffolds of bacterial cellulose and chitosan (BC-Chi) for its effective application in diagnosis of ovarian cancer. Usage of BC-Chi scaffolds led to improved mechanical and biological properties as well as utilization of anticancer activity. The scaffolds were characterized using a number of techniques including FTIR and FESEM analysis. The FTIR spectroscopy analysis confirmed the impregnation of chitosan onto the BC matrix which improved its mechanical properties and water-holding capacity along with delaying the water release rate from the matrix. These properties are desirable in an ideal scaffold to be effective in biomedical applications. SEM analysis revealed that BC matrix possessed a 3-D structure. Surface phase-contrast microscopic analysis was used in morphological studies which disclosed that A2780 cell lines had solid adhesive power towards cancer cells. Thus, strong adherence of cells on BC-Chi scaffolds suggested enhanced biocompatibility of scaffolds. An improvement in the adhesion and penetration of cell lines onto the scaffolds was observed through cross-sectional staining. WST-1 assay suggested high viability and proliferation. The H&E actin staining resulted in higher level of cell proliferation using BC-Chi scaffolds. This led to lower amount of cell aggregate formation indicating strong cell-scaffold interaction. Hence a comparatively high level of cell lines infiltration was observed. The results suggested BC-Chi scaffolds having great potential for in vitro culturing of cancer cell lines.

4.2.3 Shape memory material Adaptive medical implants, self-tightening sutures, self-retractable and removable stents, etc. are some of the applications of shape memory material in biomedical field. Utilization of natural rubber (NR) with bacterial cellulose whiskers (BCW) was explored by Yin et al. (2018) for its application in production of shape memory materials. NR/BCWs nanocomposite films were fabricated for this purpose through acid hydrolysis. This was done to retain crystalline parts to form nanowhiskers and to remove amorphous regions of BC. TEM, SEM, XRD, FTIR, etc. were some of the characterizations applied to nanocomposite films. The XRD patterns confirmed that the high crystallinity was due to the removal of amorphous regions of BC. The FTIR spectra peak has been shown in Table 4.2. This suggested the presence of intermolecular hydrogen bonds between cellulose chains. The morphology of BCWs suggested slender rod like shape of nanocomposite films. Mechanical properties indicated the development of a 3-D network of whiskers linked through hydrogen bonding and an abundance of immobilized rubber chains about the surfaces of filler. The water uptake capacity has a high diffusion coefficient of water because of the water diffusing along hydrophilic channels. This was due to high hydrophilicity of BCWs which led to an increase in hydrophilicity of composite films.

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4.2.4 Preventing deterioration of salmon muscle and slowing down the lipid oxidation Tsai et al. (2018) explored the biomedical application using silymarin-zein (SMN-Zein) nanoparticles/BC nanocomposite films. The SMN-Zein nanoparticles were firmly attached to BC nanofibers. The FTIR spectrum of BC composite displayed characteristic bands shown in Table 4.2. The XRD diffraction patterns illustrated that crystallinity of BC fibers was not at all affected because of the interaction between SMN-Zein nanoparticles and BC fibers. Wetting properties of SMN-Zein/BC nanocomposite films suggested increase in the hydrophobicity of film surfaces. This causes packaging films to be moisture resistant which acts as an excellent obstruction against water penetration causing improved shelf life. The nanocomposite films demonstrated strong antibacterial activity against Grampositive bacteria such as S. aureus and was less effective against Gram-negative bacteria such as E. coli and P. aeruginosa.

4.2.5 Lipase immobilization Kim et al. (2017) investigated the potential of alginate/BC nanocomposite beads for lipase immobilization. The average diameter of alginate/BC hydrogel beads was found to be in the range of 2.5 3.6 mm. The average diameter and volume of alginate/BC hydrogel beads increased by 144% and 300%, respectively. This was attributed to swelling and high water-holding capacity of alginate/BC beads. SEM results revealed that BC was present inside the alginate beads until 24 h. After that, cellulose fibers were released to the bead surface. After cultivation time of 72 h, number of cellulose fibers and mats layered on the bead surface was found to be increased. The characteristic FTIR peaks are mentioned in Table 4.2. The results suggested that specific interactions take place between hydroxyl groups and carboxyl groups. The XRD patterns represented four peaks at 2θ 5 14.3 , 16.7 , 22.6 , and 34.5 , which corresponded to crystalline planes of cellulose. Alginate/BC nanocomposite beads also presented with an unreported peak at 2θ 5 24.0 which may represent formation of a new crystalline structure. The water vapor sorption capacity is responsible for high water-holding ability of alginate/BC nanocomposite beads. The water vapor sorption capacity was found to be 38.88 (g/g dry bead). Biodegradability of beads was supported with the fact that cellulose present inside the beads could be degraded by cellulase producing microorganisms. Thus, alginate/BC nanocomposite beads have a high potential in biomedical applications.

4.2.6 Tissue engineering Chiaoprakobkij et al. (2011) fabricated bacterial cellulose/alginate (BCA) composite sponges for effective utilization in biomedical field. The fabricated sponges were subjected to elemental analysis using X-ray fluorescence (XRF)

4.2 Biomedical applications of bacterial cellulose

spectroscopy. It resulted in absence of sodium (Na) peak suggesting complete removal of NaOH from BCA sponge. Also, calcium content of 0.88 2.96 weight % in the BCA sponges was observed due to alginate content. The peaks identified through FTIR spectra study are presented in Table 4.2. SEM micrographs revealed porous structure with 3-D interconnection throughout the sponges. It also told that asymmetric structure consisted of a top skin layer and sponge-like porous layer. The dense outer layer helps to prevent bacterial invasion and to avoid wound dehydration, whereas the porous layer provides drainage of wound and mechanical strength. Tensile strength and elongation at break were found to be decreasing with increasing the alginate content. Hence, it was inferred that intermolecular interactions might reduce the crystallinity and mechanical strength of the composite material. For water uptake ability, it was observed that BCA sponges swelled rapidly after immersion in water and were stable in distilled water. It was concluded that BCA sponge possessed exceptional characteristic properties for utilization in oral tissue regeneration. Poly(3-hydroxubutyrate-co-4-hydroxubutyrate) and bacterial cellulose biocomposite scaffolds [P(3HB-co-4HB)/BC] were engineered by Zhijiang et al. (2012) for tissue regeneration purposes. FESEM analysis revealed that scaffolds had multipore size distribution. Medium pores with diameter of 20 μm were uniformly dispersed on the surface. Micro pores with diameter 500 nm were found inside the wall. A 3-D network structure was obtained. Porosity of the scaffolds was found to be 91%. The findings of FTIR spectroscopy are shown in Table 4.2. All these characteristics bands and peaks of pure BC and pure P(3HB-co-4HB) together combined forms the FTIR spectra for P(3HB-co-4HB)/BC composite scaffolds. XRD analysis for pure P(3HB-co-4HB) resulted in two strong peaks at 2θ value of 13 and 17 degrees assigned to (020) and (110) orthorhombic unit cell. On the other hand, XRD pattern for P(3HB-co-4HB)/BC composite scaffolds is similar to XRD patterns of pure P(3HB-co-4HB) and cellulose II but with decreased intensity. Mechanical properties of P(3HB-co-4HB)/BC composite scaffolds suggested a visible yielding point but very close to breaking point. Values for tensile strength, elongation at break and Young’s modulus were found to be 46 MPa, 13.5% and 0.88 GPa respectively. The water contact angle for scaffolds was found to be reduced to 33.6 degrees and surface tension value was 59.2 mN/ m2. These results indicated high hydrophilicity of scaffolds. Scaffolds represented better biodegradability. After degradation for 30 days, weight loss ratio was 12% of original weight. It was concluded that the fabricated scaffolds were found suitable for tissue engineering uses. Favi et al. (2013) explored the potential of EqMSC (equine derived bone marrow mesenchymal stem cells)-BC scaffolds for applications in tissue regeneration. Scaffolds were subjected to MTS assay analysis to determine viability of EqMSCs on BC. It displayed extraordinary metabolic rates as a function time and denote a high viability and proliferation configuration. The linear regressions of the 1.0 3 104, 2.0 3 104, 3.0 3 104, and 4.0 3 104 cells seeded on BC were 0.998, 0.991, 0.966, and 0.950, respectively, indicating a linear response between cell

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number and absorbance at 490 nm, further confirming the proliferation of EqMSCs under these conditions. Alkaline phosphatase staining of cell patterns confirmed that EqMSC-BC retained their structure and stem cell-like properties. SEM images revealed that EqMSC cells adhered to nanofibrous BC matrix and maintained a fibroblast membrane morphology. The study concluded that EqMSCs can adhere to, are metabolically active, viable, and retain the potential to differentiate into osteocytes and chondrocytes on BC matrix. Hyaluronic acid on bacterial cellulose (BC/HA) hybrid membranes were employed by Lopes, Riegel-Vidotti, Grein, Tischer, and Faria-Tischer (2014) for applications in tissue engineering. Different characterization techniques such as FTIR, XRD, NMR, AFM, etc. were applied to these membranes. XRD patterns of BC/ HA membranes displayed peaks at 14.6, 16.9, and 22.8 degrees referred to (100), (010), and (110) planes of cellulose I attributed to triclinic unit cell of allomorph Iα. It was suggested that membranes produced on third and sixth day after fermentation displayed highest crystallinity. Presence of HA was confirmed by 13C CP MAS NMR. The spectrum displayed both anomeric carbons of the glucuronic acid and the N-acetyl glucosamine at 101.6 ppm. Signals from HA were also observed through methyl group present at 23 ppm, N-acetyl present at 55 ppm, and carbonyl carbon present at 174 ppm. New signals observed at 30, 33, and 41 ppm were attributed to peptides/proteins. SEM micrographs exhibited a mesh like morphology of hybrid membranes. Surface morphology was also determined using AFM which revealed that fibrils were attached to each other in hybrid membranes. Wetting experiments revealed smoother and more hydrophilic surface. TGA analysis was carried out for a temperature range of 150 C 450 C over a period of 6 days. It was inferred that hybrid membranes were found to be more thermally stable. Yin, Stilwell, Santos, Wang, and Weibel (2015) explored the possibility of application of porous bacterial cellulose using agarose microparticles (pBC-M) in biomedical applications. Agarose can be termed as a polysaccharide which is extracted from red algae. Agarose has exceptional biocompatibility, physical and chemical stability and hydrophilicity. It can be used in biomolecule separation and microencapsulation. pBC-M characterized using SEM resulted in micrographs depicting a dense network of nanofibrous BC. For pBC-M micrographs, a uniform and homogenous distribution of pores with diameter 300 500 μm was observed. To analyze the feasibility of pBC-M in tissue engineering applications, surface of microparticle samples were cultivated using human P1 chondrocytes. Chondrocyte cells grew on the surface of BC and were not able to penetrate inside the polymer whereas these cells entered pBC-M scaffolds and dispersed throughout the polymer. This happened due to the smaller average pore size of the network as compared to cell dimensions. Chondrocytes cultivated on pBC-M scaffolds resulted in high cell viability of 85% 99% over a period of 14 days. Cell morphology of chondrocytes cultivated on the scaffolds was studied using confocal microscopy for 1, 7, and 14 days. After 1 day, cells formed cell body extensions while after 7 days, cells exhibited spindle shape indicating attachment

4.2 Biomedical applications of bacterial cellulose

to BC fibers. After 14 days, cells displayed a stretched morphology confirming even distribution throughout the porous scaffolds. Analysis of mechanical properties was performed for pure BC and pBC-M scaffolds. Values of Young’s Modulus and stress at break for pure BC were recorded to be 14.7 and 2.4 MPa and for pBC-M were recorded to be 5.4 and 0.52 MPa, respectively. It was suggested that values of mechanical properties of pBC-M were lying in the ideal range required for mechanical properties of materials used for tissue engineering. Thus, it was concluded that pBC-M can be a suitable material for cartilage repairing. Bacterial cellulose/silk fibroin (BC/SF) nanocomposites were developed by Oliveira Barud et al. (2015) for tissue engineering applications. The nanocomposites were subjected to various characterizations. The results of FTIR spectra study are shown in Table 4.2. FTIR spectra of BC/SF nanocomposites can simply be considered as summation of FTIR spectras of BC and SF separately. XRD peaks for pure BC were observed at 15 and 22.5 degrees assigned to native cellulose type 1. Freeze dried SF exhibited XRD peaks at 11.8, 19.8, and 22.6 degrees assigned to silk 1 crystalline plane. XRD patterns of BC/SF nanocomposites displayed no significant changes when compared to XRD patterns of BC and SF. BC, SF, BC/SF nanocomposites were all subjected to TGA analysis. Two mass losses were found in the TGA curve of BC. First mass loss was almost 4.6% which occurred at 200 C due to evaporation of surface water. Second mass loss was found to be 80% which occurred at 280 C due to decomposition and depolymerization of dehydrocellulose. Two mass losses were found in the TGA curve of SF. First mass loss was almost 7.3% which occurred at 120 C due to water loss. Second mass loss was found to be 52% which occurred at 180 C 500 C due to breakdown of amino acid residues and cleavage of peptide bonds. TGA curve of BC/SF nanocomposites represented three mass losses. First loss was almost 7% occurred at 200 C due to water losses. Other two occurred at 200 C 500 C. Images obtained from FESEM analysis displayed 3-D nano-fibril network of porous structure for BC matrix. SEM images for BC/SF: 25% and 50% nanocomposites revealed sponge-like structures whereas BC/SF: 75% nanocomposite exhibited less porous structure. Overall, a well interconnected porous structure of nano-filaments entangled with each other was observed as morphology for BC/SF nanocomposites. Pore size of the scaffolds were found to be 102 6 5.43 μm through porosity studies. Water solubility results obtained explained 0%, 3%, and 8.6% solubility of BC/SF: 25% nanocomposite, BC/SF: 50% nanocomposite and BC/SF: 75% nanocomposite in distilled water, respectively. Water uptake capacity is another important parameter for characterization. It discusses the property of a material to diffuse water which allows transport of nutrients and growth of new cells necessary for tissue regeneration. BC/SF: 50% nanocomposite scaffolds had a water uptake capacity of 216%. All scaffolds were able to absorb water within 1 min and get saturated within 1 h. Average cell viability for BC/SF: 50% nanocomposite was found to be 123.81%. No genotoxicity or cytotoxicity was found in the scaffolds which rendered it perfectly suitable for applications in tissue engineering.

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Aki et al. (2020) employed 12 wt.% polyvinyl alcohol (PVA) /0.25 wt.% hexagonal boron nitride (hBN) /(0.1, 0.25, 0.5 wt.%) BC composite scaffolds for testing its applications in bone tissue engineering. The composite scaffolds were found suitable for biomedical applications after confirmation through various characterization techniques that were applied to the scaffolds. SEM micrographs revealed that 12 wt.% PVA composites exhibited a uniform and homogenous structure. Twelve weight percentage PVA/0.25 wt.% hBN composites were found to be having smooth surface. It was also observed that 12 wt.% PVA/0.25 wt.% hBN /0.5 wt.% BC composites had small pores of 265.68 6 15.39 μm suggesting successful vascularization and nutrient transport in tissue engineering applications. DSC thermographs suggested that 12 wt.% PVA composites were highly crystalline in nature because they had a melting point close to 230 C. It was also observed that addition of BC and hBN to 12 wt.% PVA composites caused a slight shift in the melting point of the composites. Physicochemical properties of various composites were also studied. Viscosity of PVA composites was found to be increasing with the addition of hBN and BC concentration. Twelve weight percentage PVA and 12 wt.% PVA/0.25 wt.% hBN composites possessed a 30% difference in viscosity values. No significant difference in the density values of the composites were found. It was found that an increment in BC concentration led to increase in the surface tension values. 0.127 6 0.05 MPa was observed as the highest value for tensile strength in case of 12 wt.% PVA/0.25 wt.% hBN/0.1 wt. % BC composite scaffolds. Values of elongation at break tended to increase with the introduction of additives to the various scaffolds. Another observation was made that BC and hBN caused increase in the ductile nature of the composites due to the hydrogen bonding. Maximum swelling degree was observed for 12 wt. % PVA composites. It was also suggested that highly rigid structure of BC caused increase in water absorption and permeation and limited the swelling behavior. Biocompatibility was verified using human osteoblast cells which resulted in increased proliferation and better extracellular matrix compatibility through 12 wt.% PVA/0.25 wt.% hBN/0.5 wt.% BC composite scaffolds. A reduction in the cell viability was observed on addition of BC concentration to PVA/hBN composites. It was concluded that the composite scaffolds have a great potential in bone tissue engineering applications.

4.2.7 Implantable devices in regenerative medicine Bacterial cellulose-gold nanoparticles (AuNPs)—poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) namely (BC-AuNPs-PEDOT:PSS) composites were successfully employed by Khan, Ul-Islam, Ullah, et al. (2018) for their potential application for implantable devices in regenerative medicine. FESEM, TEM, and AFM characterizations were applied for determination of size, structure, and arrangement of the composite. SEM images indicated a random arrangement of microfibrils which resulted in pore formation on the surface of BC matrix. It also displayed that PEDOT:PSS molecules completely filled up

4.2 Biomedical applications of bacterial cellulose

the empty spaces in BC-AuNPs nanocomposites. TEM images further confirmed the results obtained from SEM images while also discussing the fact that PEDOT: PSS acts as connecting sheets between the BC fibers. AFM analysis was used to investigate the topography of nanocomposites. A smooth topography is observed indicating completely filled up empty spaces between AuNPs and BC fibers by PEDOT:PSS. XRD technique is used to determine the crystalline features of nanocomposites. Lower crystallinity is observed in the composites due to the disturbances in the hydrogen bonding between BC chains. FTIR analysis is used to determine the functional groups and nature of bonds present in the composite. A broad OH peak was observed at 3627/cm referring to strong hydrogen bonding interactions between OH groups of BC and anions and cations of PEDOT:PSS. Water displacement was justified by lower intensity band of H-O-H observed at 1630 cm. FTIR spectra confirmed that the formation of composites was based on hydrogen bonding. High electrical conductivity of 16.65 6 1.27 S/cm was observed due to the penetration of PEDOT:PSS into the composites. Biocompatibility analysis suggested that successful adhesion of cells was observed after incubation. Even after 3 days, cells continued to grow and proliferate confirming filopodia formation and interconnection. Cytotoxicity analysis displayed no reduction in cell proliferation. This suggested that formation of composites does not cause any toxic effects, thus justifying the potential application of BC-AuNPs-PEDOT:PSS composites in implantable devices for biomedical applications.

4.2.8 Drug delivery Abeer, Amin, Lazim, Pandey, and Martin (2014) explored the potential of acrylated AbA-g-bacterial cellulose hydrogel (AcAA-g-BC) for its use in drug delivery applications. Numerous characteristic and characterization studies were carried out for the study. Solid state CP/MAS 13C NMR analysis was conducted on the hydrogel samples. Four hundred megahertz 13C NMR spectrum was observed for acrylated AbA-g-BC hydrogel. Peak at 62 ppm refers to C6 of anhydro-glucose unit of cellulose. It also represents substituted hydroxyl groups. Typical peaks at 72 and 74 ppm referred to C2, C3, and C5, whereas peaks at 82 and 89 ppm referred to C4 carbons. Peak at 107 ppm corresponded to C1 while peaks at 145, 124, 121, and 55 ppm corresponded to C2, C3, C4, and C5 carbons, respectively. The results obtained from NMR spectra confirmed the formation and successful grafting of hydrogel. SEM images concluded that at higher radiation doses, the pore network tends to decrease. This was attributed to the possibility of cross-linking at increased radiation doses and higher AA concentrations. A sponge-like morphology was observed for the hydrogels formed. It was also observed that the pore size obtained in this study was found similar to the pore size of hydrogels used traditionally in drug delivery. Gel fraction of hydrogel was found to be in the range of 73.0 6 1.0 to 83.0 6 1.9 and was attributed to the presence of AbA. Increasing radiation dose led to an increase in the gel fraction while it was not affected at all

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on increasing concentration of AA. Swelling studies revealed that acrylated AbAg-BC hydrogel was pH responsive. Swelling ratio was found to be lowest at pH 2.0 and increased with pH. It also referred to potential application of hydrogels formed with controlled drug release at intestinal pH. It was observed from XRD analysis that peak at 2θ 5 16.5 degrees referred to crystallographic plane of BC while broad peak at 22.5 degrees referred to (002) plane. But in case of hydrogels, two different peaks at 2θ 5 10 and 16 degrees and a sharp peak at 22.5 degrees were observed. Characteristic peaks of BC were not available in the final hydrogel. Hence, it confirmed that AbA was effectively grafted onto hydrogel structure because of the decreased crystallinity. Differential scanning calorimetry analysis was done to find out the glass transition temperature (Tg). It was observed to be 80 C and was only available at endothermic peaks. The T value was higher because of the high molecular weight of hydrogels due to the presence of AbA. Also, another reason for high value of T was attributed to water resistance. Hence, it was suggested that AbA imparted water repellence to the hydrogel. Cell viability test revealed cell viability percentage to be 72% and it decreased with an increment in AA concentration. It was also suggested that AA concentration leads to acidic environment followed by cell destruction. On the concluding note, a great potential of the hydrogels formed was reported in drug delivery applications.

4.2.9 Bone healing Zimmermann, LeBlanc, Sheets, Fox, and Gatenholm (2011) examined the usage of mineralized BC for bone healing purposes. FESEM analysis revealed some interesting information regarding scaffolds. It was observed that tube out and pellicle BC scaffolds had many open surface morphologies which promoted crystal growth along BC fibrils. It was also observed that crystals were not able to completely cover up the fibrils. The tube in BC scaffold demonstrated a more extensive amount of crystallization on the surface. XRD pattern for pure BC had peaks at 2θ values of 14.5, 23.1, 29.4, 36.0, 39.4, 43.2, 47.5, 48.5, and 57.4 degrees, while XRD pattern for mineralized BC consisted of peaks at 14.4, 22.6, 27.4, 29.4, 31.8, 36.0, 39.4, 43.2, 45.5, 47.5, 48.5, and 56.5 degrees. Osteoprogenitor cell morphology illustrated that F-actin of cells formed a network over mineralized BC resulting in improvised adherence to the surface. This effect was not observed in pure BC. Various characterizations applied to mineralized BC supported its excellent usage in bone healing.

4.2.10 Wound dressing Single sugar α-linked glucuronic acid-based oligosaccharide combined with bacterial cellulose (SSGO/BC) composites were employed by Ul-Islam, Khan, and Park (2012b) for wound dressing applications. The composite was extensively characterized based on different concentrations of SSGO. FESEM analysis

4.2 Biomedical applications of bacterial cellulose

revealed the surface morphology of composite samples as reticulated fibril arrangement. The micrographs displayed that the fibrils were loosely arranged with larger pores of BC. It was also noted that thickness, density, and compactness increased with increment in SSGO concentration. BET analysis was performed to determine the pore size, pore volume and surface area of fabricated composites. The total surface area for BC0 was found to be 178 (m2/g) which decreased to 168 (m2/g) for BC1, 135 (m2/g) for BC2, and 104 (m2/g) for BC4. Similarly, total pore volume for BC0 was 0.505 cc/g which decreased to 0.144 (cc/g) for BC1, 0.124 (cc/g) for BC2, and 0.091 (cc/g) for BC4. Also, average ˚ for BC0 which decreased to 57.12 A ˚ for BC1, 58.06 A ˚ pore diameter was 309 A ˚ for BC2, and 49.48 A for BC4. Overall BET analysis suggested a decreasing trend in values with increasing SSGO content. Water-holding capacity was 106.43 times its dry weight for BC0 which decreased to 100.36, 91.84, and 85.31 times its dry weight for BC1, BC2, and BC4, respectively. Hence, water-holding capacity decreases with increasing SSGO content. The characteristics of SSGO/BC composites suggested its exceptional properties suitable for applications in wound dressing material. Lin et al. (2013) utilized BC combined with chitosan (BC-Ch) for wound dressing applications. Surface morphology of BC-Ch composites was assessed through SEM micrographs. It resulted in heavily compact membranes due to the presence of chitosan. It was suggested that chitosan may have penetrated into pores of BC forming a denser network and decreased pore size. The FTIR spectra identified various peaks as listed in Table 4.2. The FTIR spectra thus verified the presence of chitosan. Mechanical properties of BC-Ch composites were found to be 10 MPa, 29% and 132 MPa for tensile strength, elongation at break and Young’s modulus, respectively. This inferred adequate toughness of composite material for applications in wound dressing. Antibacterial activity was determined for the composites using gram-negative E. coli and gram-positive S. aureus for 24 h. A growth inhibition rate of 99.9% was observed for both bacterial samples. Thus, results suggested that addition of chitosan led to high antibacterial efficiency. The composite samples were also subjected to various water tests. Addition of chitosan to BC caused decrease in the swelling ratio. Water absorption capacity was found to be 97%. Water evaporation rates were also found to be higher as it took 48 h for water to be completely evaporated. After the analysis it was suggested that BC-Ch composites were able to maintain a suitable moisturized environment for wounds. On the concluding note, moist environment supports penetration of active substances, protects wounds against further bacterial infections and provides painless removal of wound surface after recovery. Hence, an ideal wound dressing should be able to maintain a proper moist environment for wound healing. BC-Ch composites incorporated these properties successfully, thus, they can be effectively used for wound dressing materials. Potential of bacterial cellulose-silver nanoparticles (BC-Ag) nanocomposites for biomedical applications was extensively explored by Shao et al. (2015) through various characterization techniques. The surface morphology was studied

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using SEM micrographs. It represented a denser network for BC-Ag nanocomposites where AgNPs were present in the form of white spots. EDS analysis also confirmed existence of silver in BC matrix. FTIR spectroscopy for BC-Ag nanocomposites suggested that peaks shifted to a lower value indicating presence of strong interactions between OH groups of BC and AgNPs. Optical properties of nanocomposites were assessed through UV-VIS spectroscopy. Absorption at 414 nm was observed attributed to surface plasmon resonance of metallic AgNPs. This also confirmed the existence of silver in nanocomposites. Thermal properties resulted in two significant weight losses for BC-Ag nanocomposites. It was found that Tmax decreased from 363.2 C for BC to 256.6 C 286.4 C for BC-Ag nanocomposites. Reason for this was attributed to catalysis of CO2 elimination and acceleration in degradation process. XRD patterns of BC-Ag nanocomposites resulted in four characteristic peaks at 2θ values of 38.1, 44.3, 64.4, and 78.0 degrees attributed to (111), (200), (220), and (311) planes of metallic AgNPs introduced inside BC. Antibacterial activity for nanocomposites were studied at 37 C after contact time of 1 h. It was tested against gram-negative bacteria namely E. coli and Gram-positive bacteria namely S. aureus, B. subtilis, and C. albicans. After the analysis, it was found that BC-Ag0.05 nanocomposites were able to reduce E. coli, S. aureus, B. subtilis, and Candida albicans by 98.36%, 99.98%, 100%, and 89.6%, respectively. These results indicated exceptional antibacterial properties for BC-Ag nanocomposites. Hence, it was concluded that BCAg nanocomposites could serve as good antibacterial wound dressing materials such as bandages. Shao, Liu, Wu, et al. (2016) employed bacterial cellulose-silver sulfadiazine composites (BC-AgSD) for assessing its applications in biomedical field. Surface morphology was investigated using SEM micrographs which suggested a 3-D structure with ribbon shaped microfibrils network of BC matrix while BC-AgSD composites were presented as white spots on a denser network structure. Increasing the amount of AgSD caused the surfaces to become smoother and more compacted. FTIR spectroscopy revealed numerous characteristic peaks for BC, AgSD, and BC-AgSD composites as shown in Table 4.2. In case of BCAgSD composites, the peak intensities of AgSD were found to be increasing with an increase in AgSD loadings. A significant peak was observed at 3391/cm corresponding to free N-H. XRD analysis is used to investigate crystalline properties of materials. The XRD pattern of BC-AgSD exhibited six different peaks at 2θ values of 8.6, 10.01, 14.46, 16.62, 18.32, and 22.66 degrees which indicated that AgSD was successfully incorporated inside BC matrix. It was observed that different AgSD loadings caused the peaks of BC to become weak but were still evident indicating meaningful formation of composite. Thermal properties of composite revealed two significant weight loss stages. It was found that temperature decreased from 369.0 C for BC to 353.8, 348.7, 345.6, 340.0, and 331.4 C for BC1, BC2, BC3, BC4, and BC5, respectively. Cytotoxicity studies were performed to investigate the effect of AgSD in the BC matrix on proliferation of HEK293 cell line. It was observed that all materials possessed negligible toxicity

4.2 Biomedical applications of bacterial cellulose

and decrease in cell viability with increase in AgSD loadings. Also, AgSD caused no proliferation of HEK293 cells even at elevated concentrations. Antibacterial activity of composites was also analyzed. It was found that increasing the amount of AgSD loading caused rapid increase in the inhibition zones and after some time became stable. BC5 was found to be possessing best antibacterial activity with inhibition diameters of S. aureus and C. albicans to be 17.3 and 18.6 mm, respectively. It was concluded that BC-AgSD possess excellent antibacterial properties, thus, can be effectively used for applications in wound dressing. Potential of tetracycline hydrochloride loaded bacterial cellulose composite membranes (BC-TCH) was examined by Shao, Liu, Wang, et al. (2016) for its applications in wound dressing purposes. SEM, FTIR, antibacterial analysis were some of the characterization techniques applied to the composite membranes. SEM images revealed a 3-D structure with ribbon shaped microfibrils network of BC matrix while TCH particles were presented as white spots on the composite membranes. FTIR spectroscopy revealed various characteristic peaks as listed in Table 4.2. Studies of TCH release profiles from BC-TCH composite membranes suggested that increasing the concentration of TCH caused an increase in the TCH release profiles. Antibacterial activity of composites was also analyzed. It was found that increasing the amount of TCH loading caused rapid increase in the inhibition zones and after some time became stable. BC0.5 was found to be possessing best antibacterial activity with inhibition diameters of E. coli, S. aureus, B. subtilis, and C. albicans to be 45.7, 38.5, 34, and 12.1 mm, respectively. Thus, BC0.5 composite membranes represented excellent antibacterial growth activity and reduced the growth of E. coli, S. aureus, B. subtilis, and C. albicans by 99.98%, 100%, 100%, and 99.99%, respectively. Hence, it can be concluded that BC-TCH composite membranes have great antibacterial properties, thus, having a great potential in reducing infection and inflammation. Lucyszyn et al. (2016) discovered the usage of reconstituted bacterial cellulose films (RBC) as wound dressing materials. For this, various characterizations were applied to RBC films such as SEM, cytotoxicity, mechanical properties, etc. SEM micrographs were employed to assess the morphological changes taking place in the biocomposite films. A network type structure was displayed by SEM images. RBC was presented in the form of fibers of both macro scale and micro scale. Static contact angle (SCA) measurements were done to analyze behavior of RBC films in different liquid conditions. It was found that RBC did not possess high hydrophobicity which can be explained through low stability in water and NaCl solution. Addition of AG and GHXG led to increase in hydrophobicity of RBC films. XRD analysis of RBC films resulted in peaks at 2θ angle such as 14.4 (100), 16.8 (010), and 22.8 (110), corresponding to cellulosic crystallographic planes. RBC CrI value was found to be 75.9%. Inclusion of AG and GHXG resulted in no peak shifts. Mechanical properties were vastly studied for RBC films and RBC films introduced with hydrocolloids. It was observed that introduction of hydrocolloids (AG and GHXG) to RBC films caused an increase in the mechanical properties of RBC due to polysaccharide adhesion effect. Addition of

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AG/GHXG to RBC caused 20% difference in Young’s Modulus and Strain at break. Fifty percent difference was observed by addition of AG and GHXG separately to RBC films for tensile strength. Cytotoxicity studies revealed interesting results where L929 cells were used for analysis after an incubation period of 72 h. It was observed that AG caused a decrease in cell viability by 30% and 23% at 3300 and 1667 μg/mL, respectively. Also, GHXG decreased cell viability by 16% and 9% at 3300 and 1667 μg/mL, respectively for L929 cells. Cell proliferation assay suggested that overall cell viability increased by 83% for AG/GHXG20 films as compared to GHXG20 and it increased by 88% for AG/GHXG40 films as compared to GHXG40. It was suggested that L929 cell proliferation was due to increased hydrophobicity, decreased porosity, and increased surface heterogeneity. Thus, it was concluded that RBC have a great potential as wound dressing material. Bacterial cellulose-tannic acid-MgCl2 (BC-TA-Mg) composites were successfully utilized by Zhang et al. (2020) for its applications in wound dressing. The composites were characterized using various techniques such as SEM, FTIR, XPS, cytotoxicity, antibacterial studies, etc. SEM results revealed that the BC matrix presented a porous network structure. Tannic acid and MgCl2 particles were attached to BC matrix in the form of small white particles. FTIR spectra revealed several characteristic peaks. This is shown in Table 4.2. All these peaks were also found in the FTIR spectra of BC-TA-Mg composites confirming successful introduction of TA onto BC matrix. The XPS analysis suggested that electrons were transferred from Mg21 to BC and TA. This confirmed chelation of BC, TA, and Mg21. Cytocompatibility of BC-TA-Mg composites revealed an increment in the cell viability as a result of increased concentration of Mg21. The highest cell viability was found to be 77.65 6 2.01% for the composite BC-TA6Mg. The reason was attributed to the fact that increase in concentration of Mg21 resulted in the chelation of BC, TA, and Mg21. Antibacterial activity for these composites was found to be larger in case of Gram-positive bacteria as compared to Gram-negative bacteria. Best antibacterial activity was observed for the composite BC-TA with a diameter of inhibition zone to be 17.30 6 0.53 mm against S. aureus, 15.07 6 1.05 mm against P. aeruginosa, and 14.39 6 0.80 mm against E. coli. A slight decrease in the antibacterial activity was observed on increasing the concentration of Mg21. Wang et al. (2020) explored the effects of BC functionalized with silk sericin (SS) and hyaluronic acid (HA) on wound dressing applications in the form of in situ as well as ex-situ modified BC-HA/SS composites. Different characterization techniques were applied to the composites such as FTIR, SEM, TGA, XRD, etc. FTIR analysis was used to describe various characteristic peaks for BC, SS, HA, and BC-HA/SS composite. This is presented in Table 4.2. In the case of composite samples, it displayed all the characteristic peaks of BC, SS, and HA indicating successful agglomeration of BC onto SS and HA. In addition, with this, a sharp peak at 1043/cm was found in ex-situ modified BC-HA/SS composite referring to enhancement in hydrogen bonding. SEM micrographs were used to determine the

4.3 Conclusion

morphology of composites. In situ modified BC-HA/SS composites displayed thin interwoven mesh fibrils of about 30 nm width while ex-situ modified BC-HA/SS composites displayed wider interwoven mesh fibrils of 50 100 nm. Surface morphology was also studied using AFM analysis. In situ modified BC-HA/SS displayed a smoother surface with 23.20 nm roughness resulting in more hydrophilic surface. On the other hand, ex-situ modified BC-HA/SS exhibited roughness of 30.38 nm. Crystalline structures of composites were described using XRD. Degrees of crystallinity were found to be 88.44% and 93.15% for in situ and exsitu modified BC-HA/SS composites, respectively. TGA analysis was used to determine the thermal stability of the composites. The first endothermic peaks were observed at 127.42 C and 134.67 C contributing 9.94% and 8.97% weight loss for in situ and ex-situ modified composites, respectively. Second endothermic peak was observed at 225 C contributing 11.38% and 6.12% weight loss for in situ and ex-situ modified composites, respectively. Endothermic peaks at 351.65 C and 406.53 C were observed which were attributed to thermal degradation of SS and depolymerization of glucose. These results suggested that HA was well agglomerated on in situ modified composite whereas SS was agglomerated well on ex-situ modified composite. Also, exothermic peaks were observed at 453.92 C and 479.18 C for in situ and ex-situ modified composites, respectively. Observations from TGA analysis suggested that ex-situ modified composite has much higher crystallinity and much better thermal stability as compared to in situ modified composites. No cytotoxicity was detected for the composites. Also, exsitu modified composites exhibited higher tensile strength and higher moisture content which was recorded to be 1.60 6 0.16 MPa and 79.06% 6 0.16%, respectively.

4.3 Conclusion Researchers have developed BC for myriad biomedical applications. This chapter discussed the implementation of BC in wound healing and dressing, diagnosis of ovarian cancer, shape memory material, preventing deterioration of salmon muscle, slowing down the lipid oxidation, lipase immobilization, tissue engineering, implantable devices in regenerative medicine, drug delivery, and bone healing. The morphology, microporous structure, microfibrillar, adhesion, 3-D structure, etc. of BC can be efficiently recognized with SEM/FESEM analysis. FTIR is capable to identify different characteristic peaks/groups present or impregnated in BC matrix. TEM and AFM characterizations were applied for determination of size, structure, and arrangements of fibrils within BC. TEM images have been used to confirm the results obtained from SEM images. XRD and TGA techniques have been used to study crystallographic planes and thermal stability of BC. DSC analysis is used to find out the glass transition temperature of BC while SCA measurements are done to analyze its behavior in different liquid conditions.

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BET analysis is performed to determine the pore size, pore volume, and surface area of BC and its fabricated composites. Other characterization techniques associated with BC found in literature were XRF, 13C CP MAS NMR, and XPS. Major mechanical strength properties studied for BC identified were tensile strength, elongation at break, and Young’s modulus. Other properties investigated for BC were discovered to be antimicrobial activity, photocatalytic activity, hydrophilicity, water contact angle, surface tension value, physical and chemical stability, pore size, electrical conductivity, cell viability test, water absorption capacity, optical properties, cytotoxicity, cytocompatibility, etc. It can be concluded that getting acquainted with suitable characterization techniques for BC is very important for researchers to determine its apt biomedical applications. However, it is also important to consider the limitations of a particular characterization technique for BC.

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CHAPTER

Engineering scaffolds for tissue engineering and regenerative medicine

5

Ibrahim Fatih Cengiz1,2, Rui L. Reis1,2 and Joaquim Miguel Oliveira1,2 1

3B’s Research Group, I3Bs Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal 2 ICVS/3B’s PT Government Associate Laboratory, Guimara˜es, Portugal

5.1 Introduction Orthopedic tissues are three-dimensional (3D) structures composed of cells and the extracellular matrix. Tissue grafts have been used with certain indications to treat lesions in the body. Given the challenges and shortcomings associated with both autografts and allografts (Pereira et al., 2019), the use of biomaterials has been proposed in addition to use biologics (Cengiz, Oliveira, et al., 2017; Cengiz, Oliveira, & Reis, 2018b; Cengiz, Pereira, Espregueira-Mendes, Reis, & Oliveira, 2019) and regenerative strategies (Cengiz, Silva-Correia, et al., 2017a; Cengiz, Oliveira, & Reis, 2014; Cengiz, Pereira, Espregueira-Mendes, Oliveira, & Reis, 2017; Pereira, Cengiz, et al., 2018). Engineered biomaterials, in particular scaffolds, have been changing the clinical practice for the treatment of lesions (Cengiz, Pereira, et al., 2018). Most of the regenerative strategies involve the use of scaffolds that are biomaterials processed into 3D structures to support and guide cellular activities to facilitate, guide, and modulate tissue regeneration via extracellular matrix synthesis of the cells. The ability of self-healing is considerable in vascularized tissues, while vascularization remains to be an outstanding challenge of tissue engineering targeting large defects. Different tissues have different characteristics regarding their matrix, cells, and biomechanics. Moreover, it is also known that tissues are heterogeneous in terms of the aforementioned features (Cengiz, SilvaCorreia, et al., 2017b; Cengiz, Pereira, Peˆgo, et al., 2017; Pereira et al., 2014; Pereira, Cengiz, Silva-Correia, Cucciarini, et al., 2016). Therefore engineering scaffolds is of critical importance since the outcomes of the application of the scaffolds depend on the scaffold itself and its interaction with the cells and body. While there is a range of scaffold producing and engineering methods with all their advantages and limitations, 3D-(bio)printing has impacted the field of tissue engineering more than other methods thanks to its ability to provide patient-specific Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00030-9 © 2023 Elsevier Inc. All rights reserved.

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complex scaffolds in which biomaterial(s) with cells can be localized in 3D as designed. Herein, the relevant recent works on tissue engineering scaffolds that are proposed for meniscus, cartilage, and bone have been overviewed.

5.2 Scaffolds properties and characterization Scaffolds are 3D structures obtained via processed biomaterials that are engineered to support cell culture for regenerative applications to treat tissue lesions with certain features (Fig. 5.1) (Cengiz, Pereira, Silva-Correia, et al., 2017; Pereira et al., 2015; Pereira, Cengiz, Silva, Reis, & Oliveira, 2020; Pereira, Cengiz, Silva-Correia, Ripoll, et al., 2016). As an overall evaluation, it is a challenge to decide about the superiority of a biomaterial over others because the performance of the obtained scaffold depends on various factors associated with the engineering of the biomaterials and final applications. Fig. 5.2 illustrates the development of an implant through different levels of characterization and improvement to meet the needs before any trial in humans. Scaffolds can be categorized into several groups considering different aspects, such as origin (Reddy, Ponnamma, Choudhary, & Sadasivuni, 2021): naturally derived (Celikkin et al., 2017; Filippi, Born, Chaaban, & Scherberich, 2020) (including decellularized matrices (Taylor, Sampaio, Ferdous, Gobin, & Taite,

FIGURE 5.1 Overview of the major properties to be considered when designing a scaffold.

5.2 Scaffolds properties and characterization

FIGURE 5.2 Overview of the development of an implant through rigorous characterization and improvement to meet the preclinical needs.

2018)) or synthetic (Jenkins & Little, 2019); and composition: polymers (Jafari et al., 2017), ceramics (Ribas et al., 2019), metals (Tan, Tan, Chow, Tor, & Yeong, 2017), or composites (Turnbull et al., 2018). Commonly used natural polymers include but are not limited to collagen (Bahrami, Baheiraei, & Shahrezaee, 2021; Beketov et al., 2021; Kim & Kim, 2019), silk-based (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019; Ribeiro, Pina, Canadas, et al., 2019; Ribeiro, Pina, Costa, et al., 2019), gellan gum (Choi et al., 2020; Pereira, Silva-Correia, et al., 2018; Trucco et al., 2021), gelatin (Huang et al., 2021; Leucht, Volz, Rogal, Borchers, & Kluger, 2020; Sun et al., 2022), hyaluronic acid (Teng et al., 2021; Yan et al., 2020; Zhai et al., 2020), alginate (Sathish et al., 2022; Wulf et al., 2021; Zheng, Wang, Bai, Xiao, & Che, 2022), and chitosan (Li et al., 2021; Schmitt et al., 2021; Zuliani et al., 2021). Synthetic polymers include but are not limited to polycaprolactone (PCL) (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019; Dewey et al., 2021; Sani, Rezaei, Khoshfetrat, & Razzaghi, 2021), poly(lactic acid) (Ashwin et al., 2020; Patel, Dutta, Hexiu, Ganguly, & Lim, 2020; Tan et al., 2021), and poly(vinyl alcohol) (Fatahian, Mirjalili, Khajavi, Rahimi, & Nasirizadeh, 2020; Januariyasa, Ana, & Yusuf, 2020; Liu, Chen, Liu, Tian, & Liu, 2019). Bioactive glasses

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(Heras et al., 2020; Kang et al., 2020; Saatchi, Arani, Moghanian, & Mozafari, 2021), hydroxyapatite (Cao et al., 2020; Chen et al., 2021; Mondal et al., 2020), zirconia (Askari et al., 2020; Gaddam, Brazete, Neto, Nan, & Ferreira, 2021; Pereira, Cengiz, Maia, et al., 2020), and tricalcium phosphate (Duan, Ma, Song, Li, & Qian, 2021; Liu, Chen, Chen, & Zeng, 2021; Zhu et al., 2021) are examples of the widely used ceramic biomaterials; while titanium-based scaffolds (Huang, Pan, & Qiu, 2022; Pereira, Cengiz, Maia, et al., 2020; Yang et al., 2021) are often studied metallic scaffolds which can also be 3D-printed. Table 5.1 presents selected examples of commercial products for cartilage lesions. Depending on the Table 5.1 Examples of commercial products for the treatment of cartilage lesions. Product

Company

Biomaterial 1 cells

References

Bioseed-C

BioTissue Technologies (Freiburg, Germany) Co.don (Teltow, Germany)

Polylactin/ polydiaxanon/ fibrin 1 autologous chondrocytes No scaffold 1 autologous chondrocytes

Kreuz et al. (2011), Ossendorf et al. (2007), Zeifang et al. (2010)

Arthro Kinetics Biotechnology (Krems, Austria)

Murine (rat tail) type-I collagen hydrogel 1 autologous chondrocytes Fibrin/hyaluronic acid 1 autologous chondrocytes Agarose/alginate hydrogel 1 autologous chondrocytes Bovine type-I collagen 1 autologous chondrocytes

Chondrosphere (ACT3D-CS/ ARTHROCELL 3D) CaReS-1S

Biocart II

Cartipatch

NeoCart

RevaFlex (DeNovo ET) Novocart 3D

Histogenics (Waltham, Massachusetts) Tissue Bank of France (Lyon, France) Histogenics (Waltham, Massachusetts)

ISTO Technologies (St. Louis, Missouri) TETEC Tissue Engineering Technologies (Reutlingen, Germany)

Becher et al. (2017), Fickert et al. (2012), Siebold, Suezer, Schmitt, Trattnig, and Essig (2018) Petri et al. (2013), Schneider et al. (2011)

Eshed et al. (2012), Nehrer, Chiari, Domayer, Barkay, and Yayon (2008) Selmi et al. (2008)

Anderson et al. (2017), Crawford, DeBerardino, and Williams (2012), Crawford, Heveran, Dilworth Cannon, Foo, and Potter (2009) McCormick et al. (2013)

No scaffold 1 allogeneic juvenile chondrocytes Bovine type-I collagen/ Niethammer et al. (2014, chondroitin 2017), Zak et al. (2014) sulfate 1 autologous chondrocytes

Source: Reproduced from Cengiz, I. F., Pereira, H., de Girolamo, L., Cucchiarini, M., EspregueiraMendes, J., Reis, R. L., & Oliveira, J. M. (2018). Orthopaedic regenerative tissue engineering en route to the holy grail: Disequilibrium between the demand and the supply in the operating room. Journal of Experimental Orthopaedics, 5(1), 1 14 with permission. Copyright © 2018 Cengiz et al., licensed under CC BY-NC 4.0 (http://creativecommons.org/licenses/by/4.0/).

5.2 Scaffolds properties and characterization

application on the target tissue, the choice of naturality, source, and composition are considered, since all biomaterials come with advantages and disadvantages. Typically, naturally derived scaffolds may provide a relatively higher level of bioactivity where cellular activities are supported relatively superior [e.g., silk fibroin (Sun, Gregory, Tomeh, & Zhao, 2021)] while synthetic scaffolds [e.g., PCL (Siddiqui, Asawa, Birru, Baadhe, & Rao, 2018)] have high reproducibility, customizable mechanical strength, and biodegradation while having relatively lower bioactivity. Depending on the processing methods, natural biomaterials may have inferior mechanical properties, or variation between batches, while the existing literature shows that via engineering, some known shortcomings could be overcome and superior scaffolds can be obtained such as scaffolds with increased suturability (Cengiz et al., 2020; Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019) and room-temperature gelation (Silva-Correia et al., 2013). Since the extracellular matrix of the tissue is synthesized by the cells, in the tissue engineering strategy the cells that can synthesize the specific matrix are used together with the scaffolds, where the scaffolds have certain roles. Both in vitro and in vivo (Fig. 5.3; Ricci et al., 2021), the function of cells (for

FIGURE 5.3 In vivo application of a multilayer collagen-hydroxyapatite scaffold. (A) An osteochondral lesion in the medial femoral condyle, (B) preparation of the implant site—9 mm deep regular box shape, (C) templating, (D) scaffold sizing, (E) press-fit implantation, and (F) bleeding from the subchondral bone. Reproduced from Ricci, M., Tradati, D., Maione, A., Uboldi, F. M., Usellini, E., & Berruto, M. (2021). Cellfree osteochondral scaffolds provide a substantial clinical benefit in the treatment of osteochondral defects at a minimum follow-up of 5 years. Journal of Experimental Orthopaedics, 8(1), 1 11, with permission. Copyright© 2020 Ricci et al., licensed under CC BY-NC 4.0 (http://creativecommons.org/licenses/by/4.0/).

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instance, the properties of the synthesized extracellular matrix) depends on the features of the scaffold since cells sense the characteristics of the scaffold (Li, Xiao, & Liu, 2017; Zajac & Discher, 2008; Zonderland & Moroni, 2021), including the mechanical properties, chemical properties, surface properties, and microstructure (Cengiz, Oliveira, & Reis, 2018a), which may facilitate in an in vivo application the creation of a microenvironment through recruited endogenous stem cells’ paracrine activity (Caplan, 2007; Gunawardena, Rahman, Abdullah, & Abu Kasim, 2019; Karp & Teo, 2009; Kusuma, Carthew, Lim, & Frith, 2017; Zhou, Yamamoto, Xiao, & Ochiya, 2019), Given these, the challenges associated with scaffold features should be addressed via engineering.

5.3 Fabrication of scaffolds 5.3.1 Scaffold fabrication methods Several scaffold fabrication methods have been described in the literature, which were shown in Table 5.2. Each of these methods has pros and cons, and numerous biomaterials have been utilized as comprehensively reviewed by Collins et al. (2021), Koyyada and Orsu (2021), Cheng et al. (2019), Cidonio, Glinka, Dawson, and Oreffo (2019), and Sun et al. (2020). The scaffold fabrication method is selected considering the physical and chemical properties of the biomaterial to be used and the features of the obtained scaffold using that method. 3D-(bio)printing requires certain rheology of the biomaterial to be printed to make up the 3D structure, and using a patient-specific model, it is possible to produce scaffolds with correct size and shape, with control of positions of biomaterials and cells within the structure. On the other hand, conventional methods such as freeze-drying or solvent casting and particulate leaching are relatively less demanding methods, but do not provide directly a patient-specific scaffold unless using a patientspecific mold. Moreover, 3D-(bio)printing provides a larger degree of controllability of the microstructure than the conventional methods. Typically, the commercially available scaffolds are re-sized during the surgical intervention because the size/shape of the implant should match with the implantation site. The future strategies should address the current outstanding issues and limitations and the performance of novel tissue engineering scaffolds should be shown in clinical trials. 3D-(bio)printing methods (Table 5.3) stand out from the conventional methods by being able to provide scaffolds that are anatomically correct in terms of size and shape using a digital 3D model, heterogeneous in terms of structure and/or cellularity, and alive from the first moment since bioinks contain both scaffolding biomaterial and cells. The selection of scaffolding biomaterial and the manufacturing method are of critical importance to fulfill several needs that are associated with the application for the targeted tissue because not all biomaterials/fabrication methods can meet the requirements. Based on the targeted tissue, a clinically relevant scaffold should be suitable in

5.3 Fabrication of scaffolds

Table 5.2 Overview of scaffold fabrication methods and the principles. Scaffold fabrication method 3D-(bio)printing

4D-printing

Freeze-drying

Electrospinning

Gas foaming

Main principle

Main feature

References

Layer-by-layer fabrication of scaffold alone (3D-printing), or with cells (bioprinting) based on a digital model of the scaffold via methods that are based on (including but not limited to) extrusion, droplet, or laser. It should be noted that there are also other methods under the rapid prototyping umbrella. Similar principle as 3D(bio)printing with the critical difference of an additional dimension of time, meaning that upon a stimulus, the morphology/function of the printed constructs can change over time. A porous structure is obtained via sublimation of the solvent

A 3D structure that is patient-specific in terms of size and shape with control of the positioning of biomaterial and cells.

Cengiz et al. (2020), Liu, Peng, et al. (2021), Zhang, Eyisoylu, Qin, Rubert, and Müller (2021)

A 3D-printed scaffold with the ability to alter a feature in a stimulusdepended manner.

Kim et al. (2020), Miao, Zhu, Castro, Leng, and Zhang (2016), Wang, Yue, et al. (2020)

Interconnected porosity with a range of pore sizes typically adequate for cell culture. Fibrous structures in a nano/micro scale.

Grenier et al. (2019), Safari et al. (2022), Zhang, Zhou, et al. (2019)

A fibrous structure is obtained by the collected fibers around a rotating collector, where the fibers were obtained via electrostatic forces from an electrically charged polymer. Solvent-free way of Porosity within the obtaining 3D porous foaming agent structures. (typically sodium bicarbonate) containing polymer is obtained due to the bubbles occurring as a result of escaping the gas (typically carbon dioxide).

Islam, Laing, Wilson, McConnell, and Ali (2022), Ma et al. (2021), Samadian, Khastar, Ehterami, and Salehi (2021)

Dattola et al. (2019), Kurakula and Koteswara Rao (2020), Manavitehrani et al. (2019)

(Continued)

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Table 5.2 Overview of scaffold fabrication methods and the principles. Continued Scaffold fabrication method Solvent casting and particulate leaching

Main principle

Main feature

References

A porous structure is obtained via leaching away of particles (typically salt) in a suitable liquid (typically water) after solvent evaporation.

The mold used for casting indicates the shape and size of the scaffold, while the microstructure could be altered by altering the size of the used particle. Modulation of the 3D porous structure via control of process parameters related to polymer, solvent, and temperature.

Sabzi, Abbasi, and Ghaleh (2020), Shalchy, Lovell, and Bhaskar (2020), Xie et al. (2021)

Phase separation

A polymer-rich phase is obtained via thermally induced phase separation, and then a porous structure is obtained by solvent removal. Decellularization A porous matrix is obtained via removal of cells from natural tissues.

Chen et al. (2020), Samadian et al. (2020), Wang, Kang, et al. (2020)

The extracellular matrix Goldberg-Bockhorn of the native tissue is et al. (2022), Lee, preserved. Olmer, Baek, D’Lima, and Lotz (2018), Wu et al. (2021)

Table 5.3 Advantages and limitations of 3D-(bio)printing techniques (Koyyada & Orsu, 2021; Sun et al., 2020; Zhang, Yang, Johnson, & Jia, 2019; Zheng et al., 2019). Advantages

Challenges and limitations

Extrusion

Droplet

Laser

Availability of commercial printers More popular Availability of suitable biomaterials Multiple biomaterials can be printed Not suitable for lowviscosity biomaterials Lower precision Shear stress may negatively influence the cells

More economic printers Can work with lowviscosity biomaterials Fast

More precise Nozzle-free

Limited range of biomaterials Low cell density Cells may be damaged due to heat Clogging may occur in the nozzle

Expensive system Slower process Low capability to build up in 3D

5.3 Fabrication of scaffolds

terms of volume/size and shape, have required mechanical properties including suturability for certain tissues, have suitable biodegradation profile, provide the required amount of vascularity, have a suitable macro/micro/nano-structure and topography, allow native-like neotissue formation over time, and integrate sufficiently with the surrounding tissues.

5.3.2 Patient-specific scaffolds Patient-specific scaffolds (Cengiz et al., 2016; Cengiz, Pereira, Pitikakis, et al., 2017; Oner et al., 2017) have been recently available thanks to the progress on 3D-printers, bioinks, and software allowing the development of 3D models from clinical imaging modalities (Fig. 5.4). Moreover, patient-specific cells can be used either by seeded onto the 3D-printed scaffold or incorporated into the bioink that has a balance between polymer content and cell viability, based on the pressure to be used in extrusion-based bioprinting (Fig. 5.5; Cidonio et al., 2019). In addition to the need for scaffolds to be patient-specific, there are other requirements that should be met including mechanical properties, for example shape memory and suture retention strength, controlling infiltration, and vascularity through new blood vessel formation via controlling microstructure and the 3D design of the scaffold. Cengiz et al. (2020) proposed a novel scaffold system that

FIGURE 5.4 Overview of the development of patient-specific implants.

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FIGURE 5.5 The paradigm of cell printing using a bioink. (A) The bioink comprising a biomaterial with encapsulated cells that were cultured in vitro. (B) 3D diagram illustrating the balance between the polymer content, cell viability, and pressure that is of critical importance in bioprinting and biomechanical and biological performance of the construct. A (top) and B (bottom) were reproduced (with a minimal adaptation that involves the addition of letters [“A” and “B”] at the right-bottom corner of each figure) from Cidonio, G., Glinka, M., Dawson, J., & Oreffo, R. (2019). The cell in the ink: Improving biofabrication by printing stem cells for skeletal regenerative medicine. Biomaterials, 209, 10 24, with permission. Copyright © 2019 Cidonio et al., licensed under CC BY-NC 4.0 (http://creativecommons.org/licenses/by/4.0/).

5.3 Fabrication of scaffolds

is called entrapped-in-cage where silk fibroin is entrapped in a PCL cage that is 3D-printed mainly utilizing the mechanical properties of PCL and biological properties of silk fibroin. Also, silk fibroin scaffolds that were reinforced with 3D-printed PCL mesh had an increased suturability, while allowing tissue infiltration and formation of new blood vessels in the scaffold (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019). The process of fabricating patientspecific tissue engineering constructs can be organized into three major steps from the engineering point of view as follows: 1. Acquisition of medical image dataset related with the lesion that is aimed to be treated with a tissue-engineered scaffold. From the available clinical imaging modalities, magnetic resonance imaging (MRI) has been frequently used during the normal course of medical examination. Technically, computed tomography (CT) images can also work, but for the patient, MRI is safer than CT. However, to produce patient-specific implants, sufficient resolution and a 3D isotropic sequence imaging covering the entire defect that will provide a dataset should be achieved. The isotropic sequence indicates that medical images have the same resolution in all planes. For orthopedic tissues, static medical imaging can be used. Depending on the targeted tissue, such as bone, meniscus, or intervertebral disc (IVD), either T1- or T2-weighted MRI can be selected; the critical point is to distinguish/segment the targeted volume of interest from the rest of the volume. Thus a specific imaging protocol must be developed that will serve later for the creation of the 3D digital model. While other image file types could be used, a Digital Imaging and Communication in Medicine dataset would be more standardized with associated metadata. Duration of the MRI acquisition is also another parameter that should be considered. An acquisition for the 3D model creation needs a much longer time than a typical routine image and a different protocol should be used in the MRI system. Another challenge is that the patient should remain still during the acquisition to obtain a correct image dataset. 2. Segmentation of region of interests and eventually the volume of interest is a step that can be manual, semiautomatic, or automatic, based on the available software and protocol of digital extraction for the targeted tissue. Using either semiautomatic or automatic segmentation, manual revisions may be needed to enhance the accuracy of the 3D model to be obtained. In the case that the image dataset is not ideal for the segmentation, either the image acquisition should be repeated or the artifacts should be cleaned if possible. Following the segmentation, the 3D model that would be used for the 3D-(bio)printing should be created. A surface rendering process can be using the segmented image dataset. The obtained raw model that is typically a triangle mesh can be further refined digitally through smoothing, closing of any minor holes or open structures if there are. Before printing the structure, the 3D model should be in a format recognized by the printer that will be used, that is, in stereolithography (.stl) that is a typical format used in 3D-(bio)printing.

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3. The settings and the parameters related to the 3D-(bio)printing should be done considering the model and the biomaterial/bioink to be used, including but not limited to: a. the alignment of the model; b. need of any supporting structures for the printed model; c. the time between each layer; d. printing speed; e. layer thickness; f. if applicable: nozzle type, pressure, cell density, and crosslinking pathway; g. internal pattern (2D and 3D organization of biomaterial and/or cells); h. introduction of heterogeneity (or not) within the architecture of the structure; and i. sterility of the biomaterials/bioink and/or the final scaffold/construct. Following the consideration and optimization of the critical factors mentioned above, the 3D-(bio)printed constructs can be obtained, and depending on the strategy, the construct can be implanted as it is, or maturated in a typical incubator or in a bioreactor.

5.4 Conclusion Millions of people suffer from orthopedic lesions/disorders where one or more tissues are affected. While neither all lesions are symptomatic, nor all lesions should be surgically treated; moreover, upon a strong indication for the use of a scaffold, a single scaffold could not meet all the needs. Thus lesion-specific, patientspecific scaffolds are of critical importance. It has been recognized that controlling a wide range of properties within a scaffold would affect the cell behavior and the clinical outcome. 3D-(bio)printing has been changing the concept of scaffolds in tissue engineering, with the shift from biomaterial only scaffolds to living scaffolds via the use of bioinks. While the majority of the scaffolds being used currently in the clinics are produced conventionally, the future is patient-specific scaffolds that can be produced using 3D digital models and 3D-(bio)printing. The current outstanding issues are the mismatch/gap between the formed tissue and the native tissue in terms of biocomposition and biomechanics. Still, the use of scaffold depends on indications and contraindications defined for the lesion in question.

Acknowledgments The authors thank the financial support under the Norte2020 project (NORTE-08-5369FSE000044). The author I.F.C. thanks the TERM RES-Hub, Tissue Engineering and Regenerative Medicine Infrastructure project, funded by the Portuguese Foundation for

References

Science and Technology (FCT), and the funding through the project 2IQBIONEURO (ref. 0624_2IQBIONEURO_6_E). The FCT distinction attributed to I.F.C. under the Estı´mulo ao Emprego Cientı´fico program (2021.01969.CEECIND) is also greatly acknowledged.

Declaration of conflict of interest The authors declare that there are no conflicts of interest to declare.

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Recent trends in polymeric composites and blends for three-dimensional printing and bioprinting

6

Sriya Yeleswarapu, K.N. Vijayasankar, Shibu Chameettachal and Falguni Pati Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India

6.1 Introduction Three-dimensional (3D) printing has emerged as a powerful technique to fabricate objects of any size, shape, and complexity (Wang, Jiang, Zhou, Gou, & Hui, 2017). The versatility it offers with the materials or ease of fabrication made this technology extremely user-friendly in recent years. High throughput is another most attracting feature which allowed researchers to choose 3D printing over conventional fabrication methods. With its precise depositions, wide range of material usability, and complex patterning abilities it has become one of the pivotal fabrication techniques which can produce personalized products. However, since it is in its infancy, most of the objects that are being developed are still in investigation and therefore have not completely impregnated into clinical practice. To translate objects or scaffolds that are 3D printed, the initial criterion that must be considered is the material used for fabrication (Diment, Thompson, & Bergmann, 2017). Material plays a critical role in determining the functionality of the objects with regards to its interaction with the host (Diment et al., 2017). Considering this, a range of materials have been investigated such as metals, polymers, ceramics, fibers, etc. for their use in 3D printing of scaffolds or objects (Ahangar, Cooke, Weber, & Rosenzweig, 2019). However, the properties of each material are very distinct from one another, and all the ideal characteristics cannot be achieved with one single material. Hence, considering properties of the materials based on their application is quite necessary. For instance, material to fabricate extracorporeal objects such as prosthesis or orthosis should be mechanically stiffer to bear the loads and for materials as implants requires many other properties such as biodegradation, aiding tissue regeneration, promoting cellular attachment and proliferation at physiological conditions (Horst et al., 2010; Klute, Kallfelz, & Czerniecki, 2001). Lack of availability of materials with ideal

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00004-8 © 2023 Elsevier Inc. All rights reserved.

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properties led to the idea of developing composites which improves material characteristics in a synergistic manner. Although metals and ceramics are very prominent materials for many applications in biomedical field, lack of degradability and limited processability have forced researchers to explore the potential of polymers for similar applications (Ulery, Nair, & Laurencin, 2011). Polymers provide a wide scope for tuning of parameters by altering them physically or chemically (Liu & Ma, 2004). Also, the printability of polymers can be altered making them apt materials for biomedical applications using 3D printing. Though physical and chemical properties of polymers can be modulated as per need, properties that a polymer can impart to scaffold are often limited, thus demanding composite materials which can widen the range of tunability. It is due to these limitations, such as incomparable mechanical strength, discomfort, toxic degradation, etc., 3D printed scaffolds or objects are still not available to patients (Mason, Visintini, & Quay, 2016; Yang et al., 2018); hence, this led to the finding of many composite materials so as to translate 3D printing research. These modified polymeric composites have been gaining attention as they offer many advantages such as lightweight, high fatigue strength, corrosion resistance, durability, biocompatibility, and mechanical strength (Rajak, Pagar, Kumar, & Pruncu, 2019).

6.2 Need of synergistic approach in polymeric materials For materials to be used in biomedical applications, properties such as degradation, biocompatibility, biodegradation, and cell-favoring environment also have to be considered apart from mechanical features, although the latter is also key (Kim et al., 2011). Biomedical applications such as development of prostheses, orthoses, dental implants, bone substitutes, stent development, etc., are in major focus using 3D printing technology since most of the mentioned applications require personalization. Out of the mentioned products, prosthesis and orthosis come under extracorporeal objects while all the others have applications in vivo. As the area of application varies, it is quite critical to employ suitable materials for fabrication. For example, limb prostheses or foot orthoses that are externally fastened should be made of materials that are robust, corrosive resistant, water resistant, sustain heavy loads, bioinert, etc. On the contrary, materials for implants or bone substitutes have to be extremely biocompatible, cell-friendly, biodegradable, promote or allow tissue regeneration with reasonable mechanical stiffness. Moreover, the material should be printable, which implies they have to allow modification into many forms such as filaments, powders, or liquids suitable for 3D printing. Polymeric materials are widely used materials in the field of tissue engineering and regenerative medicine due to their availability, low melting points, high ductility, low price, and high flexibility. Moreover, polymers offer high degree of modification that can be modulated as per the application and is a major

6.3 Blends and composites of natural and synthetic polymers

advantage (Liu & Wang, 2020) Additionally, availability of polymeric inks in various forms makes them suitable for 3D printing applications. Many synthetic and natural polymers find their place developing products for biomedical research and applications (Gasperini, Mano, & Reis, 2014; Liu & Wang, 2020). However, objects that are developed with pure polymers are restricted for conceptual use and prototypes as they have inferior mechanical properties. With low mechanical properties they usually do not satisfy the functional characteristics that are required. The mechanical and structural strength of pure polymers can be extended only till a particular limit, which probably might not be sufficient for real-world biomedical applications. And hence, combining two or more polymers or adding other materials to polymers to form a composite is being investigated extensively to attain suitable characteristics by means of synergistic effect. The main aim of composites preparation is to modulate and enhance the material properties in terms of processability, printability, mechanics, stiffness, stability, and bioactivity (Guvendiren, Molde, Soares, & Kohn, 2016). Primarily 3D printing focused on fabricating objects with pure polymers but advances in technologies quickly lead to development of composite inks that are 3D printable. Biodegradable as well as nonbiodegradable polymers are available for 3D printing. However, most of the applications focus on using biodegradable polymers which may be synthetic or natural depending on their origin. Natural polymers provide a cell-friendly microenvironment to the encapsulated cells due to the presence of bioactive molecules. Cell-specific activities such as attachment, migration, proliferation, and differentiation (Gasperini et al., 2014) are often promoted by them. However, due to their inferior mechanical strength, 3D structures require quick crosslinking mechanisms (physical, chemical, or ionic crosslinking) to stabilize (Gasperini et al., 2014). Synthetic polymers are man-made polymers which are produced by modulating physical properties and chemical structures (Liu & Wang, 2020). The mechanical aspects can be modified as per requirement and mostly synthetic polymers tend to have superior mechanical strengths in contrast to natural polymers (Liu & Wang, 2020). However, due to the production process that involves use of harmful organic solvents, heat, and toxic substances, final polymers are mostly bio-inert and lack attachment sites for cell adhesion and other activities (Liu & Wang, 2020). Therefore composite derived from combining these natural and synthetic materials can be appreciated as they possess both characteristics.

6.3 Blends and composites of natural and synthetic polymers Synthetic polymers such as polylactic acid (PLA), polycaprolactone (PCL), polyurethane, acrylonitrile butadiene styrene (ABS) and poly D,L-lactic-co-glycolic acid (PLGA) and natural materials such as collagen, alginate, chitosan, silk, and

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gelatin are most used materials (Simionescu & Ivanov, 2015). Synthetic polymers lack bioactivity and natural polymers lack mechanical strength. Composites developed with combining these give a new set of characteristics features to the material which can be an advantage to the biomedical community. A summary of mostly used composites is mentioned in the next section with focus on the describing modulated parameters that have a positive impact when compared to the effect obtained by using material individually. The additive added for composite preparation may vary from ceramics, biomolecules, carbon fiber, natural, and synthetic polymers.

6.3.1 Synthetic polymers based composites Thermoplastics such as PLA, PCL, PLGA, and ABS are widely used synthetic polymers for fabrication of orthopedic, dental implants and prostheses applications due to their superior mechanical properties, easy processability, and huge availability (Liu & Wang, 2020). As modifying polymers to form filaments, powders, or viscous liquid is quite effortless, 3D printing techniques mostly prefer polymers. Moreover, it is due to their innate characteristic to liquefy at certain temperatures and solidify at lower temperatures depending upon their melting points, they are mostly preferred in fused deposition modeling (FDM) and extrusion-based printing techniques (Liu & Wang, 2020). PLA is an aliphatic biodegradable polyester which also possess high strength, high modulus, and is nontoxic and biocompatible in nature and is widely used in medical industry (Garlotta, 2001; Guvendiren et al., 2016). PLA can be made into filaments and can easily be molten at 200 C 230 C that makes it an excellent material for FDM 3D printing (Guvendiren et al., 2016). However, PLA undergoes thermal degradation at temperatures greater than 200 C which tends to affect the bulk properties of the scaffold. Also, it is brittle and has inferior compressive strength in comparison to bone. Ceramic additives such as calcium phosphate glass (Serra, Planell, & Navarro, 2013), hydroxyapatite (Senatov, Niaza, Stepashkin, & Kaloshkin, 2016), and natural polymers such as chitosan (Almeida et al., 2014) and alginate have a tendency to improvise pure PLA in terms of roughness, cell adhesion, hydrophilicity, mechanical and osteointegration properties. Inclusion of nutshells from walnuts, macadamia, and almonds to form a wood polymer composite enables its use as a material to fabricate lightweight prostheses and orthoses (Song, He, Han, & Qin, 2020). Similarly, PCL is another mostly used polymers that is biodegradable, chemically inert, and biocompatible with mediocre mechanical properties (Guarino, Gentile, Sorrentino, & Ambrosio, 2017; Guerra, Cano, Rabionet, Puig, & Ciurana, 2018). Also, the hydrophobic nature along with lack of binding sites do not promote cellular adhesion and proliferation which eventually leads to lack of regeneration with PCL alone (Liu & Wang, 2020). And hence, to impart biological properties to scaffolds, and to improve wettability, bioactive glass, hydroxyapatite, tricalcium phosphate, pristine graphene, alginate, and chitosan were added to PCL (Kundu, Shim, Jang, Kim, & Cho, 2015; Lee, Yu, Jang, & Kim, 2008; Li et al., 2014; Park, Lee, & Kim, 2011;

6.3 Blends and composites of natural and synthetic polymers

Shim et al., 2017). Furthermore, the degradability of PCL can also be modulated based on the molecular weight, due to which it is a material of choice for fabricating stents and drug delivery systems (Hollander et al., 2016). PLGA is another biodegradable polymer used to fabricate sutures as it has a property to undergo hydrolytic degradation (Gentile, Chiono, Carmagnola, & Hatton, 2014). It is a copolymer derived from lactic acid and glycolic acid which exhibits controlled degradation based on the ratio of monomeric units used to prepare the copolymer and thus has both hydrophilic and hydrophobic properties (Gentile et al., 2014). Despite biocompatible, it inhibits osteoconduction and hence pro-osteo materials such as ceramics and bioglass are mixed with PLGA to form a composite (Babilotte et al., 2021; Pan & Ding, 2012; Shuai, Yang, Peng, & Li, 2013; Yun et al., 2009). Inclusion of TiO2 into PLGA improves mechanical properties making it suitable for load-bearing applications (Rasoulianboroujeni et al., 2019). The scope of using ABS as a material for load-bearing application in biomedical applications is also enormous since the mechanical strength of ABS is high due to the presence of styrene units (Zuniga et al., 2015). Tensile strength of ABS can further be improved with addition of carbon fibers but improper optimization of additive might compromise toughness, ductility, and yield strength of plastic (Wang et al., 2019). And hence while preparing a composite, immense optimization and standardization is very critical to achieve the desired features and of course to achieve reproducibility.

6.3.2 Natural polymers based composites Natural biopolymers such as collagen, alginate, chitosan, silk, and gelatin are immensely explored as materials in biomedical and tissue engineering applications (Cui & Boland, 2009; England, Rajaram, Schreyer, & Chen, 2017; Hong et al., 2020; Shi et al., 2017; Stratesteffen et al., 2017; Yang et al., 2018; Yu, Zhang, Martin, & Ozbolat, 2013). The polymer network resembling native microenvironment provided by these biopolymers aids embedded cells to improve their functionality by nourishing them with appropriate biochemical cues (Guvendiren et al., 2016). Because of this, scaffolds developed with natural biopolymers create a positive impact on the overall functionality and regeneration of tissue in vivo. However, to use them as bioinks suitable for 3D printing, certain properties such as flowability, rapid gelation, shape fidelity, integrity, etc. have to be ascertained and evaluated. And hence, composites developed with natural polymers along with other materials are encouraged as natural polymers help maintain the cellfavorable properties while other stiff polymers or ceramics preserves the shape and mechanical stability along with providing the cells with biomechanical cues. Collagen constitutes the major portion of protein present in the extracellular matrix (ECM) of the tissue (Frantz, Stewart, & Weaver, 2010). Collagen is a natural choice of biomaterial because it is highly biocompatible and integrin receptors on cell membranes tend to recognize the RGD (tripeptide consists of Arginine, Glycine, and Aspartate) peptide domains on collagen, thereby

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enhancing cell adhesion and proliferation. Also, the viscosity of collagen is highly dependent primarily on its pH and temperature. Implying, at physiological pH and temperature of around 7.5 C and 37 C, collagen fibrils start to organize themselves to form a stable microstructure and thus collagen in liquid state forms crosslinked hydrogel. This interesting innate property of collagen makes it a bit inappropriate material for 3D bioprinting as the crosslinking is not so fast to develop a 3D stable structure or it is not so slow to flow out of nozzle. Often collagen starts to form gel with slight change in temperature due to which nozzle orifice is clogged and material does not come out. And since the viscosity of collagen is low in liquid state, encapsulated cells accumulate at the bottom during printing and patterning ability of 3D printer is lost. And hence mixing materials such as genipin which reacts with lysine groups of collagen, alginate which crosslinks with CaCl2, agarose, incorporation of nano-HA or synthetic polymer such as PCL is preferred to improve the mechanical properties and to ensure shape fidelity of the collagen-based scaffolds (Campos et al., 2019; Isaacson, Swioklo, & Connon, 2018; Yang et al., 2018). Gelatin, derived from collagen, is another prominent material used for 3D printed tissue engineering applications (GomezGuillen, Gimenez, Lopez-Caballero, & Montero, 2011). It is a thermoresponsive material that stays in solid form at low temperatures and forms liquid at higher temperatures whose behavior is quite contradictory to collagen (Gomez-Guillen et al., 2011). Stable constructs with shape integrity can be obtained with gelatin when printed at low temperatures, but once the printed scaffold reaches a temperature more than its melting point (usually near physiological temperatures) it turns into liquid compromising the shape and structure of the printed scaffold. Materials such as alginate, silk, oxidized nanocellulose, and methacyrlated hyaluronic acid (HA) helps gelatin-based scaffolds to be mechanically stable even at higher temperatures (Shubhra et al., 2010; Xu, Wang, Yan, Yao, & Ge, 2010). Chitosan, a chitin derivative biocompatible polysaccharide which also possess antibacterial, antifungal, analgesic, and hemostatic properties, because of its positive charge (Croisier & Jerome, 2013), lacks mechanical strength similar to other natural polymers. To impart mechanical strength to chitosan scaffolds, it is used in combination with either PCL, PLA calcium phosphate, gelatin, or alginate (Akkineni et al., 2015; Chen et al., 2014; Cheng & Chen, 2017; Dadhich et al., 2016; Gu, Tomaskovic-Crook, Wallace, & Crook, 2017; Wu, 2016). Silk is another prominent material which is known for its mechanical as well as biocompatible properties. Due to its ability to promote cellular activity it is mostly used as a material for fabricating bone, cartilage, ligaments, etc. Since the mechanical toughness and strength of silk are superior when compared to any other natural biomaterial, it is often used in combination with alginate, gelatin, collagen, or hyaluronic acid (Park et al., 2011; Singh, Bandyopadhyay, & Mandal, 2019; Wei et al., 2019). Also, the biological properties of silk are also extracted while using silk in combination with synthetic polymers. Synthetic polymers such as PLA and PCL have been used along with silk for fabrication of bone clips and bone tissue engineering (Vyas et al., 2021; Yeon et al., 2018). Very recently, the potential of

6.3 Blends and composites of natural and synthetic polymers

decellularized ECM (dECM) as a bioink is explored extensively. Since it is derived from tissue, though sufficient biochemical cues are provided to cells, the mechanical properties of material are inferior. Therefore improving the mechanical properties of dECM has high scope of research, and combining collagen, silk, alginate, and gelatin to form a dECM-based composite is being investigated (Gao et al., 2017; Gao, Park, Kim, Jang, & Cho, 2018; Hiller et al., 2018; Lee et al., 2018). When two materials are combined to form a composite, the disadvantages of one material are overcome by another and vice versa which turns out to be an advantage for the biomaterial research, as the composite is now more applicationfriendly. Fig. 6.1 shows few examples from literature illustrating 3D printed

FIGURE 6.1 Image illustrating 3D printed scaffolds printed with natural polymer-based composite along with live/dead stains showing biocompatibility and functionality of the scaffold. (A) 3D printed alginate/agarose scaffold with (B) live/dead of chondrocytes, (C) phalloidin-stained actin filaments (Yang et al., 2018), (D) 3D printed scaffold with silk fibroin/gelatin/ hyaluronic acid/tricalcium phosphate composite, (E) live/dead of adipose stem cells, (F) alkaline phosphatase (ALP) activity (Wei et al., 2019), (G) 3D printed alginate/collagen composite, (H) live/dead of chondrocytes, (I) phalloidin-stained actin filaments (Yang et al., 2018), (J) top view of a 3D printed scaffold, (K) viable primary Schwann cells encapsulated in the strand, (L) dorsal root ganglion (DRG) neurites aligned along 3D printed fibrin-factor XIII-HA strands (England et al., 2017), (M) 3D bioprinted corneal stroma equivalent, (N) live/dead staining keratocytes, (O) brightfield image of 3D bioprinted corneal structure (Isaacson et al., 2018), (P) silk gelatin based 3D printed scaffold with live/dead stain staining of chondrocytes, (Q) live/dead image of the cells (R) immunohistochemistry (IHC) staining for macrophages (Singh et al., 2019), (S) gelatin, alginate, fibrinogen based 3D scaffold, (T) CD31 1 stem cells, (U) CD31 1 cells along with Oil red O stain (Xu et al., 2010), (V) collagen decellularized extracellular matrix (dECM) and silk fibroin scaffold, (W) live/dead of osteoblasts, and (X) phalloidin-stained actin filaments (Lee et al., 2018).

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scaffolds fabricated with composites to achieve enhanced mechanical properties without compromising the cellular functionality.

6.4 3D printing techniques employed to print polymeric materials Fabrication of 3D object is possible with 3D printing technology which deposits material in a layer-by-layer strategy (Pati et al., 2014). Fast ongoing advancements in this field have enabled development of complex geometries with various materials. Due to the wide library of materials available which can be printed, this technology has also made its mark in biomedical and tissue engineering sectors. Although the concept of fabricating 3D structures is similar in every 3D printer, the printing technique and material being used, makes them applicationspecific. The various established 3D printing techniques that are available are (1) fused filament fabrication (FFF), also known as fused deposition modeling (FDM), (2) nozzle-based extrusion, (3) selective laser sintering (SLS), Vat-based polymerization techniques which include (4) stereolithography (SLA) and (5) digital light processing (DLP), and (6) laser-assisted bioprinting (LaBP) as shown in Fig. 6.2. Polymers or polymeric composites can be printed with these techniques, due to the flexibility offered by polymers to tune their parameters.

FIGURE 6.2 Illustrating various 3D printing techniques that are commonly used for printing polymeric composites characterized based on the form of starting material used during printing. (A and E) Filament-based extrusion-based 3D printing techniques, (B and F) powder-based 3D printing techniques, (C and G) liquid polymers based 3D printing techniques, and (D) laser-assisted bioprinting for hydrogel printing applications.

6.4 3D printing techniques employed to print polymeric materials

6.4.1 Extrusion-based 3D printing Extrusion is a process of expelling the material out through an orifice by applying a force. This is the same principle applied to a syringe to expel the material out of the barrel. Extrusion-based 3D printers are also based on the same principle with a control over material deposition which is in turn based on the design modeled in modeling software such as AutoCAD or solid works. However, the form of the material that is fed into the nozzle chamber slightly varies. There are two kinds of printers available which works on this basis, and they are FFF or FDM and 3D plotting.

6.4.1.1 Fused deposition modeling FFF/FDM-based printers are widely used printers to fabricate 3D objects. It consists of a nozzle which is attached with a heating element. As the material in the form of filament approaches the orifice of the nozzle with the help of rotors, it melts due to the heat, gets extruded out of the nozzle, and is deposited on the platform (Zein, Hutmacher, Tan, & Teoh, 2002). During material deposition, subsequent layers are laid one above the other (Chia & Wu, 2015). Movement of nozzle and build platform may vary from manufacturer to manufacturer. Few printers have platform capable of moving in X-Y-direction while the nozzle moves only in Z-direction while few printers may have stable build platform and nozzle can move in all three XYZ-directions. Thermoplastics are ideal materials for FDM printing as they show a liquid-like behavior at temperature greater than melting point and almost immediately form solid when that temperature is decreased (Chia & Wu, 2015). Moreover, filament production with thermoplastics is quite straightforward. The material fed into the printer is in the form of filament and many synthetic thermoplastics such as ABS, PLA, PCL, and poly(propylene fumarate) (PPF) are common materials that are made into filaments and are used to print objects (Chiulan, Frone, Brandabur, & Panaitescu, 2018; Hutmacher et al., 2001; Zein et al., 2002). Materials choice can also be extended to composites made of PLA/ABS, graphene/PCL, PPF/ PCL, and PCL/PEG for FDM-based printing. Using these materials biomedical applications such as prosthetics and orthosis, bone implants were fabricated (Burn, Ta, & Gogola, 2016; Chiulan et al., 2018; Park et al., 2018; Placone & Engler, 2018; Teixeira, Aprile, Mendonca, Kelly, & da Silva, 2019; Zuniga et al., 2015). FFF technology has become the most common form of 3D printing technology due to its advantages such as low cost, ease of operation, wide range of material availability, and ability to deposit multiple materials. However, since the starting material is in the form of filament, composites developed should also be made into filaments while maintaining the material homogeneity. Moreover, since the processing temperatures of the material extrusion are very high, temperaturesensitive biological factors as well as viable cells cannot be incorporated into the material (Chia & Wu, 2015).

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6.4.1.2 3D plotting Extrusion-based printing technique deposits material in the form of continuous strands onto the substrate (Gu, Fu, Lin, & He, 2020). It is widely used technique for bioprinting because of its flexibility and affordability (Gu et al., 2020). This approach is also very popular due to the availability of wide range of materials that are compatible with this technique (Landers, Hu¨bner, Schmelzeisen, & Mu¨lhaupt, 2002). This technique basically works on the principle of expelling ink from the nozzle on application of mechanical or pneumatic forces. This extruded ink is deposited onto the substrate to form a desired architecture. Based on the technique that is utilized to drive the material out of the nozzle, this extrusionbased bioprinting is divided into three types, namely, pneumatic based, piston driven, and screw driven (Gu et al., 2020). Pneumatic based uses compressed air to push the material, while piston driven and screw driven use a mechanical force for ink ejection. Piston is connected to a guide screw which is in turn connected to a motor, to translate rotational motion of motor to linear motion, thereby pushing the material out of the nozzle (Gu et al., 2020). This technology is very versatile and finds applications with materials that have a wide range of viscosities. Also, it is a reliable technique to use in fabricating viable scaffolds in tissue engineering, as it is compatible with cells and various biomaterials (Landers et al., 2002). Many materials and composites such as collagen (He et al., 2018), gelatin (Wang et al., 2006), alginate (Fedorovich et al., 2012), gelatin/chitosan (Chang, Nam, & Sun, 2008; Ng, Yeong, & Naing, 2016), dECM/alginate (Gao et al., 2017), gelatin/fibrinogen (Xu et al., 2007), gelatin/alginate (Yan et al., 2005), collagen/chitosan (Ma et al., 2003), and nanocellulose/alginate (Nguyen et al., 2017), various dECM-based bioinks derived from many tissues such as adipose (Pati et al., 2014), heart (Pati et al., 2014), liver (Lee et al., 2017), cartilage (Pati et al., 2014), and skin (Kim et al., 2018) can be used with this technique. The usability of this technique in many applications can be enhanced with slight modifications to the nozzle, for instance coaxial nozzle design. However, the printed structure is highly dependent on the nozzle diameter, print speed, viscosity of bioink, force being applied, ink extrusion pressure, temperature, etc. (Gu et al., 2020). Additionally, the clogging of nozzle is said to be a major setback, yet this technique is widely used all over the world (Azad et al., 2020).

6.4.2 Vat polymerization This is an approach that uses the principle of photopolymerization of photosensitive materials on exposure to precisely controlled laser light. The two basic types of printing modalities that utilizes this approach are SLA and DLP. The basic setup in both the modes comprise of a transparent tank to hold resin (photosensitive material), photo-optics assembly, and platform to hold the printed structure (Quan et al., 2020). Typically, material is poured into the resin tank and based on the design, laser light is directed onto the resin. At places where laser light is showered in X-Y-direction, liquid material in that area solidifies forming one layer of the

6.4 3D printing techniques employed to print polymeric materials

printed structure on the platform (Wang, Goyanes, Gaisford, & Basit, 2016). Role of the platform is to move in Z-direction after one layer of printing is finished, making space for the uncrosslinked material to fill the gap and is ready for second layer polymerization (Melchels, Feijen, & Grijpma, 2010). Resolution of the printed structure is dependent on the laser spot size as well as on the movement of the platform in Z-direction. Other parameters such as laser power, scanning speed, laser wavelength, exposure time, and postprocessing affect the precision of printed structure in this technique (Melchels et al., 2010). The major difference between SLA and DLP is that SLA uses a pointed laser as light source, whereas DLP uses a divergent beam of light as source and hence a complete layer gets cured at one time in DLP technique (Quan et al., 2020). Because of light beam, the DLP technique is a much smoother and faster technique than SLA-based system in terms of construct fabrication time (Gu et al., 2020). The primary characteristic that a material should possess for it to be used for Vat-based 3D printing techniques is light sensitivity. Most of the available polymers are not light-sensitive, and hence, materials are modified to make them light-responsive. To behave as photosensitizers, materials such as gelatin, poly(ethylene glycol) (PEG), and silk were modified to form gelatin-methacryloyl (GelMA), poly(ethylene glycol diacrylate) (PEGDA), and silk-methacryloyl (SilMA) (Kim et al., 2018; Ma et al., 2016). Starch methacrylation was also explored to evaluate its potential as a material for DLP-based 3D printing strategy (Noe`, Tonda-Turo, Chiappone, Sangermano, & Hakkarainen, 2020). Recent literature reports the use of methacrylated chitosan as a material for vat polymerization. (Tonda-Turo et al. 2020). It is worth noticing that many materials that can undergo methacrylation can be used as potential materials for Vatbased 3D printing technologies. Materials such as PPF/diethyl fumarate (DEF), PPF/DEF-HA, PDLLA (Poly(D,L-Lactic Acid))/HA, and poly(ethylene glycol) dimethacrylate (PEG-DMA) (Lee et al., 2007; Luo, Fer, Dean, & Becker, 2019) were used with this technology for bone and cartilage tissue engineering (Bose, Vahabzadeh, & Bandyopadhyay, 2013; Cui & Boland, 2009). Even materials such as PLA and polyurethane diacrylate are also printed with SLA technique (Danilevicius et al., 2015; Petrochenko et al., 2015). SLA-based technology is popularly used to fabricate high-resolution, intricate, precise complex geometry due to the optical source and assembly contained with the printer. However, the starting material used in these techniques might not be cytocompatible, and hence, these are mostly used for scaffold fabrication which are further used to seed cells postprinting (Gu et al., 2020). Yet, because of possibility of modifying polymers, nowadays these modified polymers are widely explored as materials for DLP and SLA-based techniques from the conventional proprietary resins.

6.4.3 Powder bed fusion Powder bed fusion is a 3D printing technique that uses material in powder form unlike the other printing techniques which uses material in liquid form such as SLA, DLP, or 3D plotting. There are two different techniques that use material in

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powder form, and they are SLS and binder jetting. However, the technique with which powder material is fused together to form a 3D structure varies.

6.4.3.1 Selective laser sintering SLS is a powder-based 3D printing technique which uses material in powder form. The principle of operation behind this technique is sintering of material when it is exposed to a high-power laser. Powdered material is rolled onto the base platform and a high-power laser beam is directed onto the material as per the CAD design. The particles in the area that is exposed to laser fuse and bind together forming the first layer of the 3D structure. Once the first layer is done, the platform lowers, and fresh unbound powdered material is rolled onto the surface. This process continues until the final layer of structure gets printed (Gu, Meiners, Wissenbach, & Poprawe, 2012). The 3D printed object is retained inside the powdered material and the unused material can be dusted to obtain the actual part. Unbound material that does not fuse together can be reused. Print finish depends on parameters such as laser power, scanning speed, and size of the particle of the powdered material (Gibson & Shi, 1997). The main advantages of this technique are it offers support-free fabrication, fabrication of high-resolution objects, and material availability. However, void formation due to improper fusion needs to be tackled to avoid print failures. Theoretically most of the commercially available polymers can be used for SLS in powder form; however, sintering modifies properties of polymers, which limits their use for SLS technology. PCL and polyamide are widely used polymers for SLS technique (Muzaffar et al., 2019). Literature reports that materials such as PLLA and PHA can also be used as materials for SLS technology (Chiulan et al., 2018).

6.4.3.2 Binder jetting or powder liquid 3D printing Binder jetting or powder liquid 3D printing is also a similar to SLS technique but with a slight modification. A binder in liquid form is pumped and dropped on the powder layer according to the 3D design (Saroia et al., 2020). Binder allows particle agglomeration wherever it is selectively deposited to bind the particles together. As soon as the first layer is finished, platform is lowered down again to fill the surface with a fresh material ready to form second layer (Sachs, Cima, & Cornie, 1990; Saroia et al., 2020). The process continues till the last layer of the part is fused. Similar to SLS, printed object is retained in the unbound material and can be easily removed. Postprocessing of printed objects by using furnace cycles helps to improve the strength (Diegel, Withell, de Beer, Potgieter, & Noble, 2012). Complex structures with good print fidelity and stability can be obtained with the technique. Structures with variable mechanical strength can also be obtained with this technique by modulating the ratio of binder to powder (Gokuldoss, Kolla, & Eckert, 2017). Moreover, as sintering of particles is not involved as seen in SLS, and hence voids or crack formation due to residual stress is negligible. Various parameters such as binder viscosity, binder ejection rate, deposition speed, powder size, and more importantly the interaction of powder

6.5 Application of value-added polymers

and binder play a critical role in determining the success of the printed object (Gokuldoss et al., 2017). Nonetheless, parts fabricated with this technology have inferior mechanical strength and have coarse architecture when compared to parts fabricated with SLS. Materials such as PLLA, lactose, and methyl cellulose are made into powders, and cationic methacrylic ester copolymer, ammoniomethacrylic acid copolymer, and polyvinylpyrrolidone were used as binders developing tablets to release drugs (Deng et al., 2007; Rowe et al., 2000).

6.4.4 Laser-assisted bioprinting LaBP is a droplet-based, noncontact, nozzle-free technique to precisely deposit material along with cells onto the substrate (Gu et al., 2020). It basically consists of a laser pulse source, a ribbon with a coating of bioink and a receiving substrate (Jana & Lerman, 2015). The ribbon is made up of an energy-absorbing material often made up of gold or titanium, and is sandwiched between glass and bioink layer (Catros et al., 2011). The principle behind this 3D printing technique is that a laser source is directed onto the glass which is holding energy-absorbing ribbon and bioink. This ribbon absorbs the energy, and a high-pressure bubble is formed due to the local evaporation. Further this bubble pushes material containing cells onto the substrate (Gu et al., 2020; Guillemot, Souquet, Catros, & Guillotin, 2010). Laser assisted bioprinting (LAB) technique is very promising way to develop structures at high speed with high precision (Serra et al., 2006). Its ability to deliver single cell allows deposition of various cell types in spatial volume to replicate the complex coculture conditions in the native structure (Barron, Krizman, & Ringeisen, 2005; Guillemot et al., 2010). Furthermore, problems such as damage to substrate and cross-contamination are almost negligible due to its noncontact approach (Gu et al., 2020) and is said to be more suitable for in situ bioprinting.

6.5 Application of value-added polymers Biomaterials developed by polymeric composites act as a template to support cell attachment, migration, proliferation, and many other cellular activities due to the micro-morphology (O’Brien, 2011). Moreover, materials that comply with the tissue mechanical properties aid in rapid integration which helps in faster regeneration. The use of biomaterial composites with 3D printing techniques enhances the features of fabricated scaffolds. Structural features such as complex geometry, well-defined porosity, high resolution, defined cell positioning, structural similarity is imparted to the scaffolds. With these enhanced features, the cellular responses and degradation can be even more improved, thereby enhancing tissue integrity, and hence raising the chances of acceptance and regeneration in vivo (O’Brien, 2011). Many applications ranging from hard tissues such as scaffolds for dental implants, scaffolds for bone regeneration, to soft tissues like patches

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for skin regeneration along with antibacterial and wound healing abilities, extracorporeal devices such as prostheses and orthoses, and implantable devices such as stents are being fabricated with polymeric composite materials. Biocompatibility and biodegradability by actively promoting cell attachment, migration, proliferation, and eventually differentiation are considered as primary elements that an implant is supposed to possess. The differentiation of migrated cells on the implant allows cells to deposit their own ECM, thus helping in regeneration of the lost tissue. Also, the rate of degradability plays a key role so as to support the newly formed tissue. Due to cell-favorable properties, ceramics often find place in developing dental implants. However, due to few disadvantages of ceramics such as brittleness, inferior mechanical properties, and low fracture strength, they are used in combination with polymers (Miyazaki, Kawashita, & Ohtsuki, 2015). Properties such as improved cell migration, proliferation, and alkaline phosphate along with enhancement in scaffold’s surface roughness, porosity, and wettability can be imparted to PCL scaffold by combining it with β-tricalcium phosphate (β-TCP) (Park et al., 2017). Addition of TCP to polymers also facilitates early revascularization, good structural integrity, and better osteointegration which aids in speeding up the process of bone regeneration in vivo (Khojasteh et al., 2013; Li et al., 2014). Similarly, inclusion of carbon fiber to polyetheretherketone (PEEK) has led to the development of composite that has mechanical properties similar to cortical bone (Han et al., 2019). Hydroxyapatite is another promising material that is used along with polymers such as PLA and PCL as a composite to improve many properties such as tensile and flexural strengths, cell attachment and proliferation (Chen et al., 2019; Jiao et al., 2019; Mondal et al., 2020; Zhang et al., 2016, 2017). Altogether, composites developed from ceramics and polymers have enormous potential toward development of bone or dental implants to aid in faster regeneration of the damaged tissue, by promoting properties in synergistic manner. Prostheses and orthoses are other applications where polymeric composites play a crucial role in imparting critical properties to the products (Scholz et al., 2011). Prostheses are artificial assistive devices that assist a missing body part lost to trauma or injury or any disease condition to retrieve functionality. Similarly, orthoses are also artificial external assistive devices that are intended to support the limbs or spine or to correct the posture by preventing unnecessary movements. There are many different types of prostheses depending on the site of injury, namely, prostheses for lower limb, upper limb, shoulder, craniofacial, transradial, neck, foot, transtibial, transfemoral, etc., and orthoses for spine, arm, cervix, wrist, knee, hip, ankle, foot, etc. They must be designed and fabricated to cater the functional needs of the individual depending on the site of placement. Due to which few characteristics such as load bearing, high mechanical strength, and water resistant, lightweight is considered quite necessary. Since, a single material cannot provide all the necessary characteristics, often composites are considered for such applications. Most of the synthetic polymers intended for this do possess mechanical strength, but to enhance them composites such as ABS

6.6 Current challenges and possible solutions

reinforced with polycarbonate, carbon and Kevlar fibers into ABS, PLA, and nano carbon powder and polyurethane/PLA are being investigated (Jain & Tadesse, 2019; Tao, Shao, Li, & Shi, 2019; Wang et al., 2019). This concept of polymeric composites is also used for fabricating soft tissues such as skin. Combination of polymers such as chitosan/PLGA, alginate/PVA, collagen/PCL, and PCL/HA have been reported to promote many properties such as biocompatibility, neo tissue formation, improved cell responses, lightweight, and moisture retention (Dai, Williamson, Khammo, Adams, & Coombes, 2004; Shalumon et al., 2011; Wang et al., 2013). Functionalization of PVA can be improved with addition of natural material silk, as it promotes wound closure even in diabetic condition (Chouhan, Janani, Chakraborty, Nandi, & Mandal, 2018). The abovementioned examples illustrate that on addition of natural materials to synthetic material, bio favorable properties of synthetic polymers can be improved. On the other hand, the viscosity profile of silk that can be used for skin tissue engineering applications using 3D printing is enhanced by addition of PEG (Zheng et al., 2018). It is worth observing that many properties such as water uptake, printability, promoting cellular activity, and neo-vascularization can be imparted to materials by adding bioactive materials to synthetic polymers. Bacterial nano-cellulose (BNC) is a potential material that is used in fabrication of artificial blood vessels, but it possesses inferior mechanical properties. Hence, addition of PVA to BNC improves many mechanical properties such as compliance like an artery, water permeability, suture retention, and burst pressure which are very critical (Tang, Bao, Li, Chen, & Hong, 2015). The polymeric composite made of PLA and chitosan was found to be promoting cardiomyocyte activity and also enhanced production of sarcomeric α-actinin and troponin 1, thereby promoting myocardium regeneration (Liu et al., 2018). Another notable material that has been very popular in 3D bioprinting and tissue engineering area is dECM derived from specific tissue (Pati et al., 2014). But due to its weak mechanical properties it is often used in combination with other natural polymers. dECM obtained from vascular tissue in combination with alginate promoted proliferation, differentiation, and neovascularization, and also allowed printability of hydrogel (Curley et al., 2019).

6.6 Current challenges and possible solutions Pertaining to the biomedical applications such as extracorporeal objects, and implants or tissue engineered scaffolds, the existing 3D printable materials that are in research have few limitations. The primary concern for 3D printing is lack of availability of printable materials with appropriate physical properties. Material properties such as biocompatibility, biodegradability, or resorption, load bearing, and mechanically tough are very essential when a material is being considered; however, compatibility among materials is also key when we consider composites as material for fabrication (Fu, Feng, Lauke, & Mai, 2008). Hence, it is very

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crucial to select materials that can be easily combined with one another. For example, if surface property of one material is hydrophobic and the other is hydrophilic, there could be improper binding among materials and nonhomogenous composite would lead to formation of voids, or fiber pull outs, etc. The next challenge is that it is desirable to fabricate products that are light in weight for many applications, which can be attained by adding carbon fibers or fillers (Saroia et al., 2020). However, the extent of additives added should be optimized, because as the ratio of additives increases, primary mechanical properties of the base material might be compromised. Additionally, excessive incorporation of fillers might lead to nonhomogenous mixing, and the composites are prone to void formations, cluster formations by filler materials, and cracks (Mehdikhani, Gorbatikh, Verpoest, & Lomov, 2019). Any improper binding and lack of interaction between polymers in a composite might lead to voids and cracks which would diminish the mechanical properties drastically. Another crucial criterion is selection of optimal particle size of the additive to achieve homogenous distribution of the additive (Fu et al., 2008). This is also important when we use the composite as a starting material for extrusion-based 3D printing technique, since particle larger than nozzle diameter will clog the nozzle. Structural instability due to inferior mechanical properties is the major challenge that natural polymers pose while fabricating scaffolds or constructs. Therefore, viscosity of the material and its potential to crosslink after laid down is very critical and challenging. Most of the materials that encompass living cells often lack this property due to which they tend to spread as soon as they are laid down; hence fabricating 3D structures with natural materials alone is quite difficult. Apart from addressing challenges posed due to materials, there are few hurdles that are created by 3D printers too, which are inevitable. In FDM printers, the material undergoes instantaneous heating and immediate cooling leading to formation of voids inside the structure which is very prominent (Saroia et al., 2020). Similarly, in SLA or SLS technologies, there could be some binding variation in vertical direction versus horizontal direction as the layers get exposed to gradient of temperatures. All these parameters would contribute to the anisotropic properties of the scaffolds, and hence, postprocessing time and optimum printing conditions play a substantial role in determining the success rate of printed scaffold. The complexity furthermore increases, when 3D bioprinting has to be done, implying incorporation of living cells for developing in vitro tissues. All the material and printing parameters must be very well optimized and standardized to ascertain the viability of the cells in printed construct. The scope of developing newer composites to suit applications is very wide. Although many materials that are being used nowadays are biocompatible, developing a construct with material that has optimal degradation, nontoxic leaching substances, immunological response, etc., with suitable mechanical behavior is still underway. Due to the advancements, the flexibility of choosing material to develop composites for 3D printing is improving. And hence, it is worth speculating that in few years down the line, composites that are suitable for biomedical applications will come into translation.

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6.7 Conclusion Though the currently available synthetic or natural polymers are 3D printable they may not match up with all the desired characteristics for many applications. While the biomedical applications that could be developed with 3D printing technology are enormously increasing and, their requirements could only be equaled up by utilizing various combinations of material composites. Many polymeric composites that can provide unique and distinct properties are being evaluated at laboratory and research level for fabricating products that are mechanically and functionally stable. To explore the potential of 3D printing technology, selection of materials plays a key role, and its optimization is inevitable. The idea of composite preparation widens the practical use of many materials to redefine their use for specific applications. Overall, it can be summarized that the concept of composites as material for 3D printing allows development of personalized products that are patient-specific with appropriate desired product characteristics.

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Polymers for additive manufacturing and 4D-printing for tissue regenerative applications

7

Bhuvaneshwaran Subramanian1,
, Pratik Das2,
, Shreya Biswas2, Arpita Roy3 and Piyali Basak2 1

School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India 2 School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India 3 Polymer Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology (ISM), Dhanbad, Jharkhand, India

7.1 Introduction Traditional methods of manufacturing an object involve subtractive approaches, that is, removal of material through milling, machining, carving, shaping, etc. In additive manufacturing (AM), objects are created by depositing layer upon layer using a hardware system guided by a computer-aided design (CAD) software. Each of these successive super-thin layers bonds to the partially melted preceding layer. Architectural files (in stl format) are created by “slicing” the model object into ultra-thin layers. This information is then used by the CAD to direct the path of a nozzle or print head for precisely depositing the layers in place. Particular areas are melted by laser or electron beams so that they join after cooling down to give the object its final predetermined shape (Junk & Kuen, 2016). Hideo Kodama pioneered the initiation of AM in the 1980s. Kodama, then working at the Nagoya Municipal Industrial Research Institute, Japan, published detailed protocol and information on the production of a solid model using AM (Gokhare, Raut, & Shinde, 2017). In 1987, AM first appeared coupling with stereolithography (SL) from 3D manufacturing systems. Stereolithography (SL) refers to the process of curing layers of UV light-sensitive polymers. In 1986, Charles Hull patented this stereolithography as the first rapid prototyping system, which reduced the manufacturing time considerably and was thus eventually commercialized. The first commercially available AM system was SLA-1 (stereolithography apparatus), which was later upgraded to the extensively popular version


Contributed equally to the work.

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00001-2 © 2023 Elsevier Inc. All rights reserved.

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SLA 250 model, which is capable of precisely manufacturing complex designs, even to the minutest details (Martinez, Basit, & Gaisford, 2018). New generation machines started appearing from the year 2000. “Quadra”, a 3D inkjet printer capable of depositing hardened photopolymer using 1536 nozzles and UV light source, was introduced by Objet Geometries of Israel. Later on, Objet upgraded its “Quadra,” which could make polycarbonate, acrylonitrile butadiene styrene (ABS), and polyphenylsulfone parts. “Prodigy” was marketed by Stratasys, which ABS could manufacture plastic parts. T612 systems were introduced by Solidscape in 2013, and they could produce wax patterns for investment castings (Wohlers & Gornet, 2014). In the year 2013, the technology of 4D printing first came into being (Miao et al., 2017), and since its inception, this technology has rapidly grown and evolved towards betterment. 4D printing can be considered as one of the disruptive technologies of recent times, capable of producing intricate, stimuli-responsive 3D structures (Fig. 7.1). The main factor that distinguishes 4D printing from 3D printing is the additional dimension of time. Hence, 4D printing is only possible with materials that are temperature/humidity/pressure-responsive along the passage of time. Tissue and organ engineering has been greatly benefitted by the introduction of AM and 4D printing technologies. Sometimes 4D printing has been described as an

FIGURE 7.1 Building blocks of 4D printing development. Adapted from Sun, Y.-C., et al. (2019). 4D-printed hybrids with localized shape memory behaviour: Implementation in a functionally graded structure. Scientific Reports, 1, 113.

7.2 Polymers for 4D printing

FIGURE 7.2 The difference between 3D and 4D printings. Adapted and reconstructed from Bodaghi, M., et al. (2019). 4D printing self-morphing structures. Materials, 12, 81353. Available from https://doi.org/10.3390/ma12081353.

advanced form of 3D printing. The functionality, physical features, including shape, varies with respect to time. Parameters like humidity or temperature may cause changes in the 3D structure with time, and this forms the basis of the additional dimension of 4D printing (Haleem & Javaid, 2019). As with AM, here also CAD involving complex mathematical modeling is used for designing the object to be printed (Momeni, Liu, & Ni, 2017) (Fig. 7.2). The medical world has significantly benefitted from the discovery of 4D printing technology, particularly in the fields of tissue engineering (Hendrikson et al., 2017; Miao et al., 2016; Tamay et al., 2019). Very precise and particular medical devices such as shape-memory based personalized endoluminal devices (Zarek, Mansour, Shapira, & Cohn, 2017) have been successfully manufactured using 4D printing technology. This chapter focuses on summarizing the research reports on 4D printing technology and the polymeric materials used for the application of tissue engineering (Fig. 7.3).

7.2 Polymers for 4D printing AM is a well-known procedure through which layer by layer complex structures can be fabricated. In the case of AM, the 4D printing technology is an up-to-date technology that has drawn potential attention of the modern scientific community

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FIGURE 7.3 Illustrative diagram showing different stimulus for 4D printing and applications of 4D printing in medical filed. Adapted and reconstructed from Morouc¸o, P., et al. (2020). Four-dimensional (bio-) printing: A review on stimuli-responsive mechanisms and their biomedical suitability. Applied Sciences, 24, 9143.

for its high capacity towards the fabrication of complex structures, environmental stimuli-sensitive 3D structures and hence can be significantly used for the application of tissue and organ engineering applications (Kianian, 2017; Miao et al., 2017; Mitchell, Lafont, Hoły´nska, & Semprimoschnig, 2018). In the modern era, there are various smart materials like polymers and polymeric composites, which have been introduced for 4D printing. Among them, some materials to be highlighted are hydrogels, shape memory polymers, elastomer actuators and stimuli-responsive polymers (Roy et al., 2020).

7.2.1 Hydrogels Hydrogels were found to be widely used in 4D printing technology. Hydrogels are cross-linked polymeric networks that have the ability to absorb a large amount of water molecules in their network and also exhibit excellent cytocompatibility (Roy, Maity, Bose, Dhara, & Pal, 2019). The added dimension in the case of 4D printing of stimuli sensitive hydrogels is well defined as the variation of the swelling ratio, which exerts change in the shapes of the materials in response to the external stimuli (Roy, Maity, Dhara, & Pal, 2018; Shiblee, Ahmed, Khosla, Kawakami, & Furukawa, 2018; Zhao et al., 2018). In this regard, temperature and hydration sensitive cellulose-based hydrogels were synthesized and reported,

7.2 Polymers for 4D printing

which has been used as composite ink for 4D printing. For the preparation of cellulose composite hydrogel material, a hydrophilic mixture of carboxy-methyl cellulose polymer, clay platelets, and cellulose fibers were utilized. Further, the hydrophilic mixture was crosslinked (self-crosslinking as well as crosslinking with poly acrylic acid and citric acid) to generate a tissue-engineered scaffold (Mulakkal, Trask, Ting, & Seddon, 2018). Furthermore, a pH-sensitive antimicrobial hydrogel scaffold was also reported, which have been used with stereolithography to produce scaffolds for tissue regeneration. The scaffold exhibited outstanding antibacterial properties against S. aureus. This pH sensitive hydrogel was fabricated using Irgacure 819 (IRG 819) as a photoinitiator and acrylic acid (AA) was crosslinked with copolymer of polyethylene glycol dimethacrylates (PEGMA) using various types of dimethacrylate crosslinkers (Garcia et al., 2018). In this context, there was another report of fabrication of thermoplastic polyurethane hydrogel using 4D printing to produce complex tessellated origami structures. This multimaterial trilayer contained hydrophobic polyurethane materials, which was sandwiched around a hydrophilic polyurethane core. The hydrogel was fabricated using dehydrated techophilic thermoplastic polyurethane (TPU) pellets using a filament extruder with specifications of 2.85 mm ( 6 0.05 mm) diameter at 190 C. This material had unique property to bend and open up depending upon hydration of the scaffold (Baker et al., 2019). In another study, there was a report on ultrafast 4D printing of ionic strength responsive hydrogel. This unique hydrogel was fabricated using visible lightmediated polymerization of monomers (hydroxyethyl acrylate, hydroxyethyl methacrylate, potassium 3-sulfopropylmethacrylate) using IRG 819 as photoinitiator and polycaprolactone diacrylate (PCLDA) as crosslinker. This work demonstrated that the photoinitiator initially created a digital stress circulation in the synthesized 2D polymeric film, postfabrication upon release of that stress, the polymeric scaffold was converted into a 3D scaffold. These hydrogels can potentially be utilized for tissue engineering applications under suitable circumstances (Han et al., 2018; Huang et al., 2017; Roy & Maity, 2021). Another interesting report was obtained for the hydrogel nanocomposites for 4D printing which was the development of a magnetic field responsive hydrogel. This magnetic nanocomposite hydrogel was used as ink with the help of extrusion. The hydrogel was fabricated via polymerization of acrylamide monomer and carbomer (used as a rheological modifier) and Fe3O4 nanoparticles (used as a ferromagnetic substance). This carbomer centric 3D printing method enlightens new directions for advanced level bioprinting (Chen et al., 2019).

7.2.2 Shape memory polymers SMPs are special kinds of polymers that can transform their shape under the influence of some external stimuli (Salimon, Senatov, Kalyaev, & Korsunsky, 2020). In this context, a modern approach microstereolithography was introduced in the field of 4D printing. This methodology helped to generate high-resolution

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shape memory polymers of various materials and architectures. Thus generated highly potential materials were useful for the AM. As an example, a polymer was fabricated and reported using free radical photo polymerization of benzyl methacrylate and crosslinking using various crosslinkers like di(ethylene glycol) dimethacrylate, bisphenol A ethoxylate dimethacrylate and poly (ethylene glycol) dimethacrylate, etc. to form network like structures (Salimon et al., 2020). On the other hand, there was also an example of SMPs that was a photo sensitive polymeric scaffold. This matrix was produced using tert-butyl acrylate and crosslinked with the help of di(ethylene glycol) diacrylate crosslinker using photoinitiator phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) (Choong, Maleksaeedi, Eng, Wei, & Su, 2017). The synthesized material deformed when cooled and again regained its shape when heated; hence worked very efficiently as shape memory polymer. Another similar kind of work was reported recently that was postsynthesis of the polymeric material; some nanofiller like nanosilica have been introduced to the matrix to improve the overall strength of the material. On the other hand, silica also helps to generate high speed in printing (Choong et al., 2020). Recently, a novel material that helps in the digital light synthesis of shape memory polymers was reported. Interestingly it was fabricated within 30 s, which has significant control in geometries as well as in the shape memory properties. This scaffold had been fabricated using isobornyl acrylate as the monomers and 1, 6-hexanediol diacrylate as the crosslinker. Prior to the fabrication, homogenous mixture of printing precursors has been achieved to confirm the dissociation of the photoinitiator (bis (2, 4, 6-trimethylbenzoyl)- phenylphosphineoxide). For the fabrication of the material, the light of 400730 nm wavelength had been irradiated. Furthermore, the 4D printing was carried out using nano-photonics (Zhang et al., 2019). Likewise, an efficient material containing dynamic imine bonds via crosslinking of methacrylate monomer with 2-(methacryloyloxy ethyl 4-formylbenzoate) utilizing hyperbranched cross-linker was also fabricated and reported. The biggest advantage of this material was its self-healing nature. This was also very efficiently used for 4D printing (Miao et al., 2019).

7.2.3 Elastomer actuators An elastomer is a polymeric material that possesses viscoelastic properties. 4D printed elastomer actuators can efficiently be used in various AM applications like in artificial muscles, bioinspired robots, and so on (Lau, Shiau, & Chua, 2020). In the modern research of elastomer actuators, one unique discovery is liquid crystal elastomer (LCE) ink. The LCE was a new 4D printing programming technique. The LCE ink was developed by mixing 1, 4-bis-[4-(6-acryloyloxyhexyloxy)-benzoyloxy]-2-methylbenzene, n-butylamine and Irgacure 651 at 110 C. The parameter programmed 4D printing opted could be used in wider applications like the development of various software tools (Ceamanos et al., 2020). With the advancement of research, heat-responsive composite hybrid polymeric actuators were fabricated using epoxy-acrylate and also used in 4D printing technology

7.2 Polymers for 4D printing

(Yu et al., 2017). Besides, scaffolds with multifaceted applicability like soft robotics, actuators, shape-changing patterns, chronologically folding box, the hinge were developed using LCE ink was prepared by using aza-Michael addition reaction of acrylate with thiol (Roach, Kuang, Yuan, Chen, & Qi, 2018). In another study, there is an example of the development of a controlled orientation gradient 4D scaffold using 4,40 -Bis(6-hydroxyhexyloxy)- biphenyl and 4-(6hydroxyhexyloxy)cinnamic acid as monomers. These materials were polymerized under N2 atmosphere to form the printable polymeric ink for 4D printing. The scaffold has been used as temperature-responsive 4D printed multiple actuators (Zhang et al., 2019). In this context, a photo-responsive liquid crystalline elastomer actuator containing azobenzene was fabricated using n-butylamine, 1,4-Bis-[4-(6-acryloyloxyhexyloxy)benzoyloxy]-2-methylbenzene, and 4,40 -Bis[9 (acryloyloxy)nonyloxy]azobenzene in 1: 1: 0.12 mole ratio. These 4D printed materials have lifted the objects which possessed many times more weight than their own weight. This behavior demonstrated a high capability to yield effective work. One more advantage of this material was that it could be excited using UV as well as blue lights. The use of blue and UV irradiation permits the fine-tuning of produced forces that could be sustained even in a gloomy situation. Hence, this material has a very well prospect to be used for light-induced robotics applications (Ceamanos et al., 2020).

7.2.4 Thermoresponsive polymers Thermoresponsive polymers alter their behavior in response to alterations in the temperature of the environment (Tamay et al., 2019). At present, stimuliresponsive polymers, especially thermoresponsive polymers, have drawn much attention in the field of 4D printing. In this regard, a recent report was obtained on segmented 3D printed thermogel scaffold for implants, soft robotics, and other biomedical applications. The scaffolds were fabricated using thermopolymer (poly N-isopropyl acrylamide) (NIPAM) and acrylamide (Liu et al., 2019). A 3D printed thermo as well as pH-sensitive scaffolds were developed using triblock polymer of methacrylate poly(ethylene oxide) block poly(propylene oxide), block poly(ethylene oxide) and SL combination (Dutta & Cohn, 2017). Triblock polymer is a special type polymer that is made up of the linear attachment of three blocks of homopolymers. This 3D printed triblock copolymer exhibited a potential dual (pH and temperature) sensitive phenomenon and could be very beneficial in the production of medical devices, as they demonstrate the superior capability to alter its space depending on the change in pH and temperature. Like the triblock copolymers, the interpenetrating polymeric networks (IPN) were also developed and reported. These types of polymeric substances have drawn attention due to their easy synthetic steps and biocompatibility. As an example, alginate and poly NIPAM based IPN was fabricated for 4D printing to fabricate temperaturesensitive smart valves for water regulating purposes (Bakarich, Gorkin, Panhuis, & Spinks, 2015). Another ink for 4D printing was prepared using polyether/

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polyurethane (PE-PU) long-chain polymers, crosslinked with the help of UV exposure to prepare scaffolds. The UV polymerization was achieved with the help of α-ketoglutaric acid as initiator, N, N0 -methylenebisacrylamide as crosslinker and NIPAM as a monomer. Temperature-sensitive hydrogel hinges were also fabricated from several ink constructs. Thus using this simple structure, hydration and heat-responsive, more complex structures and devices can be fabricated in future (Naficy, Gately, Gorkin, Xin, & Spinks, 2017).

7.3 Application of 4D printing technology 7.3.1 Engineered tissue constructs In the recent era, 3D printing has evolved into an incomparable technology for bio-based manufacturing (Ngo, Kashani, Imbalzano, Nguyen, & Hui, 2018; Yan et al., 2018). A number of scientific evidence are present to establish the fact that 3D printing technology has a great potential for engineering artificial organs or functional tissues to heal nonfunctioning, abnormal or necrotic tissues. 4D printing, on the other hand, integrates time-dependent dynamic characteristics to the advanced fabricated platforms for tissue engineering applications (Chu et al., 2020; Miao et al., 2017).

7.3.1.1 Soft tissue regenerative implants Soft tissue anatomically includes skin, adipose, musculoskeletal, liver, lung, kidney, and ocular tissues. Soft tissues are highly flexible, and their functions depend on the mechanical properties of the tissue. All these mechanical properties are again dependent on the composition and arrangement of the extracellular matrix (ECM). Soft tissue damage is a major concern as it may lead to scarring or disfigurement and loss of bodily function. In order to overcome the limitations of current therapies related to damage or diseased soft tissues and organs, various new strategies have been developed for soft tissue engineering (Gokhare et al., 2017; Junk & Kuen, 2016). In a recent study, 4D printed hydrogel was reported for the construction of selffolding and hollow constructs with a very small diameter of 20 μm. Biopolymers like Alginate and hyaluronic acid has been employed here for the hydrogel construct. The constructs were biocompatible and could undergo reversible changes with the change in Ca21 ion concentration. Moreover, the constructs supported the survival of cells with a negligible reduction in cell viability (Kirillova, Maxson, Stoychev, Gomillion, & Ionov, 2017). Another study pointed out the use of 4D printed photocurable silk fibroin (Sil-MA) hydrogel to be used in soft tissue engineering of the trachea. The construct was found to be highly cell-friendly and biocompatible and was printed using digital light processing (DLP). The interior and exterior properties were

7.3 Application of 4D printing technology

modulated in physiological conditions in order to control the shape change of the bilayer 3D printed bilayered Sil-MA hydrogels. Finite element analysis (FEA) simulations were employed in order to explore possible changes within the complex structure. The 4D construct showed proper integration with the host trachea after 8 weeks, and formation of both cartilage and epithelium was noticed (Martinez et al., 2018). One of the recent studies pointed out near-infrared light (NIR) responsive 4D printed nanocomposite for probable use in soft tissue engineering and paving the way towards the 4D printed brain model. A smart epoxy was used with significant shape memory property, which was doped with graphene to generate a nanocomposite that exhibited an exceptional photothermal effect. The synthesized nanocomposite was highly responsive towards NIR stimulus, and its transformation was controllable dynamically and remotely in a spatiotemporal manner. Owing to the high electroconductive and optoelectronic properties of the nanocomposite, the constructed 4D neural cell-laden exhibited exceptional neural stem cell growth and differentiation (Wohlers & Gornet, 2014). Similarly, a NIR light-responsive 4D construct was fabricated using polyethylene glycol diacrylate (PEGDA) by DLP printing process for cardiac tissue engineering. 4D cardiac constructs were fabricated with aligned myofibers and adjustable curvature such that biomimetic structures of the myocardial tissue in both macro- and micro-scale can be obtained. This 4D construct was able to promote uniform cell growth and distribution throughout the curved structure. The 4D construct possessed remotely and active controllable transformation in a spatiotemporal manner and thus making it suitable as a cardiac construct. The 4D printed construct was found to be highly biocompatible when tested using humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), human bone marrow mesenchymal stem cells (hMSCs), and human umbilical vein endothelial cells (hECs) and the cell got uniformly distributed throughout the curvature and exhibited excellent myocardial maturation (Miao et al., 2017). Other than this, 4D printed multiresponsive structures has also been constructed for advances in nerve tissue engineering. Shida Miao et.al; constructed a 4D bio-printed structure using stereolithography (SL)-based technique. Materials used was mostly natural and photo-cross-linkable, like soybean oil epoxidized acrylate, SOEA. In order to achieve multiresponsive 4D construct a combined design of shape memory effect and stress-induced shape alteration was proposed and employed. The shape memory construct derived from natural biopolymers was able to initiate an additional “thermomechanical programming” shape transformation as well, thus pointing towards its multifunctionality. The introduction of nanomaterials amplified the overall 4D effect. A concept of a reprogrammable nerve guidance channel was validated using hMSCs, which in turn can differentiate into neural cell types. Thus this 4D construct with multifunctional characteristics will pave a way towards nerve regeneration (Haleem & Javaid, 2019).

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7.3.1.2 Hard tissue regenerative implants Although bone tissue has the property to heal on its own; however, its selfhealing property depends on the defect size (Szpalski, Wetterau, Barr, & Warren, 2012). Due to this, the healing of large bone defects is still afflicting surgeons (Marelli et al., 2011). In this regards, bone and tissue engineering has been explored vastly (Ansari, 2019). 4D printing technology has been utilized by researchers to fabricate scaffolds for promoting bone and dental healing by creating specific dynamic microenvironments (Khorsandi et al., 2020). Hydroxyapatite (HAp) and polylactide based scaffold was reported to show a temporary shape under compression, and upon temperature change to . Tg, the scaffold could restore its structure. These scaffolds could be used as a selffitting bone implant and could improve medical services (Senatov et al., 2016). 4D printing, on the other hand, gives an option for customized bone tissue engineering scaffolds which can overcome some common problems like implantation of scaffolds in defects with irregular shapes. A 4D printed scaffold with photothermal-responsive shape memory property was constructed using β-tricalcium phosphate/poly(lactic acid-co-trimethylene carbonate) (TCP/ P(DLLA-TMC)) along with incorporating of black phosphorus nanosheets and osteogenic peptide, thus resulting in a nanocomposite scaffold. The scaffold was highly dynamic and was appropriate to serve the purpose of a multifunctional bone tissue engineering scaffold. Application of NR irradiation caused a change in temperature of the scaffold and thus allowing shape reconfiguration and making the scaffold fit irregular bone defects. The inclusion of peptides into the scaffold resulted in enhanced osteogenesis in bone defect sites. A study of the scaffold in an animal model (rat cranial bone defects) showed improved and compact integration of the reconfigurable scaffolds along with enhanced new bone development (Momeni et al., 2017). A 4D printed, biomimetic hierarchical scaffolds showed high biocompatibility and dynamicity, which paved a path for regenerative medicine and tissue engineering. The dynamic scaffold was fabricated using castor oil and polycaprolactone (PCL) triol and was crosslinked using poly(hexamethylene diisocyanate) (PH). The printed scaffold was highly porous, indicating proper cellular growth and differentiation. The scaffolds were found to be highly biocompatible with mesenchymal stem cells (MSCs), and they also exhibited proper attachment, proliferation, and differentiation. High mechanical stability and superior biocompatibility make these types of scaffolds extensively suitable for bone tissue engineering (Miao et al., 2016). Notably, it is important to mention that the addition of functionalized mineral additives (tricalcium phosphate, HAp), decellularized bone matrix and trace elements (Mg, Zn, Sr, Ag, Si, and Sr) into 4D printed scaffolds could further improve their osteoinductive and osteoconductive properties (Kulanthaivel, Agarwal, Rathnam, Pal, & Banerjee, 2021; Qu, Fu, Han, & Sun, 2019; Ribas et al., 2019; Sawkins et al., 2013) (Fig. 7.4).

7.3 Application of 4D printing technology

FIGURE 7.4 Applications of 4D printing in hard tissue engineering. (A) Application of thermosensitive hydrogels for 4D bone tissue regeneration, (B) Application of shape memory and shape responsive material for 4D bone tissue regeneration, (C) Biomimicry for 4D tissue engineering. Adapted from Wan, Z., et al. (2020). Four-dimensional bioprinting: Current developments and applications in bone tissue engineering. Acta Biomaterialia, 101, 2642.

7.3.2 Medical devices Vascular stenosis is a major complication for cardiac patients, and the trending increasing cases of the prevalence has been reported to be a serious threat for the people (Im, Jung, & Kim, 2017). In vascular stenosis, the lumen of the artery has been reported to narrow due to plaque formation along the arterial inner wall, resulting in atherosclerosis (Sigwart, Puel, Mirkovitch, Joffre, & Kappenberger, 1987). The presently available nondegradable metal stents generally used to treat vascular stenosis have been reported to increase the occurrence of restenosis due to the proliferation of intimal smooth cells. To overcome this complication biodegradable vascular stents fabricated using 4D printing technology have been proposed to be an

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excellent solution (Fu, Liu, & Hu, 2018; Huang, Zhang, Scarpa, Liu, & Leng, 2018; Park et al., 2015; Yakacki et al., 2007). In a study, personalized 4D printed stents were developed using shape memory PLA with negative Poisson’s ratio for application as a vascular implant. Fluid-structure interaction and stress distribution during the shape recovery process for the stents were mostly based on the finite element method. This study has also reported that the shape memory behaviors of the 4D vascular stents fabricated were vital in stimulating narrow blood vessels. It was also reported that these 4D stents significantly assisted in the recovery of vascular stenosis conditions (Lin, Zhang, Liu, Liu, & Leng, 2020). A study in the recent past showed the use of both shape memory and shape healing properties of polymer using 3D printing. The ink was prepared using a photocurable resin composed of aliphatic urethane diacrylate (AUD; containing 33 wt.% of isobornyl acrylate) and n-butyl acrylate (BA) and a semicrystalline polymer, that is, PCL. The printed construct was a highly stretchable (600% with in-plane isotropic properties) semiinterpenetrating polymer network (semi-IPN) elastomer and was printed using a direct-ink-write (DIW) approach. The highly stretchable elastomer with the dual property was further demonstrated to be used as a vascular repair device. The unique property of this material makes this a good choice for various biomedical applications as well (Tamay et al., 2019). Similar multifunctional material was developed by Hongqiu Wei et.al. A 4D active shape-changing structure was fabricated by the direct wire printing technique. The material used was poly-lactic acid-based ink which was crosslinked using ultra violet rays. Fe3O4 and benzophenone (BP) were further introduced in order to achieve remotely actuated characteristics and excellent shape memory, respectively. The printed material showed excellent shape memory behavior with various configuration transformations, and the addition of iron oxide enhances the property of the material by integration of fast, remotely actuated, and magnetically guidable properties. The multifunctional property and the flexibility of this material make this a perfect candidate for a self-expandable intravascular stent. These types of multifunctional materials pave a new direction for additional development in the field of 4D printing, biomedical devices, soft robotics, microsystems, and beyond (Hendrikson et al., 2017). Other than vascular stents, recent advancements in the fabrication of tracheal stents using thermosetting materials have also been reported. A group demonstrated a heat-driven lumen device as a tracheal stent which becomes functional with the increase in temperature. UV-LED stereolithography printing technique has been used for printing methacrylated PCL precursor based material which turned out to be highly biocompatible and exhibited excellent dynamic property and shape memory (Zarek et al., 2017).

7.3.3 Drug delivery implants Drug release at a suitable time and at the appropriate location is of utmost importance in the area of pharmaceuticals and drug delivery. This is the major emphasis of the drug development industry. 4D printing technologies make it possible to

7.3 Application of 4D printing technology

optimize conditions that can help to control the spatial and temporal delivery of therapeutic agents (Hsu & Jiang, 2019). Utilizing the 4D bioprinting design of various implants can be made possible, which can self-fold or unfold either to engulf and discharge drugs or cells into the system in a programmable way (Dai et al., 2019; Larush et al., 2017). A recent study has reported that NIR-responsive double network shape memory hydrogel could be used to fabricate antimicrobial scaffold. The scaffold has been constructed using Pluronic F127 diacrylate macromer (F127DA), poly(lactide-co-glycolide) (PLGA) and graphene oxide, which served as an energy converter to ultimately convert NIR radiation to thermal energy. The scaffold on testing has been reported to show no cytotoxicity. Restoration of the folded hydrogel into the original shape has been achieved by irradiating it under NIR for about 240 s. The change in the surface area played the controlling factor for drug release property owing to the change in the shape of the scaffold. Thus on distorting the temporary shape, the surface area becomes smaller hence reducing the drug release (Dai et al., 2019). An activated drug delivery system was constructed using alginate fibers and pH-responsive material using 4D printing technologies. The printed material acted as a porous sensor. Simultaneously, alginate fibers loaded with gentamicin has been printed, which acted as drug-eluting stents. Using the printed sensors and the drugeluting stents, a dressing material was fabricated. This dressing material showed potential effects in controlling chronic and acute injuries occurred due to trauma, surgery, or diabetes. The whole system was designed in such a way that upon the exposure of the fabricated material on the wounded or infected site, there will be a change in pH which will be captured by the sensor that will activate the drugeluting stents to release the active pharmacological ingredient at the respective site of the pH change to provide antibacterial activity. Besides, various attempts are going to modify the system to make it more beneficial and to detect more specific bacterial markers (Mirani et al., 2017). A pH-responsive drug release system has also been formulated using DLP technology by using pH-independent fluorescent dye sulforhodamine B as a model drug. The fabricated scaffold has been reported to exhibit improved swelling and rapid drug release under suitable conditions (Larush et al., 2017). Another study exhibited a drug delivery model using shape memory hydrogels with internal structure (SMHs), which was fabricated using pluronic F127 diacrylate macromer (F127DA) and sodium alginate. The 3D printed structure was comprised of a dual network structure: Stable network and reversible network. These shape memory hydrogels were found to be highly biocompatible with fibroblast cells, and the drug-releasing rate was found to be more rapid than other conventional drug-loaded hydrogels (Zarek et al., 2017). All these studies indicate that 4D additive technology provides a way to construct both simple and complex structures that can control localization, release rate, and smart delivery of drugs (Wang & Kohane, 2017). A list of important tissue engineering work related to 4D printing has been summarized in Table 7.1.

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Table 7.1 Application of 4D printing in tissue engineering and various medical field. Medical application 4D additive printing in smart stent

Description

Material used

Advantages

References

• 4D printing technology can be used to create stents that would stretch and assume the desired form with the aid of the patient’s body heat • This new approach works efficiently to save a patient’s life from a potentially life-threatening operation • Shape changing polymer with respect to time and temperature

Formulation of polycaprolactone (PCL)-based resin for personalized endoluminal medical devices- PCL diol, stannous octoate, isocyanatoethyl methacrylate, 2,4,6-trimethylbenzoyldiphenylphosphineoxide as the photoinitiator Triple shape memory polymers— polyurethane-based filaments, fabricated using fused deposition modeling printing technology

• Personalized endo-luminal medical devices x Since the stent is almost identical to the arcade configuration and placement of the cartilaginous rings, this design strategy based on customized assemblies can reduce migrations, which are a common cause of tracheal stent failure x The shrunk SMP structure’s low profile allows for a less harmful launch • Triple shape memory polymers: x Superior adaptive systems with the ability to self-bend x An elasto-plastic reaction that acts hyper-elastically at low temperatures when acting elasto-plastically at high temperatures in the massive deformation regime x A new method for making self-shrinking/tightening staples. And self-bending grippers/stents

(Bodaghi, Damanpack, & Liao, 2018; Zarek, Mansour, Shapira, & Cohn, 2017)

4D printing technology of organ fabrication

• This new technique will be used to fabricate complex 3D organs • It can be used to print organs using the patient’s own cells and can potentially save a life • It is a potential option for meeting shortages of organs during the crisis

Wood filament combined with acrylonitrile butadiene styrene filamentNylon hectorite clay, nanofibrillated cellulose, and N-isopropylacrylamide monomers or N, N-dimethyl acrylamide, soybean oil epoxidized acrylate,

Multi-material 4D printing

• A layer-by-layer process, by use of UV curable polymer • It’s a brand-new method for printing surgical devices for personalized smart multimaterial printing • The 3D printed body, clearly reveal many body pieces

For supporting dyspnea (a major breathing problem)

• 4D printing technology is now being used as life support to save lives of newborns who are suffering from severe breathing problem (dyspnea)

For linear chain builder (LCB) methacrylate-based polymer (benzyl methacrylate) has been used, LCB and multifunctional oligomers, di-methacrylate, bisphenol, poly (ethylene glycol) for crosslinking purpose ethoxylate di-methacrylate, and Di (ethylene glycol) di-methacrylate has been used PCL

• The fabricated scaffolds will completely restore their original form from a temporary shape set at other temperatures, at human body temperature (37 C) • A high degree of cellular adhesion • A surgical system that is only minimally invasive • Development of 4D printed organs that conform to changing or increasing tissues, particularly for pediatric applications • Tailorable flexibility • Enhanced thermomechanical properties • Multifunctionality

(Miao, Castro, & Nowicki, 2017; Saunders, 2017)

• The 4D printed structures are hollow and porous, designed to spread open as a child grows with age

(Choi, 2015; Haq, 2015)

(Akbari, Sakhaei, & Kowsari, 2018; Ge, Sakhaei, & Lee, 2016)

(Continued)

Table 7.1 Application of 4D printing in tissue engineering and various medical field. Continued Medical application

Description

Material used

• 4D printing technologies have made it possible to fabricate dynamic medical implants that can change shape over time and as babies grow up they don’t face any major problem and can keep breathing

4D printing technology-based tissue engineering and fabrication of smart medical device

• 4D printing gives a potential opportunity to fabricate shapeshifting materials and objects that could be used for human application • It shows a promising prospect when it comes to medical implants and tissue technology, which could modify their form in the body • It’s used to regenerate tissues where the mechanical characteristics of the body alter dynamically when the muscle, bone, and cardiovascular tissue become active

• Polyether urethane • Poly(ethylene glycol) diacrylate, luminol (3aminophthalhydrazide), collagen from calf skin type I

Advantages • The devices improved breathing and expanded to allow the airways • The air splint can degrade within the body after there is suitable growth of the trachea. Hence this will give a new path for curing tracheobronchomalacia • The changes in the morphology of adherent cells could be initiated using a single mechanical stimulus • These 4D printed scaffolds could find use in supporting the regeneration of tissues with dynamically varying mechanical characteristics, for example, cardiovascular tissue • Customized design • These 4D printed scaffolds could be implanted in • Patients by minimally invasive surgery • This technique enables the printing of both cells and programmed calcification together to enable the in vitro reconstruction of cellularized bone defects • Enzyme incorporation

References

(Hendrikson, Rouwkema, & Clementi, 2017; Mandon, Blum, & Marquette, 2017)

Printing of essential organs like heart, kidney, and liver

• 4D printing in the future will allow the use of intelligent material to manufacture the heart, kidney, and liver. Hence dynamicity will be maintained • Ability to print such components that are very flexible, fit, and match genetically perfect

• 4D printed heart: HeartPrint flex material • 3D printed organs: natural materials like alginate, gullan gum, cellulose, and synthetic materials include PCL, Silicone Pluronic F127 Intensive research is going on 4D printed organs

Advanced skin grafts or artificial skin

• 4D printing has made it possible to print skin graft which looks original and realistic with matching the color complexions of the patients • Also beneficial for patients with severe burn as these printed skin grafts can quickly integrate within the body and start growing like an original

Skin bioprinting: Collagen type I embedded with Mouse NIH3T3 Swiss albino fibroblast and human immortalized HaCaT Keratinocyte cell lines Fibrinogen/collagen hydrogel

• Better visual input and tactile information to comprehend complicated heart abnormalities • Translucent material for the printing enables the inner structures to be easily visualized • The heterogeneous structure may be manufactured at the same time and diverse cell maturations are accomplished • Accurate stratification • Better properties of skin properties • The potential of reconstructing vascular network • Direct cell incorporation in ECM matrix

Fabrication of smart medical devices using 4D printing technologies

• This technique is capable of producing complicated intelligent, 3D-printed medical devices with outstanding functionality • Adjusted according to the time required for the surgical procedure

Semi-IPN based materials: PCL, EBECRYL 8413 SMPs, shape memory ceramics, shape memory gels, and other shape memory hybrids materials, for examples polylactide, thermoplastic polyurethane, and UV-cured thermoset polymers such as VeroWhite Plus RG835 Photocrosslinkable polyethylene glycol

• Remarkable functional characteristics, such as highstrain shape memory and shape memory assisted selfhealing • Extensive material having inplane isotropic characteristics and the objects can be stretched by upto 600% • Heterogeneous 4D printed parts can be printed together

(Gosnell, Pietila, & Samuel, 2016; Yi, Lee, & Cho, 2017)

(He, Zhao, & Zhang, 2018; Khoo, Teoh, & Liu, 2015)

(Castro, Meinert, Levett, & Hutmacher, 2017; Kuang, Chen, & Dunn, 2018; Pei & Loh, 2018; Zhao, Yu, & Li, 2018)

(Continued)

Table 7.1 Application of 4D printing in tissue engineering and various medical field. Continued Medical application

4D printing for complex surgery

Description

• 4D printing technologies are used to generate a haptic model that represents the body’s movement and appearance • For extremely difficult surgeries that other manufacturing technologies cannot accomplish, it may likely be embraced in the future using 4D printing • Producing a model by using various smart material could be made possible with the help of real-time CT and MRI scan which in turn can precisely reproduce any type of body movement • Any type of anatomical details can be depicted in a precise and accurate manner using this technology

Material used

Photopolymer or epoxy resinPhenylbis (2, 4, 6-trimethyl benzoyl) phosphine oxide (as a photoinitiator), Polymer base: -methacrylate-based polymer, Sudan I and Rhodamine B working as photo absorber

Advantages • The joint folding direction can be controlled by the radial orientation of the 4D printed sheets concerning the adjacent structures • In order to enhance clinical outcomes and for more successful procedures, superior spatiotemporal anatomical information could be extracted using this enhanced technology. Thus this would assist surgeons to a large extent • This technique enables the design of time-dependent sequential shape recovery of a structure fabricated using 4D techniques • This technique could be of potential use for denture positioning and denture retention and upgrading the existing dental implants • For a difficult procedure, the surgeon can foresee the problem and better comprehend it

References

(Chae, Hunter-Smith, & De-Silva, 2015; Hegde & Hsiao, 2016; Javaid, Haleem, & Kumar, 2019; Lee, An, & Chua, 2017)

• Additive manufacturing technologies that could be a major support for dentistry are helping in saving time as well as its cost is also lower • With the use of 4D flow MRI, high-resolution imaging of the complete Fontan circulation could be made possible, including anatomy, and blood flow in just a single 1015 min acquisition • It is conceivable that the surgeon can update the detailed patient-specific heart defect model with interactive anatomy-editing instruments, such as SURGEM and GeoMagic studio • The approaches allow for the use of 4D flow in patients with Fontan circulation for the refining of surgical techniques to quantify many variables of clinically important

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7.4 Conclusion In the fields of tissue engineering and biomaterials, three-dimensional printing is quickly becoming a critical tool. By creating patient-specific devices with the required shape and organization, it transformed the biomaterials industry. Biomedical devices have long used stimuli-responsive materials, such as metals and polymers, and the combination of these two properties creates 4D printing, which introduces incredibly useful, viable, dynamic, and responsive systems in tissue engineering applications. 4D printing is a revolutionary technology. Despite decades of research, only a few responsive materials have been developed for 4D printing and are now being used in tissue engineering. Biocompatible, noncytotoxic, and preferably biodegradable materials are required for this purpose (resorbable). It’s also essential that they’re capable of dynamic processes in physiological environments, as well as displaying certain mechanical strength. The stimulus used for such application must be safe according to the standard and easy to control when applied to the body, which is an important consideration. It’s best to stay away from things like pH extremes and extremely high temperatures. Only a small number of dynamic polymers meet all of the criteria due to the high standards. Furthermore, tissues in nature are exposed to a wide range of stimuli, whereas the majority of materials described to date respond only to a single stimulus. In order to improve 4D printing technology, more effort and expertise should be put into developing new and multifunctional 4D inks. As it stands, 3D and 4D printing methods continue to keep scientists busy as they try to develop new biomaterials and biomedical instruments. While there are many different types of stimuli out there, the materials currently respond to a small subset of them. Thus, the development of new materials with multiple sensitivities for use in enhancing the dynamic nature of devices remains a difficult problem.

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CHAPTER

Bioprinting of hydrogels for tissue engineering and drug screening applications

8

Ece O¨zmen
, O¨zu¨m Yıldırım
and Ahu Arslan-Yıldız Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey

8.1 Advancements in bioprinting technology Every year, millions of people suffer from tissue or organ deficiency related diseases, and number of donors is far less than the need. Transplantation saves lots of lives; however, immune rejection limits transplantation (Lanza, Langer, Vacanti, & Atala, 2020). Hence tissue-engineering approaches have been developed for treatment of damaged tissues and fabricating artificial organs. Scaffolds with or without cells are fabricated by using conventional or advanced manufacturing techniques, which provide the proper cell culture conditions, and maintain cell viability and proliferation (Langer et al., 1995). In addition to transplantation purposes, tissue engineering approach is used to create 3-dimensional (3D) cell culture systems, which model disease and are utilized for drug discovery. Drug discovery and drug screening studies are generally performed on 2-dimensional (2D) cell culture and animal models. However, in 2D cell culture models, cells grow as a monolayer and do not represent the native 3D tissues efficiently. On the other hand, there are biochemical differences between animal models and human physiology and obtained results cannot be compared with clinical data directly (Onbas, Bilginer, & Yildiz, 2021). To overcome these disadvantages, 3D cell culture model has emerged and is generally used for modeling diseases or drug screening purposes. 3D cell culture models can be fabricated by using both scaffold-free and scaffoldbased techniques. Scaffolds are generally fabricated by freeze-drying (Guzelgulgen, Ozkendir-Inanc, Yildiz, & Arslan-Yildiz, 2021; Rnjak-Kovacina et al., 2015), solvent-casting (Mikos, Bao, et al., 1993; Mikos, Sarakinos, Leite, Vacant, & Langer, 1993), electrospinning (Arica, Guzelgulgen, Yildiz, & Demir, 2021; Bilginer, Ozkendir-Inanc, Yildiz, & Arslan-Yildiz, 2021; Tu¨rker, Yildiz, & Yildiz, 2019), and bioprinting (Arslan-Yildiz et al., 2016; Kang et al., 2016). Compared to other biofabrication methodologies bioprinting is an emerging advanced manufacturing technology, which enables the creation of a designed shape in 3D by printing the bioink material that encapsulates cells or other


These authors contributed equally and were written in alphabetic order.

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00028-0 © 2023 Elsevier Inc. All rights reserved.

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biological molecules (du Chatinier, Figler, Agrawal, Liu, & Zhang, 2021; Mei, Rao, Bei, Liu, & Zhao, 2021; Singh, Choudhury, Yu, Mironov, & Naing, 2020). In the field of tissue engineering and regenerative medicine, bioprinting is a widely used advanced manufacturing technique with its reproducibility, high throughput, and controllability in an automated way (Gungor-Ozkerim, Inci, Zhang, Khademhosseini, & Dokmeci, 2018). It is possible to produce tissue scaffolds with bioprinting technology, as well as to produce functional and living 3D structures by encapsulating cell into bioink (Hospodiuk, Dey, Sosnoski, & Ozbolat, 2017; Matai, Kaur, Seyedsalehi, McClinton, & Laurencin, 2020). Creating a scaffold or functional tissue via bioprinting process requires utilizing the (1) bioprinting strategies, (2) bioprinting parameters, and (3) bioinks as illustrated in Fig. 8.1. Based on the working principle of bioprinting strategies, either laser-assisted bioprinting (LAB), droplet bioprinting, or extrusion-based bioprinting, the intended use and bioink requirements are varied (Arslan-Yildiz et al., 2016; Murphy & Atala, 2014; Pati, Gantelius, & Svahn, 2016). In LAB: a laser beam that is pulsating in a controlled time span, a surface where cells and bioinks will be positioned, and an absorption layer are utilized for bioprinting of cells and 3D structures. The absorption layer, which is made of gold or titanium, is transparent to the laser beam and covered with bioink material to encapsulate and protect cells and other biological molecules such as proteins (Pati et al., 2016). Resolution of the LAB can be tuned by changing the energy and the spot diameter of the laser beam. The depth of absorption layer, thickness of bioink layer, and

FIGURE 8.1 A schematic representation of the major components of bioprinting.

8.1 Advancements in bioprinting technology

bioink viscosity are the other significant parameters for the efficiency of LAB operation. By tuning the required parameters, LAB allows reaching microscale resolution with computer aided geometrical control (Guillotin et al., 2010). Since LAB is a nozzle-less type of bioprinting operation, the bioprinting of high cell densities and high viscosity of bioinks without causing any clog formation can be achieved (Arslan-Yildiz et al., 2016; Hribar, Soman, Warner, Chung, & Chen, 2014). On the other hand, it is not possible to use LAB for bioprinting of large 3D structures due to the limited spot area of the laser beam. Presence of metallic residues in postprinting structures, the relatively higher price of device, and higher cytotoxicity because of UV exposure are other drawbacks of LAB (Pati et al., 2016). In tissue engineering, LAB have several utilizations for cell printing (Guillotin et al., 2010), patterning (Bourget et al., 2016; Catros et al., 2011; Devillard et al., 2014), modeling (Michael et al., 2013), and regenerative medicine applications (Guillemot et al., 2011; Keriquel et al., 2017; Ke´roure´dan et al., 2019). Droplet bioprinting, in other words inkjet-based bioprinting, is a noncontact technique which is used for printing multiple cells and proteins layer-by-layer by depositing small droplets on the targeted surfaces (Pati et al., 2016). According to the droplet generation strategy, droplet bioprinting is classified into two subgroups: thermal and piezoelectric droplet bioprinting. Droplet bioprinting has been preferred during the last decades due to its high printing speed, high resolution, and cost-effectivity (Gurkan et al., 2014). High cellular viability is achieved by this approach, however it is not suitable for printing high cell densities and highly viscous hydrogels. Since only low viscosity hydrogels can be printed, the created 3D structures cannot provide desired mechanical properties. This limits the efficient achievement of completely functional and 3D usage suitable constructs (Ferris, Gilmore, & Wallace, 2013; Kim, Choi, Kim, Choi, & Cho, 2010). In the field of tissue engineering, droplet bioprinting is mostly utilized for modeling (Christensen et al., 2015; Xu, Chai, Huang, & Markwald, 2012), stem cell research (FaulknerJones et al., 2015; Gurkan et al., 2014), drug screening (Rodrı´guez-De´vora, Zhang, Reyna, Shi, & Xu, 2012), and cancer studies (Fang et al., 2012; Xu et al., 2011). Extrusion-based bioprinting is the third bioprinting approach that aims to print cells encapsulated by hydrogel bioink as designed 3D structures onto target surface. Extrusion-based bioprinting can be classified into two subgroups on the basis of working principle: pneumatic, and mechanical (screw and piston). In each principle, bioink is printed by applying pressure to overcome the surface tension on the nozzle tip. Extrusion-based bioprinting allows printing of high cell density with an adequate cell viability and high viscosity bioinks within a sufficient speed. Properties of 3D construct can easily be tuned by changing pressure, printing speed, layer height, nozzle diameter, etc. Despite these advantages, the printing of high viscosity bioinks requires higher pressure, which results in a decrease in cellular viability, and causes deformation in cellular integrity. In addition, printing resolution is worse than the aforementioned approaches, it is nearly about 200 μm (Malda et al., 2013). Bioprinting speed is higher in this approach

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compared to other methodologies and this technique allows printing constructs in desired dimensions without any limitation. Mechanical properties of printed constructs can be improved by using highly viscous bioinks. In literature, there are studies utilizing the extrusion-based bioprinting with the purpose of cell printing (Almeida et al., 2014; Horva´th et al., 2015), 3D cell culture (Yu, Zheng, Chen, Chen, & Hu, 2014), disease modeling, and cancer research (Faulkner-Jones et al., 2015; Gebeyehu et al., 2021). In addition to the bioprinting strategies, bioprinting parameters are also crucial in performing successful bioprinting of bioink materials (Webb & Doyle, 2017). They are classified into three major groups; (1) process parameters which include pressure, distance, flow rate, laser spot diameter, tip diameter, layer height, etc.; while concentration, viscosity, and crosslinking are included in (2) bioink parameters; and the third group includes (3) environmental parameters, like temperature. Process parameters for bioprinting applications vary for each bioprinting strategy. In LAB, volume of the printed material depends directly on the laser pulse energy although laser wavelength, laser beam focus diameter, focal distance, and velocity of the scanning mirrors are parameters affecting printing. During printing via LAB, the amount of energy required to provide microdroplet ejection to occur is known as the threshold energy and should be in range of 120 μJ/pulse (Guillemot, Souquet, Catros, & Guillotin, 2010). In droplet bioprinting, due to thermal actuation that is achieved by a heating element, and vapor bubble that is obtained from the temperature increase, the temperature is the most important process parameter that affects the resolution. For piezoelectric droplet bioprinting, a voltage is applied to the piezoelectric material, and by this way a droplet is formed. Therefore by changing the applied voltage, the size and shape of the printed droplet can be tuned (Cui, Nowicki, Fisher, & Zhang, 2017). On the other hand, extrusion pressure, nozzle diameter, flow rate, and speed of movement are the most influential process parameters for resolution of extrusion-based bioprinting. The volume of extruded bioink raises with increasing pressure and flow rate. Reduction in nozzle diameter and increase in movement speed results in a decrease in ejected bioink volume and increase in resolution (Ng, Yeong, & Naing, 2016; Webb & Doyle, 2017). Bioink is the third and most important component of bioprinting process. There are two types of bioinks classified; scaffold-free bioink which includes only cell aggregates and biological molecules, and scaffold-based bioink which contains synthetic or natural polymeric material for encapsulation of cells (Hospodiuk et al., 2017).

8.2 Bioinks Bioinks serve as a scaffold and can carry living cells, biochemical factors, extracellular matrix (ECM) components, as well as proteins. Success of the bioprinting

8.2 Bioinks

mostly depends on the properties of bioinks. Bioink properties, bioink concentration, rheological properties of bioink, viscosity and surface tension are studied under solution parameters for bioprinting applications. The viscosity of the bioink can be in a range between 30 mPa/s and 60 3 107 mPa/s (Mandrycky, Wang, Kim, & Kim, 2016) where the viscosity range of the bioink should be 1300 mPa/s for LAB, and 3.512 mPa/s for droplet bioprinting to avoid clog formation through the nozzle and tip. In extrusion-based bioprinting applications, non-Newtonian fluids whose viscosity depends on the shear rate are preferred. Hydrogels that have shear thinning property become extrudable via the alignment of polymer chains by pressure application (Jungst, Smolan, Schacht, Scheibel, & Groll, 2016). These extrudable shear-thinning hydrogel bioinks can preserve their shape and postprinting fidelity. Increase in hydrogel concentration causes an increase in viscosity, which results in an increase in printing consistency and leads to a more controllable printing of fine filaments by increased printing pressure (Paxton et al., 2017). However, an increased concentration and viscosity can cause clogging in the nozzle of the extruder (Kyle, Jessop, Al-Sabah, & Whitaker, 2017). On the other hand, low viscosity and concentration may cause free flowing of bioink from the nozzle without application of pressure and may lead to soft constructs that cannot sustain their shape after printing (Duan, Kapetanovic, Hockaday, & Butcher, 2014). Therefore the viscosity of the bioink should be in the proper range as aforementioned. Bioinks should also meet some other requirements depending on the applied bioprinting strategy. As summarized in Fig. 8.2, bioinks should have shear thinning and thixotropic behavior for extrusion-based bioprinting applications. Low surface tension and low adhesion properties are required for bioink to be bioprintable as a continuous filament. Rapid gelation and shape retention abilities are also needed to retain the designed structure postprinting (Hospodiuk et al., 2017). In droplet bioprinting, low viscosity and nonfibrous nature are important

FIGURE 8.2 Bioink properties and requirements for different bioprinting strategies (Reprinted by copyright permission from, Hospodiuk et al., 2017).

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requirements for the bioink that is going to be used, to eject droplets and prevent continuous filament formation (Gudapati, Dey, & Ozbolat, 2016; Hospodiuk et al., 2017). The bioink also should have rapid gelation and moderate surface tension properties so that the surface tension of bioink could be overcome for easy droplet formation. These properties make a bioink extrudable under low pressure, so that the cellular viability would not be affected by the application of high pressure. On the other hand, fast gelation mechanism to solidify without causing any spreads is the most important property of the bioink that is required for the LAB strategy. Bioink should also have the ability to adhere onto the intermediate layers, and it should possess low surface tension and viscoelasticity (Wang, Jin, Dai, Holzman, & Kim, 2016). Compared to other strategies the main difference in LAB is the bioink that should have the ability to absorb the kinetic energy supplied by laser beam pulsation. In the case of photopolymerization involved LAB applications, bioink should be photocurable. Moreover, it is crucial for the photo-initiator to be nontoxic and water-soluble to conveniently use the bioink for tissue engineering applications. Bioink should also possess the proper stability and the appropriate mechanical strength for the intended use (Hospodiuk et al., 2017). In addition to each aforementioned property, bioinks should be soft and fluid enough to be manipulated easily during printing process, and hard enough to preserve the printing pattern after printing (Leberfinger et al., 2019). In addition to this, bioink must also promote cell adhesion, be nontoxic, biocompatible, insoluble during cell culture. Depending on the application, biodegradability of the bioink might also be an important property in this context. Besides, it is important that the bioink is easily processed, cost-effective, and commercially available (Hospodiuk et al., 2017). In 3D bioprinting applications, hydrogels that are natural or synthetic polymeric structures are one of the most common bioink materials. The choice of hydrogel to be used as a bioink depends on the rheological properties of the hydrogel and the bioprinting strategy (Hospodiuk et al., 2017). Hydrogel bioinks will be discussed in detail in the following section.

8.3 Hydrogel bioinks Hydrogels are 3D, crosslinked, network-like polymeric structures (Ahmed, 2015). Either chemical or physical crosslinking can be achieved by formation of covalent bonds or physical cohesion forces such as hydrogen bonding, van der Waals forces, or hydrophobic interactions. Hydrogels are classified into two categories as chemical and physical hydrogels based on their crosslinking features (Maitra & Shukla, 2014). They can absorb great amounts of water compared to their mass but remain insoluble because of their crosslinked nature (Peppas, Bures, Leobandung, & Ichikawa, 2000). Due to these desirable properties

8.3 Hydrogel bioinks

(e.g., high amount of water holding capability, biocompatibility, tunable biodegradability, porous structure, elasticity, etc.), hydrogels have great potential to be utilized in biological applications, especially in tissue engineering (Hong et al., 2020; Serafin, Murphy, Rubio, & Collins, 2021; Su et al., 2021) and drug screening studies (Gebeyehu et al., 2021; Monteiro, Gaspar, Ferreira, & Mano, 2020; Xie, Gao, Fu, Chen, & He, 2020). Hydrogels can be classified into two main groups as natural and synthetic, based on polymer sources (Ahmed, 2015). Natural hydrogels are derived from natural polymers or decellularized tissues. They have many advantages such as biocompatibility, biodegradability, and low toxicity (Catoira, Fusaro, Di Francesco, Ramella, & Boccafoschi, 2019). Hydrogels derived from natural polymers are commonly preferred in tissue engineering and regenerative medicine applications, since they have similar chemical/biological content and physical structure of ECM as human tissues (Vieira, da Silva Morais, Silva-Correia, Oliveira, & Reis, 2017). Natural polymers can be grouped as protein-based polymers and polysaccharide-based polymers. While, collagen, gelatin, elastin, fibrin, and silk fibroin are common examples of protein-based natural polymers, alginate, glycosaminoglycans (GAGs), chitosan, and cellulose can be given as examples of polysaccharide-based polymers (Catoira et al., 2019). Beside the natural ones, synthetic polymer-based hydrogels are widely studied in the literature, due to their tunable properties (Koksal et al., 2020). Compared to natural hydrogels, the most important difference is that synthetic hydrogels can be engineered to have the desired mechanical features depending on the application (Chai, Jiao, & Yu, 2017). Considering the properties of natural and synthetic hydrogels individually, both have advantages and disadvantages. To overcome the disadvantages, composite hydrogels that are composed of two or more polymers, are frequently utilized. When different polymers are combined as composite hydrogels, the composite hydrogel may exhibit different features and different physicochemical properties than the individual polymers that are blended. Therefore, composite hydrogels are mostly preferred to obtain desired features for some specific applications (Buwalda, 2020; Rodrı´guez-Rodrı´guez, Espinosa-Andrews, VelasquilloMartı´nez, & Garcı´a-Carvajal, 2020). Hydrogels are complex materials, which consist of solid (a crosslinked polymer network) and liquid (water) components. The amount of the solid and the liquid in hydrogel content affects the final features of the hydrogel. Therefore water-holding capacity is one of the most important properties of a hydrogel. Hydrogels are generally defined by their degree of swelling when exposed to water. Hydrogel swelling depends on polymerwater interactions. Fundamentally, more hydrophilic polymers form stronger interactions with water. There are three forces that are important to expand the hydrogels network; polymerwater interactions, electrostatic interactions, and osmotic pressure which are called swelling forces. Basically, swelling of a hydrogel can be defined as limited solubility. Elastic forces, which originate from the network crosslinking, prevent the complete dissolution of the polymer. The balance between elastic forces and

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swelling forces determines the equilibrium which controls the hydrogel’s swelling. Swelling properties of a hydrogel vary based on many factors such as polymer source, network density, nature of the solvent, polymer solvent interactions, etc. The swelling capacity of a hydrogel depends on the amount of space between polymer chains in the hydrogels’ network that are available to accommodate water. The amount of this space can be regulated by crosslink density. Crosslink density is determined by the concentration of the crosslinker that is used in the crosslinking process. Using high concentration of the crosslinker in the crosslinking process would cause shorter distances between two crosslinks on the same polymer chain. Therefore the amount of space for holding water would decrease with the increasing crosslink density (Peppas, 2010). Swelling of hydrogels are generally expressed as swelling ratio by the following equation: Sð%Þ 5 Wt W20W0 3 100, where W0 is the dry weight of the hydrogel (initial weight) and Wt is the weight of swollen gel at given time (t) (Imren, Gu¨mu¨s¸derelio˘glu, & Gu¨ner, 2006). Mechanical properties are also important parameters for the final hydrogel product. Hydrogels should have mechanical features that would let them maintain their physical structure for a certain period of time during a specific application. Hydrogels are highly popular biomaterials in biomedical applications due to their high water holding capacity, softness, and elasticity (Calo´ & Khutoryanskiy, 2015). However, they have weak mechanical properties, especially in their swollen state (Chen et al., 2016). To improve mechanical properties of hydrogels, several methodologies had been used. For example, desired mechanical strength could be achieved by tuning the degree of crosslinking. Increasing the crosslinking density makes the hydrogel stronger. However, if the crosslinking density is too high, it decreases the elongation property of the hydrogels and causes it to have a more brittle structure (Mishra, Rani, Sen, & Dey, 2018). Another strategy to improve mechanical properties of hydrogels is to blend them with other materials such as nanoparticles (Dannert, Stokke, & Dias, 2019) and nanofibers (Hassanzadeh et al., 2016). Zaragoza et al. used silica nanoparticles to improve mechanical properties of polyacrylamide hydrogels. Their composite system showed that interaction between nanoparticles and polymers resembles a pseudo crosslink. This polymer-nanoparticle interaction provided an increase in elastic modulus and enhanced the mechanical properties of the hydrogel (Zaragoza, Fukuoka, Kraus, Thomin, & Asuri, 2018). Jang et al. used nanofibers to improve strength and durability of hydrogel. In that study nanofibers were electrospun into thin hydrogel solution. Results showed that the comprehensive strength and stiffness of nanofiber-reinforced hydrogel were enhanced to B221% and B434% compared to pristine hydrogel (Jang, Lee, Seol, Jeong, & Cho, 2013). On the other hand, interpenetrating polymer networks (IPNs) are commonly used to provide mechanical strength to hydrogel. Basically, IPNs are defined as a composite structure that is formed by two or more polymer networks interlaced with each other (Karak, 2009). Suo et al. designed an IPN by using GelMA and chitosan hydrogels to improve the mechanical properties. Characterization results of this study showed that IPN structure improved the mechanical features of

8.3 Hydrogel bioinks

GelMA-chitosan composite compared to the pristine chitosan or GelMA (Suo et al., 2018). Biocompatibility is another important feature of hydrogels. It is important to maintain biocompatibility while fabricating materials for biomedical applications. One of the most common and clear definitions of biocompatibility is the ability of a material to perform within an appropriate host response in a specific application (Williams, 1987). To investigate the biocompatibility of materials, several tests can be performed including in vitro cell viability assays, animal trials, etc. (de Moraes Porto, 2012). Biocompatibility of hydrogels is extremely important for tissue engineering and regenerative medicine applications due to the interaction of natural tissue and hydrogels. In crosslinking step sometimes harsh and toxic chemicals are used as a crosslinker, which affects biocompatibility of the hydrogel adversely. Moreover, initiators, organic solvents, stabilizers, emulsifiers, and unreacted monomers which are utilized in polymerization and hydrogel synthesis may be toxic to the host. In this case purification step becomes crucial to remove those toxic residuals from hydrogels (El-Sherbiny & Yacoub, 2013). Biodegradation of hydrogel is a key parameter for the intended use. In tissue engineering applications, controlled biodegradation is mostly expected from hydrogel scaffolds. Hydrogel scaffolds should degrade simultaneously with the secretion of native ECM from cells. Scaffolds provide mechanical stability to cells during tissue formation. If the scaffold degrades before the formation of ECM, cells lose their supporting material and cannot form the expected 3D tissue structure properly (Madduma-Bandarage & Madihally, 2021). Biodegradation behavior of hydrogel scaffolds can be tuned by blending two or more biopolymers that have different degradation rates. Moreover, crosslinking density is a significant factor, which influences the biodegradability of hydrogels. High crosslinking density decreases the biodegradation rate of the hydrogels (Caliari & Harley, 2011). Rapid gelation ability of a hydrogel is an important parameter for printability as it was for bioinks and can be achieved by physical or chemical crosslinking. Physical crosslinking ability eases the gel formation by reducing the risk of chemical residues and does not affect the biocompatibility of the hydrogel. Physical crosslinking of hydrogels can be achieved by self-assembly, hydrophobic interactions, and hydrogen bonding. It provides proper environment for gelation and cell encapsulation (Jungst et al., 2016). Ionic crosslinking is the formation of hydrogel network electrostatically in the presence of opposite ionic charges. Ionic crosslinking is a reversible type of crosslinking mechanism. In addition, hydrogen bonding and hydrophobic interactions can be manipulated by changing the temperature of the hydrogel. The viscosity and rheological behavior of gels can be changed by increasing or decreasing temperature by this manner. Some gels start to show organized chain conformation with decreasing temperature and develop physical gel properties (Janmaleki et al., 2020). On the other hand, in some gels that have hydrophilic and hydrophobic parts, increasing temperature favors the gelation, since hydrophilic parts dissolve at lower temperatures the viscosity of the gel decreases. For these gels, an increase in temperature causes the polymeric

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interactions to be stronger than hydrophobic interactions, and this results in the gelation of hydrogel (Rodrı´guez-Herna´ndez, Pe´rez-Martı´nez, Gallegos-Infante, Toro-Vazquez, & Ornelas-Paz, 2021). The other gelation mechanism of hydrogels is chemical crosslinking, which is achieved by covalent bonding, and provides more mechanical strength to the hydrogels. Crosslinkers or photo-initiators are generally used for chemical crosslinking. However, the presence of crosslinkers can affect the biocompatibility of hydrogels, which is a highly undesired property especially for tissue engineering applications (Naahidi et al., 2017). A higher crosslinker and photo-initiator concentration increase the mechanical strength of crosslinked hydrogel and toxicity while decreasing the swelling property. By controlling all parameters, proper crosslinker and photo-initiator concentration can be tuned, and cytotoxicity can be decreased (Fedorovich et al., 2009; Hennink & van Nostrum, 2012). Hydrogels to be used as bioink should have some of the defined features as described above. In bioprinting applications of tissue engineering, as illustrated in Fig. 8.3, natural, synthetic, and hybrid hydrogels have been commonly utilized

FIGURE 8.3 (A) Natural hydrogel bioinks: bioprinted alginate hydrogel bioink as a hollow cylinder (left); bioprinted hyaluronic acid before crosslinking (right); (B) Synthetic hydrogel bioinks: bioprinted pluronic F127 hydrogels; (C) Hybrid hydrogel bioink: Lattice structure of chitosan modified with meth acryloyl groups (CHIMA) and acrylamide (AM). (A, left) Reprinted by copyright permission from Gao, He, et al. (2015); (A, right) Reprinted by copyright permission from Kiyotake et al. (2019); (B) Reprinted from Suntornnond et al. (2016); (C) Reprinted by copyright permission from He et al. (2021).

8.4 Applications of hydrogel bioinks

during last years (Arslan-Yildiz et al., 2016). Gao and coworkers used alginate hydrogels as bioink for bioprinting of tubular structures that are based on the fusion of hollow fibers (Fig. 8.3A, left). According to mechanical tests and cell viability results, the bioprinted structures produced by hollow alginate filaments were promising to overcome drawbacks about nutrient delivery in tissue engineering applications (Gao, He, Fu, Liu, & Ma, 2015; Gao, Schilling, et al., 2015). In a study by Kiyotake et al., hyaluronic acid (HA) hydrogels were used to characterize the printability of bioink, based on different properties including viscosity, yield stress, and storage modulus recovery. Fig. 8.3A (right) illustrates the printed hydrogel structures with different concentrations of HA (8% and 10%, respectively). As shown in Fig. 8.3A, polymer concentration had affected the printability of bioink. While the bioprinted structure that included 8% HA had clean and defined edges, the structure that produced by 10% HA bioink had sharper, fractured, and irregular edges (Kiyotake, Douglas, Thomas, Nimmo, & Detamore, 2019). In addition to natural hydrogels, synthetic hydrogels are commonly used as bioink material in literature (Aydogdu et al., 2019; Kim, Kim, Hong, Park, & Park, 2021; Piluso et al., 2021). Fig. 8.3B shows bioprinted pluronic F127 hydrogel structures. The main goal of this proof-of-concept paper is to investigate the effects of printing parameters on the properties of a simple and accurate model and to contribute to the development of new bioinks (Suntornnond, Tan, An, & Chua, 2016). Besides the usage of natural and synthetic hydrogels individually, hybrid hydrogels are used to improve properties of bioinks, such as shape fidelity and mechanical strength. He and coworkers modified chitosan by using methacryoyl group and combined chitosan with acrylamide hydrogel to improve poor mechanical stability. Mechanical strength and stability could have been tuned by changing the concentration of acrylamide. As depicted in Fig. 8.3C, acrylamide provided elasticity to the bioprinted structures and they were not damaged when they were stretched (He et al., 2021). In another study, Bednarzig and coworkers developed a promising bioink that consisted of alginate di-aldehyde and gelatin for biofabrication of colorectal tumor-like structures. Developed hybrid hydrogel bioink provided mechanical strength and better cell attachment to the scaffolds (Bednarzig et al., 2021). The aforementioned studies and more are summarized in Table 8.1.

8.4 Applications of hydrogel bioinks Bioprinting technology has a wide-range utilization field like tissue engineering and regenerative medicine (Aljohani, Ullah, Zhang, & Yang, 2018; Gao & Cui, 2016), cancer research (Chaji, Al-Saleh, & Gomillion, 2020), high-throughput assays (Mazzocchi, Soker, & Skardal, 2019), transplantation (Ozbolat, 2015; Ravnic et al., 2017), and drug screening (Lee, Abelseth, De La Vega, & Willerth, 2019; Ma et al., 2018) as illustrated in Fig. 8.4. Printing of designed scaffold and

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Table 8.1 Hydrogels used as a bioink for 3D bioprinting. Material

Bioprinting strategy

Gelatin methacryloyl (GelMA) Poly 3,4ethylenedioxythiophene: Polystyrene sulfonate (PEDOT:PSS)

Pneumatic extrusion 3D bioprinting

Tyramine-modified poly (γ-glutamic acid) [hyaluronic acid (HA), alginic acid, cellulose nanofibrils, methylcellulose] Pluronic F127

Extrusion-based 3D printing

Pneumatic extrusion-based bioprinting

Chitosan Acrylamide

Laser assisted bioprinting

Polyethylene glycol (PEG)

Extrusion-based bioprinting

Chitosan Guar gum

Extrusion-based 3D bioprinting

Gly-Arg-Gly-Asp (GRGD) peptide modified Pluronic-F127 hydrogel Phenylboronic acidmodified laminarin Alginate

Self-fabricated extrusion-based 3D bioprinting system Dual-head extrusion 3D bioprinter

Advantages

References

• Tunable mechanical property • Tunable conductivity • High resolution • High cytocompatibility • Promising for complex structures • High printability • High cytocompatibility • High mechanical strength

Spencer et al. (2019)

• Model for developing novel bioinks • Proof of concept • High mechanical strength and stability • High cytocompatibility • Smooth and uniform cylindrical strand fabrication • High shape fidelity • High cell viability • Well defined cell morphology after bioprinting • Promising to fabricate complex 3D structures • Good printability • Potential to be used in the field of the health care applications • Stable printability • Solid configuration • High cell viability

Suntornnond et al. (2016)

• Bioprinting under physiologically relevant conditions

Amaral, Gaspar, Lavrador, and Mano (2021)

Kim et al. (2021)

He et al. (2021) Piluso et al. (2021)

Cleymand et al. (2021)

Chen et al. (2021)

(Continued)

8.4 Applications of hydrogel bioinks

Table 8.1 Hydrogels used as a bioink for 3D bioprinting. Continued Material

Bioprinting strategy

Alginate Gelatin

Inkjet bioprinting

PEG GelMA

Inkjet bioprinting

Alginate

Extrusion-based bioprinting

HA

Extrusion-based bioprinting Extrusion-based bioprinting

Gellan gum Poly (ethylene glycol) diacrylate (PEGDA)

Alginate di aldehyde Gelatin

Extrusion-based bioprinting

Gelatin Alginate

Extrusion-based bioprinting

HA

Extrusion-based bioprinting

Advantages • Suitable rheological properties for bioprinting • Homogeneous cell distribution (postprinting) • Good printability • High cell viability • Potential to mimic native tissue • Improved mechanical properties • Precise cell deposition • Layer-by-layer approach without extra steps for cell encapsulation • Potential for nutrient delivery • High cell viability • High mechanical strength • Proof of concept • Excellent printability with living cells • Improved mechanical strength • Decreased shear stress (shear thinning property) • High mechanical strength • Improved cell attachment • Controllable cell orientation and differentiation • Rapid stabilization of printed filaments • Bioprinting without supporting material

References

Jiao, Lian, Zhao, Wang, and Li (2021) Gao, Schilling et al. (2015)

Gao, He et al. (2015)

Kiyotake et al. (2019) Wu et al. (2018)

Bednarzig et al. (2021)

Distler et al. (2020) Ouyang, Highley, Rodell, Sun, and Burdick (2016)

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FIGURE 8.4 Application areas of bioprinting technology. Compiled by copyright permission from A´vila, Schwarz, Rotter, & Gatenholm (2016), Kim et al. (2020), Park et al. (2017), Si et al. (2019), Xie et al. (2021), Axo A2, Axolotl Biosystems Ltd, Turkey.

controlled positioning of the cells overcome most of the disadvantages of other scaffold production methodologies (Ozbolat, Peng, & Ozbolat, 2016). Bioprinting of living tissue is easily and more practically achieved by using hydrogel-based bioinks. The convenience of hydrogels for each bioprinting strategy and encapsulation of cells make them proper bioink material for bioprinting of functional tissues, tumor models, and tissue grafts (Sun et al., 2020). Bioprinting of tissue-like structures have been successfully performed for years. In this section, the applications of bioprinted hydrogels will be discussed for tissue engineering and drug screening purposes. The creation of functional tissues or organs can be achieved by using scaffoldfree or scaffold-based techniques. In bioprinting applications, both techniques can

8.4 Applications of hydrogel bioinks

be performed by using the proper bioink. Cell pellets, aggregates, and tissue strands are used for bioprinting of scaffold-free constructs (Hospodiuk et al., 2017). On the other hand, hydrogel bioinks are utilized to create scaffolds for cell seeding and create living tissue consisting of bioprinted cells and scaffolds by encapsulating cells into bioink. In literature, as given in Fig. 8.5 there are varied applications of 3D bioprinted hydrogel for bone (Abdollahiyan, Oroojalian,

FIGURE 8.5 (A) Bioprinting of alginate bioink for bone tissue engineering application (right), micro-CT image of bioprinted 3D cell-laden mineralized scaffold (left); (B) Bioprinting of nanofibrillated cellulose and alginate for patient-specific auricular cartilage regeneration (left), 28-days culture of human nasal chondrocytes-laden hybrid bioink (right); (C) 3D bioprinted facial skin reconstruction by using polyurethane (PU), keratinocyte and fibroblast-laden hydrogels composed of hyaluronic acid (HA), glycerol, gelatin, and fibrinogen; (D) bioprinted contractile cardiac tissue composed of fibrin-based composite hydrogel bioink; (E) transected rat spinal cord, and bioprinted iPSC-derived spinal NPCsladen scaffold for spinal cord regeneration by using alginate and fibrin-based bioink, and its top and side-view images. (A) Reprinted by copyright permission from Zhang et al. (2020); (B) Reprinted by copyright permission from A´vila et al. (2016); (C) Reprinted by copyright permission from Seol et al. (2018); (D) Reprinted by copyright permission from Wang et al. (2018); (E) Reprinted by copyright permission from Joung et al. (2018).

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Mokhtarzadeh, & de la Guardia, 2020; Salah, Tayebi, Moharamzadeh, & Naini, 2020), cartilage (Daly et al., 2017; Raut, Agrawal, Bagde, Fulzele, & Syed, 2021; You, Eames, & Chen, 2017), cardiac (Liu et al., 2021; Wang et al., 2021), neural (Lee et al., 2018; Yu, Zhang, & Li, 2020), skin (Weng et al., 2021), and vascular tissue engineering (Zhang & Khademhosseini, 2020) and listed in Table 8.2.

8.4.1 Bone tissue engineering 3D bioprinting is an advantageous methodology for production of anatomically designed tissue constructs to replace bone and trigger remineralization. The bioink is expected to possess osteoinductive and osteoconductive properties. In literature, there are varied studies reporting the bioprinting of hydrogels for bone tissue regeneration (Abdollahiyan et al., 2020). Demirtas et al., bioprinted MC3T3-E1 preosteoblast cells by encapsulating them into chitosan and nanostructured hydroxyapatite (HA) composite hydrogel. The rheological behavior of chitosan and HA were suitable for extrusion-based bioprinting and bioink induced mineralization, providing good mechanical property, and increased cellular viability. Storage modulus was 874 kPa and elastic modulus was 14.97 6 3.99 kPa which is in a suitable range for bone tissue engineering (Demirta¸s et al., 2017). In another study, Bendtsen and coworkers also bioprinted a composite hydrogel consisting of alginate, polyvinyl alcohol (PVA) and HA by using extrusion-based bioprinting. Viscoelastic properties of alginate/PVA/HA composite hydrogel resulted in high shape-fidelity while alginate and HA protected cells lead to increased cell viability. It is concluded that the developed bioink has the potential to heal bone defects (Bendtsen et al., 2017). ECM derived materials and bioinks were also commonly used for bioprinting of bone tissues. For instance, ECM-derived HA was methacrylated and bioprinted by Poldervaart et al. In this work, photo-crosslinking of bioink was performed and human bone marrow-derived mesenchymal stromal cells (MSCs) were encapsulated. The fabricated construct had induced osteogenic differentiation, and MeHA bioink was appeared to be a promising bioink for fabrication of bone substitutes (Poldervaart et al., 2017). In a different report, Zhang and coworkers bioprinted alginate and gelatin by encapsulating human mesenchymal stem cells (hMSCs) into bioink to fabricate 3D bone-like tissue (Fig. 8.5A). They assessed the osteogenic differentiation of stiff (1.8% alginate) and soft (0.8% alginate) scaffolds and found that increased concentration of alginate makes the scaffold softer and induces osteogenic differentiation. The soft scaffold also provided increased cellular viability and these features made it a suitable bioink for bioprinting of cell-scaffold complexes for bone tissue engineering applications (Zhang et al., 2020).

8.4.2 Cartilage tissue engineering Cartilage tissue is a type of connective tissue that does not have self-renewal ability due to lack of blood vessels and limited cellular density. Therefore any

Table 8.2 Hydrogel bioinks for tissue engineering applications.

Bone tissue engineering

Cartilage tissue engineering

Bioink material

Bioprinting strategy

Gelation mechanism

Cell line

Advantages

References

Chitosan Nanostructured Hydroxyapatite (HA)

Extrusionbased bioprinting

Temperature dependent gelation

MC3T3-E1 preosteoblast cells

Demirtas, ¸ Irmak, and ˘ Gümüsderelio ¸ glu (2017)

Alginate Polyvinyl alcohol HA

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Mouse calvaria 3T3-E1 (MC3T3) cells

Methacrylated hyaluronic acid (MeHA) Alginate gelatin

Extrusionbased bioprinting Extrusionbased bioprinting

Photo-crosslinking (Irgacure 2959)

Human bone marrow derived mesenchymal stromal cells (MSCs) Human mesenchymal stem cells (hMSCs)

• Induce mineralization • Good mechanical property • Improve cell viability • High shape fidelity • High cell viability • Induce bone defect healing • Induce osteogenic differentiation • Induce osteogenic differentiation • Increase cell viability

Zhang et al. (2020)

Nanocellulose Alginate

Extrusionbased bioprinting

Markstedt et al. (2015)

Collagen Agarose Alginate

Extrusionbased bioprinting

CaCl2

Primary chondrocytes from articular cartilage of newborn Sprague Dawley

Chitosan

Extrusionbased bioprinting

Air drying Warm drying Vacuum drying (NaOH)

ATDC5 (Mouse teratocarcinoma cells)

• Improve shear thinning properties • Provide shape fidelity • Printable at room temperature and low pressure • Improve mechanical strength • Increase cell proliferation • Proof of concept

Temperature dependent gelation and ionic crosslinking (CaCl2) CaCl2

Human nasoseptal chondrocytes (hNC)

Bendtsen, Quinnell, and Wei (2017) Poldervaart et al. (2017)

Yang et al. (2018)

Sadeghianmaryan et al. (2020)

(Continued)

Table 8.2 Hydrogel bioinks for tissue engineering applications. Continued

Cardiac tissue engineering

Bioink material

Bioprinting strategy

Gelation mechanism

Cell line

Advantages

References

Nanofibrillated cellulose Alginate (A)

Extrusionbased bioprinting

CaCl2

Human nasal chondrocytes

Ávila et al. (2016)

GelMA Fibronectin Laminin Collagen methacrylate (ColMA) Alginate PEG Fibrinogen (PF)

Extrusionbased bioprinting

Photo-crosslinking

Human induced pluripotent stem cell

• Facilitates the biofabrication of patientspecific constructs • Provide homogenous cell distribution • Holds excellent shape postprinting • Leads to redifferentiation of hNCs • Induce the differentiation of hiPSCs into cardiomyocytes • Increase cell viability

Extrusionbased bioprinting

CaCl2 ionic crosslinking Photo-crosslinking

• Perfect shape fidelity • Repeatable construction • High precision

Maiullari et al. (2018)

Fibrinogen Gelatin HA

Extrusionbased bioprinting



Human umbilical vein endothelial cells (HUVECs) and iPSC-derived cardiomyocytes Primary cardiomyocytes from infant rat hearts

• Provide uniformity • Provide contractile and beating features • High electrical conductivity

Wang, Lee, Cheng, Yoo, and Atala (2018)

HA Alginate

CaCl2

Human embryonic kidney 293 (HEK-293) cells

Kupfer et al. (2020)

Rastin et al. (2020)

Neural tissue

Skin tissue engineering

Ti3C2 MXene nanosheets

Methylcellulose Alginate Gelatin Chitosan

Vascular tissue engineering

Polyurethane HA Glycerol Gelatin Fibrinogen GelMA PEGDA Nanosilicates GelMA Sodium alginate 4-arm poly (ethylene glycol)-tetraacrylate

Extrusionbased bioprinter

Extrusionbased bioprinting Extrusionbased bioprinting

Gallium nitrate

Human foreskin fibroblast cells

pH-dependent crosslinking (NaOH)

Human foreskin fibroblast cells

Extrusionbased bioprinting

Thrombin solution

Extrusionbased bioprinting Coaxial extrusion bioprinting

• • • • • • •

Low toxicity High biodegradability Excellent printability High shape fidelity High resolution Antibacterial features High cell viability

Rastin et al. (2021) Ng et al. (2016)

Human epidermal keratinocytes and human dermal fibroblasts

• Multilayered skin structure • High shape fidelity • High resolution • Provide a functional and quick reconstruction of facial skin

UV

Human umbilical vein ECs, human umbilical artery smooth muscle cells

• Provide shear thinning property • Increase cell viability

Gold et al. (2021)

CaCl2 UV

HUVECs, human MSCs

• Support the spreading and proliferation of the cells • Tunable mechanical properties • Potentials in engineering large-scale vascularized tissue constructs

Jia et al. (2016)

Seol et al. (2018)

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damage on it leads to a chronic condition and reduces the life quality of the patient. Autologous chondrocyte implantation, microfracture, and osteochondral grafts are current treatments for cartilage tissue degeneration (Singh, Bandyopadhyay, & Mandal, 2019). Although these treatments provide some improvements for regeneration, they still cannot ensure a complete recovery. There is a need for alternative and promising treatments. Emerging of 3D bioprinting technologies paved a way for creating designed cartilage tissue with proper features. Avila et al. used cell-laden nanofibrillated cellulose and alginate bioink to fabricate artificial auricular constructs (Fig. 8.5B). Results such as providing homogeneous cell distribution, maintaining post printing shape and leading to redifferentiation human nasal chondrocytes confirmed that developed hybrid bioink is promising for auricular cartilage tissue engineering and many other bio´ vila et al., 2016). Markstedt and coworkers, combined medical applications (A nanocellulose and alginate to develop a novel bioink that has a potential to be used in cartilage tissue engineering. In addition to fast crosslinking ability of alginate, nanofibrillated cellulose improved shear-thinning properties that provide shape fidelity while printing. As a result, a novel bioink that is printable at room temperature and low pressure was developed. In this study, optimal ratio of the nanocellulose and alginate was indicated as 80:20 (w/w) based on rheological properties and compression tests, as well as shape fidelity. This bioink formulation was used for bioprinting of chondrocytes, and cytotoxicity results showed that the developed bioink is highly biocompatible and suitable for cartilage tissue engineering (Markstedt et al., 2015). In another study, Yang et al. developed two types of composite hydrogel bioinks with the aim of using them in cartilage tissue engineering. These bioinks consisted of agarose and collagen, and they were used with the purpose of improving mechanical properties of alginate hydrogel. According to characterization results of the bioprinted scaffolds, both polymers showed improved mechanical properties. Also, collagen in the hydrogel provided additional benefits, such as better cell adhesion and increased cell proliferation. In addition to the increase in expression of cartilage specific genes (Acan, Sox9 and Col2a1), it decreased expression of Col1a1 gene, which is responsible for de-differentiation of chondrocytes. This ensures maintaining the phenotypes of cartilage. Due to its bioactivity and mechanical strength, the developed collagenalginate hydrogel was considered as a good bioink candidate for cartilage tissue engineering application (Yang et al., 2018). In the study of Sadeghianmaryan and coworkers, three different drying techniques; air drying, warm drying, and vacuum drying, were used to investigate the effect of crosslinking on mechanical properties of bioprinted hydrogel. Firstly, hydrogel concentration was optimized according to rheological properties and viscosity of varied chitosan hydrogel concentration. Discontinuous flow has been observed at lower concentration while clogging occurred at higher concentration. Mechanical properties are determined by elastic modulus. Based on the mechanical test results, air-dried hydrogel scaffolds had highest elastic modulus for minimum pore size. Also, interconnectivity of pores within air-dried scaffolds was confirmed by SEM analysis and high cell

8.4 Applications of hydrogel bioinks

viability was observed proving the biocompatibility of the scaffolds. According to all aforementioned results, 3D bioprinted chitosan hydrogel scaffolds that were fabricated by air drying method appeared to be the most promising construct for cartilage tissue engineering (Sadeghianmaryan et al., 2020).

8.4.3 Cardiac tissue engineering Creating a fully functional cardiac tissue or complex structure of artificial heart, which has the ability to pump blood is challenging. However, cardiac tissue engineering is progressing recently in terms of modeling cardiac tissue. Cardiac tissue models have started to replace animal models for the discovery of therapeutics. Although 3D bioprinting has limited studies in cardiac tissue engineering, it takes precedence over conventional and advanced manufacturing techniques with higher control during the scaffold production. Kupfer and coworkers tried to develop a bioink that induces the differentiation of hiPSCs into cardiomyocytes and provides high cell viability. Bioink consists of GelMA, ColMA, and noncrosslinked ECM proteins such as laminin and fibronectin. Results proved that ECM proteins included in bioink maintain stem cell proliferation and trigger differentiation. Long-term culturing resulted in the formation of contiguous muscle wall up to 500 μm thickness and the developed bioink was proved to be a suitable candidate for cardiac tissue engineering applications (Kupfer et al., 2020). In another study reported by Maiullari and coworkers, a bioink formulation composed of alginate (ALG, 4%) and polyethylene glycol-fibrinogen (PF, 1% w/w) was used for bioprinting of HUVECs and iPSCs derived cardiomyocytes. Crosslinking of ALG was performed by using CaCl2 solution, while photo-crosslinking was utilized to crosslink PF. Results showed that the developed bioink provided perfect shape fidelity and created myocardial-like tissue structures with repeatable construction and high precision (Maiullari et al., 2018). In another report, Wang et al. developed a contractile cardiac tissue by 3D bioprinting of fibrin-based composite hydrogel bioink that contains fibrinogen, gelatin, and HA dissolved in DMEM. They also used a sacrificial hydrogel and a supporting polymeric frame for bioprinting of a simple aligned cardiac construct (Fig. 8.5D). All bioprinting processes were carried out at 18 C to allow gelation of gelatin. After 3 days from bioprinting, the contractile feature of the cardiac tissue construct was assessed, the synchronous beating was observed starting from the first week, and until the third week contraction force was measured to be about 7.26 mN with the addition of epinephrine. This study has concluded that cardiac tissue models could be created by 3D bioprinting, since it provides cellular organization, uniformity, improved contractile, and beating features than scaffolds fabricated by other methodologies (Wang et al., 2018).

8.4.4 Skin tissue engineering Skin consists of three main layers which are epidermis, dermis, and hypodermis. These layers include different cells and ECM components that are responsible for

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different functions. Obtaining multilayered structure of skin is the main goal of tissue engineering to mimic native skin tissue properly (Vig et al., 2017). Seol et al. used a composite material that consisted of PU, keratinocytes laden hydrogel, and fibroblast laden hydrogel to fabricate a face mask as a skin substitute (Fig. 8.5C). Result of this proof of concept study shows that bioprinted facial skin masks provided functional and quick wound healing and reconstruction of skin (Seol et al., 2018). Collagen is the most abundant protein that is included in the native ECM structure (Shoulders & Raines, 2009). Yet, its poor printability and requirement for toxic solvents are the common challenges of this biomaterial (Diamantides et al., 2017). Ng and coworkers used gelatin that is denatured version of collagen to develop native skin mimetic scaffolds. In this study, negatively charged gelatin was blended with positively charged chitosan polymer to obtain polyelectrolyte-complex hydrogel. Chitosan promoted cell attachment on the scaffolds due to the excessive positive charges, as well as the contribution on the gelation process. As a result, a 3-layered construct (approximately 400 μm) was bioprinted with high resolution and fidelity. This thickness was considered enough to mimic full of the epidermis and a part of the dermis. In addition, cytocompatibility results confirmed that the utilization of gelatin-chitosan hydrogel scaffold in skin tissue engineering applications is convenient (Ng et al., 2016). In addition to skin tissue engineering, bioprinted hydrogels are also used for contributing wound-healing applications. Bacterial infections are the most common and important challenges for wound-healing processes. To eliminate bacterial infection risks, antibacterial tissue scaffolds (Zakeri-Siavashani et al., 2020) and wound dressings (Simo˜es et al., 2018) have been started to be used. Rastin and coworkers developed a novel cell-laden bioink that consists of methylcellulose and alginate hydrogels. In this study researchers used gallium ion as a crosslinker, where gallium ions form ionic interactions between alginate chains to form hydrogel structure. In addition to its crosslinker role, gallium has an antibacterial feature. Therefore, it is highlighted as the key component of this study. Human foreskin fibroblast cell-laden methylcellulose alginate hydrogels were bioprinted and crosslinked in the gallium nitrate solution to produce antibacterial scaffolds. When cytocompatibility results were examined, cell viability of gallium crosslinked scaffolds was very close to cell viability results of calcium crosslinked samples. Considering all characterization results, antibacterial cell-laden methylcellulose alginate bioink was a promising candidate for skin tissue engineering due to its excellent printability, biocompatibility, and antibacterial potential (Rastin et al., 2021).

8.4.5 Vascular tissue engineering As the purpose of tissue engineering is to create a fully functional organ to replace the damaged or lost organs, vascularization has great importance. Creating an organ with the required vasculature could not be possible with conventional scaffold production methodologies (Huang, Li, et al., 2017; Huang,

8.4 Applications of hydrogel bioinks

Zhang, Gao, Yonezawa, & Cui, 2017). In recent years, 3D bioprinting has advanced in vascular tissue engineering and generating vasculature for artificial organs. Gold et al., developed a novel hydrogel-based bioink which consists of GelMA, PEGDA, and nanosilicates. Here, nanosilicates provided electrostatic interactions, and hydrogen bonds were formed between polymer chains and nanosilicates, which resulted in colloidal structure. Generally, GelMA and PEDGA have limitations for 3D printing methodology because of their Newtonian characteristics. In this case, addition of nanosilicates brought shear thinning property and improved the printability of the bioink. In addition, acquired shear thinning behavior prevented damaging of encapsulated human umbilical vein ECs (HUVECs) and human umbilical artery smooth muscle cells (HUASMC) in the bioink during the printing process. Cell viability results indicated that bioprinted cells keep their healthy phenotype and stay viable for approximately 1 month after bioprinting process. The reported cell-laden bioink was able to be bioprinted as anatomically accurate, and results confirmed that bioprinted vascular structure mimicked native human vascular tissue, properly. In conclusion, the bioprinted vascular mimetic structure has a potential to be used as 3D vascular disease model for drug development and screening applications (Gold et al., 2021).

8.4.6 Neural tissue engineering Neurodegenerative disorders, brain, and spinal cord damage are frequently encountered health problems, and modeling these diseases is crucial for understanding their mechanism and providing the required treatment. Bioprinting of hydrogel bioinks is used for modeling spinal cord injury and regenerating the spinal cord as illustrated in Fig. 8.5E (Joung et al., 2018). In addition to this, researchers have tried to develop conductive hydrogel bioinks for varied neural tissue engineering applications (Bordoni et al., 2020; Rastin et al., 2020; Vijayavenkataraman, Vialli, Fuh, & Lu, 2019). Although conductivity is one of the critical features of scaffolds that are used in neural tissue engineering applications, hydrogel bioinks are generally not conductive. Addition of some biomaterials, such as metal nanoparticles, carbon-based nanomaterials, or conductive polymers to the hydrogel bioinks provides electrical conductivity to bioink. Rastin and coworkers developed a novel cell-laden electroconductive bioink for neural tissue engineering applications. Addition of Ti3C2 MXene nanosheets provided electrical conductivity to cell-laden bioink that consists of HA and alginate. MXenes are novel metal carbide nanomaterials that possess large specific surface area, high electrical conductivity, low toxicity, and biodegradability features. Due to these excellent features, they are promising materials for biomedical applications. In the study, Ti3C2 MXene nanosheets were preferred due to their ease of dispersion in aqueous hydrogel solution. According to characterization results, developed electroconductive bioink was electrically conductive besides its excellent printability with high fidelity and resolution. In addition, MXenes improved the mechanical features due to interactions between MXenes and polymers in the

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hydrogel. Cell viability studies indicated that viability of encapsulated human embryonic kidney 293 (HEK-293) cells in the hydrogel bioink is similar to that of in vitro studies that were performed by seeding cell to the hydrogel scaffolds. Considering all results, this study confirmed that the developed bioprinted electroconductive scaffold is an excellent candidate for neural tissue engineering applications (Rastin et al., 2020).

8.4.7 Drug screening For decades, 2D cell culture and animal models have been used for drug screening applications despite their limitations. Although 2D methods are convenient, 2D cultures provide monolayer cell culture that exhibit different cell morphologies, polarity, receptor expression, cell-ECM and cellacell interactions, as well as different chemical and physical properties, when compared to what is observed in vivo (Ravi, Paramesh, Kaviya, Anuradha, & Solomon, 2015). In addition, animal testing has long been used in science to study complex biological phenomena that cannot be investigated using 2D cell cultures. However, it appeared that there are more differences between animal models and human patients because of the vast divergence between their physiologies and anatomies, which brings many uncontrollable variables in the animal experiments. Moreover, experimental complexity, ethical concerns, and high costs are other common issues and disadvantages of animal testing. Considering all these limitations, animal models and 2D cell culture systems are not considered to be as effective as once assumed anymore for disease modeling and drug research. On the contrary, 3D cell culture systems proved to be effectively usable for disease modeling due to their superior properties such as successfully mimicking the cell-cell and cell- ECM interactions and providing relevant drug response compared to in vivo conditions (Chenchula, Kumar, & Babu, 2019). There are conventional and advanced methodologies to produce 3D scaffolds. Many approaches, such as solvent casting with particulate leaching, thermally induced phase separation (TIPS), and freeze drying which can be used to fabricate scaffolds in tissue engineering are considered conventional. Recently, some advanced techniques, such as electrospinning, 3D bioprinting, and combination molding techniques, have been developed to fabricate scaffolds that can mimic the ECM (Zhao et al., 2018). 3D bioprinting method can be used to produce artificial tissues and disease models by fabricating scaffolds with controlled spatial heterogeneity of physical properties, cellular composition, and ECM structure. This methodology has potential to create artificial functional constructs for drug screening and toxicology research (Arslan-Yildiz et al., 2016). In literature, there are several drug screening applications (Table 8.3) that utilized 3D bioprinted constructs. For example, Zhao and coworkers developed a 3D tumor model by using 3D bioprinting methodology. In the study, HeLa cells were used to create a cervical tumor microenvironment and they are encapsulated in the hydrogel bioink that is composed of fibrinogen, alginate, and gelatin. Morphological analyses results indicated that HeLa cells in the bioprinted

Table 8.3 Hydrogels used as bioink for drug screening applications. Bioink material

Bioprinting strategy

Gelation mechanism

Cell line

Drug

References

Fibrinogen Alginate Gelatin Vitrogel

Extrusionbased bioprinting

Thrombin CaCl2

HeLa (cervical tumor cell line)

Paclitaxel

Zhao et al. (2014)

Extrusionbased bioprinting

—

Docetaxel Doxorubicin Erlotinib

Gebeyehu et al. (2021)

Fibrin Alginate Genipin

Extrusionbased bioprinting

Thrombin CaCl2 Chitosan

Lung cancer (NSCLC-PDX, H460, HCC827and A549) and breast cancer (MDAMB-231WT), and bladder cancer (RT4) cell lines U87MG human glioblastoma cells

Lee et al. (2019)

Alginate Gelatin Fibrinogen Thrombin Gelatin Fibrin Pluronic Alginate Pluronic

Extrusionbased bioprinting

Ionic crosslinking (CaCl2) Transglutaminase

Glioma stem cell line SU3 Human glioma cell line U87

Glioblastoma-reprogramming cocktail [Forskolin, ISX9, CHIR99021, I-BET 151, DAPT (FICBD)] Temozolomide

Extrusionbased bioprinting

Thrombin Transglutaminase

Proximal tubule epithelial cells

Resazurin, Cisplatin, Cyclosporine A

Homan et al. (2016)

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Murine C2C12 cells

Cardiotoxin

Gelatin Alginate Matrigelt

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Intrahepatic cholangiocarcinoma cells (ICC)

Sorafenib, Cisplatin, 5Fluorouracil

Mozetic, Giannitelli, Gori, Trombetta, and Rainer (2017) Mao, Yang et al. (2020), Mao, He et al. (2020)

Dai, Ma, Lan, and Xu (2016)

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hydrogel constructs formed spheroid structures. Moreover, 3D bioprinted constructs allowed long-term cell proliferation due to increased cellcell and cellmatrix interactions like real tumor tissue. Furthermore, tumor-related protein expression levels in 3D constructs were found to be higher than 2D culture, and it confirmed that 3D bioprinted constructs successfully mimicked native tumorigenic tissue. To investigate drug resistance of HeLa cells in the 3D constructs Paclitaxel was preferred as an antitumor drug. When it was compared to 2D cell culture, 3D cervical tumor model was more resistant to Paclitaxel and it showed the importance of the dimensionality on drug screening studies (Zhao et al., 2014). Recently another approach reported by Gebeyehu et al., a commercial hydrogel (VitroGel) was modified with the aim of improving printability, rheological characteristics, and stability without requiring crosslinking strategies such as UV exposure, sudden changes in temperature, or chemical reactions. Thus, they overcame the challenges that caused cell death during bioprinting and solidification steps. Characterization results showed that RGD modified hydrogel had the best characteristics with high shape fidelity along with high cell viability. In this study, the efficacies of the docetaxel, doxorubicin, and erlotinib which are anticancer drugs were investigated on various cancer types by bioprinting 3D models of RGD modified hydrogel bioink. Drug screening results revealed that developed 3D bioprinted tumor models were more resistant than 2D cell culture models. According to these results, researchers concluded that the developed bioink was very convenient for 3D bioprinting process due to its rheological features and biocompatibility (Gebeyehu et al., 2021). In another work, Lee and coworkers proposed a novel fibrin-based 3D printed construct for modeling glioblastoma multiforme, which is one of the deadliest cancer types. In the study, a microfluidic print head was used to decrease the effect of shear stress which causes cell death during the bioprinting process. The microfluidic print head allows printing cell-laden bioink and crosslinker simultaneously. To create a glioblastoma multiforme model, U87MG (human glioblastoma) cells were encapsulated into bioink. Live dead assay images were taken shortly after the bioprinting process showed that the cell viability was quite high. These results proved that cells were not harmed by shear stress during the bioprinting process. Also, in the following days, single cells started to form spheroid structures that mimic native tumor structure, and high cell viability was maintained up to day 12. In addition, high expression levels of glioblastoma multiforme markers confirmed the convenience of 3D cancer model. Furthermore, developed cancer model was used for screening of novel glioblastoma multiforme treatment methods, which were only screened by 2D models previously. Drug screening results revealed that developed 3D cancer model was more resistant than 2D cell culture. These results supported that developed 3D printed construct mimicked native tumor structure and it was promising to model other complex diseases for drug development and screening applications (Lee et al., 2019).

8.5 Challenges of bioprinted hydrogels in tissue engineering

8.5 Challenges of bioprinted hydrogels in tissue engineering and drug screening Although there are several advancements for using 3D bioprinting in tissue engineering and drug screening, the development of a perfect bioink is still unmet. Features of a perfect bioink were discussed in detail at Section 8.2. Briefly, bioink materials should have fast gelling features, convenience for simple as well as nontoxic crosslinking mechanisms, and should be capable to be bioprinted with a low amount of pressure application to prevent cell death. Having a proper mechanical property regarding intended use, and good printability are also other features that a bioink should meet (Leberfinger et al., 2019). Hydrogels are commonly used and promising bioink materials in the field of tissue engineering. However, printability analyses of each hydrogel are yet to be performed. As listed in Table 8.2 and Table 8.3, gelatin, GelMA, alginate, and chitosan are the most preferred hydrogels for 3D bioprinting. In literature, many printability studies and their bioprinting parameters are readily optimized for above mentioned hydrogels and their derivatives. There are also various hydrogels whose bioprinting parameters need to be optimized for each bioprinting strategy. Printability of some hydrogels such as collagen is not possible at all without dissolving them in harsh and toxic solvents (Diamantides et al., 2017). Hence, cellular viability is an important challenge in such cases. Viscosity is another critical parameter, but can also be a challenge for bioprinting process. The rheological properties and viscosity of hydrogels have significant importance on printability and shape fidelity. If the viscosity of hydrogel is low, it can flow out from the cartridge; on the contrary, it can clog the tip of the extruder if the viscosity is too high. To prevent this, examination of the rheological properties and viscosity of hydrogels have great importance (Gao et al., 2018). Another challenge is decreasing of cellular viability based on high pressure applied during the bioprinting of cell-laden hydrogels. Extrusion of hydrogel from the cartridge requires application or generation of high pressure, the applied pressure cause stress on cells, and this results in reduced cellular viability. Therefore the ability of the hydrogel to be able to be extruded at low pressure is critical for cell-laden hydrogels (Nair et al., 2009). The mechanical strength of hydrogel bioinks is one of the challenging limitations especially for some specific tissue engineering applications. Regarding the intended use of scaffold, the hydrogel should meet some specific biomechanical properties. However, since hydrogels can retain water and are softer than most of the other synthetic polymeric materials, they are widely used for replacement of soft tissue. The stiffness of hydrogel can be achieved by increasing the crosslinking density and molecular weight (Huang, Li, et al., 2017; Huang, Zhang, et al., 2017). However, it can also result in decreased cellular viability because cells get entrapped into the polymer network, which limits diffusion of nutrients and oxygen (Chimene, Kaunas, & Gaharwar, 2020). To overcome the challenge of low

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mechanical strength, composite bioinks can be preferred. Composites or polymer blends can provide mechanical stability while natural components of these blends provide a proper microenvironment for cell adherence and growth. This can be achieved either by bioprinting of blended composite scaffold or heterogeneous layer-by-layer printing (Mao, He, et al., 2020; Mao, Yang, et al., 2020). Resolution is one of the most important problems encountered in tissue and organ bioprinting with hydrogels. Although LAB has a higher resolution than extrusion-based bioprinting and droplet bioprinting, encapsulation of cells into hydrogel decreases the resolution and this prevents the bioprinting of fine filaments (Ozbolat & Yu, 2013). Despite the final construct having the desired shape, it cannot conserve the shape postprinting and can spread or shrink. To prevent this, the resolution of utilized strategy needs to be improved. Lastly, as in all tissue engineering methodologies, the inability of vascularization creates an important limitation. Maintaining the viability of bioprinted tissue or organ model cannot be possible without vascularization (Ozbolat & Yu, 2013). As a result, bioprinting of hydrogels for tissue engineering and drug screening purpose has still several challenges and limitations, which could be eliminated by further research and experimentation.

8.6 Conclusion and future perspectives Creating fully functional tissue constructs and artificial organ-like structures have great importance for tissue engineering and regenerative medicine applications, also modeling diseases would provide better opportunity to reduce the cost during drug discovery process. 3D bioprinting of hydrogels has numerous advantages for scaffold/tissue fabrication and has great potential in utilization for bone, cardiac, cartilage, neural, skin, vascular tissue engineering, and drug screening as given in Section 8.4. On the other hand, hard processibility of some hydrogels and lack of their optimized bioprinting parameters, low mechanical strength of hydrogel bioinks, low resolution in the case of cell-laden bioink, and the inability of vascularization limits the successful bioprinting of hydrogels for fully functional tissue constructs. Developing new generation bioinks with improved features and creating composite hydrogel scaffolds to provide better mechanical properties and increased cellular viability (Schwarz et al., 2020; You, Chen, Cooper, Chang, & Eames, 2018; Zehnder, Sarker, Boccaccini, & Detsch, 2015) would possibly eliminate the obstacles (Chimene, Lennox, Kaunas, & Gaharwar, 2016) that were explained in the previous section. With the advancements in both bioprinting technology and bioinks, it is expected that clinical applications of 3D bioprinted tissue/organ transplantation dream will come true. In addition, in terms of drug discovery and drug screening, it is anticipated to reduce the cost by creating disease models using the 3D bioprinting approach (Peng et al., 2017). As expected in the tissue engineering perspective,

References

developments in 3D bioprinting technology and hydrogels will pave the way for personalized medicine, reduce the cost for drug discovery. Besides, in recent years, bioinks have started to enter product portfolio of many companies, and it is foreseen that the new generation bioinks to be developed will take their place in the market as in great demand commercial product either for tissue engineering and regenerative medicine studies or transplantation (Guvendiren, 2019; Roskos, Stuiver, Pentoney, & Presnell, 2015).

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Smart polymers for biomedical applications

9

Deepti Bharti1, Indranil Banerjee2, Preetam Sarkar3, Doman Kim4 and Kunal Pal1 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Bioscience & Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India 3 Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 4 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

9.1 Introduction The response toward any external stimulus as a mechanism to adapt to the changing environments is an existing phenomenon for most living systems. With the exact mindset, many polymer scientists are working progressively to search and develop a polymeric system that can behave similarly to the living system. The search has landed researchers in a particular class of polymers with unique chemical and physical properties, which has found applications in various biomedical fields like drug delivery and tissue engineering. These polymers are commonly associated with names like “environment-sensitive polymer,” “smart-polymer,” “stimuli-responsive polymer,” and “intelligent polymers” (Kumar, Srivastava, Galaev, & Mattiasson, 2007). These polymers will be further referred to as smart polymers (SP) for clarity. In simple terms, SPs have the potential to overcome and adapt to even trivial environmental changes. These environmental conditions are a set of thermodynamic constraints like pressure, temperature, pH, and concentration (Galaev & Mattiasson, 2019). Therefore SPs are thermodynamic systems that undergo reversible changes through changes in the chemical structure of phases inside the range of different environmental constraints. The system of SP is exclusive not because of its ability to produce rapid macroscopic changes but the reversible nature of these transitions. Hence, scientists have a vast opportunity to explore polymer chemical structure, compositions, architectures, and environmental conditions before designing a suitable SP for any application. The solubility, degradation, structural and molecular rearrangement, along with the balance of lipophilic and hydrophilic groups of the polymers, are a few more essential considerable information (Ribeiro & Flores-Sahagun, 2019). SP responds to either Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00010-3 © 2023 Elsevier Inc. All rights reserved.

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single or multiple external stimuli like pH, temperature, biological molecules, light, etc. The variation in the polymers and their structural conformations can either occur gradually or abruptly. The response of the polymers against the different stimuli is observed in terms of variation in the macromolecular structure, swelling characteristics, structural collapsing, or simply a solution to gel transition (Aguilar & Roma´n, 2014). The transition from sol to gel is attributed to the hydrophilic group of the polymer, which adsorbs water. It can be seen that the response of SPs is always an effort to correlate with the living systems. This is further supported by the evidence that most polymers have a transitional temperature of B37 C (Galaev &. Mattiasson, 2019). It is the physiological temperature where many biological molecules like proteins, polysaccharides, and nucleotides function typically. The environmental stimuli can be classified majorly into three groups: physical stimuli, chemical stimuli, and biological stimuli, which is also the basis for categorizing SPs (Ribeiro & Flores-Sahagun, 2019). Any physical stimuli will lead to changes in the molecular and structural dynamics of the polymeric system. Changes in response to temperature, pH, magnetic and electric fields, and light are a few examples of physical stimuli. The chemical stimuli will alter the molecular interaction between the polymeric chains or the chain and solvent system. An alteration in the concentration or ionic strength can be considered as chemical stimuli. The physical and chemical stimuli-based SPs are often associated with chemical contamination, cell damage, and low biological specificity (Xu, Liu, & Yan, 2017). A relatively new area of research is with the biological stimuli that will improvise the enzymatic reactions that might help recognize the targeted cell receptors. Sometimes SPs are even designed to understand the influence of more than one physiological or environmental stimuli. Conjugating many natural and synthetic biomolecules often design the SPs to their backbone (Hoffman & Stayton, 2020). These conjugations usually act as targeting signaling molecules or simply as a key for cellular entry. All the environmental alterations mentioned above can be accounted for developing SPs for drug delivery systems and, in fact, substrates for culturing cells for tissue regeneration. Fig. 9.1 represents different stimuli that are often explored for the development of SPs. One of the most explored applications of SPs has been observed in the case of drug delivery. The SP-based micro-/nanocarriers that encapsulate drugs/biologics are used for controlled or targeted delivery (Xu et al., 2017). For drug delivery, many formulations constituting SPs like Bion Tears, Polygel, and Genteal Gel, are already in the market with appreciable performance (Thrimawithana, Rupenthal, Young, & Alany, 2012). Many researchers have recently explored SP to design scaffolds for tissue regeneration in bone, cartilage, etc. (Haryanto & Khan, 2021). SP formulated as gels can also be implanted at the damaged tissue site in a minimally invasive manner. This chapter has made an effort to guide the readers through a complete understanding of the functioning of SP. The main aim will be to summarize the conceptual working of the various environmental stimuli attributed to the polymer’s “smart” behavior. The chapter will cover a wide range of stimuli, that is,

9.2 Temperature-sensitive smart polymers

FIGURE 9.1 Various stimuli for the development of smart polymers.

temperature, pH, light, enzyme, and their specific role in regard to the SP. Additionally, it will cover the recent advancements of the applications of SPs in biomedical, especially drug delivery, tissue repair, and regeneration.

9.2 Temperature-sensitive smart polymers Temperature-sensitive smart polymers (TSSPs) became a topic of discussion in the late 1960s after the first report of thermal phase transition in poly(N-isopropyl acrylamide) (pNIPAAm) (Heskins & Guillet, 1968). Since then, TSSPs have been most explored by polymer scientists, particularly in the field of biomedicine. At a critical solution temperature (CST), these SPs display sensitive behavior through changes in their structural and functional properties. Based upon the response of the polymers, CST may be defined either as lower critical solution temperature (LCST) and upper critical solution temperature (UCST) (Teotia, Sami, & Kumar, 2015). The phase diagram of polymer weight fraction versus temperature (Fig. 9.2) depicts mono-phase and biphase regions which are helpful in the identification of LCST and UCST (Gibson & O’Reilly, 2013). The temperature at

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FIGURE 9.2 Schematics of the phase diagram of polymer solution: (A) lower critical solution temperature and (B) upper critical solution temperature. Reproduced from Gibson, M. I., & O’Reilly, R. K. (2013). To aggregate, or not to aggregate? considerations in the design and application of polymeric thermally-responsive nanoparticles. Chemical Society Reviews, 42(17), 7204 7213. https://doi.org/10.1039/C3CS60035A under Creative Commons license.

which the phase separation takes place is called cloud point, a quantifiable macroscopic event of the transition process (Gibson & O’Reilly, 2013). The LCST depicts the lowest temperature of the phase diagram below which the polymers in aqueous solvent remain monophasic (soluble). On the contrary, the polymeric system displaying single-phase above a specific temperature is classified under UCST. Similar to any process to become thermodynamically stable, the polymer dissolution also requires a negative value for Gibbs free energy (ΔG) (Teotia et al., 2015). For an overall negative value of ΔG (ΔG 5 ΔH 2 TΔS), the term of enthalpy (ΔH) and entropy (ΔS) should also be negative. The negative value of ΔH assists in polymer dissolution and is related to the hydrogen bonding between the polymer and the solvent (Pattanashetti, Heggannavar, & Kariduraganavar, 2017). Similarly, the negative ΔS value comes from the organized arrangement of water molecules around the polymer. A rise in the temperature beyond LCST results in the system’s increased entropy. Hence, the overall value of ΔS exceeds ΔH that leads to the breakage of hydrogen bonds and further separation of phases. In comparison to UCST, the LCST polymers are explored by scientists for the majority of the applications. pNIPAAm, poly(N,N-diethyl acrylamide), poly(N-vinyl alkylamine), and poly(N-vinyl caprolactam) are a few of the commonly used thermo-sensitive polymers (Aguilar & Roma´n, 2014).

9.3 Applications of temperature-sensitive smart polymers The biomedical applications of temperature-sensitive SPs are well explored in drug delivery, tissue engineering, and regenerative medicines. Among various SPs

9.3 Applications of temperature-sensitive smart polymers

under this category, pNIPAAm is majorly explored due to its solubility in aqueous solutions and LCST of 32 C (Mu & Ebara, 2020). The sensitivity of this polymer toward the CST can be correlated to its structure which is made from amide linkage (hydrophilic) and isopropyl portion (hydrophobic) represented in Fig. 9.3 (Yang, Fan, Zhang, & Ju, 2020). The synthesis and functionalization of pNIPAAm occur through the free radical polymerization of monomers. One of the beneficial roles of pNIPAAm occurs in culturing of the mammalian cell. This specific application is required to recover cells from the surface to which they adhere for growth and proliferation. Since the techniques of mechanical scraping or enzymatic treatments may prove harsh for the cultured cells, the development of temperature-sensitive substrates might serve a beneficial role here. The vapor phase deposition of pNIPAAm on polystyrene-coated plate showed adherence of most cell types above the LCST for proper growth and proliferation. However, below the LCST the surface allows the cells to detach due to the hydration of pNIPAAm coating (Canavan, Cheng, Graham, Ratner, & Castner, 2004). The immunological assay revealed that proteins like fibronectin, laminin, and collagen were found to be closely associated with the detached cells. However, some collagen is allied with the surface.

FIGURE 9.3 (A) The molecular structure and mechanism of thermo-sensitivity of pNIPAAm. (B) The schematic representation of thermo-responsive behavior of the stimuli-responsive hydrogel-based drug delivery system. (A) Reproduced from Yang, L., Fan, X., Zhang, J., & Ju, J. (2020). Preparation and characterization of thermoresponsive poly(N-isopropylacrylamide) for cell culture applications. Polymers, 12(2), 389. https://doi. org/10.3390/POLYM12020389 under Creative Commons license. (B) Reproduced from Li, L., He, Y., Zheng, X., Yi, L., Nian, W., & Abadi, P. P. (2021). Progress on preparation of pH/temperature-sensitive intelligent hydrogels and applications in target transport and controlled release of drugs. International Journal of Polymer Science, 2021. https://doi.org/10.1155/2021/1340538 under Creative Commons license.

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The thermoresponsive behavior of SP for drug delivery can be understood through Fig. 9.3B (Li et al., 2021). The closeness of LCST of pNIPAAms to physiological temperature makes them suitable for drug delivery applications. Alginate-g-pNIPAAm copolymers have been studied in this regard for the controlled release of hydrophobic anticancer drugs (Liu, Song, Wen, Zhu, & Li, 2017). The clear idea here is that the temperature-sensitive hydrogel will yield self-assembled micellar structures at 37 C, followed by the dissolution of the polymer in the biological fluid. Thus the drug was released in an encapsulated form in the micelles formed by the polymers, which even showed enhanced cellular uptake when tested in vitro in multidrug-resistant cells. Chitosan is a natural polymer derived from the deacetylation of chitin. It is often united with polymers holding thermoresponsive characteristics for the synthesis SP. Carboxymethyl chitosan (CMCh) modified pluronic gels were studied for localized delivery of paclitaxel with reported LCST of B29 C (Ju, Sun, Zi, Jin, & Zhang, 2013). In the fabricated pluronic gel system, glutaraldehyde-mediated cross-linking took place to generate a stable network with better mechanical strength. The formed hydrogel displayed excellent drug holding ability, sustained release, reduction in hepatic metabolism, and improved anticancer ability. Another application of thermo-sensitive SP is seen in gene therapy. Gene therapy serves as a corrective treatment for defective genes, which can potentially cause different genetic diseases. The significant role will involve the delivery of the therapeutic gene (deoxyribonucleic acid, DNA) to the damaged site, where repair and replacement will occur. The negative charge of the DNA will face hindrance from the negatively charged cellular membrane before it finally reaches the nucleus. Accordingly, the gene needs to be packed in a suitable carrier/vector for its delivery. For most gene-targeted diseases, the temperature is considered a factor for the successful release of the gene from the carrier (Liu, Yang, Xiong, & Gu, 2016). A thermoresponsive nanogel was prepared through radical graft copolymerization of pNIPAAm to PEI. The nanogels were found effective in anticancer gene therapy (Cao et al., 2015). PEI is a low immunogenic polymer. Its cationic nature can assist in condensing DNA molecules in small size, which assists in endocytosis. pNIPAAm enhances the cellular uptake when the temperature rises the LCST. A tumor suppressor gene, that is p53, was efficiently loaded and delivered through the synthesized temperature-responsive PEI/pNIPAAm (Cao et al., 2015). A list of thermoresponsive polymers and their potential applications is represented (Table 9.1).

9.4 pH-sensitive smart polymers The different human tissues and their anatomical location exhibit a variation in the pH. The SPs which respond to the change in the variations of environmental pH are categorized as pH-sensitive SP. These responses occur in terms of

9.4 pH-sensitive smart polymers

Table 9.1 List of few thermoresponsive polymers and their applications. S. No.

Polymer

Polymer type

Therapeutic agent

1.

Cytosinepolypropylene glycol

LCST (35 C)

Doxorubicin

2.

β-Cyclodextrin-g(PEG-vpNIPAAm)

LCST (35 C)

Paclitaxel

3.

Poly(lactide-coglycolide)-b-PEGb-PLGA

LCST (37 C)

4.

Chitosan/ glycerophosphate

LCST (37 C)

Codelivery of Dox, cisplatin, and methotrexate Chondrocytes

5.

Chitosan

Doxorubicin and vaccinia virus vaccine

Application

References

Efficient for drug loading and sustained release when tested for small cell lung cancer cell line Complete inhibition of tumor growth in vivo in mice having a drugresistant tumor Inhibition of tumor growth and tumor necrosis Capable of maintaining high cell viability and expression of cartilagespecific genes Displayed a synergistic antitumor effect when combined with the vaccinia virus

Cheng, Liang, Liao, Huang, and Lee (2017)

Fan et al. (2018)

Ma et al. (2015)

Zhang et al. (2019)

Han et al. (2008)

LCST, Lower critical solution temperature; PLGA, poly(lactic-co-glycolic acid); pNIPAAm, poly(Nisopropyl acrylamide), PEG, polyethylglycol.

modification to the different properties of the polymers like structure, solubility, surface property, etc. (Ofridam et al., 2021). The fundamental structure of a pHsensitive polymer includes an ionizable group that is connected to the hydrophobic polymeric backbone. The pH-sensitive SPs are similar to polyelectrolytes and consist of acidic or basic groups. These acidic or basic functional groups respond to the external environment by accepting or donating the protons, respectively. The elementary categorization of pH-sensitive SPs is done based on the surface charge possessed by these polymers. Broadly, they are categorized into two groups: polyacid polymers consisting of anionic functional groups, and polybasic polymers consisting of cationic functional groups. Some of the common examples of polyacid polymers include poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMA). In contrast, the polymers like PEI and poly

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(dimethylaminoethyl methacrylate) are categorized as polybasic polymers (Roy & De, 2014). Polyacid polymers consist of an acidic group like carboxylic acids and sulfonic acids. At high and neutral pH values, they tend to lose protons, thereby forming a negatively charged polymer chain; however, at low pH, it accepts protons (Kocak, Tuncer, & Bu¨tu¨n, 2016). On the contrary, polybasic polymers are enriched with basic functional groups like amines and imidazole. They form positively charged polymer at low pH by accepting the protons (Kocak et al., 2016). Therefore the protonation and deprotonation of these groups tune the net charge on the polymers (Bazban-Shotorbani et al., 2017). The increase in overall net charge causes a transition of the chains from a collapsed state to an expanded state. On the other hand, a decrease in the net charge will cause a change to a collapsed state. The pH-sensitive polymers are explored for a wide range of applications because of their biocompatibility. Eudragit L and Eudragit S from Ro¨hm Pharma GmBH, and CMEC (derived from cellulose) produced by Freund Sangyo Co., Ltd. are few commercialized pH-sensitive polymers (Aguilar & Roma´n, 2014). This pH sensitivity depends on the structure of the ionizable groups and other environmental factors like polymer composition, ionic strength, and properties of the polymeric backbone (Moo Huh, Kang, Lee, & Bae, 2012). In response to a change in the environmental pH, the net charge of polymeric structures like hydrogels typically displays swelling or deswelling behavior. The changes mentioned above occur at specific pH for a specific polymer, thus called critical pH (pH ) (Bazban-Shotorbani et al., 2017). pH depends upon the pKa value, which is defined as the pH at which half of the ionizable groups of polyelectrolytes are ionized (Bazban-Shotorbani et al., 2017). Selecting a polymer with the desired pH based upon the environmental condition is crucial in developing these polymers. Few of the early studies on pH-sensitive polymers were focused on the delivery of bioactive agents to the gastrointestinal (GI) tract, which depicts a great fluctuation in the pH. The copolymerization method allows the designing of polymers with a desired pH . Additionally, hydrophobic modification of polyelectrolytes can also be used to shift the pH value. Thus a complete understanding of the polymers’ ionizable group, their chemical structure, and an estimation of their pKa value is required for the development of pH-sensitive polymers.

9.4.1 Applications Researchers have explored the existence of pH variations within the human body to design smart delivery systems. The topical or transdermal route for drug delivery is considered a safe and patient-friendly way to deliver drugs to the human body. The pH of human skin is around 5; however, in melanoma, the pH varies from 5.5 to 7. A pH-responsive poly(lactic-co-glycolic acid) (PLGA)/chitosan nanogel was synthesized to achieve sustained delivery of anticancer drug 5-fluorouracil to the dermal tissue (Sahu, Kashaw, Jain, Sau,

9.4 pH-sensitive smart polymers

& Iyer, 2017). The drug was incorporated in the PLGA matrix by “solvent evaporation emulsification” followed by layering a chitosan coat (Sahu et al., 2017). The drug release pattern showed a sustained release due to PLGA particles. Further, pH-sensitive behavior and site-specific drug release characteristics of the formulations were revealed through the study. The formulated gel improved the penetration of the drug into the stratum corneum. The developed formulation was found to be a suitable candidate for transdermal drug delivery against skin cancer. Delivery of drugs through the oral route is also considered a convenient approach for the administration of drugs. However, there is a possibility that drugs administrated through the oral route might face some hindrance at the later stage when it reaches to GI tract. Additionally, many therapeutic peptides and proteins will also prove to be unstable under the stomach’s acidic environment. Hence, pH-sensitive oral delivery systems can be designed to protect the therapeutic peptides from the harsh acidic environment. One such example is the use of pH-sensitive alginate nanogels for the oral delivery of peptides. In this regard, alginate nanogel was developed using a microfluidic platform (Bazban-Shotorbani et al., 2016). Alginate nanogel behaves as an anionic polyelectrolyte, and its swelling is dependent upon its degree of ionization. The proposed system was found to be a promising approach for the encapsulation of polypeptides and their sustained release. Silica nanoparticles (SNPs) are an excellent drug carrier because of their large specific surface area, better mechanical stability, and low cell toxicity. A pH-sensitive polymer poly(2(diethylamino)ethyl methacrylate) (PDEAEMA), when grafted with SNP, has shown an improvisation of the therapeutic efficacy (Fig. 9.4) (Xu, Li, & Wang, 2019). This polymeric system was explored to deliver quercetin, whose anticancerous application is often limited due to its poor bioavailability. The formulated delivery system remained compact under the physiological pH (pH 5 7.4) and did not release the entrapped drug molecules. However, when the pH turns slightly acidic (pH 5 5.5), the system disintegrates because of the amine groups’ protonation present in PDEAEMA (Xu et al., 2019). This results in the release of quercetin from the polymer micelle. The pH variation at the wound site is also dynamic. However, this variation in the pH is often not given due diligence while designing a wound dressing material. A smart dressing material can be created using pH-sensitive polymers, acting in accordance to wound healing. The cationic nature of chitosan has been revealed to be constructive in this regard. Chitosan has a pKa value of 6.5, forming a stable gel at basic pHs and a swollen or dissolved polymer in the acidic environment (Chen et al., 2004). The swelling property of chitosan is beneficial in the initial stages of healing, where it improvises cell infiltration, proliferation, and oxygen permeability. Zhu and Bratlie (2018) have reported the synthesis of a pH-sensitive hydrogel using methacrylate chitosan. The fabricated gel released antiinflammatory factors during the initial stages of healing and further accelerated the healing process. pHsensitivity chitosan-based systems are beneficial in tissue repair and regeneration

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FIGURE 9.4 Synthesis and pH-dependent release from SNPs-g-PDEAEMA. Reproduced from Xu, L., Li, H.-L., & Wang, L.-P. (2019). pH-sensitive, polymer functionalized, nonporous silica nanoparticles for quercetin controlled release. Polymers, 11(12), 2026. https://doi.org/10.3390/ POLYM11122026 under Creative Commons license.

applications. In this regard, self-assembled CMCh and amorphous calcium phosphate hydrogel served as an ideal scaffold material for bone tissue regeneration (Zhao et al., 2019). The formed hydrogel supported proper growth, proliferation, and differentiation of mesenchymal stem cells (Table 9.2).

9.5 Photosensitive polymers Photochemistry has a central role in many biological processes, including photosynthesis, circadian cycle regulation, and sight. Light as a stimulus has recently gained attention in the biomedical field, where researchers are trying to explore its potential to regulate biological functions. The possible reason can be the spatiotemporal localization of light and its ability to activate a system even from outside. The photosensitive polymers display a reversible nature in their chemical and structural properties when exposed to light. An adequately engineered photosensitive polymer with appropriate knowledge of photochemistry can widen the opportunities of altering biological processes with great precision. There are several advantages associated with using light as a stimulus. Firstly, light includes a wide range of available wavelengths ranging from ultraviolet to infrared; secondly, it provides a possible four-dimensional control over the material’s responsiveness. Thirdly, the control over the administered light dose under in vivo conditions assists in functional regulations (Ruskowitz & DeForest, 2018).

Table 9.2 pH-responsive polymers and their range of applications. Loaded therapeutics

pH

Application

References

Metronidazole

Acidic

Treatment of Helicobacter pylori infection

Amoxicillin

Acidic

Treatment of H. Pylori infection

Dexamethasone

Slightly basic

Divinyl sulfone

Isoliquiritigenin

Neutral

Delivery at the lower (gastrointestinal) GI tract to treat inflammatory bowel disease and ulcerative colitis Inhibition of the growth of acne

El-Mahrouk, Aboul-Einien, and Makhlouf (2015) Risbud, Hardikar, Bhat, and Bhonde (2000) Das and Subuddhi (2015)

—

DNA

Basic

Gene therapy

Polymers

Cross-linker

Chitosan

Cross-linked with tripolyphosphate Cross-linked with glutaraldehyde Tetraethyl orthosilicate

Chitosan and poly(vinyl pyrrolidone) Acrylic acid grafted guar gum/β-cyclodextrin Hydroxyethylcellulose/ hyaluronic acid Poly(Nisopropylacrylamide)-copoly(acrylic acid)-co-poly (caprolactone)

Kwon, Kong, and Park (2015) Hu, Zhang, Zhang, Xu, and Zhuo (2009)

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These advantages altogether allow light-sensitive polymers for a wide variety of applications. Additionally, the photochemical reaction does not require extra reagents apart from the light stimulus, and they also have limited by-products (Bertrand & Gohy, 2016). The functional aspects of photosensitive polymers depend upon the extent and rate of change that occur due to light triggering and the reversibility of the process when the triggering light source is withdrawn (Cui & Del Campo, 2014). The fundamental structure of photosensitive polymer includes a photosensitive moiety and a bulk polymer (Upadhyay, Thomas, Tamrakar, & Kalarikkal, 2019). Photoresponsive polymers are synthesized by incorporating a chromophore, a light-sensitive functional group, in the polymer chain. The type of active group decides the reversibility of the process. Additionally, these groups are crucial in determining the dynamic behavior of the synthesized polymer. Azobenzene, spirooxazine, spiropyran, and fulgide derivatives are a few examples of chromophores (Schattling, Jochum, & Theato, 2013). These chromophores are capable of absorbing specific wavelengths of light. When the polymer solution is irradiated with light, the chromophores undergo photoinduced isomerization, dimerization, or cleavage, thus converting light into a chemical signal (Fig. 9.5; Ferna´ndez & Orozco, 2021). The photoisomerization process changes the molecular structure among the two isomers when kept under light irradiation (Upadhyay et al., 2019). Azobenzene and spiropyran are typical examples that let the polymer undergo photo-isomerization. It is a reversible process and results in changes in the polymer’s physical properties (e.g., color, refractive index, conductivity, etc.) (Ercole, Davis, & Evans, 2010). Photodimerization includes a chemical reaction between the photoexcited and an unexcited molecule within the same molecular species (Upadhyay et al., 2019). Like photo-isomerization, dimerization is also a reversible process and commonly incorporates photosensitive behavior in the polymer chain. Various molecules like cinnamylidene acetate and nitrocinnamate exhibit the photo-dimerization process (Zheng et al., 2001). Photocleavage, as the name suggests, involves the breaking of molecules under the influence of light. They comprise molecules that are unstable under the influence of light. Due to their said advantages, photoresponsive polymers have been explored in the biomedical field as photo-actuators, therapeutic hydrogels, optical devices, etc.

9.5.1 Applications Among the various applications of photosensitive polymers, their role in targeted in vivo drug delivery is appreciable. One example in this regard is the synthesis of polymers as photochromic vesicles using poly(ethylene oxide)-b-PSPA, in which SPA stands for spiropyran (Wei, Gao, Li, & Serpe, 2016). Inside the polymeric vesicle, there occurs a photoinduced isomerization between the spiropyran and merocyanine. This transition is helpful in tuning the permeability of the

9.5 Photosensitive polymers

FIGURE 9.5 Photoresponsive molecules for photo-triggered targeting. Reproduced from Ferna´ndez, M., & Orozco, J. (2021). Advances in functionalized photosensitive polymeric nanocarrier. Polymers, 13(15), 2464. https://doi.org/10.3390/POLYM13152464 under Creative Commons license.

vesicle membranes, impermeable to slightly permeable (Fig. 9.6). Such a system has shown a sustained release upon irradiation with the shorter wavelength ultraviolet (UV) radiation (Wei et al., 2016). The usage of photosensitive polymers has been explored to aid the wound healing process, which can also protect against on-site infection. In this regard, methacrylate hyaluronan polyacrylamide (MHA PAAm) hydrogels, integrated with silver nanoparticles, have been explored by a research group (Tang et al., 2020). Hyaluronic acid has a photo-crosslinking feature and is abundantly present in human tissues. The formed hydrogel attained an antimicrobial and hemostatic

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FIGURE 9.6 Photochromic polymersomes displaying photo-switchable and reversible bilayer permeability. Reproduced from Wei, M., Gao, Y., Li, X., & Serpe, M. J. (2016). Stimuli-responsive polymers and their applications. Polymer Chemistry, 8(1), 127 143. https://doi.org/10.1039/C6PY01585A under Creative Commons license.

activity through a two-step process. Under UV irradiation, a free radical polymerization starts over both MHA and PAAm which forms hydrogel upon photocrosslinking. In the later stage, biodegradable gelatin (holding an amine group) was coupled with carboxylic group of MHA and finally synthesizing skin adhesive with antibacterial hemostatic activities (AHAs) (Tang et al., 2020). AHAs boosted tissue formation at the wounded site, improved vascularization and collagen formation. The in vivo study displayed a faster wound closure in the infected rat models. Another successful application of these polymers was seen in cancer chemotherapy. A spiropyran and polyethylene glycol (PEG)-based hybrid nanoparticle was loaded with docetaxel and was evaluated for tissue penetration and drug release (Tong, Chiang, & Kohane, 2013). The triggered UV (365 nm) light assists in a reversible change in the nanoparticle volume and thus influences the release. The light irradiation converts the spiropyran to merocyanine (zwitterion). The merocyanine is less stable than spiropyran and, therefore, under the influence of visible light or simply in darkness, the system reverts, resulting in an increased size of nanoparticles (Tong et al., 2013). This photo-switching assists in repeated dosing (Table 9.3).

9.6 Enzyme-responsive polymers

Table 9.3 Photoresponsive polymers in the biomedical fields. Polymer

Light irradiation effect

Poly[S-(o-nitrobenzyl)L-cysteine-ethylene glycol] (PNBC-PEO)

Photocleavage

Collagen hydrolysate gelatin and methacrylate

Photopolymerization

Lutrol-F127

Photopolymerization

Polyethyleneglycol (PEG)-star-2-(4-nitro-3benzyl carbonate camptothecin) phenoxyethyl methacrylate

Photocleavage

Application

References

Release of anticancer drug doxorubicin in a controlled manner with a change in light irradiation time The polymeric hydrogel was employed for transplantation of nucleus pulposus cells to support regeneration Higher cell viability and the possibility of the osteogenic differentiation Controlled loading of camptothecin. Possible traceable intracellular release and distribution

Liu and Dong (2012)

Silva-Correia et al. (2013)

Fedorovich et al. (2009)

Li et al. (2018)

9.6 Enzyme-responsive polymers Enzymes are a fairly new class of stimuli that polymer scientists have explored. They are crucial in various biological processes and metabolic pathways. Enzymes control a large number of dynamic processes occurring inside the human body. The idea of incorporating enzyme responsiveness to the biomaterial brings the artificial system close to the biological system. The enzyme-responsive polymers (ERPs) can alter functional changes in the polymer by responding to the small biomolecules. This widely accepted definition of ERP includes polymers whose structure and function change upon direct actions of selected enzymes (Zelzer, 2014). Various ERPs can perform innumerable tasks efficiently in their biological niche. However, this can even be a disadvantage of these polymers as the enzyme’s activity will be specific to a particular microenvironment. ERPs tend to undergo macroscopic changes through selective enzyme catalysis (Asha, Srinivas, Hao, & Narain, 2019). These catalysis reactions are substrate-specific. A few of the most significant advantages of ERP include no requirement of the trigger from the external environment, higher selectivity, and the ability to work under mild conditions (Cabane, Zhang, Langowska, Palivan, & Meier, 2012). ERPs can exist in the form of solutions, gel, self-assembled structures, nanoparticles, films, etc. (Asha et al., 2019). The design of ERP can occur either through

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the use of enzyme degradable polymer or simply by amending the polymeric chain with enzyme responsive, functional groups. Typically an ERP includes two subunits, that is an enzyme recognized specific substrate and another component that controls the reversible/irreversible transitions (Asha et al., 2019). ERPs need to meet three general requirements for efficient functioning. Firstly, the polymers need to have an incorporated substrate or moiety specific to an enzyme for recognition (Chandrawati, 2016). For most proteolytic enzymes, the recognition moiety is peptide or linker chains that are conjugated with one particular amino-acid sequence. Next, the substrate accessibility to the enzyme should be considered. The substrate accessibility is responsible for guiding the kinetics of the enzymecatalyzed reaction. And last is the translation of enzyme-induced response to the modification in the material’s properties like degradation or morphological alterations (Chandrawati, 2016). The intrinsic biocompatibility of enzymes allows the exploitation of ERPs for drug delivery, tissue regeneration, diagnostics, and selfhealing materials. The therapeutic agents or bioactive molecules can be incorporated through the covalent attachment or physical encapsulations for the potential application of ERP in the mentioned fields.

9.6.1 Applications Many natural and synthetic polymers are modified for enzyme responsiveness, specifically for the application of drug delivery. Hydrolase enzyme has a vital role in the hydrolysis of many biomolecules (protein, lipids, nucleic acids, etc.). Proteases, esterases, and glycosylases are three significant subclasses of hydrolase enzymes. Many diseases like Alzheimer’s, atherosclerosis, and several viral infections (e.g., influenza) are often associated with increased proteases. The design of hydrolase-sensitive polymer will function in a manner that it can specifically be cleaved by a protease and thus release the drug at the targeted site. Wilson, Salas, and Guiseppi-Elie (2012) showed a controlled and targeted delivery of pro-drug (cleavable peptide) using α-chymotrypsin (protease) inside a poly-2-hydroxyethyl methacrylate hydrogel system (Wilson et al., 2012). The peptide sequence was cleaved by the chymotrypsin, and the release of the drug occurred due to the alteration in hydrophobicity of the hydrogel. ERPs can even be employed for targeted delivery in cancer chemotherapy. In this regard, a phosphatase-responsive supramolecular spherical assembly was designed by a group of researchers that mainly utilized noncovalent interactions for its assembly. This structure was based upon the complexion of calixarene with adenosine triphosphate (Wang, Guo, Cao, & Liu, 2013). Calixarene is crucial in providing rigidity and stability to such structures and constitutes sites for enzymatic reactions (Lee, Lee, & Jiang, 2004). These spherical assemblies are quite responsive to phosphatase, which is overexpressed in many cancerous cells and thus can be employed as delivery vehicles in cancer therapy. The application of ERP is not limited to the delivery of therapeutic drugs but is also considered for peptide delivery. Antimicrobial peptides are although efficient in their activity; however, their application is often limited due

9.6 Enzyme-responsive polymers

to inactivation from serum proteases, and high cost. An enzyme-responsive polyion complex nanoparticle was synthesized for a selective delivery of antimicrobial peptide, that is PEI, which was tested in presence of infectious Pseudomonas aeruginosa (Fig. 9.7) (Insua et al., 2016). It was shown that the nanoparticle and peptide can be easily degraded by the enzyme elastase (LasB), which is secreted from the pathogen P. aeruginosa. The tissue repair and regeneration potential of ERPs are again an appreciated field. The presence of avascularity in the cartilage tissue often limits its ability of self-regeneration. The aggrecanase-degradable hydrogel was synthesized and tested with chondrocytes for application in cartilage repair. Cartilage tissue comprises elastic collagen II fibrils and proteoglycans, majorly aggrecan. The aggrecan consists of two major sites for its proteolysis termed as interglobular domain (IGD). Therefore the designed hydrogel was based on a specific cleavage site within IGD (Durigova, Nagase, Mort, & Roughley, 2011). The encapsulated cells in the hydrogel were capable of degrading the hydrogel and promoted the growth of hyaline-like cartilage, which is desired for cartilage regeneration (Skaalure, Chu, & Bryant, 2015). Matrix metalloproteinases (MMPs) are yet another class of hydrolases that are often explored for this purpose. Schneider, Chu, Randolph, and Bryant (2019) reported MMP-sensitive PEG hydrogel for their beneficial role in cartilage regeneration (Schneider et al., 2019). Since growth factors are essential for repair and regeneration, the formulated hydrogel was incorporated with transforming growth factor-β. Prepared MMP-sensitive hydrogel was able to form tissue representative of hyaline cartilage and was rich in aggrecan, decorin, biglycan, and collagen type II (Schneider et al., 2019) (Table 9.4).

FIGURE 9.7 Assembly and oxidative cross-linking of PIC nanoparticles from P1SH (Ac-C-E-GLA-E-COH) and antimicrobial branched PEI. Degradation of PIC nanoparticles by LasB and subsequent PEI release. PEI, Polyethyleneimine; PIC, polyion complex. Reproduced from Insua, I., Liamas, E., Zhang, Z., Peacock, A. F. A., Krachler, A. M., & Fernandez-Trillo, F. (2016). Enzyme-responsive polyion complex (PIC) nanoparticles for the targeted delivery of antimicrobial polymers. Polymer Chemistry, 7(15), 2684 2690. https://doi.org/10.1039/C6PY00146G under Creative Commons license.

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Table 9.4 Enzyme-responsive polymers and their applications. S. No. 1.

2.

Polymers Poly(N-[2(acryloyloxy)ethyl]-N[p-acetyloxyphenyl]N,Ndiethylammonium chloride) Naphthalene-nitric oxide (NO) hydrogel

Responsive enzyme

Applications

References

Esterase

Intraperitoneal gene therapy and reduced adverse effects

Qiu, Gao, Liu, Wang, and Shen (2018)

Glycosidase

Controlled release of NO improved the therapeutic efficiency of mesenchymal stem cells (MSCs). This aids in the betterment of myocardial infection Helped direct differentiation of MSCs

Yao et al. (2015)

3.

Polyethylene glycol (PEG)

MMP

4.

PEG and poly (styrene)

Azoreductase

5.

Chondroitin sulfate and PEG

Transglutaminase factor XIII

The enzymeresponsive cleavage has potential application in the colon treatment Promote tissue healing and regeneration

Anderson, Lin, Kuntzler, and Anseth (2011) Rao and Khan (2013)

Anjum et al. (2016)

MMPs, Matrix metalloproteinases.

9.7 Conclusion In recent years SPs have evolved along with great potential in the biomedical field. These are a particular class of polymers that undergo a rapid transition in their physicochemical properties based upon environmental changes. Alteration in the surrounding environment like pH, temperature, light, and magnetic or electric fields acts as a trigger for the polymeric systems. The majority of the researchers have explored various environmental triggered pH, temperature, light, and bioenzymes for the design and synthesis of SPs. The mentioned stimuli are known for their better responsiveness under in vivo conditions, biocompatibility, nontoxicity, biodegradability, and possibility of dose quantification. This chapter provided a glimpse of SPs and their utility as an intelligent biomaterial in a wide range of applications. The various examples discussed under individual topics above give a clear picture regarding the versatility of these materials. Design of

References

drug delivery system through SPs can improve precision delivery of bioactives and hence the efficiency of the treatment of diseased conditions. Such delivery systems have found interest in specific applications like cancer and gene therapy as well. Additionally, the role of SPs in tissue repair and regeneration is crucial, as SPs are designed to modulate hypersensitivity without hampering the immune system. SPs hold great potential and an exciting future in biomedicine, and therefore researchers keep developing and creating new SPs that are responsive to various environmental stimuli.

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CHAPTER

Chitosan-based nanoparticles for ocular drug delivery

10

Kunal Pal1, Bikash K. Pradhan1, Doman Kim2 and Maciej Jarze˛bski3 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of International Agricultural Technology and Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea 3 Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznan´ ´ Poland University of Life Sciences, Poznan,

10.1 Introduction The human eyes being a protected organ, restrict the bioavailability of the drugs after the drug formulations are applied over the ocular surface (Li et al., 2018). Hence, it is challenging for pharmaceutical researchers to deliver a drug to the anteroposterior segments of the eye (Rodrigues et al., 2018; Suri, Beg, & Kohli, 2020). In this regard, several studies have explored the nanoparticle-mediated ocular drug delivery system and got efficient results (Alkholief et al., 2019; Taghe, Mirzaeei, Alany, & Nokhodchi, 2020). Various polymers, including natural polymers, have been explored to develop nanoparticle-mediated ocular drug delivery systems (Jumelle, Gholizadeh, Annabi, & Dana, 2020; Mittal & Kaur, 2019a, 2019b; Tan & Ho, 2018). Among these, chitosan is one of the most prevalent polymers that has been explored with some success (Arafa, Girgis, & ElDahan, 2020; Bao et al., 2021; De Gaetano et al., 2021). Chitosan, a cationic linear polysaccharide, has been greatly used to design formulations for nanotherapeutic applications to treat ocular diseases (Dai et al., 2020; Kazemi & Javanbakht, 2020; Yang, Cabe, Nowak, & Langert, 2022). The success of the chitosan formulations as ocular delivery systems has been related to the mucoadhesive property of the chitosan (Dubashynskaya et al., 2020; Irimia, Dinu-Pıˆrvu, et al., 2018; Sun et al., 2022). The mucoadhesive properties of chitosan could be related to the presence of the amino groups (Coutinho, Lima, Afonso, & Reis, 2020; Laffleur & Ro¨ttges, 2019; Pauluk, Padilha, Khalil, & Mainardes, 2019). These amino groups interact with the sialic acid residues that are in abundance in mucosal linings in the human body (Hejjaji, Smith, & Morris, 2018; Kolawole, Lau, & Khutoryanskiy, 2019; Pauluk et al., 2019). It has been found that the ocular retention of the chitosan-based nanoparticles can be tailored by crosslinking Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00023-1 © 2023 Elsevier Inc. All rights reserved.

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(Abruzzo et al., 2021; Samprasit, Opanasopit, & Chamsai, 2021). The ocular retention, in turn, improves the penetration of the chitosan nanoformulations within the eye, thereby significantly improving the bioavailability of the drugs (Irimia, Ghica, et al., 2018; Li et al., 2018). Further, chitosan is biodegradable and biocompatible (nontoxic) (Islam, Dmour, & Taha, 2019; Mart˘au, Mihai, & Vodnar, 2019; Zhang et al., 2021). Accordingly, the human body degrades the chitosan nanoparticles without forming any toxic by-products penetrating the ocular tissue. In this chapter, the anatomy of the eye and types of ocular diseases will be discussed initially. Thereafter the properties of chitosan and some potential applications of chitosan nanoparticle based formulations will be briefly discussed.

10.2 Anatomy and protection mechanism of eye The human eye is nearly a spherical organ located in the orbits (Foletti et al., 2019; Glarin et al., 2021). Fig. 10.1 summarizes the different parts of the eye.

FIGURE 10.1 Schematic diagram of the anatomy of the eye and the diseases associated with the eyes. Reproduced from Vichare, R., Garner, I., Paulson, R. J., Tzekov, R., Sahiner, N., Panguluri, S. K., . . . Biswal, M. R. (2020). Biofabrication of chitosan-based nanomedicines and its potential use for translational ophthalmic applications. Applied Sciences, 10(12), 4189. https://www.mdpi.com/2076-3417/10/12/4189 under creative commons license.

10.2 Anatomy and protection mechanism of eye

Human eyes consist of anterior and posterior segments (El Basha, Furuta, Iyer, & Bolch, 2018). A layer of transparent tissue called the cornea is present in the outermost part of the anterior segment of the eye. It is in continuation with the sclera, an opaque white tissue (Lee, Low, Kim, & Teoh, 2022). The sclera helps to maintain the shape of the eye and protects it from the outer environment. On the other hand, the cornea allows visible light to enter the inner part of the eye. The eye’s anterior chamber is the front part that lies between the cornea and lens (Erdem et al., 2021). Iris forms an aperture known as the pupil, which helps to accommodate the eye in bright and dark regions by controlling the aperture opening. The iris is followed by the anterior portion of the lens, which is connected with the ciliary body. The ciliary body helps to control the curvature of the lens so as to focus the light onto the macula of the retinal tissue (Geiger et al., 2020; Yui, Kunikata, Aizawa, & Nakazawa, 2019). The posterior part of the lens forms the interface with the vitreous humor of the posterior chamber. In other words, the anterior and posterior chambers of the eye are segregated by the lens (Jacobson & Bohnsack, 2021; Nagae, Sawamura, & Aihara, 2020). The retinal tissue is present at the rear end of the posterior chamber (Peynshaert, Devoldere, De Smedt, & Remaut, 2018). The macula is the part of the retinal tissue where the image is formed (Bagewadi, Parameswaran, Subramanian, Sethuraman, & Subramanian, 2021; Inana et al., 2018). The retinal tissue forms the optic nerve, which collects information from the retinal tissue and transfers the same to the central nervous system (Mesentier-Louro et al., 2021). The eyes are prone to various ocular diseases. According to the location of the disease, the ocular diseases are categorized into the anterior and posterior segment diseases. Some of the diseases of the anterior segments include cataracts, corneal ulcers, keratitis, and conjunctivitis (Hoshi, Todokoro, & Sasaki, 2020; Stamate, T˘ataru, & Zemba, 2021). Among these diseases, cataract is one of the most commonly occurring ocular diseases across the globe. The cataract is characterized by the clouding of the lens, which is usually transparent. If left untreated, it will induce blindness in the patients and has been reported to be the leading cause of blindness (Vichare et al., 2020). Some posterior segment diseases include retinal detachment, glaucoma, macular degeneration, and endophthalmitis (Peng, Kung, Tsai, & Wu, 2021). Usually, ocular diseases are treated with topical administration of pharmaceutical formulations. However, in several cases, surgical procedures are performed. Unfortunately, there are several limitations for delivering the drugs through topical administration. The leading cause is the poor bioavailability of the drug at the site of the disease (Lynch et al., 2019). This is due to the loss of the drug formulation due to the formation of several barrier mechanisms (Fig. 10.2). Some of the barriers to the drug delivery to the eyes that hampers the bioavailability include tear film barrier (consist of an outer hydrophobic layer followed by an aqueous layer), tight epithelial junction of the cornea, reflex blinking, metabolism in ocular tissue, nasolacrimal drainage, efflux pumps, blood retinal barrier, and blood aqueous barrier (Bachu, Chowdhury, Al-Saedi, Karla, & Boddu, 2018; Bı´ro´ & Aigner, 2019). Hence, there is poor uptake of the drug molecules.

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FIGURE 10.2 Anatomy of the eye highlighting the different ocular barriers. BAB, Blood aqueous barrier; BRB, blood retinal barrier. Reproduced from Bı´ro´, T., & Aigner, Z. (2019). Current approaches to use cyclodextrins and mucoadhesive polymers in ocular drug delivery—A mini-review. Scientia Pharmaceutica, 87(3), 15. https://www.mdpi.com/ 2218-0532/87/3/15 under creative commons license.

10.3 Properties of chitosan Chitosan is a polycationic polysaccharide. The backbone of the polysaccharide is linear. Chitosan is obtained from crustaceans (e.g., lobster, shrimp, and crab), insects (e.g., butterfly, and fly), and fungi (e.g., Aspergillus niger and Penicillium chrysogenum) (Fig. 10.3A) (Bastiaens, Soetemans, D’Hondt, & Elst, 2019; da Silva Alves, Healy, Pinto, Cadaval, & Breslin, 2021). The chitosan is synthesized from another natural linear polysaccharide, chitin. Chitin is converted to chitosan by the process of partial deacetylation (Harmsen, Tuveng, Antonsen, Eijsink, & Sørlie, 2019). The process of deacetylation can be carried out either by the chitin deacetylase enzyme or the alkaline deacetylation process (Fig. 10.3B) (Bastiaens et al., 2019). Chemically, the chitosan polysaccharide is composed of β-(1 4)-linked D-glucosamine and Nacetyl-D-glucosamine monomeric units (Resmi & Beena, 2021; Sahira Nsayef Muslim, 2018). The distribution of these monomeric units is random. The existence of a free amine group in N-acetyl-D-glucosamine is the reason behind the cationic nature of the chitosan. Chitosan can form electrostatic complexes with negatively charged polymers and other chemical entities (e.g., sodium tripolyphosphate) (Hosseini, Soleimani, & Nikkhah, 2018) due to its cationic nature. Inherently chitosan is nontoxic to human tissues. Further, it is biodegradable and biocompatible. Also, the polysaccharide exhibits antimicrobial and antioxidant properties (Fig. 10.4;

10.3 Properties of chitosan

FIGURE 10.3 (A) Sources of chitosan and (B) conversion processes to synthesize chitosan from chitin. Reproduced from da Silva Alves, D. C., Healy, B., Pinto, L. A. D. A., Cadaval, T. R. S. A., & Breslin, C. B. (2021). Recent developments in chitosan-based adsorbents for the removal of pollutants from aqueous environments. Molecules, 26(3), 594. https://www.mdpi.com/1420-3049/26/3/594 under creative commons license.

FIGURE 10.4 Inherent properties of chitosan that makes it suitable for biomedical applications. ˇ & Leitgeb, M. (2019). Chitosan-based (nano)materials ˇ c, ˇ M., Knez, Z., Reproduced from Kravanja, G., Primozi for novel biomedical applications. Molecules, 24(10), 1960. https://www.mdpi.com/1420-3049/24/10/1960 under creative commons license.

Kravanja, Primoˇziˇc, Knez, & Leitgeb, 2019). These inherent properties of chitosan make it an excellent material for biomedical and pharmaceutical applications (Ali & Ahmed, 2018).

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Though chitosan exhibits several advantageous properties, some of the properties are more beneficial in ocular delivery than others. The most important properties of chitosan that play an essential role in ocular delivery include mucoadhesion, antibacterial, and penetration enhancement (Zamboulis et al., 2020). The mucoadhesive property of chitosan is exhibited due to the presence of hydroxyl and amine functional groups. These functional groups can form noncovalent bonds (ionic and hydrogen-bonding interactions) with the mucin, which is the primary constituent of the mucosal layers. Again, these noncovalent interactions improve the adhesion of chitosan formulations over the mucosal layers, including the ocular surface. Mucin is a negatively charged polymer, which is ascribed to the presence of free carbohydrate-bound ester sulfate residues and the carboxyl groups of sialic acid groups (Zamboulis et al., 2020). The interactions of chitosan nanoparticles with the mucosal layers and subsequent drug delivery mechanism is summarized in Fig. 10.5 (Mohammed, Syeda, Wasan, & Wasan, 2017). As mentioned in the previous paragraph, there is an ionic interaction between the mucosal layer and the chitosan nanoparticles. This interaction significantly

FIGURE 10.5 Schematic showing the interaction of chitosan nanoparticles with the mucosal layer and subsequent drug delivery. Reproduced from Mohammed, M. A., Syeda, J. T. M., Wasan, K. M., & Wasan, E. K. (2017). An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics, 9(4), 53. https:// www.mdpi.com/1999-4923/9/4/53 under creative commons license.

10.3 Properties of chitosan

improves the bioadhesivity of the chitosan nanoparticles over the mucosal surface, thereby improving their ocular residence time by preventing lacrimal elimination. The improved residence time of the chitosan nanoparticles over the ocular surface increases their chances of transcellular transport within the ocular tissues. Transcellular transport is made possible due to the disruption of the tight junctions (Hong, Yoo, Kim, & Lee, 2017). The schematic representation of the transcellular transport of the chitosan nanoparticles is depicted in Fig. 10.6. Chitosan can elicit antimicrobial activity in various ways (Ke, Deng, Chuang, & Lin, 2021). The high molecular weight chitosan can chelate with the ions and nutrients around the microbes. Accordingly, the microbes will be deprived of the required ions and nutrients to survive and grow. Further, such chitosan molecules can interact with the lipoteichoic acids present in the peptidoglycan layer of Gram-positive bacteria, thereby weakening their activity. Also, the chitosan with higher molecular weight can disrupt the cell membranes of Gram-positive bacteria, Gram-negative bacteria, and fungi (Ke et al., 2021). It causes the leakage of the internal contents of the microbes, thereby resulting in their death. On the other hand, the chitosan molecules with a low molecular weight influence penetrate the cell wall of the microbes. After their penetration, they hinder the functions of DNA/RNA. The protein synthesis within the microbes can also be significantly affected. In fungi, low molecular weight chitosan has been reported to inhibit

FIGURE 10.6 Schematic representation of the transcellular transport exhibited by chitosan nanoparticles. Reproduced from Hong, S.-C., Yoo, S.-Y., Kim, H., & Lee, J. (2017). Chitosan-based multifunctional platforms for local delivery of therapeutics. Marine Drugs, 15(3), 60. https://www.mdpi.com/1660-3397/15/ 3/60 under creative commons license.

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FIGURE 10.7 Schematic representation exhibiting the antimicrobial activity of chitosan molecules against: (A) Gram positive bacteria, (B) Gram negative bacteria, and (C) fungi. Reproduced from Ke, C.-L., Deng, F.-S., Chuang, C.-Y., & Lin, C.-H. (2021). Antimicrobial actions and applications of chitosan. Polymers, 13(6), 904. https://www.mdpi.com/2073-4360/13/6/904 under creative commons license.

mitochondrial activity (Ke et al., 2021). Fig. 10.7 depicts the mechanism involved with the antimicrobial activities of chitosan.

10.4 Some recent applications of chitosan nanoparticles in ocular delivery Clarithromycin-loaded chitosan nanoparticles have been prepared by (Bin-Jumah et al., 2020). The drug clarithromycin is a well-established antibiotic and is characterized as a broad-spectrum macrolide antibiotic. It has been reported that the antibiotic is widely used to treat various bacterial keratitis infections.

10.4 Some recent applications of chitosan nanoparticles

The nanoparticulate formulations showed sustained drug release properties. Further, the corneal permeation of the drug was considerably increased as compared to the clarithromycin solution. An improvement in the corneal permeation was reasoned to the increased precorneal residence time. Even though the corneal permeation of the drug was enhanced, the formulations did not cause damage to the corneal tissues. The developed formulation was proposed to treat bacterial conjunctivitis. In Shinde, Joshi, Jain, and Singh (2019), authors have reported the synthesis of N-trimethyl chitosan nanoparticles for the ocular delivery of flurbiprofen, a nonsteroidal antiinflammatory drug. The solubility of flurbiprofen was first improved by developing a complex with hydroxyl propyl-β-cyclodextrin. Thereafter the complex was loaded into the chitosan nanoparticle matrices. These nanoparticulate systems were capable of transmucosal delivery of flurbiprofen over a prolonged period of time. Ameeduzzafar, Imam, Abbas Bukhari, Ahmad, and Ali (2018) have developed a levofloxacin-loaded chitosan nanoparticle for ocular drug delivery. Levofloxacin is a well-established antibacterial agent that has been successfully explored to treat ocular infections. The nanoparticles were prepared by the ionotropic gelation method. Herein, the authors used sodium tripolyphosphate as the ionic crosslinker. The optimized nanoparticle formulation was converted into an in situ gel-forming formulation. As the encapsulation efficiency of the drug within the nanoparticles was very high, it improved the loading efficiency of the drug. It was found that the corneal residence of the chitosan nanoparticles was enhanced when converted into an in situ gel formulation. Further, the chitosan nanoparticles did not induce irritation of the ocular tissue. The proposed formulation showed sufficiently high antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Also, the proposed formulation achieved a shelf life of 2.16 years, which is high enough to promote its commercialization. In a similar study, gentamycin-ferrying chitosan nanoparticles were used to develop in situ gel formulations (Alruwaili et al., 2020). The in situ gel was pH-sensitive. It was prepared using the polymer carbopol 974P, a well-known pH-sensitive polymer. This polymer has been extensively used to develop several pharmaceutical products, including ocular delivery systems. In a recent study, Shahab, Rizwanullah, Alshehri, and Imam (2020) have developed chitosan-decorated polycaprolactone nanoparticles (Shahab et al., 2020). The developed nanoparticles, prepared by single-step emulsification technique, were explored to improve ocular drug delivery of dorzolamide. Dorzolamide is used to reduce the ocular pressure in open angle-type glaucoma. The drug has also been used to treat ocular hypertension. The release of the drug from the nanoparticles followed a biphasic release pattern. During the first 2 h, a burst release of the drug was observed. Thereafter a sustained release of the drug for 12 h was revealed. The results showed an improved corneal permeation of the drug. It can be explained by the higher mucoadhesive properties of the developed nanoparticulate formulations. Finally, the nanoparticles were found to be safe for ocular administration.

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Table 10.1 Recent publications on the applications of chitosan-based nanoparticle formulations for ocular drug delivery.

Literature

Composition of the formulation

Nanoparticle preparation method

Ocular disease focused

Zhao et al. (2017)

Timolol maleate loaded galactosylated chitosan

Ionic crosslinking

Glaucoma

Silva et al. (2017)

Chitosanhyaluronic acidbased nanoparticles containing ceftazidime

Ionotropic gelation

Bacterial keratitis

Wang et al. (2018)

Chitosan-coated solid lipid nanoparticles encapsulated with methazolamide

Emulsificationsolvent evaporation method

Glaucoma

Imam, Bukhari, Ahmad, and Ali (2018)

Levofloxacinloaded chitosan nanoparticle

Ionic gelation

Ocular infection

Bin-Jumah et al. (2020)

Clarithromycinloaded chitosan nanoparticles

Iontophoretic gelation

Ocular infection

Inference from the study The proposed formulation showed a sustained release effect and enhanced penetration and retention in the cornea compared to the commercially available eye drop. The nanoparticle formulation presented showed the necessary mucoadhesive property desirable for efficient drug delivery. The nanoformulation also possesses good antimicrobial properties. The proposed formulation showed an efficient result in the ocular delivery of methazolamide. A significant decrease in the intra-ocular pressure was also observed. The optimized formulation shows nonirritability for the cornea. Also, the formulation is found to possess a higher antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. The optimized formulation showed a small particle size, high encapsulation, and a sustained drug release profile. (Continued)

10.5 Conclusion

Table 10.1 Recent publications on the applications of chitosan-based nanoparticle formulations for ocular drug delivery. Continued Nanoparticle preparation method

Ocular disease focused

Literature

Composition of the formulation

Sobhani, MohammadiSamani, and Arazi (2020)

Chitosansulfacetamide sodium nanoparticles

Iontophoretic gelation

Ocular infection

Yu et al. (2020)

Dexamethasoneglycol chitosan nanoparticles

Iontophoretic gelation

Ocular diseases

Inference from the study The proposed formulation showed that sulfacetamide sodium did not lead satisfactory drug release profile during the 24 h test. The proposed nanoparticle formulation showed good ocular tolerance and longer precorneal duration than the aqueous solution.

In an interesting study, lipid nanoparticles were coated with chitosan (Eid, Elkomy, El Menshawe, & Salem, 2019). The coated nanoparticulate systems were explored for the ocular delivery of ofloxacin. It was observed that 66% of the drug permeated through the cornea. The authors reported that chitosan coating significantly improved trans-corneal permeation and bioavailability. It is due to an increased mucoadhesive property. Table 10.1 lists some of the recent publications based on chitosan-based nanoparticle formulations.

10.5 Conclusion Chitosan is a naturally occurring polymer and is obtained from chitin by the process of deacetylation. This polymer is a versatile polymer and has been explored in various applications, including food, pharmaceutical, and biomedical. The main reason behind the versatility of this polymer is its inherent biocompatible, biodegradable, and mucoadhesive properties. Also, chitosan is nonirritant to the human cells and tissues and does not provoke an immunological response. Further, the polymer has been found to enhance the transmucosal penetration of the drug molecules. Due to these properties, it has found applications in ocular drug delivery. Chitosan and its derivatives can considerably improve the ocular residence time, thereby improving the bioavailability of the drugs at the ocular tissue. Accordingly, there is an increased therapeutic effect of the drugs. The effect is more pronounced when the polymer is used to develop nanoparticles. This therapeutic

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effect of chitosan nanoparticles is due to their small size. Also, the surface area of such nanoparticulate systems is quite high, which in turn increases the mucoadhesive property of the polymers. Even though various synthesis methods of chitosan nanoparticles are present, the ionotropic gelation method is the most common method. Among the various ionic crosslinker, sodium tripolyphosphate has quite often been employed. This is because the development of chitosan nanoparticles using sodium tripolyphosphate is very convenient. Such nanoparticles have found applications as sustained release systems for ocular drug delivery. The ocular residence of these nanoparticle formulations can be further increased by converting them into in situ gelforming systems. Researchers have reported many encouraging results based on chitosan coating over other polymeric nanoparticulate matrices. In gist, it has been found that chitosan and its derivatives can be explored to develop novel ocular delivery systems with improved therapeutic efficacy.

References Abruzzo, A., Giordani, B., Miti, A., Vitali, B., Zuccheri, G., Cerchiara, T., . . . Bigucci, F. (2021). Mucoadhesive and mucopenetrating chitosan nanoparticles for glycopeptide antibiotic administration. International Journal of Pharmaceutics, 606, 120874. Ali, A., & Ahmed, S. (2018). A review on chitosan and its nanocomposites in drug delivery. International Journal of Biological Macromolecules, 109, 273 286. Alkholief, M., Albasit, H., Alhowyan, A., Alshehri, S., Raish, M., Kalam, M. A., & Alshamsan, A. (2019). Employing a PLGA-TPGS based nanoparticle to improve the ocular delivery of Acyclovir. Saudi Pharmaceutical Journal, 27(2), 293 302. Alruwaili, N. K., Zafar, A., Imam, S. S., Alharbi, K. S., Alotaibi, N. H., Alshehri, S., . . . Elmowafy, M. (2020). Stimulus responsive ocular gentamycin-ferrying chitosan nanoparticles hydrogel: Formulation optimization, ocular safety and antibacterial assessment. International Journal of Nanomedicine, 15, 4717. Ameeduzzafar., Imam, S. S., Abbas Bukhari, S. N., Ahmad, J., & Ali, A. (2018). Formulation and optimization of levofloxacin loaded chitosan nanoparticle for ocular delivery: In-vitro characterization, ocular tolerance and antibacterial activity. International Journal of Biological Macromolecules, 108, 650 659. Available from https://doi.org/10.1016/j.ijbiomac.2017.11.170. Arafa, M. G., Girgis, G. N., & El-Dahan, M. S. (2020). Chitosan-coated PLGA nanoparticles for enhanced ocular anti-inflammatory efficacy of atorvastatin calcium. International Journal of Nanomedicine, 15, 1335. Bachu, R. D., Chowdhury, P., Al-Saedi, Z. H. F., Karla, P. K., & Boddu, S. H. (2018). Ocular drug delivery barriers—Role of nanocarriers in the treatment of anterior segment ocular diseases. Pharmaceutics, 10(1), 28. Available from https://www.mdpi.com/ 1999-4923/10/1/28. Bagewadi, S., Parameswaran, S., Subramanian, K., Sethuraman, S., & Subramanian, A. (2021). Tissue engineering approaches towards the regeneration of biomimetic scaffolds for age-related macular degeneration. Journal of Materials Chemistry B, 9, 5935 5953.

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Appraisal of conducting polymers for potential bioelectronics

11

Rimita Dey1 and Pallab Datta2 1

Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India 2 Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India

11.1 Introduction Discoveries in polymer chemistry showing that polymers can conduct electricity open new path for application of polymeric materials in electronics industries. Before the 1970s all carbon-based polymers were considered electrical insulators. Thereafter electronic polymers are discovered which facilitate in energy conversion and storing in various systems like rechargeable batteries, solar cells, and ultracapacitors. Electronic polymers are highly appreciated because of optical properties, high electrical conductivity, mechanical flexibility, and affordable cost (Granero, Wagner, Wagner, Razal, & Wallace, 2011; Weng, Pan, Wu, & Chen, 2015). Intrinsically conductive polymers (ICPs) are obtained by a two-stage process—(1) the monomer is transformed into a conjugated double-bonded polymer; and (2) the extra positive or negative charge is added by a doping method. To conduct the entire process in single step, excess charge is introduced during polymerization process. Conjugated polymers are doped via photochemical, chemical or electromechanical route. Charge transfer between dopant and polymer is the characteristics of doping reactions. The most common method of chemical synthesis is step growth condensation polymerization to produce polyaniline, polyparaphenylene, and polythiophene (and its derivatives). Polyacetylene is generally synthesized by polymerizing acetylene using Ziegler
Natta method. Polymers (like polyaniline, polypyrrole, and polythiophene) are prepared by electrochemical oxidation methods where the monomer is anodically polymerized on electrode surface using appropriate solvent or electrode medium. The chemical structures of the repeating units of conducting polymers can exhibit much variation (Min, Patel, & Koh, 2018) and some of them are shown in Fig. 11.1. Polymer film is synthesized by the oxidation reaction. Undoping of the film can be done by changing the current flow. The resultant doped polymers can be easily stripped off from the surface of the electrode in coated and self-supporting film form and Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00014-0 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 11.1 Chemical structures of repeating units of some common conducting polymers. Reproduced from Min, J.H., Patel, M., & Koh, W.-G. (2018). Incorporation of conductive materials into hydrogels for tissue engineering applications. Polymers, 10.

thus electrochemical polymerization method is more preferable to chemical methods. Photochemical synthesis techniques for synthesis of ICPs are not preferable but they are applied for polymerizing pyrrole and some derivatives of thiophene. Thin polymer films can be fabricated from dedicated solid surfaces such as conductive indium tin oxide
plated glass. Distinctive coexistence of electrical and mechanical properties can be found in conjugated polymers. Most conjugated polymers are insoluble and infusible in nature due to their chemical structure. But those materials are preferred that are fusible and soluble in common solvents, enhancing easy transformation of the conductive polymers into convenient products. The polymers can be synthesized with different solid-state structures and according to the structures and properties of different polymers associated with electronic devices, the charge transport property of amorphous material occurs by several hopping events. The conformational defects of each chain determine the structure of polymers. For example, due to very slow equilibration, the definite shape of less molecule polymers like poly(p-phenylene vinylene) (PPV) becomes highly organized, and this does not affect the association between conformation and electronic configuration (Qin and Troisi, 2013). In the crystalline regions of the electronic polymers, the chains are folded and orderly arranged. The flow of charge through electronic polymer is hindered by interspherulite boundaries because of reduction in tie chains which connect adjoining structured regions in comparison with edges between quasiparallel lamella inside the spherulite (Street, Northrup, & Salleo, 2005). Moreover, fabrication of different morphologies like nanofiber, nanoparticles, etc. can further provide better control on the stability and morphology. Other than core shell, hollow nanoparticles and solid structure are becoming famous in recent years. Polymer-coated hollow sulfur particles (transfers the electron and prevents the spill of polysulfides) are manufactured by in situ polymerization and used in lithium
sulfur batteries as cathode materials (Li et al., 2013).

11.2 Sensors and actuators used on conducting polymers

Polyaniline (PANI) is commonly used material due to its simplistic synthetic process, tailor ability by nonredox acidic or basic doping, more environmental stability, ease in controlling electrical conductance, and probability of a large-scale fabrication and is highly studied for electrorheological, electronic, and electrochemical applications. Monodispersed PANI nanoparticles are obtained from oxidative dispersed polymerization where poly(sodium 4-styrenesulfonate) (PSSA) is employed as a dopant agent as well as polymeric stabilizer due to its activity. Apart from nanoparticles, nanofibers have been immensely explored for structural characteristics such as high curvatures and large aspect ratios that offer higher distinct surface areas and removable electrochemical activities. Nanofiber networks or arrays are used in biological materials, composite materials, and filtering membranes other than a variety of electronic materials. Regio-regular P3HT is one of the important organic semiconductors widely used in fabricating electronic devices like solar cells, field-effect transistors, or different sensors. P3HT-based nanofibers are fabricated by electrospinning and self-assembly process. Continuous and long nanofibers produced by electrospinning process are of diameters ranged from 10 nm to submicron. During fabrication of nanofibers, conducting P3HT is mixed with nonconducting polymer which decreases the electrical conductivity of P3HT. ICPs can also be converted into films having both high toughness and strength. These conducting polymers are lightweight and flexible than the other equivalent inorganic materials. Porous conducting polymers have also been explored due to high specific surface area. Pure poly(3,4-ethylenedioxythiophene) (PEDOT):PSS shows conductivity of 0.24 S/cm which is very low, but addition of nonionic surfactant stimulates the growth of PEDOT nanofibrils during coatings, which increases the conductivity (100 S/cm). After the synthesis of polymers and their fabrication in suitable form, the materials are characterized thoroughly for wide range of properties. For example, mechanical properties of the electronic devices determine their stability. For determining electronic properties of the conducting polymer, the space charge
limited conduction is considered, which means under applied voltage, the electric field from adjacent carriers dominates. Polymer-based electronic devices have high electrical conductivity. But still they are greatly sensitive to few reaction parameters, for example, temperature, impurities, and moisture content which sometimes form heterogeneous surface properties and severely hamper the electrical conductivity and reproducibility. Hence, the electronic polymers are needed to become more stable and more durable in different conditions.

11.2 Sensors and actuators used on conducting polymers Electrochemical actuator converts electrical energy into mechanical energy through electrochemical mechanisms. These actuators have huge potential in the

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field of biomimetic sensors and devices, artificial muscles, robotics (Bauer et al., 2014; Ma, Chirarattananon, Fuller, & Wood, 2013; Madden, 2007). They are made up of shape memory alloys and inorganic materials like ferroelectric ceramics so get major attention because of their rapid responsiveness and powerful stress output (Madden et al., 2004; Zhang & Li, 2012). But they are not good for practical applications because of high cost, heavyweight, and small actuation strain (,10%). Electrochemical mechanical actuators are composed of composites of polymeric materials and are widely explored since they inherit advantages of its constituents, such as low cost, large strain, high flexibility, and synthesis controllability. Depending on mechanism of actuation, the polymeric materials
based electromechanical actuators are classified into three types. The classes are electrothermally driving actuators, electrostatically driving, and electrochemically driving. In actuators that are driven electrostatically, various polymeric materials possessing large value of dielectric constant, for example, fluoroelastomers, isoprene, polyurethanes (PUs), polydimethylsiloxane (PDMS) are used as dielectric layer (Mirfakhrai, Madden, & Baughman, 2007). Under electric field of 412 kV/mm, the actuator comprising prestretched VHB-4910 elastomers from 3 M can induce maximum actuation strain of 158%. But they have a limited range of ultracharge voltage of operation upto 100 V/μm. There are two ways for reducing the operational voltage, one is directly to increase dielectric permittivity or to decrease the thickness for enhancing the electric field strength. To reduce the operational voltage, great measures are taken to increase films dielectric constant. Amalgamation of organic dipole groups, ceramic nanoparticles having high dielectric permittivity into polymer composition effectively enhances the dielectric permittivity of polymer film. For example, by solution mixing method, silicone oil
laminated TiO2 nanoparticles are blended with SEBS (poly-styrene-co-ethylene-co-butylene-costyrene) (Stoyanov, Kollosche, Risse, McCarthy, & Kofod, 2011). Chemical grafting method is used to add dipole into the elastomer matrix at molecular level rather than conventional compositing or blending method (Kussmaul et al., 2011; Risse, Kussmaul, Kru¨ger, & Kofod, 2012). Homogenous elastomer films having better efficiency are fabricated using this method and the aggregation problem is also resolved. For example, by one-step film formation mechanism, N-alkyl-Nmethyl-p-nitroaniline, is inserted into silicone cross linkers (Kussmaul et al., 2011). Since after applying external voltage, the dipoles align among themselves and the resultant silicone having a dipole content of 13.4% exhibit the electric permittivity which is 5.9 times enhanced. The actuation performance depends on the dielectric films prestretching treatment (Zhao & Wang, 2014). Circularly or spherically, uniaxially, or biaxially, the prestretching is done upon the dielectric films. The direction of actuation can be regulated by altering the directions of prestretching. However, the actuators made from prestretching film have disadvantages such as necessity of rigid frame for maintaining the stretching state. This enhances the equipment mass leading to the work/power density reduction.

11.2 Sensors and actuators used on conducting polymers

In electrostatic actuators, due to the actuation performances, the electrodes have a significant importance. Ideally, the characteristics of materials used as electrodes are high mechanical stretchability and electrical conductivity along with properties such as stable and uniform charge distribution on both of the surfaces. For electrostatic actuation, in recent years, several stretchable electrode materials are examined. In earlier days, thin metal films were used since they have high electrical conductivity. But later they lost potential due to disadvantages of small strain less than 1%. In another way, elastomer surfaces coated with palladium, gold, or titanium nanoparticles through metal ion implantation method can be used as electrode. These gold nanoparticles laminated dielectric elastomers are stretchable up to strain of 175% and exhibit area resistance (Yun et al., 2012). For preparing high conductive stretchable electrodes, metallic nanowires are inserted into the elastomeric matrix. Carbonaceous materials like carbon power, graphite, and carbon grease are inexpensive, compatible with the dielectric elastomers and highly conducting compared to metal-based electronics. Graphene and carbon nanotubes (CNTs) also have a lot of potential as electrode materials. Single-walled CNT-based electrodes are flexible and transparent and thus the resultant actuator having self-clearing property can create 200% strain in the area. The aligned CNT sheet-based actuators show large actuation strain and directional motions. In the fields of mechanical machines, robotics, and artificial muscles, electrostatically driving actuators have huge potential (Anderson, Gisby, McKay, O’Brien, & Calius, 2012). But high operational voltage ranging from kilovolts to megavolts limits their application. So, extensive research is going on to create novel dielectric elastomers. Due to low voltage, the actuators which are driven electrochemically are widely explored (Kong & Chen, 2014). These actuators comprising electrolytic layer are sandwiched between two layers of electrode. Expansion of electrode layers and asymmetrical volume shrinkage due to migration of ions under electric field results in bending motion. For fabrication of electrochemically driven actuators, composites of ionic electroactive polymers are used. Conducting polymers (CPs) and ionic polymer metal composites (IPMC) are the mostly known among them. In case of PPy in neutral state (Carpi, Kornbluh, Sommer-Larsen, & Alici, 2011), reduction and oxidation take place respectively at the cathode and anode simultaneously. In case of actuators that are driven by anions, volume starts to expand as soon as the anions are introduced into the oxidized PPy at cathode for neutralizing desertion of electron. Conversely, reverse actuation occurs which is driven by cations when the anions are too heavy to migrate in electrolyte and at the cathode volume is contracted. The CP-based electrochemical actuator is fabricated with bilayer or multilayer designs for achieving bending motion electrochemically. For electrochemical actuators the largely used CPs are derivatives of polythiophene (PTh), polyaniline (PANI), and polypyrrole. The multilayered structured actuators are fabricated by coating two film conductive polymers with a layer of electrolyte (Torop, Aabloo, & Jager, 2014). Migration rate is the number of migrating ions per unit time. In electrolyte system, migration rate linearly

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controls the actuation speed. During the electrochemical process, speed of migration of ions is slow; hence the strain rate of traditional conductive polymers is low. However, due to the conductive polymer’s structural instability during the cyclic electrochemical process, the conducting polymer-based actuators have short life span. To overcome this drawback, conductive or functional additives are added into the conductive polymer to increase the actuation performance. Unfortunately, the conductive polymer-based electrochemical actuators have disadvantages such as limited lifetime and delay in responsiveness. Especially, the conductive polymers’ mechanical performances in electrolytic medium degrade compared to the dry-state counterparts and this makes them unsuitable for practical applications (Spinks, Mottaghitalab, Bahrami-Samani, Whitten, & Wallace, 2006). Generally, actuators based on IPMC are electrochemically driven, comprising an ion exchange polymeric film coated by two electrodes. Contraction and expansion of electrodes create bending motion. Earlier, liquid electrolytes were majorly applied in the IPMC actuator’s operation system. Unfortunately, due to the complex electrochemical system and heavyweight, liquid electrolyte reduces the applications of electrochemical actuators. Additionally, due to liquid resistance and slow migration of ions, the actuation responsiveness is slow for liquid electrolyte. The solid-state electrolyte-based actuators can operate with stable actuation in air, replacing the liquid electrolytes (Lee et al., 2014). Due to limited conductivity, actuation responsiveness of the solid-state electrolyte-based actuators is comparatively low (Baughman, 2003). Recently, an electrolyte force strategy is adopted for fabrication of IPMC actuators by introducing polymeric or metallic interface (Detsi, Onck, De, & Hosson, 2013). Similarly, poly(vinyl alcohol)-based polymer electrolytes have also been developed for electrical double-layered capacitor application (Aziz et al., 2021) (see Fig. 11.2). When sulfuric acid doped PANI doped with porous gold electrodes, the resultant electrochemically driven actuator shows three times greater strain rate compared to the conventional threecomponent actuators. The layered structure of the electrochemically driving actuator is mostly investigated for generating bending actuation (Chen et al., 2011; Chen, Liu, Hu, & Fan, 2008; Zhang et al., 2014). In comparison to electrothermally driven actuation of inorganic components, the counterparts based on polymeric materials show superior properties such as large deformation, lightweight, and flexibility. The electrothermally driving polymer-based actuators are divided into two categories. First one is single-layer polymeric film comprising conducting additives. Due to electrothermal heating, these additives can contract or expand (Chen et al., 2008; Hu, Chen, Lu, Liu, & Chang, 2010; Sellinger, Wang, Tan, & Vaia, 2010). The other one is bilayer structured film with coating consisting of polymeric materials and current conducting layers (Chen et al., 2010; Liang et al., 2012; Zhang et al., 2011; Zhang et al., 2014; Seo, Kang, Kim, & Kim, 2012). When electric current is applied, the conducting layer having low value of coefficient of thermal expansion (CTE) can produce heat and the polymer layer expands. This leads to

11.2 Sensors and actuators used on conducting polymers

FIGURE 11.2 A capacitor design based on conducting polymers and their performance (Aziz et al., 2021). Reproduced from Aziz, S.B., Asnawi, A.S.F.M., Abdulwahid, R.T., Ghareeb, H.O., Alshehri, S.M., & Ahamad, T., et al. (2021). Design of potassium ion conducting PVA based polymer electrolyte with improved ion transport properties for EDLC device application. Journal of Materials Research, 13, 933
946.

dissimilar changes in volume of the electrode materials and the polymeric layers generate bending deformation. The expansion layers are generally made up of PDMS, chitosan, PU, and epoxy. CNTs have electro heating characteristics, high electrical conductivity flexibility, so they are employed as heating electrode. For example, to form highly conducting networks, the CNTs are synthesized into the polymeric film, for example, elastomers of silicone and chitosan (Chen et al., 2008). Periodic heating of the CNTs’ conducting network causes thermal contraction and expansion and when a pulse voltage is applied, the resultant composite film actuator produces tunable vibration. Aligned CNT sheets and fibers have higher mechanical performances and high electrical conductivity. Hence, in electrothermally driving actuators, they have more potential for acting as heating electrodes. Graphene and its derivatives are also used as electrodes due to their improved mechanical, electronic, and thermal performances and negative CTE. Graphene-on-organic microactuator can generate large bending vibrational motion at driving voltage less than 4 V. Within 0.02 s the bending movement gets finished which is 10
100 times quicker compared to the traditional bilayered electrothermally driving actuator. Graphene can be fabricated from graphite through chemical exfoliation mechanism (Liang et al., 2012). Resultant film of graphene is current conducting and flexible, thus it can be used as heating electrodes. CNT fibers are mostly employed for rotary and contractive actuations. The helically aligned CNT fibers have huge potential in fabrication of the electrothermally

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driving actuator. The electrothermal fiber actuators prepared using inorganic nanowires and CNTs have very high cost. Therefore they are not used for applications purpose. As a replacement the inexpensive nylon, polyethylene and polyester are converted into helical structures and then they are used (Haines et al., 2014). The resulting coiled fibers precoated with conducting components when subjected to electrothermal treatment and heating are able to produce 49% contractive strain. For preparing supercapacitors the materials employed as flexible electrodes are conductive polymers having high capacitance and electrical conductivity (Wang et al., 2011). Various conductive polymers, for example, polythiophene (PTP), PANI, and polypyrrole (PPy) are inspected. But, after charging
discharging mechanism, bare conductive polymeric materials generally become less stable. To overcome this obstacle, one procedure is incorporating conducting materials having superior thermal and mechanical stability and better electrical conductivity, for example, graphene and CNT. Properties like long cyclic stability, high electrical conductivity, and specific capacitance are exhibited by these hybrid electrodes. PANI is grown on the exterior part of CNT network which acts as template. PANI is synthesized after polymerizing aniline monomers by in situ chemical polymerization method and it forms a homogenous lamination on to the CNTs. To prepare thin, flexible, and freestanding CNT/PANI hybrid film, “skeleton/skin” procedure is followed. In case of flexible supercapacitors, without using extra polymeric substrate or metallic current collector, the CNT/PANI composite film is employed as both charge collector and electrodes (Niu et al., 2012).

11.3 Energy storage from conducting polymer Development of energy storing systems [namely, supercapacitors or lithium-ion batteries (LIBs)] is vital to utilize renewable and sustainable energy sources. Supercapacitors are able to provide greater magnitude of energy density compared to dielectric capacitors. Not only that they have better energy storage capacity and but also can dispatch ample amount of charges within a few seconds. Hence, they have greater power capacity compared to batteries. A supercapacitor comprises two electrodes immerged into electrolytic solution having appropriate divider (Jost, Dion, & Gogotsi, 2014). Several kinds of conductive polymers are used in pseudocapacitors in terms of electrode materials. These conducting polymers are fabricated either electrochemically or chemically by oxidizing suitable monomer (Li, Bai, & Shi, 2009; Yuan et al., 2013). Based on the charge storing method of conducting polymers, other than active materials, the reversible, rapid redox reactions occur at electrode
electrolyte interface, raising attention in designing nanostructured CPs. Reduction in the size of bulk CPs minimizes the transportation distance for both electrons and ions that uphold the high capacitance at high density of current

11.3 Energy storage from conducting polymer

(Zhang, Uchaker, Candelaria, & Cao, 2013). Perpendicularly ordered PANI nanowire arrays show higher capacitance (950 F/g) and can preserve upto 780 F/g even at higher current densities (Wang, Wu, Meng, & Wei, 2014). This indicates that fabrication of one-dimensionally aligned nanostructures, especially nanowires, constitutes an efficacious procedure for highly performing electrodes in supercapacitors. The reason for this is accredited to the one-dimensional nanostructured PPy that permits an effective transportation of charge and delivers a minimized ion transport distance. But, there are few demerits for applying conducting polymers as materials of electrode in supercapacitors as the electrodes generally have comparatively poor cycle stability (which is responsible for inferior mechanical stability) and low electrical conductivity throughout long chargedischarge mechanisms. Although to design different composites of conducting polymers combined with other metals, metal and metal oxides/hydroxides and carbon materials overcome the drawback. Frequently, metals are employed as matrices for CPs because of their high electrical conductivities. The resulting composite electrodes generally have great performance rate, good cycling stability, and higher energy density compared to the pure CPs (Chen et al., 2015; Huang et al., 2015; Zhang, Hu, Yao, & Ye, 2015). Amidst them, stainless steel, aluminum (Al) foil, copper (Cu), nickel (Ni) foam, and gold (Au) are mostly applied metals. For example, core composite of PPy shell/3D-Ni can be fabricated using three-dimensional nickel films as current collector (Chen et al., 2015). The electrode composite exhibits high specific capacitance (726 F/g) and superior rate at high current density compared to pure PPy. Three-dimensional metal substrates with high conductivity deliver shorter diffusion route to transport ions fast increasing efficiency of transfer of electrons. Low conductivity of pure conducting polymers has moderately limited their applications in electrode materials in practical field. Conversely, carbon materials have advantages such as higher mechanical and electrical characteristics, high stability, and cost-effective (Wang et al., 2014; Yan, Wang, Wei, & Fan, 2014). Hence, blending of conducting materials and carbon materials at the molecular levels enhances the electrical conductivity and mechanical characteristics of polymers. Porous carbon materials in several forms (Chmiola, Celine Largeot, Simon, & Gogotsi, 2010; Kajdos, Kvit, Jones, Jagiello, & Yushin, 2010; Ma, Liu, & Yuan, 2013; Pech et al., 2010; Zhang & Zhao, 2009; Zhu et al., 2011). For example, mesoporous carbon, carbon sphere, activated carbon, and carbon onion constitute advantage of having high porosity, conductivity, and surface area that make them potential candidates for fabricating electrode materials. The effects of significant collaboration between porous carbon materials and CPs result in better electrochemical characteristics higher than the independent components (Liu et al., 2014; Yan et al., 2013; Nyholm, Nystro¨m, Mihranyan, & Strømme, 2011; Wang, Tao, An, Wu, & Meng, 2013; Yan, Cheng, Wang, & Li, 2011; Zhang, Kong, Cai, Luo, & Kang, 2010). By chemical oxidation polymerization method, perpendicularly aligned PANI nanowhiskers are fabricated on the superficial layer of aligned mesoporous carbon (CML-3) and the resultant PANI/CMK-3 complex

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exhibits specific capacitance and high capacitance detention even after 1000 cycles. These improvements are electrochemical properties due to the ordered pseudocapacitance of PANI nanowhiskers which can create electrolytic ions more electrochemically accessible and decrease their transportation distance. 1D nanostructures (e.g., nanorods, nanotubes, nanowires) are coming up as new material used in supercapacitors as they impart an efficient route for transporting charges as well as minimized ionic and electronic transportation distances (Yan et al., 2014; Yu, Tetard, Zhai, & Thomas, 2015). CNTs exhibit higher aspect ratio along with higher stability and electrical conductivity compared to porous carbon material. They have open tubular network structure and superior mechanical flexibility, which helps them to become better substrate to deposit active materials. CPs in several configurations of nanosheets, nanowire/rod, and nanoparticles are homogeneously sprouted on CNT surfaces by electrostatic and π
π interactions. Other than CNTs, carbon nanofibers are easily available commercially, precisely chosen as the current collector because they have properties, for example, lightweight, flexibility, chemical stability, and superior electrical conductivity (Chen et al., 2011). Carbon cloth synthesized from nanofibers is also used for developing electrodes. It is observed that carbon cloth containing PANI nanowire dispatches extraordinarily higher specific area and gravimetric capacitance (Horng et al., 2010). The composite electrodes with magnificent electrochemical properties are more preferred as supercapacitors. Graphene as attracting 2D carbon compound shows various intensity characteristics, for example, high flexibility, high surface area, and conductivity. Hence, graphene is extensively used as a superior supporting framework to grow different CPs (Wang et al., 2014; Yan et al., 2011; Liu, Ma, Guang, Xu, & Su, 2014; Mini, Balakrishnan, Nair, & Subramanian, 2011; Xu, Sun, & Gao, 2011). For further improving electrochemical properties of electrode composites, significant labor is given for controlling the surface structure of CPs grown on nanosheets of graphene. Both graphene and CNT show higher electrical conductivity and surface areas, so their assemblage in a 3D porous structure, for example, sponge, carbon foam, framework, and aerogel incorporates their characteristics and makes highly performing supercapacitor. Three-dimensional porous nanostructures having distinct meso/micropores and high surface area make adequate contact between active materials with electrolyte and minimize the ion transportation length, which results in faster reaction kinetics, essential for superior power density. Highly ionic and current conducting active CPs decrease polarization rate and enhance the reaction kinetics, when electrical conductivity is taken into consideration. The synthesis of tertiary composites incorporates the properties of individual constituents (CP, metal oxide hydroxide, and carbon material) such that every constituent contributes their best properties in the composite (Xia et al., 2012; Grover, Shekhar, Sharma, & Singh, 2014; Tang, Han, & Zhang, 2014; Wang, Yang, Huang, & Kang, 2012; Jung, Yoon, Kim, & Rhee, 2005). It is observed that the quality of graphene/MnO2 textile electrodes is boosted up when it is wrapped with conducting PEDOT:PSS thin layer by simple dry dipping method

11.3 Energy storage from conducting polymer

(Yu et al., 2011). The film of PEDOT:PSS decreases graphene/MnO2 composite’s internal resistivity. CP film of PEDOT:PSS serves as binder materials conductive additives as well as binder materials. Redox reaction
based pseudocapacitance helps it to participate in the charge storing mechanism. Coating of the polymer intercepts disintegration and fragmentation of MnO2 and suspension of ions during continuous charging
discharging mechanism. In recent times, cable structured flexible supercapacitors evolved from three-dimensional CNT-cotton thread/ MnO2/PPy composited electrodes are fabricated by a simple three-phase mechanism, where first ink of single-walled CNT is layered on the surface of cotton threads and then PPy films and MnO2 nanomaterials are easily grown on the cotton threads layered with CNT. This is done by in situ electrochemical deposition mechanism. Compared to carbonaceous components, transition metal hydroxides/oxides are able to deliver high specific capacitance according to reversible and fast redox reactions. However, comparatively poor cycling stability and low power density make them inappropriate for practical applications. They have lower power density because of inferior current conducting nature of the metal compounds for confining speedy electron transportation. Poor cycling stability makes structure of electrode materials weaker. Hence, it can be smoothly impaired by protrusion and diminution during charging
discharging mechanism. Conversely, CPs have comparatively higher current conducting capacity. Hence the acting synergic effect between metal oxides/hydroxides and CPs enhances electrochemical characteristics. In these types of composites, the oxide/hydroxide of metals generally impart contribution in increasing specific capacitance based on morphological characteristics and redox properties. The CPs are pivotal for higher electrical conductivities and higher mechanical stability facilitating rapid transportation of electron inside the composite material for higher charge
discharge rate. Their electrochemical properties are significantly influenced by crystallinity of metal oxide/hydroxide, morphology, or surface area (Yan et al., 2014; Wei, Cui, Chen, & Ivey, 2011). Other low-cost transition oxides/hydroxides of metals, for example, Fe2O3, TiO2, NiO/Ni(OH)2, MnO2, and Co3O4/Co(OH)2 are used. Generally, core/shell coaxially aligned nanorods/wires are synthesized by two strategies. In the first step, one-dimensional metal hydroxide/oxide is prepared that acts as core and then it is coated with a coating of CPs in the form of shell via electrochemical or chemical processes. Various types of coaxially aligned metal compound/composites of CPs, for example, CoO/PPy, MnO2/PPy, and MnO2/PEDOT are effectively synthesized and exhibit superior mechanical as well as superior electrochemical characteristics (Liu & Sang, 2008; Xie et al., 2011; Yao, Zhou, & Lu, 2013; Zhou, Zhang, Li, & Liu, 2013). For instance, nanowire array of CoO combined with PPy exhibits specific capacitance extraordinarily as high (2223 F/g), and 99.8% cycling stability (Zhou et al., 2013). Mainly, the core of CoO attributes to higher energy storing capacity; on the other hand, PPy shell which is flexible, porous, and the highly conductive facilitates bothion diffusion and transportation of electron inside the core of CoO as well as protects the core from disintegrating and

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shattering during charge
discharge mechanism. But the only disadvantage is the CP prevents the exposure of electrolyte with the core of metal oxide core and thus the energy density suffers. Metaloorganic frameworks (MOFs) are a novel set of porous materials, famous for having high porosity and high specific areas. Hence these materials can be used in drug delivery, sensor, catalysis, and gas storage and separation (Furukawa, 1989). They are also employed as electrode material in supercapacitors due to improved specific surface areas and exceptional porosities that increase availability of ions by diffusion effect in MOFs electrode. Generally, supercapacitors made up of MOF are divided into two categories: (Granero et al., 2011) preparation of porous carbon or metal oxide where MOFs act as original template, and (Weng et al., 2015) for energy storing purposes by electric double layer capacitor (EDLCs) method or through redox reaction of pseudocapacitor of metal centers pure MOFs are employed as novel electrode materials (Liu, Shioyama, Jiang, Zhang, & Xu, 2010; Yang, Xiong, Zheng, Qiu, & Wei, 2014). Supercapacitor’s effectiveness is hugely regulated by the electrode materials and the electrolyte used. Energy and power density of any device proportionally change with square of operating voltage. Typically, three varieties of the liquid electrolytes are employed in supercapacitors—ionic liquids (ILs), aqueous, and organic electrolytes. Aqueous electrolytes are easy to fabricate, possess high ionic conductivity, high safety, low viscosity, nonflammability, and cost-effectiveness are the major advantages of aqueous electrolytes. Their voltage limit is B1.0 V which is lower compared to organic electrolytes having voltage window of 2.5
3 V. Thus energy density of organic electrolytes is 6
14 times higher than aqueous electrolyte. IL, organic solutions, and aqueous solutions based on traditional supercapacitors are not suitable as flexible and portable electronics. To overcome these problems, solid-state supercapacitors are applied for energy storing. In comparison with liquid capacitors, solid-state supercapacitors share merits like flexibility, high safety and lightweight (Wang et al., 2014). The solid-state electrolyte acts as the electrode divider and ionic conducting media. It has characteristics (Gao & Lian, 2014) such as high electrochemical stability, wide voltage window, stability, good formability, greater mechanical strength, low electrical conductivity at room temperatures, and higher ionic conductivity (Lu, Yu, Wang, Tong, & Li, 2014; Zhong et al., 2015). Polymer gel electrolytes exhibit comparatively higher ionic conductivity so they are the most commonly used solid-state electrolytes. They are composed of polymeric frameworks which act as host material providing superior mechanical integrity. The electrolytic salts provide ionic conduction and aqueous or organic solvent impart ion-conducting medium. The most commonly used host polymers are potassium polyacrylate, poly(acrylic acid), etc. In supercapacitors, directly polymeric materials are used as substrates. For fabricating flexible supercapacitors, polyethylene terephthalate (PET) and PDMS are widely employed as substrates. PDMS films are used to prepare translucent and flexible thin filmed supercapacitor. For increasing thermal, electronic, and mechanical stability of PDMS films, single-walled CNTs are introduced (Yuksel, Sarioba, Cirpan,

11.3 Energy storage from conducting polymer

Hiralal, & Unalan, 2014). For achieving high electrical conductivity and flexibility, conducting materials like CNT and graphene are deposited onto PET films. Other polymeric materials are also applied apart from PET and PDMS. Localized pulsed laser radiation is applied for rapid transformation of pure polyimide (PI) surface into electrical conductive porous structure made up of carbon. Using programmable laser scanning method, the interdigitated pattern of electrode is grown on PI sheets. To fabricate conducting flexible electrodes for preparing flexible supercapacitor, the PI sheets work as precursor for carbonization and a flexible substrate according to this method. This consolidated electrode exhibits better flexibility and stability in comparison with conducting material-coated polymeric substrate-based electrodes. Pure polymeric materials-like nonwoven cloths synthesized from papers and wood fibers are used as substrates that are lightweight and flexible. Flexible CNT/paper electrodes are synthesized by coating a paper with CNTs. In LIB, cathode and anode are isolated by ion conducting electrolytic solution (Lee, Yanilmaz, Toprakci, Fu, & Zhang, 2014). Reversible electrochemical redox reactions procure the capacity of LIB between two electrodes. Lithium ions travel from cathode to electrolyte and then they reach the surface of anode during charging mechanism. Subsequently, electrons move cathode to anode through external circuitry. This whole mechanism is altered in discharge process. Electrodes are composed of materials such as binders, conducting additives, and active materials and they are connected with current collectors. Separator is positioned between cathode and anode to tackle short circuit. The separator also absorbs electrolyte. Polymers have a significant role in LIB fabrication. The parameters of LIBs such as charging
discharging rate, perpetuity, capacity, voltage basically are dependent on active materials of anode and cathode. The mostly employed materials for cathode are LiFePO4, LiMn2O4, Li4Ti5O12, and LiCoO2. Subsequently, the materials used for anode are Sn, Si, and graphite. The conventional inorganic cathode materials suffer from poor structure stability and low theoretical specific capacity. Meanwhile, due to possible environmental pollution and limited resources, the massive scale fabrication of cathode materials based on transition metal is hampered. Polymer-based electrode provides greater theoretical capacities compared to inorganic materials. On the other hand, organic polymer electrodes exhibit higher power densities and higher rate performance compared to inorganic-based electrode due to rapid organic redox reactions. So, for replacing inorganic electrode materials, extensive studies are going on to explore polymeric electrode materials. The five major classes of conductive polymers are polythiophene (PTh), polyaniline (PAn), polyparaphenylene (PPP), and polyacetylene (Pac). In the past decades, these are widely explored as electrode constituents. In case of organosulfides, the S
S bond can be fragmented and reconstructed in reversible manner. In comparison with the conductive polymers, superior performance can be attained by the redox reaction. Hence, for rechargeable LIBs, organosulfides are acknowledged as preferable organic constituents. A typical example of organosulfides is PDMcT. Organic bipolar polymers of nitroxyl radical are employed as p-type polymers to achieve stable cycling performance

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and higher discharge voltage. Among them, the most widely used material is PTMA [poly(2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl methacrylate)]. Polymers of nitroxyl radical have fast reaction kinetics which is favorable for LIBs. Due to properties such as high structural diversity, fast reaction kinetics and high theorical capacity, conjugated carbonyl polymer is considered preferable organic component for LIB. The electrochemical redox reaction is simplified as a reverse reaction of the carbonyl group and an enolization reaction. For promoting the reaction, conjugated structure is crucial. Electroactive polymer materials have several merits, for example, good flexibility, good design versatility, and high capacity. But they are restrained from practical applications due to few internal limitations. Subsequently, other than CPs, polymer electrode constituents have limitations like poor electrical conductivity. During the reaction of electrode, this causes slowdown of transportation of electron. Hence, extra efforts are given to overcome this drawback and to improve electrochemical performances. One method is conductive carbon materials; for example, porous carbon, graphene, carbon fiber (CF), and CNT are coupled with the polymer and a composite electrode is formed. This removes major drawbacks. Due to properties of CFs such as chemical resistance, thermal and electrical conductivity, and high strength, they have been on focus of attraction. The fusion of polymer electrode constituents and CFs improves the electronic conductivity of insulated polymer. Following this method, the rate capability and cyclability are observed to be exceptionally upgraded. Since CNT has unparallel chemical and physical characteristics like high mechanical strength, good chemical stability, and high electrical conductivity, it is assumed as suitable constituent used for LIBs. CNTs are integrated into polymeric components which result in the formation of hybrid electrodes. Capacity of LIBs can be improved by incorporating conducting additives like CNTs into the hybrid electrodes. At the 80th cycle the resultant LIB exhibits discharge capacity of 86 mAh/g (Sivakkumar and Kim, 2007). At twice rate, it exhibits remarkable discharge capacity of 65 mAh/g. This value is higher compared to what is achieved by the combination of super-p carbon and polyaniline. Graphene shows extraordinary chemical and physical properties, for example, high stability, mechanical strength, huge theoretical specific surface area, and remarkable electrical conductivity. Hence, it is evaluated to be a potential candidate as polymeric electrode material for LIBs. For instance, via dispersion
deposition mechanism, graphene is introduced into PTMA (Guo, Yin, Xin, Guo, & Wan, 2012). By in situ polymerization method, polymer/graphene hybrid is manufactured with highly loaded active materials. Using this strategy poly(anthraquinonyl sulfide)/ graphene can be successfully prepared (Song et al., 2012). In comparison with pure polymers, nanocomposite exhibits six times higher electrical conductivity because graphene has high dispersion capability. Interlinked nanochannels present in the porous carbon materials enfold redox-active electrode constituents. They enhance the properties of polymeric electrodes majorly in two methods. In one method, the nanochannels provide accommodation to the polymers, facilitation of the electrolytic infusion, and prevention of the dissolution mechanism. In the

11.4 Energy harvesting based on polymer

second method the structure of carbon matrix enhances rapid flow of electron. But, the size of pore diameter impacts the performance of the organic-carbon hybrid. Larger pore size enables the filling of molecules.

11.4 Energy harvesting based on polymer We have limited reserves of fossil fuels. To fulfill ever-expanding need of electricity, we need to find out nonconventional sources (Cook et al., 2010; Dillon, 2010). New devices are continuously invented to harvest ambient energy in the form of heat, mechanical vibration, and light (Zeng et al., 2014). Polymers are strong, lightweight, characteristic-tunable, solution-processable material. Polymers are promising candidate for developing flexible, low cost, harvesting devices at large scale (Coakley & McGehee, 2004). Photovoltaic devices are capable to generate electric current or voltage in the presence of light and convert photo energy into electricity (Tang, 1986). Polymer or plastic solar cell having high efficiency of energy conversion (Dun et al., 2015) is used in the applications of photovoltaic devices. In a photovoltaic process, excitons are generated and free carriers are produced from incident photons absorbed by conjugated polymers. Excitons are nothing but bonded electron
hole pairs that dissociate and free carriers formed at interface of donor or acceptor and migrate into transporting materials. Afterwards, electrons are deposited in anode and holes are collected in cathode. The electrodes are present after the electron and hole extraction layer. Electrons
holes are then transferred to external circuit for the formation electricity from light (Zhu, Yang, & Muntwiler, 2009; Collavini, Vo¨lker, & Delgado, 2015). The solar cell’s performance is determined by fill factor connected with microscopic morphology of charge extraction layer, active layer, and treatment done after deposition process (Guo et al., 2013). For enhancing the photovoltaic performances of solar cell, fill factor (voltage, open-, and short-circuit current) must be upgraded. Typically, fabricated conjugated polymer is employed as donor, which is also p-type semiconductor. Polymer mixed with [6,6]-phenylC61-butyric acid methyl ester (PCBM) is used as acceptor and light-harvesting polymer is used as a donor for absorbing light as excitation source. The most popular conjugated polymers are derivatives of polythiophene (PTh), such as poly(3hexylthiphene) (P3HT). Maximum efficiency of 5% can be observed when PCBM is employed as acceptor and P3HT is used as donor. For electron injection and transport in heterojunction blend, another important component is acceptor components which are derivatives of fullerene especially PCBM and PCBM (Wienk et al., 2003). Recently, n-type conjugated polymers that produce every polymer solar cell (PSC) are developed as acceptor materials (Facchetti, 2013). The morphology basically implies the phase segregation and crystallite of conjugated polymers in heterojunction mixture. In dye sensitized solar cells (DSSC),

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different types of conducting polymers such as polypyrrole (PPy), PEDOT, PANI are used as counter electrode. Generally, PEDOT:PSS, which is a p-type conducting polymer, is used in solar cells as hole-transporting element. To eliminate the drawback of traditional DSSC being highly volatile, several p-type conductors or solid-state electrolytes are being realized for solid-state DSSCs. Piezoelectric generator or transducer transforms vibrational energy of the particles present in the material into electrical energy for driving minute devices that require less energy and can run for a long time. Piezoelectric components that are interposed between two electrodes can induce strain which creates electric field and thus voltage after applying an external force. Some polymer materials such as polyuria (Hattori, Takahashi, Iijima, & Fukada, 1996), nylon-11 (Newman, Chen, Pae, & Scheinbeim, 1980), and PVDF and its copolymer (Furukawa, 1989) are used to fabricate most of the polymer-based piezoelectric generators. Similarly, triboelectric effect is defined as the charge transfer between any two materials. Triggering potential for triboelectrification and preserving the induced charge on the surface of the dielectric is assumed as parameters in case of triboelectric generator. According to the electrostatic effect and contact triboelectrification, based on specified applications, various generator structures can be designed. Generally, these generator structures are classified into four groups—(Wang, Lin, & Wang, 2015) (1) contact mode (Fan, Tian, & Lin Wang, 2012), (2) single mode, (3) sliding mode (Li et al., 2015; Niu et al., 2013), and (4) freestanding triboelectric-layer mode structure (Niu et al., 2015). Efficiency of triboelectric generator depends on the surface area of triboelectrified materials. Typically, PDMS, Kapton (polyimide), and polytetrafluoroethylene (PTFE) are used as dielectric materials. Triboelectric generator is applied for converting mechanical energy into electric signal via electrostatic induction and triboelectrification and this process is quite similar to piezoelectric generator. For example, blending with polyvinylidene fluoride can also be explored to develop flexible devices based on conducting polymers (Sengupta et al., 2021; Sengupta, Ghosh, Bose, Mukherjee, Roy Chowdhury, Datta), as can be observed in Fig. 11.3. Another class of energy harvesting devices are thermoelectric generators that depict a different method of harvesting energy and generating electricity. Inorganic elements are generally applied in thermoelectric generators. But inorganic components are heavyweight and fragile in nature and they have high annealing temperature. As illustrated in Fig. 11.4, highly efficient, lightweight polymer materials are used as thermoelectric generators (Zhang et al., 2021). In the 1820s, thermoelectric generator, based on Seebeck effect, was innovated. Diffusion of carriers like electrons or holes generates diffusing potential which further resists diffusion via drift current. Eventually, an equilibrium is established between drift current and diffusion current, creating a temperaturedependent electric field at this junction. It is used to measure temperature. The ample availability of sunlight as thermal energy and excessive wastage of heat energy from automobiles proves that thermoelectric generators can be successfully used as low-cost energy harvesting sources. To calculate the efficiency of thermodynamic generator, the output power (Pout) is divided by the input heat

11.4 Energy harvesting based on polymer

FIGURE 11.3 A flexible piezoelectric nanogenerator fabricated using electrospinning technique of conducting polymers depicting human motion energy harvesting from (A) heel, toe movement, and wrist movement, and (B) finger twisting movements (Sengupta, Das, Dasgupta, Sengupta, & Datta, 2021). Reproduced with permission from Sengupta, A., Das, S., Dasgupta, S., Sengupta, P., Datta, P. (2021). Flexible Nanogenerator from electrospun PVDF
polycarbazole nanofiber membranes for human motion energy-harvesting device applications. ACS Biomaterials Science and Engineering, 7(4),1673
1685.

(Qin), so the efficiency (η) is formulated as η 5 Pout/Qin. Power conversion efficacy of thermodynamic generator can be calculated by using following formula (Culebras, Uriol, Go´mez, & Cantarero, 2015): pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 1 ZTav 2 1 ηmax 5 Φc pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  ΦC γ 1 1 ZTav 1 Th =Tc

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FIGURE 11.4 Representative 3D printing of flexible electrodes composed of conducting polymers: (A) design wearable thermocell, (B) fabrication of wearable thermocell, (C) flexible strapshaped thermocell, and (D) strap-shaped thermocell charge supercapacitor and light a lab timer. Reproduced from Zhang, S., Zhou, Y., Liu, Y., Wallace, G.G., Beirne, S., & Chen, J. (2021). All-polymer wearable thermoelectrochemical cells harvesting body heat. iScience, 24(12), 103466.

where Tc and Th represent the low and high temperature, respectively. Meantime, Tav is the mean of low and high temperature and ΦC 5 (Th 2 Tc)/Th is Carnot efficiency. Z is figure of merit and it is calculated as: ZT 5

α2 σ T κ

where α is the Seebeck coefficient, k is thermal conductivity of material, σ is the electrical conductivity. For enhancing efficiency of thermoelectric generators, Seebeck coefficient and electrical conductivity are concurrently increased with constant conductivity. In thermoelectric generators, p-type conductive polymers are employed. The mostly used conducting polymers in thermoelectricity are PANI, PPy, polyalkylthiophenes and its derivatives, polyacetylene, PEDOT and poly(2,7-carbazolyenevinylene) (Wei, Mukaida, Kirihara, Naitoh, & Ishida, 2015). Changing parameters

11.5 Organic light-emitting diodes

such as backbone structure and side-chain length, electrical behavior of semiconductive conjugated polymer can be effectively regulated. Electrical conductivity and Seebeck coefficient are affected by doping level of the constituents (Mengistie et al., 2015). The polymer conformation and morphology influences its electrical conductivity. By changing polymerization parameters such as electrolytes, temperature, current, density and monomer concentration, thermoelectric property can be modulated. It has been found out that various types of charge carriers influence the thermoelectric behavior of conductive polymer materials (Wang, Ail, Gabrielsson, Berggren, & Crispin, 2015).

11.5 Organic light-emitting diodes The phenomenon by which the electrical excitation causes emission of light energy is called electroluminescence. First time, polymer light-emitting diodes (LEDs) were successfully developed using PET by Heeger and his coworkers in 1992. This innovation made lead to creation of flexible display devices. Later in 1994, polymer-based white light-emitting electroluminescent device was developed by Matsumoto and Kido (Kido, Hongawa, Okuyama, & Nagai, 1994). In comparison with inorganic LEDs, polymer light emitting diodes (PLEDs) inherit various advantageous properties such as high flexibility, adaptability for large area fabrication, compatibility to solution process, low cost, and numerous applicable materials. The electrons and holes overcome the threshold of the interface when the bias voltage is exerted externally across the device. The electrons leave the cathode and enter the organic layer. Similarly, holes travel from anode to organic layer. Electrons flow through the lowest unoccupied molecular orbital (LUMO) region which is a part of the electron transporting layer (ETL) and holes flow through the highest occupied molecular orbital (HOMO) region present in the hole transporting layer (HTL). The external electric field excites the carriers and they move to the emission layer. There is emission of light as soon as the excitons bounce back to ground state. Color of emitted light depends on variation in energy between HOMO and LUMO regions of organic material. For achieving high luminous efficiency and low driving voltage, the effectiveness of injection and transportation of charge carriers are important. For achieving lower driving voltage, two factors are important. During the charge carrier injection mechanism, Ohmic contact between the organic layers and the electrodes is the first factor and during the charge carrier transportation mechanism, maximizing the mobility of carriers is the second important factor. Applied bias voltage when overcomes the interfacial gap between organic material and electrode, the charge carriers are injected. The minimum voltage applied for exceeding the interfacial gap is known as turn-on voltage. Lowering the interfacial gap increases light-emitting efficiency by incorporating buffer layers. The mobility of charge carriers of organic molecular materials is comparatively low (Campbell Scott & Malliaras, 1999). The

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electrons are confined inside single molecules. Consequently, PLEDs do not have high luminous efficiency and low turn-on voltage because injection and transportation of charge carriers act as limiting factors. Rate of injection at an electrode having limited contact is proportional to the mobility of charge carriers of organic materials. Similarly to improve charge carrier transport, ETL and HTL are introduced for realizing successful transportation of electrons and holes simultaneously. It is demonstrated that introducing a PPV layer between emissive PF layer and PEDOT:PSS to obstruct electrons results in increasing the maximum luminous efficiency twice. Polymerized perfluorocyclobutane (PFCB)-based copolymers consist of electron-rich triarylamine side chains which help in transportation of blocking electrons and holes. Amongst various types of copolymers, bis(N,N0 -diphenyl-N,N0 bis(3-butylphenyl)-(1,10 -biphenyl)-4,40 amine)-PFCB (BTPD-PFCB) has maximum capability to transport holes compared to PEDOT:PSS and the maximum triarylamine group density. Work function of BTPD-PFCB and PEDOT:PSS are 5.3 eV and 5.2 eV, respectively. Hence, for emissive materials having comparatively poor HOMO level, the number of holes injected from HTL and released in the emission layer is relatively increased. In comparison, with PEDOT:PSS, copolymers based on PFCB when employed as HTL demonstrate superior electron blocking and hole transporting characteristics. But the fabrication method of PFCB films is very complex. In place of PEDOT:PSS, thin metal oxide films like V2O5, MoO3, WO3 can be used due to several advantages. However, vacuum evaporation technique is followed for preparing the thin metal oxide films, which restricts their usage in PLED. Carbon-based materials have several applications because of improved mechanical flexibility, conductivity, and optical transmittance. Graphene oxide (GO) is an another material which can be employed as alternate of PEDOT:PSS to make HTL. As the middle layer composed of GO prevents movement of electrons from emission layer to indium tin oxide (ITO) and thus the charge carrier injection balance is improved and simultaneously avoids radiative quenching of excitons (Lee et al., 2012). When the performances of PLED with various types of HTL layers are analyzed in the form of power efficiency voltage (P-V), luminous efficiency voltage (E-V), L-V, and J-V curves, it is seen that for those PLEDs where PEDOT:PSS is used as HTL, at 12.6 V the highest luminance is 33,800 cd/m2 and at 9.6 V the highest luminous efficiency is 8.7 cd/A. However, in case of those PLEDs where rGO is employed as HTL, it is observed that at 13.0 V the highest luminance is 8300 cd/m2 and at 8.6 V the highest luminous efficiency is 5.0 cd/A. This indicates that when rGO is used as HTL, both luminance and luminous efficiency decrease compared to those with PEDOT:PSS since rGO has poor hole injection capability and electrical conductivity due to greater interfacial contact gap between HTL and emission layer. When GO is used as HTL, the properties of PLED are dependent on width of the GO films. The GO film’s width is 4.3 nm for the optimized PLEDs. In this case it is observed that at 4.4 V the highest power efficiency is 11.0 lm/W, at 6.8 V the highest luminous efficiency is 19.1 cd/A and at 10.8 V the highest luminance is 39,000 cd/m2, which are boosted by 280%, 220%, and 120%, respectively, in comparison with standard PLEDs where PEDOT:PSS is used HTL.

11.6 Electrochromic materials and devices

GO has better capability to block electrons to itself due to their large bandgap. The probability of electron hole recombination is enhanced by this property. Hence such high performance is obtained. Thinner GO films make it tough for the material for realizing full coverage. This is not good for properties to block electrons. The electrical conductivity increases in case of thicker GO films and it leaves poor impact on hole transportation mechanism. An example of application of conducting polymers as organic LEDs based on PEDOT polymer treated with benzoic acid (Kang, Kim, & Kim, 2021) and their characterization is shown in Fig. 11.5. Electron transporting or injection layers (ETL) are used in PLED for decreasing the barrier for electron injection between emission layer and cathode, implying that ETL should have good electron affinity and high ionization potential for blocking hole. Other than inorganic material and tiny organic molecules, conjugated polyelectrolytes (CPEs) effectively reduce the electron junction barrier. CPEs are more preferred in practical applications as it removes metals having lower work function like Ba and Ca, which are highly sensitive to surrounding atmosphere, decreasing the device’s longevity. Using polyfluorene backbone, poly(9,9-bis(2-(2-ethoxyethoxy)ethyl)fluorene) uniformly forms film outside the emission layer. This is a type of neutral CPE. In the form of cathode modifiers, anionic and cationic CPEs are also employed. After applying external voltage, counterions migrate to the interfacial plane existing between CPEs and emission layer in case of PLEDs where cationic CPEs are employed as ETL. Luminous efficiency of the PLED decreases due to the quenching effect of counterions. In case of PLEDs, where ETL is made up of anionic CPEs, the mobile counterions transfer to the cathode for obtaining strongly aligned dipole. Hence, a bilayered structure is created between the cathode and the mobile ions facilitating injection of electrons. As cathode modifier, anionic CPEs are relatively more appropriate than cationic CPEs. Compared to the traditional design, the inverted design eliminates the materials which inject electrons and are air-sensitive like Ca, for solving difficulties of deterioration of the device at normal room temperature. For electron injection layer present in the inverted PLED, air-stable metal oxides are generally employed. ZnO is highly preferred material amongst them since they have n-type characteristics which originate from oxygen vacancies and interstitial Zn (Bolink, Coronado, Repetto, & Sessolo, 2007). Branched polyethyleneimine ethoxylated (PEIE) and polyethyleneimine (PEI) act as layer for injecting electrons in invertedly designed PLEDs (Kim et al., 2014). PEIE and PEI are air stable and consist of amine groups which form string of dipoles between the ZnO surface and PEI. These strong dipoles are favorable for decreasing the gap of electron injection and improving its mechanism.

11.6 Electrochromic materials and devices Electrochromism is the event of reversibly changing colors shown by some materials. For fabricating electrochromic devices, several materials are employed, for example, polymers, photonic crystals, liquid crystals, and transition metal oxides

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FIGURE 11.5 Luminescence and current-density properties of organic light-emitting diodes composed of conducting polymers depicted by (A) schematic representation of bending test, voltage vs (B) current density and (C) luminance, (D) current efficiency relationship with current density, and (E) performance stability under bending test. Reproduced from Kang, H.S., Kim, D.H., Kim, T.W. (2021). Organic light-emitting devices based on conducting polymer treated with benzoic acid. Scientific reports, 11(1), 3885.

(Arsenault, Puzzo, Manners, & Ozin, 2007; Booth & Casey, 2009; Nicoletta et al., 2005). Various procedures describe the color-changing property of polymeric electrochromic material such as electrothermal chromatic transition and electro-induced oxidation reduction.

11.6 Electrochromic materials and devices

Conjugated polymers have some distinctive characteristics like color versatility and electrical conductivity and employed as ideal electrochromic materials especially in textiles, displayers, mirrors, and sensors. Under a specific voltage the chemical species reversible changes between two redox states are responsible for their reversible electrochromic behaviors. This process is called electroinduced oxidation reduction (Beaujuge, Amb, & Reynolds, 2010; Niklasson & Granqvist, 2007). Based on this, the multilayered structure of an electrochromic device comprises ion storage/electrochromic layer, electrolyte, electrochromic, substrate, and transparent conductor (Thakur, Ding, Ma, Lee, & Lu, 2012). The translucent conductor as the functional electrode adheres to electrochromic layer; electrolyte serves in ions conduction and supports redox reaction. As soon as ions are introduced in electrochromic layer, ion storing layer acts as a buffer which captures the ions from electrolytic layer. With advancement in electrochromic devices, another electrochromic layer replaces the ion storing layer. Electrochromic materials and their derivatives (namely, PPy, PANI, PTh) are mostly explored conjugated polymers. Among them, first conjugated polymer was PTh to be used as electrochromic material (Garnier, Tourillon, Gazard, & Dubois, 1983). But, the insoluble nature of PTh makes it difficult to synthesize its derivatives. Poly(3,4-ethylenedioxythiophene) (PEDOT) derived from PTh has unique electro-optical properties, high stability, and good conductivity. Exhibiting properties such as rapid switching of color, environmental stability, and low cost, PANI is another promising conjugated polymer. Hence, PANI has high potential for practical application. If the applied voltage is increased, PANI exhibits a moderate change in colors among black, blue, green, and yellow along with high reversibility and swift response. The conventional electrochromic materials can create small range of colors. To reinforce the change in colors, other electrochromic polymers are fabricated displaying purple (Reeves et al., 2004), blue (Balan, Gunbas, Durmus, & Toppare, 2008; Invernale et al., 2009; Wu, Lu, Chang, & Wei, 2007), green (Durmus, Gunbas, Camurlu, & Toppare, 2007; Gunbas, Durmus, & Toppare, 2008), yellow (Lin et al., 2015), red (Dyer, Craig, Babiarz, Kiyak, & Reynolds, 2010), and orange (Dyer et al., 2010). Conjugated polymers which can produce various colors at neutral, intermediate, and doped states are prepared (Thompson, Schottland, Zong, & Reynolds, 2000). Changing the components of monomers, the colors of copolymers are easily readily regulated (Beaujuge, Ellinger, & Reynolds, 2008). The π-conjugated polymers such as poly (3-methylthiophene) (P3MT), poly(2,5-dimethoxyaniline) (PDMA), and PEDOT are introduced into the wires made up of stainless steel for preparing electrochromic fibers by electrochemical polymerization method. After lamination of gel on electrochromic layer, stainless steel wire is crumpled. The electrochromic fibers show reversible and rapid change in colors (Li, Zhang, Wang, & Li, 2014). One of the favorable strategies is to prepare polymeric or inorganic hybrid materials. NiO/PANI (Sonavane, Inamdar, Deshmukh, & Patil, 2010), NiO/PEDOT (Xia et al., 2009), NiO/PPy (Sonavane, Inamdar, Dalavi, Deshmukh, & Patil, 2010), PEDOT/Au/CdSe (Bhandari et al., 2010), WO3/PANI (Ma, Shi, Wang, Zhang, &

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Li, 2014), PANI/Graphene (Sheng, Bai, Sun, Li, & Shi, 2011), and PEDOT/CNT (Bhandari, Deepa, Srivastava, Lal, & Kant, 2008) are few examples of inorganic hybrid or polymeric materials having exceptional electrochromic characteristics. Synergetic characteristics of individual constituents help these hybrid materials to exhibit high coloration efficiency and fast responsiveness. Polymer dispersed liquid crystals (PDLCs) are micrometer-sized crystalline droplets of liquid scattered into the polymeric matrix. Under electric field, they are able to convert themselves into opaque states from transparent states. The liquid crystal molecules are realigned by the electric field and transmittance changes reversibly. Functional device comprising thermoplastic matrices and crystalline nematic liquids, which can change its colors by electricity are developed from PDLC film (Nicoletta et al., 2005). Due to scattering of light by crystalline liquids dispersed into the polymeric matrix, these electrochromic devices are not transparent. After applying an electronic field of 1 V/μm, the crystalline liquids reconstruct themselves along the electric field at droplet surface and thus increasing transmittance to 8% from 2%. The liquid crystals retain its initial random arrangement as soon as the electric field is removed. Due to unique electrochromic characteristics, photonic crystals are widely explored. Photonic crystals inherit properties such as metallodielectric, supercapacitor structures which control propagation of electromagnetic waves setting a definition of forbidden and allow photonic energy bands. According to Bragg’s law, they show functions such as controlling photonic movement, reflecting thin wavelength bands, and exhibiting compositional colors. Since photonic crystals have exceptional properties such as highly aligned structures, multiresponsive colors, and nonbleachable compositional colors which cover the complete visible range of spectrum. According to volume variation stimulated by temperature and electro-induced redox reactions, the lattice constants present in the crystal structures change and thus color transition takes place in photonic crystals (Seeboth, Lo¨tzsch, Ruhmann, & Muehling, 2014). The PSCs possess characteristics features like flexibility, facile association with other devices, and lightweight. Plastic substrate PET films coated with ITO are used for fabrication of flexible polymeric solar cells. For fabricating flexible electrodes, as a replacement of ITO layer, conductive polymers are directly used to laminate polymeric substrates while preparing flexible electrodes. To increase conductivity of the electrodes, varieties of metal grids other than conductive polymers are employed for constructing composite layer. Silver ink is deposited on PET surface to manufacture flexible ITO free conducting PET film. For instance, flexible electrodes exhibit 4.8% of maximum power conversion efficiency and high flexibility (Nickel et al., 2014). In construction process of PSCs, polymers are also employed as working layers, other than acting as conducting layer and substrate. Flexible energy storing devices gain a lot of attraction. Selecting appropriate electrode material and designing suitable structure for achieving stable electrochemical and mechanical performances during stretching, twisting or bending are quite

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challenging. Introducing polymers or polymeric composites for flexibility enhancement is considered one efficient strategy. At academic or industrial level, supercapacitors and LIBs are widely explored.

11.7 Conclusions Conducting polymers have immense potential in the fields of sustainable and flexible electronics, flexible devices, prosthetics and drug delivery devices, corrosionresistant coatings, and biosensing applications. An extensive understanding of the mechanism of electrical conduction and doping effects is allowing the synthesis of new conducting polymers as well as adoption of surface modification techniques to functionalize biomolecules on the conducting polymers. However, developments in increasing the solubility of these polymers will be one of the key aspects to enhance their industrial applicability and processing. Similarly, stability of conducting polymers is another area which needs to be improved for wider applications. For, life cycle of supercapacitors based on conducting polymers is an area seeking improvements. Moreover, biomedical applications of conductive polymers are yet another vast unexplored area. It is imperative that researches into new conducting polymers and composites are enhanced intensively in the coming years.

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3171. Zhang, S., & Li, F. (2012). High performance ferroelectric relaxor-PbTiO 3 single crystals: Status and perspective. Journal of Applied Physics. Zhang, S., Zhou, Y., Liu, Y., Wallace, G. G., Beirne, S., & Chen, J. (2021). All-polymer wearable thermoelectrochemical cells harvesting body heat. iScience, 24(12), 103466, [Internet]. Available from https://www.sciencedirect.com/science/article/pii/S2589004221014371.

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Zhang, X., Pint, C. L., Lee, M. H., Schubert, B. E., Jamshidi, A., Takei, K., et al. (2011). Optically- and thermally-responsive programmable materials based on carbon nanotube-hydrogel polymer composites. Nano Letters, 11(8), 3239
3244. Zhang, X., Yu, Z., Wang, C., Zarrouk, D., Seo, J. W. T., Cheng, J. C., et al. (2014). Photoactuators and motors based on carbon nanotubes with selective chirality distributions. Nature communications, 5. Zhao, X., & Wang, Q. (2014). Harnessing large deformation and instabilities of soft dielectrics: Theory, experiment, and application. Applied Physics Reviews, 1, 021304. Zhong, C., Deng, Y., Hu, W., Qiao, J., Zhang, L., & Zhang, J. (2015). A review of electrolyte materials and compositions for electrochemical supercapacitors. Chemical Society Reviews, 44, 7484
7539. Zhou, C., Zhang, Y., Li, Y., & Liu, J. (2013). Construction of high-capacitance 3D CoO@Polypyrrole nanowire array electrode for aqueous asymmetric supercapacitor. Nano Letters, 13(5), 2078
2085. Zhu, X. Y., Yang, Q., & Muntwiler, M. (2009). Charge-transfer excitons at organic semiconductor surfaces and interfaces. Accounts of Chemical Research, 42(11), 1779
1787. Zhu, Y., Murali, S., Stoller, M. D., Ganesh, K. J., Cai, W., Ferreira, P. J., et al. (2011). Carbon-based supercapacitors produced by activation of graphene. Science, 332, 1537
1541.

CHAPTER

Shape-memory polymers

12 Deepshikha Rathore

Amity School of Applied Sciences, Amity University Rajasthan, Jaipur, Rajasthan, India

12.1 Introduction Shape-memory polymers (SMPs) are an incipient category of intellectual polymers, because on suitable stimulation they are capable to alter their shape in a previous manner. SMPs can attain temporary shape in the form of deformation, which is identified as second fixed shape, until deformed system is exposed to a suitable stimulus. After imposing appropriate stimulus, system gains its original shape immediately. As such polymers remember their memorized shape, hence they are known as SMPs (Lendlein, 2010). Those stimuli reactive polymers can transform their mechanical, optical, and electrical properties substantially, including shape, phase separation, surface, permeability upon slight change of atmospheric conditions such as pH, temperature, magnetic field, light, electric field, sonic field, ions, solvent, glucose, and enzyme (Meng & Li, 2013). These transformations on demand are of scientific and technological importance and can be explained in three significant processes as shown in Fig. 12.1: 1. Programming: It is the first step, in which, on applying mechanical force at eminent temperature, SMPs hold impermanent shape after changing their previous shape.

FIGURE 12.1 Schematic representation of the thermally induced one-way SME. SME, Shape-memory effect. Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00016-4 © 2023 Elsevier Inc. All rights reserved.

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2. Storage: It is the second step, in which, on cooling under stimulation temperature and removing applied mechanical force, impermanent shape of SMPs is sealed. 3. Recovery: It is the third step, in which, SMPs achieve their previous shape.

12.2 Various shape-memory polymers A variety of SMPs have been developed, in which three very common polymers (polylactide, polytetrafluoroethylene, and ethylene-vinyl acetate) were used. Those polymers exhibit shape-memory effect (SME), which are categorized in cross-linking and thermal transitions to understand their comprehensive features (Safranski & Griffis, 2017).

12.2.1 Cross-linking The various polymers are available, including several types of structures. Those structures are (1) linear, (2) branched, and (3) linked in the form of network, which depends upon the variety of monomers utilized in formation of polymers and shown in Fig. 12.2. To form the polymer network, polymer chains are linked together either by covalent bond or by physical entanglements. This linkage process restricts permanent chain dislocation and prevents chain’s mobility, due to which netpoints generate, which are permanent. Hence, the original shape establishes for the SMP. This cross-linking of molecule or polymer chain can be

FIGURE 12.2 Schematic of polymer structures: (A) Linear, (B) branched, (C) lightly cross-linked, and (D) highly cross-linked (Safranski & Griffis, 2017).

12.2 Various shape-memory polymers

accomplished very easily with the help of exposure of radiation like ultraviolet (UV) light, electron beam, gamma, etc. or heat (Hearon et al., 2013; Yakacki et al., 2008). There are two types of cross-linking: (1) chemical and (2) physical. When link between the molecules is created by covalent bonds, it is known as chemical cross-linking, for example, methacrylate or epoxies networks. On the other hand, when link between the molecules is generated by hydrogen bonding, phase separation, or physical entanglements, it is called physical cross-linking, for example, thermoplastic polyurethanes. Generally, cross-linking is measured by the cross-linking density. With the help of theory of elasticity of rubber, the cross-linking density is determined in the form of modulus by Eq. (12.1) (Pascault et al., 2002): ξ 5 3RTρ

(12.1)

where ξ is the elastic modulus, R is the universal gas constant, T is the temperature in Kelvin, and ρ is the cross-link density.

12.2.2 Thermal transitions The glass transition is kinetic phenomenon and considered a second-order phase transition—the temperature over which the amorphous polymer material comes into a flexible rubbery state from a brittle glassy state as they are heated. This temperature is known as glass transition temperature. The polymer structure keeps glassy state and it is rigid in nature with some small degree of molecular motion below the glass transition temperature. While the polymer structure becomes flexible, higher degree of molecular motion is achievable above the glass transition temperature. There are usually three techniques utilized to measure the glass transition: (1) differential scanning calorimetry (DSC), in which a step-change occurs in the heat capacity of the polymer during the glass transition; (2) dynamic mechanical analysis (DMA), in which a dramatic decrease in storage modulus signifies the onset of the glass transition or the peak of the tan delta is often used to represent the glass transition temperature, even though the glass transition occurs over a temperature range; and (3) thermomechanical analysis, in which a change in volume or a change in the coefficient of thermal expansion occurs when heating through the glass transition. The temporary shape can be locked in the SMPs with the help of cooling after glass transition, due to which modulus and viscosity increase rapidly. The glass transition temperature depends upon compositions and chemical structure of polymers; hence it varies broadly for a variety of polymers. For example, (meth)acrylates possess glass transition from 223
C to 112
C (Safranski & Gall, 2008). The shape-memory cycle can also use crystallization of polymer chains. After melting, cooling process is performed, and due to which some polymers begin to organize in the form of crystalline lamellae, they are folded upon themselves and known as stacked polymer chains. Further, these crystalline lamellae may arrange in the form of bigger crystalline spherulites. Moreover, mostly polymers possess crystalline and amorphous both regions; thus

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they come in the category of semicrystalline. Because when some sections of the polymer chains are folded and stacked in these lamellae, a section remains without stack in the amorphous region outside of the crystalline lamellae. Chain mobility is constrained during crystallization, and due to which a temporary shape becomes programable for generating SME. This is usually happened by a huge enhancement in modulus as the polymer cools after reaching glass transition (Nielsen & Landel, 1994). On the other hand, above glass transition the melting process takes place, due to which polymer spherulites and lamellae lose their arranged stacking and revert into nonarranged melt. This revert process of lamellae disorder permits the motion of polymer chains at huge scale resulting to bring original structure of SMPs. The complete features in terms of crystallization and melt transitions of SMPs can be seen by DSC. In the DSC scan, exothermic peak is associated with crystallization transition and endothermic peak is related to melt transition.

12.2.3 Categorization of shape-memory polymers Chemical structures of SMPs are responsible to categorize them with different properties. According to Mather (Liu, Qin, & Mather, 2007), SMPs can be divided into four core groups. Chemically cross-linked matrix that employs a glass transition for their activation is first classification. Chemically cross-linked matrix that employs a melting transition for their activation is second classification. Third classification is physically cross-linked polymer that employs a glass transition for their activation, while physically cross-linked polymer that employs a melting transition for their activation comes in the fourth classification.

12.3 Mechanism of shape-memory polymers The internal energy plays an important role in SMPs, because whole mechanism of SMPs depends upon internal energy. The mechanism can be assumed in the way, through analyzing an alteration in entropy, when strain energy is stored and released in the SMPs system (Lendlein & Kelch, 2002). In undeformed or original state, the SMPs possess very low entropy. When temperature of SMPs increase up to activation energy or above, they start to deform and attain higher entropy state. Here, strain energy can be locked on cooling. At this stage, SMPs achieve metastable state at high energy. Due to vanishing kinetic energy at cooling, the melt transition or glass transition inhibits SMPs to return at low or original energy state. Further, they gain activation energy on raising temperature above melt transition or glass transition. At this stage, available polymer chains come in moveable condition again by discharging stored strain energy resulted decrease in entropy and arrive back to their original state or shape. There are two significant practical aspects (1) cross-links and (2) switching segments of molecular structure

12.4 Composites using shape-memory polymers

of SMPs. With the help of cross-links, the SMPs are able to memorize their original specific shape, while reversible shape modification experiences by switching segments, which are the polymer chains existing between cross-links. In the switching segments, on increasing temperature above definite transition temperature, the polymer chains become extremely elastic. Hence, they can be deformed certainly and can produce huge strain at small stress. Hereafter, the polymer chains become immovable with securing temporary shape, when system is cooled beneath transition temperature. The one kind of transition is known as glass transition at which polymer chains become inelastic, because at this stage they leg behind the activation energy for basic movement. On the other hand, transition is recognized as melt transition at which system latches the impermanent shape by precluding the polymer chains from returning to the original perpetual shape. The schematic representation of mechanism for three types of polymers with two types of transition temperature and structures is illustrated in Fig. 12.3 (Lendlein & Kelch, 2002).

12.4 Composites using shape-memory polymers Polymer composites consist of polymer network with fillers, including various physical and chemical properties. The size and properties of fillers play a significant role in polymer composites, which can enhance the existing property of polymer composites. The functionalization of SMPs in terms of polymer composites with magnetic, optical, electrical, and biological fillers has been described in this section. The fillers can be in the particle, sheet, and tubes form.

FIGURE 12.3 Schematic representation of the molecular mechanism of the thermally induced shapememory effect for (A) a multiblock copolymer with Ttrans 5 Tm, (B) a covalently crosslinked polymer with Ttrans 5 Tm, (C) a covalently cross-linked polymer with Ttrans 5 Tg. If the increase in temperature is higher than Ttrans of the switching segments, these segments are flexible, and the polymer can be deformed elastically. The temporary shape is fixed by cooling down below Ttrans. If the polymer is heated up again, the permanent shape is recovered.

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12.4.1 Functionalization of shape-memory polymers by silicate To enhance mechanical properties of SMP nanocomposites (SMPNCs), the layered silicate has been obtained a very efficient filler (Lendlein, 2010). It has also been observed from the literature (Lendlein, 2010) that layered silicate-doped SMPNCs have been achieved high value of Young modulus and heat resistance, while lower value of inflammability and gas permeability with higher level of biodegradability. The aspect ratio can be increased from 10 to 1000 by developing layer thickness of the order of 1 nm in layered silicate. There are three different kinds of morphological feature in nanocomposites as (1) intercalated, (2) flocculated, and (3) exfoliated, designed by interaction between polymer network and filler as layered silicate. The intercalated nanocomposites were developed and characterized by introducing polymer chain into layered silicate in the form of replication distance of some nm as crystallographic manner. Whereas flocculated nanocomposites are generated because of hydroxylated edgeedge interactions, intercalated stacked silicate layers start to flocculate. Moreover, exfoliated nanocomposites are fashioned when each specific silicate layer arranges separately in the polymer network with regular distances, which entirely depends upon the relative clay composition. The aggregation of silicate in the polymer network may occur due to deficiency of interaction between hydrophobic polymer and hydrophilic silicate. However, this problem can be solved by surface modification of filler, which makes compatible interaction with polymer network resulted significant enhancement in mechanical properties. It has been found that the silicate of high aspect ratio provides large surface area to stay in contact and make bond with polymer network. This higher order of silicate and polymer interaction due to large aspect ratio of silicate generates greater barrier and mechanical properties than polymer network alone (Pinnavaia & Beall, 2000). This enhancement in mechanical properties could be accredited by elevated toughness with high strength, where silicate layers are randomly dispersed in polymer network, which propagate stress in matrix and impact strongly on the mobility of polymeric chain (Lietz et al., 2007; Yang, Zhang, Schlarb, & Friedrich, 2006). It is very well demonstrated that dispersion of silicate layers in polymer network reduces the mobility and creates short-range order in alignment of polymer chain. Hence, the development of resistance to drive polymer chain due to stress is increasing the toughness and modulus in composites (Yang et al., 2006).

12.4.2 Functionalization of shape-memory polymers by magnetic particles The functionalization of SMPs using magnetic particles can bring many features in composites with enhanced properties. Cobalt, nickel, iron, and their composites possess ferromagnetic or ferrimagnetic properties. These magnetic materials attract toward strong magnetic field and come back after removing field. Without magnetic field the magnetic domains arrange randomly, hence generate net

12.4 Composites using shape-memory polymers

magnetic moment zero. As magnetic field is applied, the magnetic domains start to arrange in the direction of magnetic field and generate strong magnetization within the domain region. In this process, ferromagnetic or ferrimagnetic particles can produce heat through eddy current and hysteresis loss in an alternating magnetic field, which depends upon different kinds and sizes of the magnetic particles (Goldman, 1990). The magnetic particles interact with the external magnetic field, SMP network, and with each other through Zeeman term, elastic deformation, and demagnetization field, respectively (Conti, Lenz, & Rumpf, 2007). When an alternating magnetic field is applied to SMP composite functionalized with magnetic particles, the temperature of the composite increases. As temperature reaches above the switching temperature of SMP network, the SMP has probability to recover its permanent or original shape. Whenever SMP could not trigger the SME via direct heating on increasing surrounding temperature, then this noncontact indirect heating technique via magnetization could be used to actuate SME. The size of the magnetic particles can alter the amount of produced heat via an alternating magnetic field because they are directly related to each other. The core size and concentration of magnetic particles can be measured using vibrating sample magnetometry (Kurchania, Rathore, & Pandey, 2015). At high magnetization frequency the nanoparticles have capability to generate enough heat needed for stimulated SME (Rosensweig, 2002). For controlling the amount of heat in the system, the applied frequency acts as a significant parameter. If the size of the magnetic particles is at the micrometer scale, then on applying magnetic field, the eddy current loss and hysteresis loss become higher and generate excess amount of heat in the low-frequency range. Consequently, agglomeration and higher size of the particles are not suggested for inducing SME specially in the medical field, because extra heating may cause serious damage of nearby tissues. Some specific ferromagnetic particles contain feature of thermoregulation because of Curie temperature (TC), which appears on producing heat by magnetic particles of suitable size in an alternating magnetic field. The ferromagnetic particles have capability to generate heat up to TC. Above TC ferromagnetic material alters in paramagnetic and cannot produce heat through the process of hysteresis loss. Because ferromagnetic material with appropriate TC acts like a thermostat, hence it could substantially decrease the risk of excess heating in biomedical applications. Below TC ferromagnetic nanomaterials (below 15 nm, single domain) possess superparamagnetic behavior, in which the energy needed to alter the direction of magnetic moments present in single domain is analogous to the surrounding thermal energy. The magnetic moments will start to reverse randomly at a significant rate. In the absence of applied magnetic field, superparamagnetic materials do not hold any substantial magnetization as ferromagnetic materials possess, hence they do not accumulate. The applied magnetic field orients all the magnetic moments in superparamagnetic (single domain) material in its own direction. This orientation enhances the strength of applied magnetic field in its vicinity. On removing magnetic field, Brownian motion begins to mix up magnetic moments resulted material’s demagnetization.

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12.4.3 Functionalization of shape-memory polymers by carbon fillers The functionalization of SMPs with carbon compounds has brought in knowledge where they use as fillers [e.g., carbon fibers (CFs), carbon black, carbon nanorods (CNRs), graphite, etc.]. All these carbon fillers are extremely conductive and reduce electric resistance of composites significantly. Hence, conductive SMPs can be produced in terms of SMPC. These SMPC has capability to trigger through small amount of heat by indirect actuation process. Due to the abovementioned carbon fillers, electricity can be conducted in the plane of all covalently attached layers to create electrons-cloud because of delocalization of outer electrons. Thus the smaller bulk electrical conductivity for carbon generated than for metals. Although metals in both nano and bulk forms are more conductive than any kind of carbon fillers, still carbon fillers have some atmospheric quality. Metals have tendency to corrode and form a nonconductive layer on their surface due to oxidation process while carbon forms two-dimensional flat graphite sheet at atmospheric pressure, in which carbon atoms are weakly bounded in the form of hexagonal rings by van der Waals forces resulting graphite has cleaving properties with softness. The layers slip very easily on another in the graphite. Hence these layers can be folded cylindrically in the form of carbon nanotubes (CNTs) either in single-walled (SWCNT) or in multiwalled (MWCNT) (Harris, 2009). Carbon nanofibers (CNFs) are also easily available in huge quantities at a suitable price. Due to very large diameter B100 nm, CNFs are different from CNTs (SWCNTB1-nm diameter and MWCNTB10-nm diameter). On the other hand, CFs are thin fibers of approximately 5- to 10-μm diameter. In CFs, carbon atoms are microscopically bonded as crystals and aligned parallel along the axis of fiber. This kind of crystalline arrangement of carbon atoms makes the fibers usually stronger in the same dimension. The functionalization of SMPs using carbon compounds brings new opportunities in the field of composite. The polymer composites using SMPs as polymer matrix and CNTs as fillers have been prepared by many researchers in the last decade. It has been observed from literature (Lendlein, 2010) that the presence of CNTs in small concentration can enhance the mechanical and thermal properties, which can also generate exceptional electrical properties with improvement in the behavior of SMPs. Due to anisotropic behavior of CNTs, they show percolative nature at small concentration as they use like fillers in the SMPs network. The diameter in the nano range and excellent electrical and mechanical properties of CNTs offer an exceptional opportunity to enhance the structural strengthening and thermal controlling of a polymer network. The functionalized SMPs with CNTs are conducting composites in terms of SMPCs and can generate SME’s actuation on applying electric field rather than by providing heat through atmospheric temperature. Hence, for controlling microaerial vehicles, SMPCs are being employed as electroactive actuators (Paik, Goo, Jung, & Cho, 2006). Some chemical properties like interfacial adhesion, homogenous distribution as well as

12.4 Composites using shape-memory polymers

compatibility of CNTs with polymer matrix have been accomplished by surface modification using mixture of sulfuric and nitric acids (Lin et al., 2003). Usually, insertion of carbon compounds as filler in the matrix of SMPs may not only considerably modify the polymeric behavior as thermoplastic elastomers but also enhance several characteristic features of composites.

12.4.4 Functionalization of shape-memory polymers by biocompatible materials The biocompatible material hydroxyapatite or hydroxylapatite (HA— Ca10(PO4)6(OH)2) is a mineral appearing in nature in the crystalline hexagonal form, in which crystal unit cell contains two molecules of Ca5(PO4)3(OH). Around 70% of inorganic HA mineral is being comprised by natural bone and teeth. Hence, HA is very vital bioactive material which uses in orthopedic, dental, and maxillofacial applications, it supports bone ingrowth and osseointegration (Jarcho, 1981). Hence, it is used as a filler while replacing bone and as a coating to promote bone ingrowth into prosthetic implants. The mechanical properties and protein adsorption capacity of the SMPCs experience great improvement when nano-HA are introduced to it (Labella, Braden, & Deb, 1994). It has been observed that proteins in mineralized tissues acted as nature’s crystal engineers, playing a pivotal role in promoting or inhibiting the growth of minerals such as hydroxyapatite. It is to be noted that pure β-tricalcium phosphate (β-TCP), Ca3(PO4)2, does not exist in nature or in any biological system and cannot be prepared by direct precipitation and hydrolysis methods. It can be synthesized by heating calcium-deficient apatite of appropriate Ca/P molar ratio above 800
C or by heating amorphous calcium phosphate. In terms of solubility and in vivo biodegradation, both HA and β-TCP were found to be different. But displayed osteoconductive properties. A carrier matrix for bioactive agents was provided by porous β-TCP material and it formed a moldable putty composition when a binder was added. The use of HA and β-TCP in wide applications in hard tissue implantations has been limited owing to their poor mechanical properties such as low strength and fracture toughness (De Groot, De Putter, Smitt, & Driessen, 1981). But composites from biocompatible SMPs and HA or β-TCP have shown improved mechanical properties resulting in the introduction of these composites in wide-ranging medical applications. The nanocomposites of hydroxyapatite (HA-Ca10(PO4)6(OH)2), poly(DL-lactide) (PDLL)/β-TCP have proved to be an outstanding biomaterial used in tissue engineering and have also been used clinically in various forms (Zheng et al., 2008). The preparation of these nanocomposites with different β-TCP is same as the preparation of PDLL/HA nanocomposites described in literature (Lendlein, 2010). As detected by laser diffraction particle size analyzer, average particle size of β-TCP used in this work was approximately 720 nm with particle distribution of 2001500 nm. Phosphate buffer saline solution at 37
C (PBS, pH 5 7.4) was used to investigate the

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hydrolytic degradation process while studying the effect of in vitro degradation on the shape-memory capability of PDLL/β-TCP nanocomposites. The specimens were removed from degradation medium and rinsed with distilled water to remove residual buffer salts at fixed intervals and then put in vacuum for drying. The SME of PDLLA/β-TCP and pure PDLLA composites with different β-TCP were examined after and before dipping in the buffer solution. Bending test was conducted for studying the shape recovery. Noticeable differences were observed in the behavior of their shape memory at different intervals of degradation time. The dependence of shape recovery ratio (RR) on degradation time as calculated according to given Eq. (12.2) (Lendlein, 2010), RR 5

180
2 final angle 3 100 180


(12.2)

The SMPCs were folded by 180
for programming. RR decreases when in vitro degradation time in PBS increases. Keeping degradation time constant, the RR for PDLLA/β-TCP composites was way more than that of pure PDLLA. RR was minutely higher for all composites at the 21st day of degradation time as reported (Lendlein, 2010). This may be due to breaking of PDLLA chains or crystal phase changes of β-TCP particles. The following reaction formula depicts how in vitro degradation of PDLLA/β-TCP composites can result in Ca2P2O7, CaHPO4, and HA phases (Yakacki et al., 2008): 4β 2 Ca3 ðPO4 Þ2 1 H2 O-Ca2 P2 O7 1 Ca10 ðPO4 Þ6 ðOHÞ2 ; Ca2 P2 O7 1 H2 O-2CaHPO4

(12.3)

Due to a plasticizer effect, reduction of Tg can take place when new inorganic phases are formed (Yang, Huang, Li, & Chor, 2005). The existence of Ca2P2O7, CaHPO4, and HA particles aids in the shape recovery ratios after 21 days of degradation time due to imparting more constraints on polymer chain dynamically (Prokop, Jubel, & Hahn, 2005). Shape memory may become undesirable as the degradation time increases. This may be due to PDLLA chains breaking and dissolution degradation of the inorganic phases in PDLLA/β-TCP composites.

12.5 Limitations of shape-memory polymers There are two fundamental limitations of SMPs. First one describes about recovery time and activation process, while second discusses about recovery force and work capacity in the following section.

12.5.1 Recovery time and activation process The recovery time of SMPs depends on difference between their activation temperature and the operating temperature. If activation temperature is very near to

12.5 Limitations of shape-memory polymers

their operating temperature, system takes only some minutes in recovery, while activation temperature is very far above from the operating temperature, system may take long time as hours or days to recover its previous shape. According to Yakacki et al. (2008), the SMPs have capability to recover within a couple of minutes if the operating temperature is nearby to the activation temperature, which is a glass transition of given material as illustrated in Fig. 12.4. Hence, for fast recovery, quick heat transfer is required because most polymers are thermal insulators. Usually, in various applications, heating is carried out in the presence of air. Hence, for rapid recovery a wet atmosphere is suitable due to maximum heat transmission (Lakhera et al., 2012a). According to preferred application, activation process has many advantages and drawbacks. The thermal activation can be comparatively improved by providing heat using energy or heat source. Thus big problem may occur as quick thermal activation, if there is no control on heat source. Here, thermal processes are attaining more emphasis rather than classic thermal processes for activation. To recover previous shape a solvent-induced activation may perform, particularly in wet biological atmospheres where utilizing a heat source is not possible. In this case, recovery of SMPs completely depends on the consuming rate and quantity of water. Consequently, it may take some minutes to hours for recovery, which may be adequate for preferred application. These activation techniques are not like mechanically sponsored activation, which basically uses mechanical energy to apply force on SMPs to bring them at low energy level from its high energy level (Safranski & Griffis, 2017; Yakacki, 2008). This mechanical activation can be performed quickly without any use of

FIGURE 12.4 Strain recovery profile (%) at operating temperature (Top 5 50
C) for three different SMP networks (1, 2, and 3) as function of time (min) with varying transition temperature (Ttrans) (Yakacki et al., 2008). SMPs, Shape-memory polymers.

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solvent or thermal energy. Nevertheless, SMPs must have capability to bear the distortion and applied forces without rupture during returning back to their previous shape.

12.5.2 Recovery force and work capacity As a consequence of forced or partially forced recovery, SMPs act as actuators driven by the stored energy from programming. Many parameters on which stored energy is dependent include temperature, strain and deformation rate, the programming conditions that determine the deformation capacity of SMPs. There is a trade-off in the failure strain as a function of programming temperature for many SMPs that are chemically cross-linked and have a glass transition (acrylics, epoxies, some polyurethanes). Hence there occurs an optimal temperature near the onset of the glass transition (Safranski & Gall, 2008) that exists for programming as shown in Fig. 12.5. The SMP should be programmed at this optimal temperature to achieve the largest recovery forces, so that most of the energy is put into the polymer during deformation (e.g., a high strength and high strain actuator). As examined by Lakhera (Lakhera et al., 2012b), mechanical work can also be performed by SMPs. It was found that under a constant amount of

FIGURE 12.5 Failure strain as a function of temperature relative to glass transition temperature for two networks at 2.5 mol% PEGDMA550. Applied for permission from Elsevier, Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and cross-linking density on the thermo-mechanical properties and toughness of (meth)acrylate shape memory polymer networks. Polymer, 49(20), 444655.

12.5 Limitations of shape-memory polymers

programming strain, mechanical work improves with cross-linking density (i.e., rubbery modulus). Under partially constrained conditions, when a bias force is applied until the fully constrained condition is reached, the recovery strain decreases in a linear manner. As the bias force decreases the recovery strain to 50% of its unconstrained value, the mechanical work displays a maximum. This factor can be of great help in the designing best of SMP actuators just by knowing the maximum amount of possible work that can be done and the distance the actuator will move during deployment. It is to be noted that these results were observed at a constant programming strain for polymers with varying crosslinking densities (i.e., different spring stiffness constants) and under compression conditions. Stress and strain change as a temperature change for SMPs, although work is a function of stress and strain. This state is usually employed to establish the recovery stress, σr , which can be applied by the SMPs. This is known as permanent-strain recovery as the quantity of strain is set up to a constant value, which can be seen by Eq. (12.4) (Safranski & Griffis, 2017), σr  Er Amax

(12.4)

where Er is the rubbery modulus and Amax is the maximum programming strain. For a given SMP, work can be maximized when programming occurs at its

Table 12.1 Thermomechanical properties of networks composed of 90 mol% tBA and 10 mol% multifunctional (meth)acrylate. Multifunctional (meth)acrylate

Tg (
C)

Er (MPa)

BPA1700 BPA540 BPA688 BPA512 BPA468 NGPDA HEXDA PEGDMA550 PETA TETA428 TETA604 TETA912 TPTA GPTA DTTA DPPHA

22.75 70.5 43.5 64.5 59.5 62.5 68.5 40.5 98 83 55 24.5 58 69.5 92 74

7.35 8.15 8.25 9.0 8.8 6.48 10.85 10.7 42.5 25 16.65 15.95 23 15.5 49.5 129.5

Source: Applied for permission from Elsevier, Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and cross-linking density on the thermo-mechanical properties and toughness of (meth) acrylate shape memory polymer networks. Polymer, 49(20), 444655.

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optimal temperature and programming strain is increased. The thermomechanical properties of networks composed of 90 mol% tBA and 10 mol% multifunctional (meth)acrylate are tabulated in Table 12.1.

12.6 Conclusion The SMPs have been discussed as actively moving materials, which come in the category of smart materials. SMPs have been defined by three steps: programming, storage, and recovery, in which SMPs alter their shape at ambient temperature, lock it on cooling, and further regain it. The molecular mechanism of the thermally induced SME for different polymers has been illustrated precisely. The functionalization of SMPs with silane, carbon nanomaterials, and magnetic nanomaterials prepares them for various applications. The biocompatible SMPs are being used in orthopedic, dental, and maxillofacial applications, and it supports bone ingrowth and osseointegration. The limitations of regain their shape have been described by strain recovery profile as function of time with varying transition temperature and failure strain as a function of temperature relative to glass transition temperature of different SMP networks.

References Conti, S., Lenz, M., & Rumpf, M. (2007). Journal of the Mechanics and Physics of Solids, 55, 1462. De Groot, K., De Putter, C., Smitt, P., & Driessen, A. (1981). Journal of Ceramic Science and Technology, 1, 433. Goldman, A. (1990). Modern ferrite technology. New York: Van Nostrand Reinhold. Harris, P. J. F. (2009). Carbon nanotube science: Synthesis, properties and applications. New York: Cambridge University Press. Hearon, K., et al. (2013). Electron beam crosslinked polyurethane shape memory polymers with tunable mechanical properties. Macromolecular Chemistry and Physics, 214, 12581272. Jarcho, M. (1981). Clinical orthopaedics, 157, 259. Kurchania, R., Rathore, D., & Pandey, R. K. (2015). Studies on size dependent strain and nanomagnetism in CoFe2O4 nanoparticles. Journal of Materials Science: Materials in Electronics, 26, 93559365. Labella, R., Braden, M., & Deb, S. (1994). Biomaterials, 15, 1197. Lakhera, N., et al. (2012a). Biodegradable thermoset shape-memory polymer developed from poly(β-amino ester) networks. Journal of Polymer Science, Part B: Polymer Physics, 50, 777789. Lakhera, N., et al. (2012b). Partially constrained recovery of (meth)acrylate shape-memory polymer networks. Journal of Applied Polymer Science, 126, 7282. Lendlein, A (Ed.), (2010). Advances in Polymer Sciences: Shape Memory Polymer. Springer.

References

Lendlein, A., & Kelch, S. (2002). Shape-memory polymers. Angewandte Chemie— International Edition, 41, 20342057. Lietz, S., Yang, J. L., Bosch, E., Sandler, J. K. W., Zhang, Z., & Altsta, V. (2007). Improvement of the mechanical properties and creep resistance of SBS block copolymers by nanoclay fillers. Macromolecular Materials and Engineering, 292, 23. Lin, Y., Zhou, B., Fernando, K. A. S., Liu, P., Allard, L. F., & Sun, Y. P. (2003). Polymeric carbon nanocomposites from carbon nanotubes functionalized with matrix polymer. Macromolecules, 36, 7199. Liu, C., Qin, H., & Mather, P. T. (2007). Review of progress in shape-memory polymers. Journal of Materials Chemistry, 17, 15431558. Meng, H., & Li, G. (2013). A review of stimuli-responsive shape memory polymer composites. Polymer, 54, 21992221. Nielsen, L. E., & Landel, R. F. (1994). Mechanical properties of polymers and composites (2nd (ed.)). New York: Marcel Dekker. Paik, H., Goo, N. S., Jung, Y. C., & Cho, J. W. (2006). Development and application of conducting shape memory polyurethane actuators. Smart Materials and Structures, 15, 1476. Pascault, J. P., et al. (2002). Thermosetting polymers. New York: Marcel Dekker. Pinnavaia, T. J., & Beall, G. W. (2000). Polymer-clay nanocomposites. New York: Wiley. Prokop, A., Jubel, A., & Hahn, U. (2005). Biomaterials, 26, 4129. Rosensweig, R. E. (2002). Heating magnetic fluid with alternating magnetic field. Journal of Magnetism and Magnetic Materials, 252, 370. Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and crosslinking density on the thermo-mechanical properties and toughness of (meth)acrylate shape memory polymer networks. Polymer, 49, 44464455. Safranski, D. L., & Griffis, J. C. (2017). Shape-memory polymer device design. Atlanta, GA, United States: MedShape, Inc. Yakacki, C. M., et al. (2008). Strong, tailored, biocompatible shape-memory polymer networks. Advanced Functional Materials, 18, 24282435. Yang, B., Huang, W. M., Li, C., & Chor, J. H. (2005). Effects of moisture on the glass transition temperature of polyurethane shape memory polymer filled with nano-carbon powder. European Polymer Journal, 41, 1123. Yang, J. L., Zhang, Z., Schlarb, A. K., & Friedrich, K. (2006). On the characterization of tensile creep resistance of polyamide 66 nanocomposites. Part II: Modeling and prediction of long-term performance. Polymer, 47, 2791. Zheng, X., Zhou, S., Yu, X., Li, X., Feng, B., Qu, S., & Weng, J. (2008). Effect of in vitro degradation of poly(D,L-lactide)/β-tricalcium composite on its shape-memory properties. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 86B, 170.

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Rapid prototyping

13

Umesh K. Dwivedi1, Shashank Mishra2 and Vishal Parashar2 1

Amity School of Applied Sciences, Amity University Jaipur, Jaipur, Rajasthan, India Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India

2

13.1 Introduction Rapid prototyping (RP) is an interdisciplinary approach in the field of manufacturing industry that has a potential to bring a revolutionary change in prototyping. The methodology employed in the design and development of parts using RP systems is novel and dissimilar from the conventional process. RP as evident by its name is a technology capable of quick fabrication of prototypes. It was developed by Charles Hull in 1986, since then the technology has become a thrust field of attention for researchers and design engineers. The first RP technology was based on the SLA process and termed as stereolithography. Because of advantages like flexibility and ease of customization over other conventional processes, these systems attract the attention of the researchers. Expiry of the patents associated with these systems opened an opportunity for progress and the technology evolved rapidly after that. There are several examples of industrial applications employing RP systems for production, for example, a group in China has developed cheap houses causing the cost of the house as low as $4000. A wide variety of materials are used in these systems for the development of parts that employ thermoplastics like polylactic acid (PLA), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), and nylon. Some costly RP processes are utilizing metal alloy powders as feed materials. Ceramics are used for the development of three-dimensional (3D) printed scaffolds. Depending upon the method these systems take up raw materials in different physical forms like powder, liquid, slurry, and filaments. A prominent application associated with 3D printing systems includes the development of custom orthoses and prostheses. According to Chang et al. micro-manufacturing (MEMS, microelectromechanical systems) is also being done using RP processes (Blok, Longana, Yu, & Woods, 2018; Chang, 2015; Chen, Jin, Wensman, & Shih, 2016; Chua, Leong, & An, 2020; Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016; Ngo, Kashani, Imbalzano, Nguyen, & Hui, 2018; Wang, Jiang, Zhou, Gou, & Hui, 2017). Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00027-9 © 2023 Elsevier Inc. All rights reserved.

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In RP, the computer-drafted designs are fabricated layer by layer and bonding them together using automatic and smart manufacturing processes; thus the final product is apparently printed, because of which this method is also termed as additive manufacturing or 3D printing. Along with mass customization, these printers do not employ any tool for part machining. Also, there is absolutely no wastage of material in the fabrication processes. On contrary, most of the conventional machining processes use the subtractive approach for fabrication that results in material wastage. MacDonald and Wicker (2016) have mentioned that, with the progress in the commercial application of RP, these systems are now considered more than just prototyping technique. Because of the mass-scale production from this technology users are reluctant to use the term prototyping, that is why it is frequently termed as rapid manufacturing or additive manufacturing (MacDonald & Wicker, 2016). While most of the authors across the world use RP as a synonym for additive manufacturing, Gurr and Mu¨lhaupt (2012) have considered computer numeric controlled (CNC) machining as one of the types of RP as shown in Fig. 13.1. CNC machining operations are rapid in production and prototyping, but the fabrication methodology is subtractive in nature leading to a contradiction to call it as one of the additive manufacturing methods. Table 13.1 elaborates about the available 3D printing technology, their year of market entry, materials employed by these systems, maximum size of the parts, and the cost at which they are purchased.

FIGURE 13.1 Classification of rapid prototyping processes according to the initial state of the processed material and the principle of layer solidification (Gurr & Mu¨lhaupt, 2012).

Table 13.1 Key characteristics for comparison (Kumbhar & Mulay, 2018). RP process SLA FDM SLS 3DP 3D bioplotting LOM

Materials Photocurable resins (acrylics and epoxies) Thermoplastics (ABS, PC) Metals and thermoplastics (PA12, PC) Thermoplastics, ceramics, metals Thermoplastics, hydrogels, ceramics Paper, polymer, metal, ceramic

Market entry

Max. part size (mm)

Dim accuracy

Cost/machine (h)

Cost/part

1987

1500 3 600 3 500

,0.05

.10.000

Medium

1991

914 3 610 3 914

0.0.1

.10.000

1991

700 3 380 3 580

,0.05 0.1

.150.000

1998 2001

4000 3 2000 3 1000 150 3 150 3 140

0.0.1 0.0.1

.20.000 .150.000

1990

550 3 800 3 500

0.0.15

.50.000

Low/ medium Medium/ high Low Low/ medium Low/ medium

ABS, Acrylonitrile butadiene styrene; LOM, laminated object manufacturing; PC, polycarbonate; RP, rapid prototyping; SLA, stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing.

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Since the last two decades, 3D printers are finding a lot of applications in biomedical and artificial implants. Some of the most crucial applications include surgical planning by the development of the replica of the organs. The printing of artificial soft tissues is one of the most significant biomedical applications achieved with the help of RP technology; others include development of implants, prosthetics, etc. (MacDonald & Wicker, 2016). Design and fabrication of multifunctional products by use of 3D printing have been achieved recently. MacDonald and Wicker (2016) have reported different types of such developments. Examples of such fabrication include the development of sensor, actuators, antennas etc. which is further discussed in the forthcoming sections. Such systems are capable of producing parts from hybrid materials. Giachini et al. (2020) have employed 3D printing technology to produce functionally graded material (FGM) with uniform stiffness gradient by employing two different materials and varying the composition of the two materials throughout the fabricated part (Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016). Fig. 13.2 shows the commercialization status of RP systems. Another noteworthy innovation in the field of RP is the design and development of fiber-reinforced composite (FRC) (Zindani & Kumar, 2019). There are several examples of manufacturing composite materials by employing rapid manufacturing processes. Matsuzaki et al. (2016) have produced jute fiberreinforced unidirectional thermoplastic composite using 3D printing technology. Composites are widely used in different applications including aircraft, automobile, biomedicals, etc. Fabrication of FRC employing additive manufacturing is the milestone in the sector of manufacturing. In the recent studies, it has been observed that the influential parameters in FRC like fiber geometry, stacking

FIGURE 13.2 Commercial relevance of rapid prototyping technologies. Revenues made concerning (A) industry and (B) field of application (Gurr & Mu¨lhaupt, 2012).

13.2 Preprocessing, the process, and postprocessing

sequence, etc. can also be altered using 3D printing. Natural FRCs employing RP systems are also at embryological stage (Krishna, Kate, Satyavolu, & Singh, 2019). In this chapter, various types of RP systems are explained; an attempt has been made to brief about the working principle and discuss all the variants in chronological order, till the latest development. Also, the associated terminology in relation to the additive manufacturing systems is defined to enhance the grasp on the content. In addition to that, the contemporary research and developments are detailed in the forthcoming sections to explore the future orientation and possibilities associated with these processes. Finally, the applications of additive manufacturing systems in vital sectors like biomedical and biomechanical engineering are also reported herein.

13.2 Preprocessing, the process, and postprocessing in rapid prototyping The fabrication methodology involved in RP undergoes the following three stages: • • •

Preprocessing The process Postprocessing

13.2.1 Preprocessing In this phase of the development, the part is usually drafted in the computerbased drafting software and the dimensioning is done. However, there are software that can produce a 3D draft directly by the images obtained after scanning the object at various orientations. This draft is then directly used for the next stage of development. Such types of methods are most suited for the development of an implant that can be used during surgical procedures. Scanning saves a lot of time for complex geometries that require intricate detailing, because of which this method finds frequent application in the field of medical science for the printing of implants (Kumbhar & Mulay, 2018). For printing a prototype, the images that are either drafted manually or developed by scanning any body organ are converted in Standard Tessellation Language (STL) format. STL file contains the draft in the form of small triangular facets, which are connected with each other producing the 3D image of the implant to be fabricated. The conversion of the computer-drafted Computer Aided Drafting (CAD) file to STL extension reduces the accuracy in the dimensions of the part to be fabricated. This compromise in the accuracy is a persistent shortcoming of 3D printing systems. The coordinates of the vertices of triangular facets and the direction of the outward normal are present within the STL file.

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Because of the facets being triangular in shape, the curved surfaces not easily obtained, to achieve the desired accuracy the number of facets needs to be increased, that results in increase in file size. It is the responsibility of the designing engineer to balance the accuracy and file size as per the application of the implant being manufactured. Usually, the STL model is used as a universal format; however, another model that works on a similar principle is also available, known as stereolithography contour (Chang, 2015). Conversion of the drafted file in STL format is followed by slicing of the STL file which is to be developed using a RR system. The slicing is achieved with slicing software. Depending upon the type of 3D printer system employed for manufacturing the product the thickness of the slices vary from 0.0254 to 0.254 mm (Chang, 2015).

13.2.2 The process During this phase, the STL file is sliced in layers within the aforementioned thickness range using the slicing software. The layer thickness is a significant parameter of the product quality. The choice of layer thickness depends upon the purpose of the part/implant being fabricated as well as the type of 3D printing system being used. In the next step, platform within the printer also termed as the printing bed starts fabricating the sliced layers successively. Workflow can be further subdivided into three steps; first one is the supply of raw material for fabrication, followed by deposition material on a predefined contour for the development of layer mostly by application of heat, laser beam, or ultraviolet (UV). The final step is the upward or downward motion of the bed for the development of next layer. These steps are repeated until the part is fully fabricated. In the case of computer-aided manufacturing, the fabrication is assisted with programmable sensors, actuators, and robots for the required operation. The placement of workpiece is manual, and the operation codes are typed. The movement of tool to the workpiece and the machining operation is encoded through the programming. This is achieved with the help of motors, servo motors, microprocessors, and microcontrollers. The machining process is accompanied with the continuous flow of coolant. The part build is achieved by machining operations like turning, milling, drilling, etc. and hence, the manufacturing process is subtractive (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012).

13.2.3 Postprocessing The fabricated part is allowed to cool/cure until it attains room temperature. Consecutively, the surface treatment is done and the method of surface treatment depends on the type of system employed for the fabrication. Fig. 13.3 shows the fabrication steps in 3D printing and conventional manufacturing processes. It is intended to perform a comparison among the former and the later. Fig. 13.3A shows the development and fabrication of product employing additive

13.3 Contemporary rapid prototyping systems

FIGURE 13.3 Diagrammatic comparison of different manufacturing processes: (A) 3D printing, (B) milling, and (C) molding (Gurr & Mu¨lhaupt, 2012).

manufacturing techniques. Fig. 13.3B is a subtractive manufacturing method involving machining operation like milling. Fig. 13.3C shows the casting process, that is one of the most orthodox methods of fabrication and manufacturing. With the advancements in additive manufacturing systems, it can be observed that the researchers have started focusing on the improvement in the quality of the product like the surface finish of the fabricated part, the dimensional accuracy, mechanical properties, etc. The improvement is achieved by the identification of the parameters that influence the quality of the end product. Depending upon the type of 3D printer, these parameters can be different, for example, printing speed in SLA, heat supplied, and nozzle diameter in FDM, voltage in laser engineered net shaping (LENS), etc. These parameters are then altered one by one to check the degree of dependency on individual parameters and figure out the optimal conditions for best results. An illustrative example of these parameters is shown in Fig. 13.4 (Blok et al., 2018).

13.3 Contemporary rapid prototyping systems According to the American society of testing and manufacturing (ASTM) F42 standard provided in the year 2009, there is a total of seven different types of

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FIGURE 13.4 Main parameters for good surface contact and temperature conditions to enable optimal polymer sintering conditions (Blok et al., 2018).

systems that can be considered under the category of RR. This standard defines the terminology, methods for testing, and provides the standard file formats along with other relevant information and concepts. The list of various RR systems is available below: 1. 2. 3. 4. 5. 6. 7.

VAT polymerization Powder bed fusion Binder jetting Direct energy deposition Sheet lamination Material extrusion Material jetting

More elaborate data about the types of RP systems available in the market are provided in Table 13.2. In the scope of this chapter, all the systems as defined in ASTM F42 standard have been detailed to enhance the understanding of the contemporary additive manufacturing technology. Systems have been classified based on the form of the raw material supplied as the input in the printer as shown in Fig. 13.5 (Kumbhar & Mulay, 2018). The classification of RP systems has been reported on the basis of materials used. Table 13.3 shows the aforementioned classification. The physical form in which these materials are fed depends on the type of 3D printing system. Table 13.4 lists the different types of systems that require their feedstock in the form of solid, powder, and liquid, respectively.

13.3 Contemporary rapid prototyping systems

Table 13.2 Examples of various AM processes on basis of raw material input (Kumbhar & Mulay, 2018). Working principle

Manufacturing process

VAT photopolymerization Material extrusion Material jetting Binder jetting Sheet lamination Powder bed fusion

Stereolithography FDM Drop-on-demand Binder jet LOM Direct metal laser sintering Electron beam melting (EBM) Selective heat sintering SLS 3DP LENS, DMD

Directed energy deposition

LENS, Laser engineered net shaping; DMD, direct metal deposition, LOM, laminated object manufacturing; SLS, selective laser sintering; 3DP, three-dimensional printing.

3D Printing

Solid Raw Material

Powder Based

SLS,SLM,3DP

Laminate Based

LOM

Liquid Raw Material

Filament Based

VAT Polymerisation

FDM,

Slurry INKJET, CONTOUR CRAFTING

SLA,

FIGURE 13.5 Classification of the available additive manufacturing processes.

13.3.1 Available rapid prototyping systems The systems that fall within the domain of the ASTM F42 and have standards for research and experimentation are available directly from the aforementioned manual. At least one system from all the seven variants has been described in the forthcoming pages of this chapter. Fig. 13.5 shows classification of different types of 3D printing systems on the basis of physical form of the feedstock for the seven different variants mentioned in ASTM F42.

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Table 13.3 Classification based on the type of material used (Kumbhar & Mulay, 2018). Feed material

Employed Additive Manufacturing process

Ceramic

Three-dimensional printing (3DP) Selective laser sintering (SLS) FDM 3DP SLS Direct metal laser sintering Stereolithography Fused deposition modeling (FDM) 3DP SLS Laminated object manufacturing

Metal

Polymer

Table 13.4 Rapid prototyping process based on form of raw material (Kumbhar & Mulay, 2018). Supply phase

Additive manufacturing processes

Solid

LOM, FDM

Powder

3DP DMLS, SLS SLA

Liquid

Materials Polymers (ABS, polyacrylate, etc.)wax, metals and ceramics with binder Ceramic, polymer, and metal powder with binder Photopolymer(epoxies, acrylate, filled resin, colorable resin)

ABS, Acrylonitrile butadiene styrene; DMLS, direct metal laser sintering; LOM, laminated object manufacturing; SLA, stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing.

13.3.1.1 Selective laser sintering Selective laser sintering (SLS) was developed in 1986 at the University of Texas by Deckard. This is a powder-based system that requires polymer material in powder form, because of which it is also termed as powder bed system. Laser beam is applied for heating and melting of the powder, on specified contour. The powder then melts and bonds to produce the first layer of the powder bed, and then descends by a magnitude of one layer thickness in the downward direction. The layer thickness of the SLS printing process thus is an influential parameter in the product quality. After the bed descends, a fresh layer of powder is rolled over the bed. The process repeats until the part is developed (Kumbhar & Mulay, 2018; Tiwari, Pande, Agrawal, & Bobade, 2015; Chitresh, Singh, & Himanshu, 2014). Gurr and Mu¨lhaupt have presented a detailed review of various kinds of

13.3 Contemporary rapid prototyping systems

material used in the process of selective laser sintering which include thermoplastic polymers like high density polyethelene (HDPE), poly ether-ether Ketone (PEEK), etc. and composites that include fillers like glass beads, nano-silica, carbon nanofiber, nano-alumina, etc. These systems are also capable of direct sintering of ceramics and metals, like zirconia, alumina, silicon carbide, and stainless steel, titanium, bronze, and nickel, respectively (Gurr & Mu¨lhaupt, 2012).

13.3.1.2 Selective laser melting The process of selective laser melting (SLM) is similar to the previous process, that is SLS but it is more efficient and frequently used for the part developed using metallic powder or sometimes a mixture of more than one metallic powder. In the process of SLM the parts fabricated exhibit properties almost similar to the conventional machining process. However, the cost of producing metal powder is high but on the other side, the intricate and complex geometry produced from RP technology can be considered as compensation to the aforementioned hard work of producing metal powder (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012; Kumbhar & Mulay, 2018). Chang (2015) has covered a detailed review about the types of processes involved in producing parts from metal, which include the technologies that use additive manufacturing from powdered metal, like LENS and electron beam melting (EBM). EBM employs titanium powder to fabricate parts; titanium is a material that is frequently employed in biomedical implants because of its biocompatibility, noncorrosive and nontoxic behavior. Fig. 13.6A is the diagrammatic representation of EBM technology, and the second part of the same figure, that is (B), is an image

FIGURE 13.6 The EBM technology of Arcam: (A) electron beam melting diagram. (B) Typical Arcam A2 system capable of producing parts with dimensions up to 7.87 in. 3 7.87 in. 3 13.0 in. (200 mm 3 200 mm 3 330 mm) (Chang, 2015).

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FIGURE 13.7 The laser engineered net shaping (LENS) technology: (A) the LENS, (B) LENS fabrication, and (C) the Optomec LENS 850 system (Chang, 2015).

of the Arcam A2 system, an EBM 3D printer capable of producing parts of size of 200 mm 3 200 mm 3 330 mm in dimension. Fig. 13.7 shows a typical setup of the laser engineered net shaping also known as LENS technology. An actual physical setup of the system is shown in the third part of Fig. 13.7. Optomec’s RP machine works on the principle of LENS technology. The size of printing envelop of Optomec LENS 850 system is (18 3 18 3 42) in., with X and Y accuracy/resolution up to a level of 0.002 and 0.020 in. in the direction of the Z-axis. The LENS technology has a nozzle that push out the metallic powder and is melted simultaneously with the help of laser and thus the fabrication proceeds. However, this system does not require a powder bed kind of arrangement in contrast to its other substitutes (Chang, 2015). The challenge associated with LENS is the limitation of part size. These are good for small parts but very large metallic parts are difficult to be produced from these systems. There is a need of large setups, capable of manufacturing large metallic parts. On the positive side, these setups are capable in producing parts of size big enough to serve the requirement for biomedical implants. So, it can be successfully used for bioengineering-based applications. The other associated challenges with these setups include the metal shrinkage issue and the estimation of the allowance for the same. Also, the part printing takes a significant amount of time which produces a temperature gradient in the direction of printing. Under the influence of this temperature gradient the overall part develops a tendency to get distorted as the cooling proceeds with time. The distortion allowance is again a complex situation to deal with. Apart from this, warping is also observed in the fabricated part after they are cooled. It is also difficult to predict the grain size that will be produced in the part after it cools down. The significant parameter which affects the part quality includes the power associated with the laser, the melting point of the metallic powder, cooling time, nozzle diameter, etc. (Blok et al., 2018; Chang, 2015).

13.3 Contemporary rapid prototyping systems

13.3.1.3 Laminated object manufacturing In the process of laminated object manufacturing (LOM), the feed material is in the form of sheets; the layer of specified contour is cut by applying laser on the specified path. The individual sheet acts as the layer and thus the thickness of the sheet becomes the layer thickness. As the printing proceeds the layers are thermally bonded together to fabricate the part. The cutting of the sheet at the specified contour is performed using a laser. The ultrasonic additive manufacturing is the only technology in available 3D printing systems that use metallic sheets and do the fabrication at low temperatures. The fabricated part is then taken to a CNC machine for postprocessing. The leftover material from the sheet can be recycled for future use. It has been observed that the surface quality produced from LOM is inferior when compared against the powder bed fusion technology. Nevertheless, the cost of raw material, as well as the printing cost, is relatively low (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012).

13.3.1.4 Fused deposition modeling (FDM) Thermoplastics such as ABS, PLA, and PC are the material employed in the process of FDM. In the fabrication of part using this technology, a solid filament of plastic is melted to semisolid paste and extruded through the nozzle to deposit on the specified contour layer by layer. This process is widely available and economically viable. A lot of beginners use this method to develop their understanding on rapid manufacturing technology using this system (Chitresh, Singh, & Himanshu, 2014). The influential parameters in this technology include the layer thickness, printing orientation, raster width, air gap, raster angle nozzle diameter, heating temperature, and printing speed. Researchers have developed parts by using multimaterials employing more than one printing nozzles having filaments of different materials (Wang et al., 2017). The details about which are discussed in the forthcoming sections. Fig. 13.8 represents the schematic diagram of the fabrication process using FDM. Ease of operation in FDM has made this technology viable for the application in both commercial and research projects, in the field of biomedical and tissue engineering.

13.3.1.5 Stereolithography Stereolithography (SLA) is the oldest RP process among all the processes mentioned in this chapter. In this process, photocurable polymer resins are employed for the development of the part; most frequently used resins are acrylic or epoxybased monomers. These monomers solidify under the exposure of UV radiation. The monomers bond to become polymer at the instant they are exposed under radiation. After this the platform descends and liquid monomer covers up the polymer layer for the formation of the next layer. The researchers have incorporated particles of ceramics to produce a ceramic-filled composite by using this process. The influential parameter in this process is the energy of the incident UV

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FIGURE 13.8 Schematic of the FDM: (1) vertically movable platform; (2) horizontally movable, heated deposition unit with nozzles; (3) model material; (4) support material; (5) feedstock of filament rolls (Gurr & Mu¨lhaupt, 2012).

FIGURE 13.9 (A) Part-building process, (B) the iPro 9000 XL system (http://www.3dsystems.com), and (C) dashboard built by using iPro 9000 XL (Chang, 2015).

beam which can even alter the thickness of the developed layer. However, the part accuracy, in this process, is very good because of which this technology finds wide application in the field ofnanocomposites. It can be employed to print parts up to a resolution of 10 µm (Chang, 2015; Chua et al., 2020; Ngo et al., 2018; Wang et al., 2017). Fig. 13.9A shows the part building process of SLA. The other parts of the same figure, that is (B) and (C), show the actual image of iPro 9000 XL and a dashboard fabricated from the same printer, respectively.

13.4 Applications

FIGURE 13.10 A schematic view of stereolithography process: (1) photopolymerizable resin, (2) movable platform in the vertical direction, (3) CO2 laser, (4) scanning resin surface by optical systems, and (5) horizontal wiping blade (Gurr & Mu¨lhaupt, 2012).

A renowned company in this field is 3D systems that offer a variety of printers, with different configurations like SLA viper, iPro 8000MP, iPro 9000 XL, etc.; the price range typically varies between $180K and $950K. The one shown in the above picture is iPro 9000 XL which is the largest platform that is widely used for commercial applications across the world. The dimension up to which it fabricates the parts can be as big as 1500 mm 3 750mm 3 550 mm (59.1 in. 3 29.53 in. 3 21.65 in.). Products like an entire dashboard or complete bumpers can be fabricated by this printer. Fig. 13.10 shows a schematic representation for the working principle involved in the photopolymerization technique. In Fig. 13.11, a demonstration of the remaining RR processes is pictorially represented to enhance the imagination of the reader. Table 13.5 describes the contemporary RR systems detailing about physical form of feed materials, involved working principal, employed material, resolution, and the associated advantages and disadvantages. The surface roughness of the fabricated part is reported in Table 13.6; a quantitative comparison can thus be performed on the surface quality that can be obtained from different types of 3D printing systems. The associated layer thickness is also reported in the table for each of the system.

13.4 Applications RP technology finds a wide variety of applications in various sectors of engineering and biomedical. Development of complicated design for molds, dental applications,

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FIGURE 13.11 Schematic representation of a typical (A) FDM setup, (B) 3DP setup, (C) SLA setup, (D) SLS setup, and (E) 3D plotting setup (Wang et al., 2017). SLA, Stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing.

fabrication of soft tissue in medical application, prosthetics, etc. has been made remarkably simple by the use of 3D printers. These parts required a complex and expensive process to develop and hence were far from the reach of people belonging to a relatively weaker economic section of the society. In addition to this, the effectiveness, accuracy, and surface finish achieved in fabrication of intricate and complex geometry form found to be superior in performance when compared to conventional methods. Apart from evident advantages like fabrication flexibility and customization the RP technology does not cause any wastage of the raw material. Also, the printers do not require any dedicated infrastructure or similar facility for operating (Tiwari, Pande, Agrawal, & Bobade, 2015; Chitresh, Singh, &

Table 13.5 Contemporary rapid prototyping techniques in summarized form (Wang et al., 2017).

Technique

State of starting of materials

FDM

Filament

Extrusion and disposition

3D

Liquid or paste

3DP

Powder

Pressurized syringe extrusion, and heat or UVassisted curing Drop-on-demand binder printing

SLS

Powder

SLA

Liquid photopolymer

Working principle

Heat-induced sintering and laser scanning Laser scanning and UV-induced curing

Typical polymer materials Thermoplastic, like PC, ABS, PLA, and nylon PCL, PLA, hydrogel

Any material can be supplied as powder, binder needed Polymer powder and PCL Photocurable resin (epoxy acrylate-based resin)

Resolution (Z-direction, µm)

Advantages

Disadvantage

50 200 (Rapide Lite 500)

Low cost, multimaterial, good strength capabilities

5 200 (Fab@home)

High printing resolution, soft material capability

Nozzle clogging, anisotropy Low mechanical strength, slow

100 250 (Plan B Ytec3D)

Material capabilities, low cost, multi-material capability, easy removal of support powder Good strength, easy removal of support powder High-resolution printing

80 (Spo 230 HS)

DWS LAB XFAB

Clogging of binder jet, binder contamination High cost, powder surface Material limitation, high cost, cytotoxicity

ABS, Acrylonitrile butadiene styrene; PC, polycarbonate; PLA, polylactic acid; PCL, polycaprolactone, SLA, stereolithography; SLS, selective laser sintering; UV, ultraviolet; 3DP, three-dimensional printing.

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Table 13.6 Layer thickness and surface roughness (Kumbhar & Mulay, 2018). S. No.

Name of process

Surface roughness (Ra), (µm)

Minimum layer thickness (mm)

1 2

3D printing Poly-jetting process LOM SLA FDM SLS

12 27 3 30

0.175 0.10

6 2 9 5

0.114 0.10 0.254 0.125

3 4 5 6

27 40 40 35

LOM, Laminated object manufacturing; SLA, stereolithography; SLS, selective laser sintering.

Himanshu, 2014). Collectively considering all these advantages of the technology finds a wide application in various fields as mentioned below: •

Medical science and bioengineering applications

The scope of 3D printing is increasing in various sectors of the technology but the development and the benefits reaped in the field of medical are remarkable. The development in this field is further divided into following subcategories: 1. Rehearsal and planning before surgery There are many examples where 3D printed replicas of internal organs have assisted in the efficient planning of surgery. But the most eye-catching example is the surgery that separated two conjoined twins. The twins were born with joined heads; hence surgery was required to separate their heads. The conventional approach of planning that involved two-dimensional pictures from X-ray and computed tomography (CT) scans was producing a surgery that required nearly 97 h to complete. Working on a surgery that will last this long was very overwhelming; also the blood supply for these many hours was not possible. Using the data from X-rays and CT scans a 3D printed replica of the conjoined head was fabricated. The surgeons planned the surgery by studying the printed model. The surgery took only 22 hand was a success.1 Other examples involved the development of replicated models for surgery planning provided by different authors. Another example is the Sandia Lab replica of the human spine (Chang, 2015) (Fig. 13.12). 2. Educational purposes Educational institutes develop physical 3D printed models of internal body organs to make visualization simpler so that the student mind can appreciate the concept. 1

http://www.turkcadcam.net/rapor/otoinsa/uyg-medikal-conjoined-twins.html.

13.4 Applications

FIGURE 13.12 Replica of the human spine for planning a surgery at Sandia Labs (Chang, 2015).

3. Dental application Use of the RR technology in surgery planning for a dental implant is also very common. 3D printing is also employed for the fabrication of crowns for teeth. Chang (2015) quotes the significance of RP technology from the anthropological point of view. If there are only one or two specimens the entire teeth can be printed using 3D printing. 4. Prosthesis customization and implants Chitresh, Singh, & Himanshu, (2014) have employed 3D printing from the fused deposition modeling (FDM) for the customization and printing of prosthetic implants. This study carried out by the author is an example of how the CAD file is directly obtained after scanning the required geometry at different orientations. 5. Tissues and scaffolds There are examples where researchers have tried and successfully printed tissue scaffolds. Sometimes these scaffolds require metals such as titanium because of their biocompatible nature in the human body (Chua et al., 2020). However, there are 3D printing methods that employ metal as raw material. Ngo et al. (2018) have cited a report from Wohlers that the biomedical market represents 11% of the total market share in the field of RR technology. According to the report in the year 2016, the total market share of RR globally is around $6.1 billion. The flexibility and ease of customization in 3D printing are very useful in the development of implants and tissues. These parts are produced faster and more easily (Ngo et al., 2018). Patient-specific implants are easy to produce as scanned models of required implants are easily accessible to the domain of doctors and the 3D printer can print the implant in almost no time. Doctors have also used replicated model of scanned body parts to plan a surgery which reduces the time of surgery and the associated risk to the health of the patient (Fig. 13.13).

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FIGURE 13.13 (A) 3D CAD, computer aided drafting model individuates a defect in the mandible from CT, computed tomography scan images; (B) motion distribution of print from the developed software. Green, blue, and red indicates the paths of PCL, polycaprolactone, Pluronic F-127, and hydrogel, respectively. (C) AM, additive manufacturing process; (D) 3D printed bony defect implant, cultured in osteogenic medium for 28 days; (E) osteogenic differentiation confirmed by Alizarin Red S staining, showing calcium deposition. (Ngo et al., 2018). .

• Aerospace 1. The aerospace industry shares approximately 18.2% of the total market share which is as per the Wohlers report cited by Ngo et al. (2018). 2. Building protective (Blok et al., 2018; Chang, 2015; Chua et al., 2020; Ngo et al., 2018; Wang et al., 2017).

13.5 Advancements in the rapid prototyping technology Since the development of RR techniques, there have been numerous advancements that can be classified into two broad categories:

13.5 Advancements in the rapid prototyping technology

13.5.1 Improvement of product quality Researchers working on this platform intend to address the shortcomings associated with 3D printing, which include distortion, part warping, dimensional accuracy, etc. To reduce these shortcomings, the influential parameters like nozzle size, printing speed, and melting temperature are addressed. Different researchers across the world have studied these parameters individually to investigate their degree of dependence on the product quality. Efforts are being made to optimize the working conditions of these parameters to improve the part quality (Blok et al., 2018; Chang, 2015; Chua et al., 2020; Gurr & Mu¨lhaupt, 2012).

13.5.2 Improvement on versatility of rapid prototyping Initially, limited materials were used in RP technology; with the advancement in the systems more materials are now used. Over the period of time, researchers have expanded the capabilities of 3D printing technology and the expansion is still going on. In the present scenario, biocompatible materials that can be used in prosthetics and dental applications are developed (Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016; Ngo et al., 2018). The development of the parts with intricate geometry from the traditional manufacturing systems is a complex job and the outcome is not as effective. Also, there was a considerable amount of material loss that increased the cost of production. That loss has been minimized specially for complex shapes which are now easily fabricated by additive manufacturing, also referred to as rapid prototyping. This has now inspired the investigators to print a fully functional system assembled directly from a 3D printer. The upcoming generation of 3D printing is expected to have the capability to directly fabricate a fully functional working component in a single run of the process. There are works done by researchers in different parts of the world that have been covered by a literature study conducted by MacDonald and Wicker (2016). There is a paradigm shift in this approach where the objective of the process will be to fabricate in contrast to the previous system of assembly. This process will be a nonassembly system producing a multifunctional end-use device. According to the chief executive officer of General Electrical, Jeff Immelt, there is an ambitious goal of producing as many as 100,000 jet engines to meet the target demand. To achieve this level of production the organization has planned to invest a sum of 3.5 billion dollars in the technology of additive manufacturing. Most of the 3D printing patents are also expiring, resulting in the expansion of this technology by the entry of new manufacturing companies employing the technology of 3D printing (MacDonald & Wicker, 2016). There are several other examples in which the research and development departments of various companies have successfully achieved this target. Those examples are explained in the forthcoming section.

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13.5.3 Multifunctional fabrication process When it comes to defining multifunctional fabrication process in rapid manufacturing technology, it can be described as an additive manufacturing process in which apart from the fabrication of any intricate shape an additional functionality is also introduced in the printed part. Along with this multicolor, density variation, stiffness gradient, etc. are embedded in the printed structure.

13.5.4 Printable and embeddable functions In addition to the basic shape printing, the next-generation 3D printing technology has evolved and became capable to embed certain types of functionality like transducing, functionally gradient, thermal management, energy storage, propulsion utility, and sensing, most of which are described as follows.

13.5.4.1 Sensors In the production of sensors using RR technology, the researcher has put a considerable amount of approaches by focusing on the following two ways to achieve this target: firstly, interrupting the process and then embedding the sensor directly into the printed part; secondly, to arrange the setup such that the entire sensor can be directly printed along with the structure. A highly stretchable sensor employing the 3D printing known carbon-stretchable sensor is printing from a carbon-based ink with an elastomer structure (Muth et al., 2014). Shemelya et al. have demonstrated the development of a capacitive touch sensing system by employing a wire-on-wire submerged with the 3D-printed thermoplastic structure. This sensor can be used as a single capacitive plate for a touch sensor (Shemelya et al., 2014).

13.5.4.2 Actuations Researchers across the world have printed several working models and prototypes of actuators. Prosthetic hands with embedded external motion have been printed. Richter and Lipson successfully printed a bio-inspired flapping wings insect by employing VAT polymerization (Richter & Lipson, 2011).

13.5.4.3 Thermal management With further advancement in multifunctional 3D printing, advance thermal management systems are also developed. Complex large surface area structures with good thermal conductivity have been fabricated using metal 3D printing techniques (Wong, Tsopanos, Sutcliffe, & Owen, 2007). Researchers are hoping to produce an advanced system with embedded heat pipes and reservoirs for material with phase change. These fabricated systems lead to the improvement in the thermal management system of 3D structures (MacDonald & Wicker, 2016).

13.5 Advancements in the rapid prototyping technology

13.5.4.4 Energy storage A study has been reported of printing a battery, setting up an example for the development of an energy storage system. Malone, Berry, and Lipson (2008) have successfully printed customizable arbitrary shaped battery system. This contained 3D printed structures after sintering (Malone et al., 2008).

13.5.4.5 Antennas and electromagnetic structures Additive manufacturing offers few distinctive features that have increased its utility and application throughout various fields of technology. One of these distinctive features is the presence of provision to intentionally embed porosity within the fabricated part. This distribution of porosity can be varied throughout the structure resulting in variation of density within the structure. This feature functionally grades the permittivity and permeability of the structure. Developers have exploited this feature in enabling the electromagnetic transitions through the interface of the material and minimize reflections which in return can be used for sculpturing EM waves in antennas. Still, the unintentional porosity is difficult to avoid which acts as a challenge in 3D printing processes that involve thermoplastics (Deffenbaugh, Rumpf, & Church, 2013; Liang et al., 2014; Rumpf, Pazos, Digaum, & Kuebler, 2015).

13.5.4.6 Propulsion Polymers manufactured from additive manufacturing deliver appropriate dielectric strength. While fine quality available copper wires possess low resistance sufficient enough to serve the purpose. Researchers were able to show this kind of propulsion by supplying high voltage in fabricated test coupons for igniting micro-pulsed plasma thrusters. This shows the utility of multipurpose additive manufacturing with wire and components placement, which benefits the manufacturing of space vehicles and their embedded component (Marshall et al., 2015).

13.5.5 Fiber-reinforced polymer composites There are several examples of the development of composite material using RR technology. These included various types of reinforcement material within the structure itself. Also, there are examples of FRC material printed from RP technology. But in most cases, the orientation of the fiber was not user-defined. However, there are examples where researchers have embedded the information about fiber orientation. Matsuzaki et al. have fabricated jute fiber-reinforced polymer composite using 3D printing technology. In the composite fabricated by them, the unidirectional alignment of jute fiber was made possible. The strength of the fabricated material was compared with one which was produced from the conventional fabrication process. The result of the comparison had shown superior performance of the unidirectionally aligned composite specimen fabricated

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FIGURE 13.14 (A) Carbon fiber-reinforced polymer. (B) Jute fiber-reinforced polymer tensile test specimens. (C) Cross-sectional view and (D) magnified cross-section of the carbon fiber composite specimen (Matsuzaki et al., 2016).

from RR. Unidirectionally aligned carbon fiber-reinforced polymer composite was also fabricated and tested against their conventionally fabricated counterparts. Fig. 13.14 shows the pictures of the fabricated specimens (Matsuzaki et al., 2016). In the research work conducted by Le Duigou et al., PLA-based Flax FRC was 3D printed to investigate the tensile strength in longitudinal and transverse directions. The specimen showed superior magnitude of the properties, when compared against the composite prepared employing conventional methodology for fabrication (Le Duigou, Barbe´, Guillou, & Castro, 2019).

13.5.6 Functionally graded materials using rapid prototyping Giachini et al. used additive manufacturing of cellulose-based material with continuous, multidirectional stiffness gradient. This process is also termed multimaterial additive manufacturing. FDM process was employed for the development of this FGM containing two different materials. Various combinations of the materials were tested with base material such as hydroxyethylcellulose and different compositions of other materials like lignin, microfibrillated cellulose, citric acid, and hydrogen chloride. The composition gradient was embedded within the system producing a continuous stiffness gradient (Giachini et al., 2020).

13.5.7 Comparison with traditional manufacturing These systems are recently developed, and the user feedback of the product is not available, making it difficult to comment about reliability or durability.

References

However, when they are subjected to the specified test as per the standards they show comparable performance with that of traditional manufacturing techniques.

13.6 Conclusion Flexibility, freedom of design, and ease of prototyping have been redefined after the development of rapid manufacturing technology. This system has evolved significantly after it was experimented for the first time. Contemporary development in the field of RR suggests that the potential of this technology is remarkably high and might partially or fully substitute the conventional manufacturing technologies. The scope of research and development in this field is wide and it is still expanding. Commercialization of this technology has now started to gain pace and is rising exponentially. With the involvement of bioengineering application, development of multifunctional devices from 3D printing, fabrication of composite material, and FGMs this technology is not only on the path of representing the pioneer of interdisciplinary achievement in the field of engineering but will also produce lots of jobs and opportunity in the future benefiting the economy. The market share of the rapid manufacturing technology is also increasing at a high pace and it is expected that it will keep on increasing. With the commercialization of the technology soon the reliability of the products and parts fabricated will become clear by the feedback provided after these are used by the end customers. These reviews from the general customer will increase the scope of research, as well as applications. Along with the same, there is a high probability that soon this technology will become less expensive and will become available for the use of the public. With further advancement in 3d printing, we can get personal 3D printer in an affordable price in the market. While, STL file for useful good and stationary will be available online by the manufacturer making additive manufacturing as user friendly as conventional printing. As far as biomedical is concerned the accuracy and the capability of RP system to produce intricate geometry when clubbed the advancement in the development of biocompatible material, it is not unrealistic to expect a groundbreaking breakthrough in the field of medical science.

References Blok, L. G., Longana, M. L., Yu, H., & Woods, B. K. S. (2018). An investigation into 3D printing of fibre reinforced thermoplastic composites. Additive Manufacturing, 22, 176 186. Chang, K.-H. (2015). Chapter 14 Rapid prototyping. In K.-H. Chang (Ed.), e-Design (pp. 743 786). Academic Press, ISBN 9780123820389.

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Chen, R. K., Jin, Y.-A., Wensman, J., & Shih, A. (2016). Additive manufacturing of custom orthoses and prostheses—A review. Additive Manufacturing, 12, 77 89. Chitresh, N., Singh, A., & Himanshu, C. (2014). Customised prosthetic socket fabrication using 3D scanning and printing. In Conference additive manufacturing society of India. Additive Manufacturing Society of India, Banglore. Chua, C. K., Leong, K. F., & An, J. (2020). Introduction to rapid prototyping of biomaterials. Rapid prototyping of biomaterials (pp. 1 15). Woodhead Publishing. Deffenbaugh, P. I., Rumpf, R. C., & Church, K. H. (2013). Broadband microwave frequency characterization of 3-d printed materials. IEEE Transactions on Components, Packaging and Manufacturing Technology, 3(12), 2147 2155. Available from https:// doi.org/10.1109/TCPMT.2013.2273306. Giachini, P. A. G. S., Gupta, S. S., Wang, W., Wood, D., Yunusa, M., Baharlou, E., . . . Menges, A. (2020). Additive manufacturing of cellulose-based materials with continuous, multidirectional stiffness gradients. Science Advances, 6(8), eaay0929. Gurr, M., & Mu¨lhaupt, R. (2012). Rapid prototyping. Polymer science: A comprehensive reference (pp. 77 99). Amsterdam: Elsevier. Krishna, V., Kate, K. H., Satyavolu, J., & Singh, P. (2019). Additive manufacturing of natural fiber reinforced polymer composites: Processing and prospects. Composites Part B: Engineering, 174, 106956. Available from https://doi.org/10.1016/j. compositesb.2019.106956. Kumbhar, N. N., & Mulay, A. V. (2018). Post processing methods used to improve surface finish of products which are manufactured by additive manufacturing technologies: A review. Journal of The Institution of Engineers (India): Series C, 99(4), 481 487. Available from https://doi.org/10.1007/s40032-016-0340-z. Le Duigou, A., Barbe´, A., Guillou, E., & Castro, M. (2019). 3D printing of continuous flax fibre reinforced biocomposites for structural applications. Materials & Design, 180, 107884. Liang, M., Ng, W. R., Chang, K., Gbele, K., Gehm, M. E., & Xin, H. (2014). A 3-D Luneburg lens antenna fabricated by polymer jetting rapid prototyping. IEEE Transactions on Antennas and Propagation, 62(4), 1799 1807. Available from https:// doi.org/10.1109/TAP.2013.2297165. MacDonald, E., & Wicker, R. (2016). Multiprocess 3D printing for increasing component functionality. Science (New York, N.Y.), 353(6307). Malone, E., Berry, M., & Lipson, H. (2008). Freeform fabrication and characterization of Zn-air batteries. Rapid Prototyping Journal, 14(3), 128 140. Available from https:// doi.org/10.1108/13552540810877987. Marshall, W. M., Stegeman, J. D., Zemba, M., MacDonald, E., Shemelya, C., Wicker, R., . . . Kief, C. (2015). Using additive manufacturing to print a CubeSat propulsion system. In 51st AIAA/SAE/ASEE joint propulsion conference (p. 4184). Matsuzaki, R., Ueda, M., Namiki, M., Jeong, T.-K., Asahara, H., Horiguchi, K., . . . Hirano, Y. (2016). Three-dimensional printing of continuous-fiber composites by innozzle impregnation. Scientific Reports, 6(1), 1 7. Muth, J. T., Vogt, D. M., Truby, R. L., Mengu¨c¸, Y., Kolesky, D. B., Wood, R. J., & Lewis, J. A. (2014). Embedded 3D printing of strain sensors within highly stretchable elastomers. Advanced Materials, 26(36), 6307 6312. Ngo, T. D., Kashani, A., Imbalzano, G., Nguyen, K. T. Q., & Hui, D. (2018). Additive manufacturing (3D printing): A review of materials, methods, applications and challenges. Composites Part B: Engineering, 143, 172 196.

References

Richter, C., & Lipson, H. (2011). Untethered hovering flapping flight of a 3D-printed mechanical insect. Artificial Life, 17(2), 73 86. Rumpf, R. C., Pazos, J. J., Digaum, J. L., & Kuebler, S. M. (2015). Spatially variant periodic structures in electromagnetics. Philosophical Transactions of the Royal Society A—Mathematical, Physical and Engineering Sciences, 373(2049). Available from https://doi.org/10.1098/rsta.2014.0359. Shemelya, C., Cedillos, F., Aguilera, E., Espalin, D., Muse, D., Wicker, R., & MacDonald, E. (2014). Encapsulated copper wire and copper mesh capacitive sensing for 3-D printing applications. IEEE Sensors Journal, 15(2), 1280 1286. Tiwari, S. K., Pande, S., Agrawal, S., & Bobade, S. M. (2015). Selection of selective laser sintering materials for different applications. Rapid Prototyping Journal, 21, 630 648. Wang, X., Jiang, M., Zhou, Z., Gou, J., & Hui, D. (2017). 3D printing of polymer matrix composites: A review and prospective. Composites Part B: Engineering, 110, 442 458. Wong, M., Tsopanos, S., Sutcliffe, C. J., & Owen, I. (2007). Selective laser melting of heat transfer devices. Rapid Prototyping Journal, 13(5), 291 297. Available from https:// doi.org/10.1108/13552540710824797. Zindani, D., & Kumar, K. (2019). An insight into additive manufacturing of fiber reinforced polymer composite. International Journal of Lightweight Materials and Manufacture, 2(4), 267 278. Available from https://doi.org/10.1016/j. ijlmm.2019.08.004.

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Self-assembled polymer nanocomposites in biomedical applications

14

Anurag Dutta1,2, Manash Jyoti Baruah1, Satyabrat Gogoi2 and Jayanta Kumar Sarmah2 1

Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India

2

14.1 Introduction Nanocomposites materials are those materials where one or more of the primary constituent phases has a dimension in the nanoscale range. Owing to widespread applicability and the tunability, they have been the center stage of attraction in the academia as well as industrial research. The reason behind this is the ability of such materials to inherit the advantageous properties of the constituent components and/or enhance it toward being a multifunctional entity. The nanocomposites in polymeric forms have offered a plethora of applications with their inherence quality of target specific designing and the ability to meet the demands of functional material world. Polymer nanocomposites usually contain an inorganic nanomaterial (usually particles, tubes, and/or wires of nanometric dimensions, or nanoclay) decorated within/over an organic matrix (polymers or biomacromolecules). The synergy between the characteristics of the inorganic component and the organic polymer aids such nanocomposite materials in showing amplified optical, mechanical, magnetic, thermal, and optoelectronic properties (Kumar & Jouault, 2013). Such synchronism and display of diversified properties have resulted in such polymer nanocomposites, finding indispensable utilities in the field of sensing (Hosu, Barsan, Cristea, S˘andulescu, & Brett, 2017), designing of solar cells (Zhao & Lin, 2012), catalysis (Marcoux, Florek, & Kleitz, 2015), electronics (Yousefi et al., 2014), biotechnology (Wang, Cui, Wang, & Li, 2016; Wang, Yang, et al., 2016), and biomedicine. The surge in the innovations in polymer chemistry and nanofabrication technologies has not only pushed the boundaries of research in the field of designing newer types of polymer nanocomposites but also in the production of multifunctional materials. This has opened an arena for the development and production of polymer nanocomposites with tailored functionalities for applications which are highly sophisticated and efficient in nature. However, this Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00003-6 © 2023 Elsevier Inc. All rights reserved.

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achievement is highly dependent upon the synthetic methodologies employed and much has already been achieved (Li et al., 2016; Matsuura, 2017; Nie, Li, Wang, & Zhang, 2016). Among the available protocols, self-assembly is one such technique that has been extensively used. The reason being the simplicity and economic efficacy of the protocols as well as the precision and the flexibility associated with them. Self-assembly is a process of spontaneous molecular arrangement of disordered entities of molecules into well-defined ordered structures via local interactions among the constituent entities themselves. Major approaches that have been reported, but not limited to, for the construction of self-assembled polymer nanocomposites are: 1. Surface modification of grafted polymer (Kumar, Bansal, Behera, Jain, & Ray, 2016; Kumar, Behera, Thakre, & Ray, 2016) 2. Spin coating (Nunes-Pereira et al., 2015) 3. Deposition (Hu et al., 2012) 4. Layer-by-layer assembly (Zhang, Tong, & Xia, 2014) Given to the widespread liberty that can be taken toward the designing of selfassembled polymer nanocomposites, the utility and employment into biomedical applications are expanding at a very healthy pace (Ahmad, Manzoor, Singh, & Ikram, 2017; Komiyama, Yoshimoto, Sisido, & Ariga, 2017; Yi, Zhang, Webb, & Nie, 2017). The foundation to this endeavor is based upon the never ending list of self-assembled biological systems that form the very basis of life alone. Be it the construction of cellular membranes via assembling the phospholipid bilayers, protein folding, or the characteristic double-helix structure of the DNA, they are all types of biological self-assembly. In fact, they can also be called as biological self-assembled nanostructures. The ligand-to-receptor interactions that form the basis of neural signal transfer and enzyme catalysis in the biological systems can also be attributed to self-assembly of the complex biological polymer nanostructures. Self-assembly also accounts for the formation of molecular crystals, various forms of colloids, miscelles, self-assembled monolayers, and phase-separated polymers. The point to note here is that such type of molecular self-assembly is the key to the emergence of life and its maintenance. Concepts for modern applications of self-assembled polymer nanocomposites have been derived from synthetic amino acids, oligo- and polypeptides, dendrimeric ensembles, polymers and pi-conjugated compounds toward construction of various nanostructures like nanotubes, fibers, micellar aggregations, and vesicles. In addition to this, people have also considered small-molecule self-assembly as building units of structurecontrolled materials. In a similar way, DNA-based nanomaterials have written their own story of being potential diagnostics and drug delivery tools. In the beginning of the 21st century, (Whitesides & Boncheva, 2002) mentioned that newer nanomaterials will find a new face with the process of self-assembly. This was based on grounds that the process is not only important toward maintaining the standard of living or for technological advancement of mankind, but also for

14.2 Methods of preparation

FIGURE 14.1 Schematic representation of the subject of this chapter: preparation of self-assembled polymer nanocomposite and their biomedical applications.

keeping it alive. Living materials such as the cell contain complex nanostructures such as the lipid membranes, protein aggregates, complex molecular machines such as the folded proteins and the nucleic acids, etc. These have shown the natural tendencies of self-assembly. Now, it can be said that self-assembly has paved a way into a diversified arenas and has provided ample opportunities toward the development of novel functional materials and building blocks of life through a close-knit exchange of concepts among them. In this chapter we shall see in detail the ways in which self-assembled polymer nanocomposites are formed and their various applications in biomedical sciences (Fig. 14.1).

14.2 Methods of preparation of self-assembled polymer nanocomposites The self-assembly process involves a fine-tuned balance between the attractive and repulsive forces, which commences the aggregation, along with an entity that gives a proper direction to the growth of the polymer nanocomposite (Fig. 14.2).

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FIGURE 14.2 The distinct forces involved in self-assembly.

While self-assembled nanocomposites can be constructed by using a number of available methods, designing self-assembled polymer nanocomposites require extra attention and complexity is involved in the due course of its formation. Two methods that have risen above others in terms of efficacy, sustainability, and ease of follow-up are polymer grafting on or from modified surface of nanoparticles and layer-by-layer assembly. The methods are briefly described below:

14.2.1 Polymer grafting on/from the modified surface of nanoparticles The technique of polymer grafting for modifying the surface of nanoparticles has been known for quite a while now (Zoppe et al., 2017). This has allowed the uniform dispersion of the nanoparticles in to the matrix of the polymer at a nanometer scale. Polymers with well-defined structural units and molecular weight along with a small polydispersity ratio are synthesized via controlled radical polymerization. Among the radical polymerization methods, atom transfer radical polymerization, well known as ATRP, is a technique that offers an efficient route toward preparing multifunctional composites with end group functionality (Siegwart, Oh, & Matyjaszewski, 2012). Another specialized form of ATRP is surface-initiated ATRP (SIATRP) (Fig. 14.3). This technique allows the polymer growth from the solid surface and is known to show several advantages in the preparation of the mentioned class of materials. The advantages associated with this type of polymerization are: 1. Ability to produce polymer chains which are covalently grafted on the solid surface. Such polymer chains can then control the functional properties of the entire nanocomposite like the interfacial properties, concentration of the polymer, and also regulate the yield of the nanocomposite synthesized.

14.2 Methods of preparation

FIGURE 14.3 Schematic representation of surface-initiated polymerization.

2. The molecular weight of the nanocomposite, its polydispersity ratio, and the composition can be tailored with considerably good control. 3. The applicability of the method to a variety of monomers under varying conditions is one of the foremost advantages of the protocol. This provides a platform to designing the substrate particles with varying functionalities. Until now, the SIATRP technique has been successfully studied with nanomaterials such as silica (Mao et al., 2017), multiwalled carbon nanotubes (Song et al., 2016), graphene and graphene oxide (GO; Ata, Banerjee, & Singha, 2016), gold (Lee, Kim, Park, Cho, & Choi, 2016), magnesium hydroxide (Liu, Feng, Chang, & Kang, 2012), and clay (Vo et al., 2016). A fine example of this technique was demonstrated by Huang et al. (2017), where ultraviolet light, in the presence of an organic catalyst, 10-phenylphenothiazine, was used to induce polymerization for modifying the surface of mesoporous silica nanoparticles (MSNs) with itaconic acid (IA) and polyethylene glycol methylacrylate (PEGMA) (Fig. 14.4). This was a novel metal-free ATRP, and the polymer nanocomposite [MSNs-NH2-poly (IA-co_PEGMA)] thus formed demonstrated a very good dispersity in both aqueous and organic media. Moreover, it was used as a potential carrier for the drug cis-platin.

14.2.2 Layer-by layer assembly technique The method based upon sequential deposition of oppositely charged species is broadly termed as the layer-by-layer assembly technique. Originally developed for polyelectrolyte systems, this technique has now found applications in almost all types of polymer growth protocols and with almost any type of component. Due to its broad scope of applicability, simplicity, versatility, and robustness, it has been accepted as a go-to choice for the synthesis of polymer nanocomposites (Ariga et al., 2014; Cui, Li, & Decher, 2016; Cui, Yang, Wang, & Wang, 2016; Xuan et al., 2017). There has been a variety of nanocomposites with diverse

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FIGURE 14.4 Schematic route for the synthesis of MSNs-NH2-poly(IA-co-PEGMA) via a metal-free ATRP. Reproduced with permission from Huang, L., Liu, M., Mao, L., Xu, D., Wan, Q., Zeng G., et al. (2017). Preparation and controlled drug delivery applications of mesoporous silica polymer nanocomposites through the visible light induced surface-initiated ATRP. Applied Surface Science, 412571 412577. https://doi.org/ 10.1016/j.apsusc.2017.04.026.

applications which were developed with the help of this technique. Some of them are summarized in Table 14.1. This technique is capable of fabricating diversified multifunctional polymer nanocomposites with ultra-high precise dimensional properties. Moreover, the content and dispersion can also be fine-tuned to a large extent. Ma, Cai, Qi, Kong, and Wang (2013) constructed a nanocomposite, which comprised of polyacrylic acid (PAA) functionalized graphene bearing poly(diallyldimethylammonium chloride) protected Prussian blue (PDDA-PB) nanoparticles (Fig. 14.5). This nanocomposite was used as a hydrogen peroxide sensor. The graphene and PB nanoparticles expressed a synergistic effect and catalytically reduced H2O2. The response toward the change in concentration of the peroxide was quick and the steady-state signal was reached within 2 s. The excellence in the properties demonstrated could be attributed to the increase in the rate of electron transfer between the electrodes and the detection molecules due to the large surface to volume ratio of the nanomaterial that was deposited on the electrode. This finally led to rapid and highly sensitive current response. This work is a noteworthy example of bio-sensing. The salient feature of this technique is that the electrocatalytic activity of the film could be designed and tuned by simply selecting the number of bilayers required or by choosing the electrically active species. Another case of layer-bylayer assembled GO nanocomposite films used in increasing the mechanical properties of poly(allylamine hydrochloride) and poly(sodium 4-styrene sulfonate) containing polyelectrolyte multilayer films, where the fibroblast cell adhesion and

14.2 Methods of preparation

Table 14.1 Some polymer nanocomposites synthesized via layer-by-layer assembly technique. S. No.

Type of polymer nanocomposite

1

Tricobalt tetroxide (Co3O4)/poly(styrene sulfonate) Graphene oxide/poly (allylamine hydrochloride) Polypyrrole/titanium dioxide (TiO2) Halloysite/polyaniline Nanoclay/ polyethylenimine

2

3 4 5

Utility

Reference

Humidity sensor

Zhang, Jiang, Sun, and Zhou (2017)

Biointerface with excellent mechanical properties Gas sensor

Qi, Xue, Yuan, and Wang (2014), Qi, Yuan, Yan, and Wang (2014) Cui, Yang, et al. (2016), Cui, Li, et al. (2016) Huang et al. (2016) Ziminska, Dunne, and Hamilton (2016)

Supercapacitor Foam coating agent with customizable properties

FIGURE 14.5 Scheme for assembling process of (PAA graphene/PDDA PB)n multilayer films. Reproduced with permission from Ma, J., Cai, P., Qi, W, Kong, D, & Wang, H. (2013). The layer-by-layer assembly of polyelectrolyte functionalized graphene sheets: A potential tool for biosensing. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 426, 6 11. https://doi.org/10.1016/j. colsurfa.2013.02.039.

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cell proliferation were studied, happens to be a beautiful example of biomedical and biotechnological advancement of assembled polymer nanocomposites. It will be quite a valid point to state that this highly tuned technique of engineering polymer matrix via incorporation of nanomaterials is an effective way of altering the physicochemistry of polymer composites and it holds promising contributions toward designing biologically active materials. In the following sections we shall discuss the applications of self-assembled polymer nanocomposites in biomedical sciences:

14.3 Applications of the self-assembled polymer nanocomposites in biomedical science As mankind saw the dawn of technological revolution, the hunger for investigation and study of the biological systems grew exponentially. This gave rise to the area of nanobiotechnology and its application in biomedical research. The arena of study expanded and so did the demand for highly precise applications of the diagnostic and therapeutic tools. Tailor-made medicines are in demand and so are the challenges in designing and commercializing them. In the earlier days of mankind conceiving the idea nanomedicine, the formulation was quite straightforward and simple. The available drugs had to be developed at nanoscale level. However, as the popularity of such medicines grew, the requirements for customization of the drugs and even personalized drugs, with in-built properties, were demanded. For this, an improved control over the structure, composition, and functionalization at the molecular level is a prime requirement. It is here that self-assembly comes into play. As this method of engineering nanomaterials is highly tunable to the level where it can even mimic the 3-D structures of biomacromolecules, many novel nanomaterials can be and has been produced to suffice the economic and ease-of-synthesis factors. Also, the diversification of nanostructures using self-assembly methods, where tailoring the monomer units give a new type of material, has paved a way toward patient-specific medicines. But this is not the end of the story. All nanomaterials thus produced via this protocol may not show the desired activities during the in vivo studies because of the complexities associated with the biological systems. So, we can only state that selfassembled polymer nanocomposites hold a lot of promise toward being the nextgeneration biomedical agents but a lot more has to be studied and achieved even today. However, the advancements made by these materials cannot be ignored for any reason, and therefore we shall discuss the varied forms in which self-assembled polymer nanoparticles have seen applications in the biomedical domains.

14.3.1 Drug delivery Nanocomposite drug delivery systems and their fabrication have been a matter of discussion and research ever since the inception of the concept. Material scientists as

14.3 Applications of the self-assembled polymer nanocomposites

well as pharmaceutical agencies have always invested a lot into the development of efficient drug carriers and target-specific active agents. This is solely because of the advantages that come with these therapeutic delivery systems. They are: 1. The particle sizes of the nanoparticles are amenable and the surface properties can be easily manipulated to suit the drug targeting. 2. The efficient binding, absorption, and capacity to carry bigger loads are inherent property of nanoparticles, due to their large surface to mass ratio. 3. Nanoparticles can easily be tuned to control the release rate and the degradation of drug in the biological system. The efficacy of drug entrapment in nanoclusters has been a remarkable feat. This has led to widespread acceptable results for the preservation of the drug in the body and also in minimizing the doses of highly potent biologically active molecules. This has greatly reduced the side effects and toxicity parameters of a drug molecule. 4. The nano drugs can be administered via different parenteral as well as enteral pathways. Following up the advantages of a nanoparticle drug, it is not any material that can be used for this purpose. There are a few criteria that have to be fulfilled by the nanomaterial to actually become a drug delivery agent. These criteria are majorly dependent on the properties of the therapeutic agent that is to be delivered and the intended activity of it. The common points that are to be kept in mind during the synthesis of such entities are as follows: 1. The delivery vehicle cannot be toxic. It has to be biocompatible and biodegradable. Moreover, it must be eliminated from the body almost immediately after serving the purpose. 2. The drug loading efficiency of these agents must be high, so as to reduce the number of dosage of the drug and also the number of cycles for which it must be administered. 3. The therapeutic agent that the delivery vehicle will be carrying cannot be modified in any form by the physiology and chemistry of the carrier, during the entrapment process. 4. The release profiles of the drug carried by these delivery agents must be consistent of a considerable period of time and even after repeated use by the patient. 5. The carriers should at least provide the bare minimal support to maintaining the stability of the drug during the entire route till it reached the target site. 6. The administration of these nano drug carriers must be simple and cause little/ no discomfort to the biological entity. 7. The preparation of the delivery system should be simple and reasonably easy. Moreover, the protocol must be reproducible, economically viable, and should bear the prospect of being upgraded for mass production. Once the above criteria are fulfilled, the nanoparticle drug carrier can be considered for delivering target-specific drugs. In addition to these, we must also walk hand in hand with the type of research that has been going around enhancing the achievements

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of biomedical sciences. Nowadays, nanocomposite drug delivery systems are not only limited to the design of the drug delivery agent/systems but to modifying their properties in such a way that the release of the drug takes place only after being triggered by a stimuli, which in turn is preferably an external one like heat or light. In this context, many nanocomposite drug carriers have been developed which convert an external stimulus into heat and thereby generate highly oxidative species at the target of choice to express the required action. These advances are termed as combinational therapies (Li et al., 2016; Molina et al., 2015; Xing et al., 2016). An elaborate exemplification of such form of drug delivery system was done by Qi et al. They designed an assembly of inorganic nanocomposite using folate ion modified lipid bilayer, which was spread on a gold nanorod (AuNR)-coated MSN-bearing photosensitizer (hypocrellin B). The nanocomposite was loaded with a high dose of photosensitive molecules and the entire modification was further mixed with selective liposomes so that the surface of the AuNR@MSN was coated. This enabled faster cellular intake and also showed remarkable endocytosis. Under the stimulation of one-time near-infrared two-photon illumination, the AuNR-mediated hyperthermia killed the cancer cells directly. Meanwhile the photosensitizer generated two kinds of reactive oxygen species (ROS), and it induced apoptosis of the cells. In addition to this, the hyperthermia induced also enhances the production of ROS (Fig. 14.6; Qin, Fei, Wang, Yang, & Li, 2015).

FIGURE 14.6 (A) The process of formulation of the biointerfacial nanocomplex. (B) Combined treatment of two-photon photothermal/photodynamic therapy for inhibiting tumor cell growth. Reproduced with permission from Qin, C., Fei, J. B., Wang, A. H, Yang, Y., & Li, J. B. (2015). Rational assembly of a biointerfaced core@shell nanocomplex towards selective and highly efficient synergistic photothermal/photodynamic therapy. Nanoscale, 7, 20197 20210. https://doi.org/10.1039/c5nr06501a.

14.3 Applications of the self-assembled polymer nanocomposites

In addition to this, several groups of researchers have also taken the advantage of existing polymeric (both synthetic and natural) materials to overcome the issues of biodegradability and toxic side effects or other health hazards (George, Shah, & Shrivastav, 2019; Sofi et al., 2018). Quite a number of reports on self-assembled drug delivery system falling into the category under discussion have shown successful encapsulation of drug molecules to enhance the desired properties such as: • • •

Bioavailability, Controlled/target-specific delivery, and Bioactivity.

Some of the drug delivery systems bearing self-assembled polymer nanocomposites as the structural form have found utility in treatment of various therapeutic conditions and also been commercialized or are under clinical trials (Table 14.2). Polysacaccharides such as cellulose, chitosan, and pullulan, have quite efficiently, aided to be the foundation of several self-assembled systems, which have found to target the colon. The property of adhesion to the mucosal surface in the small intestine has enabled the absorption of these drugs in it. Another report by Niers et al. (2007) shows the inhibitory property of a heparin-based system, amphiphilic by nature, toward the growth of blood vessels in and around tumors, thereby reducing its size. Polymers such as alginate, dextran, and chitosan also have their own therapeutic properties. Selfassembled PEG-alginate derivatives have been found effective in improving the intake of calcitonin by rats suffering from hypocalcemia (Li et al., 2012). In another instance of alginate-based self-assemblies, it was found that modifying its surface via phenylalanine ethyl esterification, the efficacy of cellular uptake as well as biocompatibility toward the human intestinal cells under in vivo conditions improved (Zhang et al., 2019). Ayub et al. (2019) reported the synthesis of a conjugated system of cysteamine and sodium alginate nanoparticles bearing disulfide crosslinks via the layer-by-layer assembly mechanism and it resulted in improving the delivery of the paclitaxel (PTX) drug, an anticancer drug for treating colon cancer. In addition to treatment of cancer, alginate nanoparticle based self-assembly nanostructures have been put to use for the delivery of antigens. Polylysine-sodium alginate nanoparticles bearing antigen-bovine serum albumin were found to show properties such as better cellular uptake, controlled release of the vaccine, and reduced cytotoxicity under in vitro conditions. They were prepared by taking advantage of the electrostatic interactions between polyelectrolyte complexes which bore opposite charges (Yuan et al., 2018). Alginate-based self-assembled nanoparticles have also found utility in the treatment of tumors which previously showed resistance to multiple drugs (Kumar et al., 2019) and as light and redox potential responsive therapeutic agent in the field of combinatorial chemotherapy, as reported by Zhang et al. (2017). Another formulation of self-assembled nanosystems is the nanogel form, which has earned popularity in the field of

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CHAPTER 14 Self-assembled polymer nanocomposites

Table 14.2 List of self-assembled polymer nanocomposites for application as drug delivery systems (Bobo, Robinson, Islam, Thurecht, & Corrie, 2016; Felice, Prabhakaran, Rodriguez, & Ramakrishna, 2014; Patra et al., 2018). Product name Livatag Lupron Depot Estrasorb

Carrier material Poly(isohexylcyanoacrylate) PLA Lecithin

Risperdal Consta

PLGA

Abraxane

Albumin

GenexolPM Adagen

PEG PLA PEG

Oncaspar

PEG

PEG-Intron

PEG

Cimzia

PEG

Omontys

PEG

Xyotax

Polyglumex

Puricase

PEG

Mylotarg

Anti-CD33 monoclonal antibody Anti-CD20 monoclonal antibody Anti-CD20 monoclonal antibody Anti-CD37 monoclonal antibody

Zevalin

Bexxar

Kadcyla

Drug/type of drug

Disease

Approval year/ phase

Doxorubicin/ anthracycline Leuprolide/ peptidic Estradiol/ esteroide Risperidone/ dopamine antagonist Paclitaxel/ anthracycline Paclitaxel/ anthracycline Adenosine deaminase/ peptidic Asparaginase/ peptidic Interferon a2b/proteic Interferon a2b/proteic Peginesatide acetate/ peptidic Paclitaxel/ anthracycline Uricase/ proteic Ozogamicin/ calicheamicins

Hepatocellular carcinoma Prostate and breast cancer Hot flushes during menopause Bipolar disorderschizophrenia

Phase II

Breast cancer

2005

Breast cancer

Phase II

Severe combined immunodeficiency

1990

Leukemia

1994

Chronic hepatitis C

2001

Crohn’s disease

2008

Anemia

2012

Lung cancer, ovarian cancer Hyperuricemia

Phase III Phase III

Leukemia

2000

Yttrium-90/ radioactive material Iodine-131/ radioactive material Emtansine/ maytansinoid

Non-Hodgkin’s lymphoma

2002

Non-Hodgkin’s lymphoma

2003

Breast cancer

2013

1989 2003 2003

(Continued)

14.3 Applications of the self-assembled polymer nanocomposites

Table 14.2 List of self-assembled polymer nanocomposites for application as drug delivery systems (Bobo, Robinson, Islam, Thurecht, & Corrie, 2016; Felice, Prabhakaran, Rodriguez, & Ramakrishna, 2014; Patra et al., 2018). Continued Product name Opaxio

Cimzia

Plegridy Adynovate (Baxalta)

Carrier material

Drug/type of drug

Disease

Approval year/ phase

Paclitaxel covalently linked to solid NPs of polyglutamate Pegylated antibody fragment

Paclitaxel

Metastatic breast cancer

2012

Certolizumab pegol

Pegylated IFN-B1 protein Pegylated factor VIII

Interferon B

Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, spondylitis Multiple sclerosis

2008, 2009, 2013, 2013 2015

Factor VIII

Hemophilia

2015

Zilretta

Triamcinolone acetonide 1 microspherical polylactic-co-glycolic acid matrix

Osteoarthritis of the knee

2017

Rebinyn

Coagulation factor IX glycoPEGylated

Hemophilia B patients

2017

IFN, Interferon; NPs, nanoparticles; PEG, polyethylene glycol; PLA, polylactic acid; PLGA, poly(lactic-co-glycolic acid); PTX, paclitaxel.

protein delivery. This system is based on the property of tunable porosity and on the principles of microfluidics. Chen, Li, Zhu, Liang, and Zeng (2019) reported similar self-assembled alginate-coated and chitosan polyelectrolyte nanocomposite system, which was utilized for pH-directed controlled insulin release. In addition to these polysaccharide systems cyclodextrins have also been incorporated into various nanoparticle systems with an aim to improve the physiochemical behaviors and thereby enhance the efficacy of the drug delivery system (Zerkoune, Angelova, & Lesieur, 2014; Zhang & Ma, 2013). One such report was made by Song et al. (2016), where conjugates of β-cyclodextrin and poly[n-isopropylacrylamide] with PTX drug incorporation, via host guest interaction was utilized as an anticancer drug which responded to temperature. The polymer unit also showed enhanced cellular uptake. Among other synthetic polymers polylactic acid, polyethylene glycol, and poly(lactic-co-glycolic acid) have been used in multiblock formation to aid therapeutic interests. Their utilities are also summarized in Table 14.2. Their utility has been approved by the Food and Drug Administration regulatory bodies from various countries. This was done owing to their biodegradability as well as biocompatibility alone.

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In addition to drug delivery, self-assembled nanocomposites are also used in the fields of bio-sensing, wound dressing, and in controlling stem cell behaviors. However, pure self-assembled polymer nanocomposites have been underexplored toward the cause of biomedical applications. Yet some of the noteworthy mentions are as follows: Qi et al. reported a GO and poly-L-lysine (PLL) composite, which acted as a coating for the bio-scaffold. The GO/PLL composite formed, supported the growth of stem cells with a high rate of proliferation. It was also found to accelerate the osteogenic differentiation of the cells and gene expression. Li et al. reported a microRNA sensor that was based on the property of amplifying the deposition of silver via synergistic photocatalysis by TiO2 photoreduction by guanine. Neutral charge probes were used for this purpose. Huang et al. also reported the preparation of nanocomposite hydrogels bearing poly(sulfobetaine acrylamide). They were used as wound dressings for chronic conditions. The sulfobetaine-based polymers were the main components and their conjugation with nanoclay, via incorporation, led to enhanced mechanical properties of the hydrogel. These hydrogel nanocomposites showed remarkable mechanical properties and high water content along with antifouling properties. Another important aspect associated is the histological examination of the wound profile. This led to the confirmation of the fact that complete re-epithelialization and formation of new connective tissues on the normal and diabetic wounds was found to take place at a good rate.

14.4 Future prospects and conclusion From the information shown above it is fair enough to comment that the field of applications of self-assembled polymer nanocomposites has been explored in a few biomedical domains. It is also found that many realms such as treatment of chronic tuberculosis, vaccination, etc. still remain underexploited but with the promises that these materials have shown over the years and the rate of progress that it has made toward achieving them, we can say that the day is near when they will cover a wide arena. Certain aspects can still be improved, such as the uniformity in the preparation of these materials. Sophisticated polymer nanocomposites can still be designed to show multifacial applications and become ideal examples of multifunctionality. Detailed studies of the types of interactions within the composite as well as with the receptor sites can be done in detailed fashion, so that the efficacy could be enhanced to better levels. These form the basis for explaining the structural activity relationships, which in turn helps in designing therapeutics for highly specialized needs. Another aspect that can be attended to is the sustainability of these materials. All in all, these materials can be expected to work wonders in the field of biomedical sciences in the very near future with major transformations and innovative applications.

References

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Song, Y., Ye, G., Lu, Y., Chen, J., Wang, J., & Matyjaszewski, K. (2016). Surfaceinitiated ARGET ATRP of poly(glycidyl methacrylate) from carbon nanotubes via bioinspired catechol chemistry for efficient adsorption of uranium ions. ACS Macro Letters, 5, 382 386. Available from https://doi.org/10.1021/acsmacrolett.6b00099. Vo, V.-S., Mahouche-Chergui, S., Babinot, J., Nguyen, V.-H., Naili, S., & Carbonnier, B. (2016). Photoinduced SI-ATRP for the synthesis of photoclickable intercalated clay nanofillers. RSC Advances, 6, 89322 89327. Available from https://doi.org/10.1039/ C6RA14724K. Wang, A. H., Yang, Y., Qi, Y. F., Qi, W., Fei, J. B., Ma, H. C., . . . Li, J. (2016). Fabrication of mesoporous silica nanoparticle with well-defined multicompartment structure as efficient drug carrier for cancer therapy in vitro and in vivo. ACS Applied Materials & Interfaces, 8, 8900 8907. Available from https://doi.org/10.1021/ acsami.5b12031. Wang, C., Cui, Q., Wang, X., & Li, L. (2016). Preparation of hybrid gold/polymer nanocomposites and their application in a controlled antibacterial assay. ACS Applied Materials & Interfaces, 8, 29101 29109. Available from https://doi.org/10.1021/ acsami.6b12487. Whitesides, G. M., & Boncheva, M. (2002). Beyond molecules: Self assembly of mesoscopic and macroscopic components. Proceedings of the National Academy of Sciences, 99(8), 4769 4774. Available from https://doi.org/10.1073/pnas.082065899. Xing, R. R., Liu, K., Jiao, T. F., Zhang, N., Ma, K., Zhang, R. Y., . . . Yan, X. (2016). An injectable self-assembling collagen gold hybrid hydrogel for combinatorial antitumor photothermal/ photodynamic therapy. Advanced Materials, 28, 3669 3676. Available from https://doi.org/10.1002/adma.201600284. Xuan, M. J., Zhao, J., Shao, J. X., Du, C. L., Cui, W., Duan, L., . . . Li, J. (2017). Recent progresses in layer-bylayer assembled biogenic capsules and their applications. Journal of Colloid and Interface Science, 487, 107 117. Available from https://doi.org/ 10.1016/j.jcis.2016.10.018. Yi, C., Zhang, S., Webb, K. T., & Nie, Z. (2017). Anisotropic self-assembly of hairy inorganic nanoparticles. Accounts of Chemical Research, 50, 12 21. Available from https://doi.org/10.1021/acs.accounts.6b00343. Yousefi, N., Sun, X., Lin, X., Shen, X., Jia, J., Zhang, B., . . . Kim, J. K. (2014). Highly aligned graphene/polymer nanocomposites with excellent dielectric properties for highperformance electromagnetic interference shielding. Advanced Materials, 26, 5480 5487. Available from https://doi.org/10.1002/adma.201305293. Yuan, J., Guo, L., Wang, S., Liu, D., Qin, X., Zheng, L., . . . Yin, R. (2018). Preparation of self-assembled nanoparticles of ε-polylysine-sodium alginate: A sustained release carrier for antigen delivery. Colloids and Surfaces. B, Biointerfaces, 171, 406 412. Available from https://doi.org/10.1016/j.colsurfb.2018.07.058. Zerkoune, L., Angelova, A., & Lesieur, S. (2014). Nano-assemblies of modified cyclodextrins and their complexes with guest molecules: Incorporation in nanostructured membranes and amphiphile nanoarchitectonics design. Nanomaterials, 4, 741 765. Available from https://doi.org/10.3390/nano4030741. Zhang, D., Jiang, C., Sun, Y., & Zhou, Q. (2017). Layer-by-layer self-assembly of tricobalt tetroxidepolymer nanocomposite toward high-performance humidity-sensing. Journal of Alloys and Compounds, 711, 652 658. Available from https://doi.org/10.1016/j. jallcom.2017.03.365.

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Thermoresponsive polymers and polymeric composites

15

Mh Busra Fauzi1, Samantha Lo1, Maheswary Thambirajoo1, Zawani Mazlan1, Izzat Zulkiflee1, Syafira Masri1, Isma Liza Mohd Isa2 and Sabarul Afian Mokhtar3 1

Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia 2 Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia 3 Department of Orthopaedics and Traumatology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

15.1 Introduction 15.1.1 Thermoresponsive polymers Thermoresponsive polymers are polymers that are widely utilized in various research applications such as tissue engineering, drug delivery systems, disease therapy, and genetic studies. In recent years, thermoresponsive polymers have increased in demand due to their capability to react and reversible alteration phase or volume transition especially in various ranges of temperatures. Hence the term “thermo” refers to temperature. As such, these polymers that react to different temperature can be considered as “smart material” as they can display different state of matters based on the temperature levels (Gandhi, Paul, Sen, & Sen, 2015; Zhang et al., 2019). Apart from these, thermoresponsive polymer system does not need any backup or activation from the human body system, namely cells, tissues, or organs, for it to respond to any biomedical applications because it functions very well in human body temperature between 35
C and 37
C (Kim & Matsunaga, 2017). Thermoresponsive polymers tend to aggregate after dissolving in a liquid medium when the temperature rises or drops. There is a space formed (miscibility gap) between the mixing components of polymers and later phase separation, which can be observed in the phase diagram of temperature versus polymer fraction volume (Zhang, Weber, Schubert, & Hoogenboom, 2017). The phase separation takes place due to hydrophobic molecules bond interconnected with the polymer sequences and thus causes polymer aggregation or precipitation in liquid medium (Kim & Matsunaga, 2017).

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00007-3 © 2023 Elsevier Inc. All rights reserved.

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Generally, thermoresponsive polymers will retain its physical state even after heating is known as lower critical solution temperature (LCST) and after heating become liquid is upper critical solution temperature (UCST). Thermoresponsive polymers can be either LCST or UCST based on their reactivity in chosen media whether in solvent or aqueous medium (Zhao, Ma, & Zhu, 2019). Fluctuation of temperature plays an important role in the macroscopic appearance of the polymers. This indicates that different concentrations of polymers, which dissolved in fluid media and form two distinct phases, manifest LCST properties while two fluid media are formed under lower temperature that is more likely to inherit UCST properties (Zhang et al., 2017). Unlike some polymers that dissolved in water solution, polymer with hydrogen bonds that can change to liquid form (in aqueous solution) is preferred by many researchers (Gandhi et al., 2015). In addition, the transformation to liquid state occurs faster in LCST than in UCST due to low temperature and interplays between hydrogen bond with other ion particles in water (Zhao, Ma, et al., 2019). Besides temperature, thermoresponsive polymers are also can be influenced by other physical and chemical stimuli, which include pH, ion concentrations, light, enzymes, shearing stress, and oxidation-reduction reaction. Similar to temperature, these stimuli also have the capabilities to modify the physical properties of polymers thus alteration in their contents and behavior (Bauri, Nandi, & De, 2018; Doberenz, Zeng, Willems, Zhang, & Groth, 2020). More than one stimulus system can be combined and applied in one polymer system to study its responsive behavior. This type of polymer is known as transient thermoresponsive polymers. These polymers are being exposed to the temperature, the polymeric structure is modified then undergoes another transition phase probably pH of a solution based on its purposes in medical applications (Vanparijs & Nuhn, 2017). Some of the known types of thermoresponsive polymers that belong to LCST or UCST are naturally derived thermoresponsive polymers such as cellulose polymers, collagen, gelatin, and chitosan. Synthetic thermoresponsive-derived polymers are poly(N-vinylcaprolactam) (PNVCL), poly (ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPAM), poly(2-oxazolines), elastin-like polypeptides, and hybrid polymers (Hogan & Mikos, 2020). Naturally derived polymers are widely acceptable for many clinical settings due to low toxicity effects, biocompatible, and on rare occasions might cause adverse immunologic effects. However, synthetic-based polymers draw major attentions due to their ability to mold the polymers into innumerable designs following the preferences of properties that suit the desired applications best (Saurabh, 2016). Basically, stimuli thermoresponsive polymers are manufactured to make membranes, films, and polymer brushes, which largely utilized in cancer treatment especially transporting drugs to the targeted area in human body (Niskanen & Tenhu, 2017).

15.1.2 Thermoresponsive polymeric composites Thermal conductive and insulating composite materials are mainly used in electronic components, aircraft, aerospace, or photonic devices as temperature control systems. The importance of this composite material is the thermal conductivity,

15.1 Introduction

which could be cost containment for thermal control resources if the conductivity level increases (Jiang et al., 2019). As the thermal heat rises due to the conductivity of the composites, this leads to insufficient heat dissipation thus distorting the devices (Hou et al., 2019). Furthermore, the traditional or conventional composites utilize less thermal conductivity though it contains sufficient amount of thermal conductive fillers (Meng et al., 2017). To cater this problem, polymeric-based composites are produced. Thermoresponsive polymeric composites are chosen compared to the traditional type of thermal conductive because these composites are malleable, extendable, mobility, and adaptive to any changes of the surrounding temperatures (Liu et al., 2018). Thermoresponsive polymeric composites also have been substituted with other substances to increase functionality for better performance. One paper elaborated to have used nanoparticles to produce thermoresponsive polymeric composites by mixing the polyethylene (PE) and polyethylene oxide (PEO) (both are semicrystalline polymer matrixes) integrated with spiky nanostructured metal known as Ni particles, which function as conductive molecules. This study was compared with carbon black (CB), which is another type of conductive molecule. The study revealed that nanospicky Ni molecules detect reversible temperature and work efficiently to speed up the conductivity whenever there are slight fluctuation in temperatures (Chen, Pfattner, & Bao, 2017a). In addition, one of the recent findings in the aspect of medical applications is the usage of thermoresponsive polymeric composite, PNIPAM fabricated with hydrogels to apply in drug delivery, tissue engineering, and wound dressing. Although thermoresponsive polymer compositebased hydrogel has similar properties as thermoresponsive polymers, it gives some defects to the applications such as a decrease in mechanical strength, biodegradability, and biocompatibility. As such, adding extra components like another type of polymers and incorporating nanoparticles could strengthen the efficiency of this composite (Xu, Liu, et al., 2020). In drug delivery system, it is pivotal to carry the drugs to the targeted site to treat the cells/tissues without causing any detrimental effects. In the latest study, the drug molecules loaded with clay like montmorillonite belong to bentonite, clay mineral used commonly in polymerization. This clay is molded into hydrogel to form polymer clay nanocomposites-based hydrogel. The composite might be useful in longterm effects to acquire desired healing based on the disease treatment (Tipa, Cidade, Vieira, Silva, & Soares, 2021). The drug particles can be incorporated into the hydrogel by using low temperature to liquefy and increase temperature above LCST through solidify process to form gelation (Xu, Liu, et al., 2020). Besides drug delivery, in tissue engineering, thermoresponsive composites such as PNIPAM (Parameswaran-thankam et al., 2018; Watson, Kasper, Engel, & Mikos, 2014) or PNVCL also can be fabricated into injectable hydrogels to make scaffolds that could mimic human extracellular matrixes (ECMs) in bone tissue transformation. However, the usage of PNIPAM in application has some drawbacks as such polymer inhibiting the role of fibrinogen in vascular or tissue injury and production of neurotoxicity to the cells. Due to this, PNVCL could be the best choice to replace PNIPAM. Even though this polymer possesses good

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physicochemical properties such as biocompatibility, biodegradability, and lack of cytotoxicity effects, the combination of it with other artificial polymers would avoid some unwanted pitfalls that include triggering a negative reaction in the immune system and cause inflammation (Parameswaran-thankam et al., 2018). Other than bone reformation, thermoresponsive polymeric composites also emerged as a promising agent to promote wound healing. In this paper, the researchers have incorporated thermoresponsive polymer in gel form with gold nanoparticles (AuNPs) and hydrogel to see the effects of skin burn on murine model. The results showed that there was improvement in skin re-epithelialization of the wounded mice. Other than those physicochemical properties that already explained earlier, thermoresponsive polymer containing hydrogel is a good agent for wound healing because it helps to retain water for dry wound and remove exudate or more water content from wet wounds. Other than these factors, thermoresponsive polymers have the capability of alternating between solid and liquid states depending on their specified temperature range. Meanwhile, AuNPs have antibacterial effects against Gram-positive bacteria especially Staphylococcus aureus, which is commonly found in burn wounds. Together with all these factors, this composite would become ideal to be used as a wound healing agent in treating skin burn (Arafa, El-kased, & Elmazar, 2018). Apart from this, thermoresponsive polymers also can be manufactured as a scaffold to support cell proliferation, cell migration, tissue reformation, transport drugs in the bloodstream, and expedites in tissue healing (Castillo-Henrı´quez, CastroAlpı´zar, Lopretti-Correa, & Vega-Baudrit, 2021).

15.2 Mechanisms 15.2.1 Protein adsorption To begin with, adsorption is defined basically as the accumulation and adhesion of particles to a surface, but without penetration. It is the first process that occurs after the implantation of a biomaterial in the human body. This phenomenon nonetheless is very complex as protein interactions with surfaces are unpredictable and are impacted by a combination of conditions, including protein structure, protein properties, and the biomaterial’s chemistry, geometry, and topography (Wilson, Clegg, Leavesley, & Pearcy, 2005). This process changes the properties of the surface and can induce structural alterations on the adsorb proteins. Cellbiomaterial interactions are mediated by the type and conformation of adsorbed proteins that can interact with specific integrins expressed by the cells. Integrins are one of the most common types of adhesion receptors (Schwartz, Schaller, & Ginsberg, 1995). At biological interfaces, the interaction of signaling moleculesproteins and proteinproteins controls plenty of other biological functions (Aiyelabegan & Sadroddiny, 2017). There are a few main components that go into protein adsorption: protein properties or composition, surface of the protein, and the protein environment. These components will be assessed individually to see how each of them affects protein adsorption.

15.2 Mechanisms

First, for the protein properties, two factors affect protein adsorption, which are the surface affinity and the concentration of the protein. The higher the concentration of the protein more likely will be in contact with the surface and consequently will bind to it. Due to their respective concentration gradients and surface affinities, a significant number of proteins compete for surface-binding sites. The surface affinity is basically how energetically favorable it is for a protein to be adsorbed to a surface. Surface affinity will also influence by many components such as the size looking at the larger protein will have more areas of contact it has with the surface, and also by the charge depending on the charge of the surface of the protein which may be attracted or repelled, together with the hydrophobicity of the proteins which tends to stick to surfaces in greater amounts. In addition, the structural ability of the proteins affects how much it adsorbs to the surface. As the protein is less stable, the more it will unfold, and the more contact points it will have with a surface. Unfolding will lead to increased active site exposure for protein surface contacts, regardless of the protein’s original structural rearrangement. Other than that, other factors that affect the structural ability such as the side chains, which may have a lot of different hydrophobic and hydrophilic side chains that might coil up and unfold and the folding rate will determine how quickly the protein will be adsorbed. Proteins’ abilities to destabilize nanostructures can be vary depending on the protein itself (Nance & McKenna, 2020). For biomaterial surfaces, many surface properties affect the interactions of proteins. First of all, the surface potential will influence the distribution of ions in the solution and will interact with the proteins. Next, topography can be one of the factors as the greater texture exposes more surface areas for interactions of proteins. Next, the composition of the surface can also be included that the chemical makeup of the surface will ultimately determine the types of intermolecular forces that govern the interactions with the proteins. Another insight will be the hydrophobicity of the surface, which tends to bind more protein. Protein adsorption can theoretically be reduced by adjusting the surface hydrophobicity (Wang, Robertson, Spillman, & Claus, 2004). When a protein structure is dehydrated, hydrophobic moieties within the protein structure form weak hydrophobic associations with the surface, preventing water molecules from interacting. As a result, the entropy of water in solution increases favorably, driving protein adsorption to the biomaterial surface (Kyriakides, 2015). Besides, the heterogeneity of the surface may cause nonuniformity of surface interaction results in domains that can interact differently with the proteins. In the final component that is the environment of the proteins, the pH of the environment will dictate how close the protein is to its isoelectric point. The closer the protein is to its isoelectric point, the more readily it will be adsorbed. The positive and negative charges of a protein are balanced and become neutral only when the isoelectric point equals the physiological pH. If proteins and surfaces have opposite charges, adsorption rates increase (Rosen & Kunjappu, 2004). Other than that, another factor of the environment that will greatly influence protein adsorption toward a surface is the temperature. As the temperature increased, the protein adsorption increases (Koutsoukos, Norde, & Lyklema, 1983). It is

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closely tied to the diffusion thermal convection and flow transport of the protein. There are many factors within the environment, the protein, and the surface itself that determine whether a protein will adsorb to a surface. This process is so multifaceted, in which protein adsorption to a surface cannot be predicted. Based on the total amount of adsorbed protein to surface over time graph, it can be observed that there is an initial high adsorption rate correlation to the diffusion control mechanisms. Next, there is an overshoot or a hump in the adsorption curve that can be explained in a variety of ways. For example, it may be the rearrangement of the proteins on the surface or that the proteins are less strongly bound may desorb, which is known as the short period of reversible binding. Only by exchanging protein molecules with those of different molecular weights and affinity for the surface can the adsorption mechanism be reversed biologically (Alves, Reis, & Hunt, 2010). Then, a plateau value is reached in the graph. For multiple proteins, they might have different affinity and different concentrations, they will have some sort of exchanging on the surface, which is known as the Vroman effect. Vroman and Adams first observed competitive protein exchange on surfaces in the 1960s, in which proteins already adsorbed on a surface from a protein mixture solution are replaced by later arriving proteins (Vroman & Adams, 1969a, 1969b). Figs. 15.1 and 15.2 illustrate the Vroman effect based on the

FIGURE 15.1 Representation of the Vroman effect. Protein B were replaced by protein A (Aiyelabegan & Sadroddiny, 2017).

15.2 Mechanisms

FIGURE 15.2 The three different processes for the exchange of proteins (exchange of earlier adsorbed proteins with other proteins). (A) Adsorbed protein A desorbs, leaving a vacancy for protein B to adsorb. (B) Adsorbed protein A was displaced by protein B, which has stronger binding affinity to the surface. (C) Protein B embedded itself in previously adsorbed protein A to form a transient complex (top); the complex then turns, exposing protein A to solution (middle); protein desorbed into the solution and protein B remained on the surface (bottom) (Hirsh et al., 2013).

protein’s surface affinity or concentration. The proteins at high concentrations with low surface affinity might take up the spots at first and over time it will reduce the amount of adsorbed and replaced by the proteins with high surface affinity. Then, proteins with the highest affinity will replace other proteins. As a whole, proteins with higher concentration will rapidly attach, and be replaced over time with greater surface affinity. All things considered, many aspects will increase the adsorption to a surface, especially to biomaterials or even thermoresponsive polymers. Generally, the aspects are usually depending on the three main components, which are the protein, the surface, and the environment of one’s protein. Under these components, there will be more parameters to be defined as protein adsorption is a very complex phenomenon with a relatively interesting topic to be explored.

15.2.2 Cells adhesion and attachments Nowadays, the usage of thermoresponsive polymers shows significant increases in biomedical applications due to the temperature-dependent

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properties. Thermoresponsive polymers can change the molecular conformation in an aqueous state at an LCST for cell culture utilization (Anderson, Abecunas, Warrener, Laschewsky, & Wischerhoff, 2017b). In tissue engineering, the type of polymers used as a biomaterial must possess unique characteristics that can support cell adhesion and attachments. Currently, the main aim in tissue engineering is to successfully fabricate the scaffold from various types of polymers to enhance cell adhesion, attachment, and proliferation. Thermoresponsive polymers are known as temperature-dependent polymers and possess hydrophobic properties. When the temperature is at LCST, the polymers become hydrophilic with optimum hydration conditions. However, when the temperature above the LCST, the polymers prone to become hydrophobic toward the water interactions (Anderson et al., 2017a). In biomedical applications, cell adhesion is the primary process that can determine the success of tissue regeneration and development. Tissue engineering involves the usage of scaffolds that are seeded with living cells to support tissue regeneration. There are two types of biomaterials used for fabrication, including natural and synthetic polymers. Generally, thermoresponsive polymers have two main applications in the tissue engineering field: a substrate to enhance cell growth and injectable gels for in situ scaffolds (Ward & Georgiou, 2011). In tissue engineering, thermoresponsive polymers must have the ability to enhance cell growth and proliferation, and fabricate scaffold with a surface that can regulate the cells’ attachment and detachment, as described in Fig. 15.3. Fig. 15.3 describes the response of cell detachment after being applied with different temperatures. Below 32
C, the thermoresponsive polymer tends to

FIGURE 15.3 Mechanism for cell detachment on thermoresponsive polymer surfaces when exposed to different temperatures (Doberenz et al., 2020).

15.2 Mechanisms

have a coil-like structure and become a more elongated shape. After the temperature increases to 32
C, the structure of the thermoresponsive polymers starts to change. Therefore cells being seeded on the surface of the thermoresponsive polymer and start to adhere. At 37
C, which indicates the optimum temperature for living cells start to attach and proliferate on the surface of the thermoresponsive polymer. However, after reducing temperature lower than 32
C, the surface of the polymer starts to change its surface properties. Thus, causing cell detachment. Therefore reducing temperature leads to hydration of polymer, low mechanical modulus, and low cell engagement (Mizutani, Kikuchi, Yamato, Kanazawa, & Okano, 2008). In addition, the formation of in situ scaffolds usually allows the delivery of nutrients and growth factors toward the cells (Ward & Georgiou, 2011). Besides, the hydrophobicity and hydrophilicity of polymers also play a role in cell adhesion and attachment. In thermoresponsive polymers, below the LCST, the polymers show hydrophilic and well hydrated. The polymers show hydrophobic properties when the polymers chain above the LCST (Anderson et al., 2017a; Tsuda et al., 2004). The optimum wettability range for the scaffold needs to be optimized to enhance cellular adhesion, cell attachment, and detachment (Mokhtarinia et al., 2018).

15.2.3 Thermoresponsive behaviors Thermoresponsive polymers are known as polymers that undergo changes of physical state in water from a soluble state to become an insoluble state after being exposed to the heated UCST or cooled LCST (Zhao, Dolmans, & Zhu, 2019). Many studies focused on the effect of thermoresponsive behavior of polymers upon tissue engineering, cells, and drug delivery. The UCST and LCST are the main categories of the phase diagram for thermoresponsive behavior. The increasing of polymer’s temperature will transition between two phase and single phase. Thus the UCST can easily be recognized (Mohammed, Bin Yusoh, & Shariffuddin, 2018).

15.2.3.1 Principle for thermoresponsive polymers showing UCST and LCST Thermoresponsive polymers are made up of both hydrophobic and hydrophilic properties. The hydrophilic properties can interact with water to form the hydrogen bond to retain the structure of the polymer chains in coil-shaped (Rashid, Zaid Ahmad, & Tajuddin, 2019). However, upon the exposure of the polymers to the temperature change above the critical solution, the temperature may change the structure of coil-shaped to become globule form. Thus the water could not access the polymers and not the formation of the hydrogen bond. Hydrogen bond plays an essential role to maintain the hydration of the polymers. In UCST, the polymers chain becomes more dehydrated and can become a nonhomogeneous mixture (Rashid et al., 2019). Fig. 15.4 shows the differences of polymer chain

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Temperature effect

Homogenous polymer chain Heterogenous polymer chain

Water molecule

Polymer chain

Hydrogen bond

FIGURE 15.4 Differences between homogenous polymer chain and heterogenous polymer chain.

between the homogenous and heterogenous structure caused by above critical limit solution temperature. The temperature at which the polymers solution starts to separate is known as critical solution temperature (CST). The temperature plays a role in phase transition for different types of polymers. LCST is the lower temperature for two phases for the coexistence curve, while UCST is the maximum temperature of the coexistence curve (Xiaoyan, Jian, Hui, Dafu, & Anna, 2009). Therefore the UCST polymers are usually more soluble compared to LCST. LCST mainly occurs among water-soluble thermoresponsive polymers. LCST occurred due to the lack of contribution of the enthalpy of mixing (ΔHmix , 0) to produce free energy being compensated by the entropic term TΔS, thus, enhance the (ΔSmix , 0) (Sponchioni, Capasso Palmiero, & Moscatelli, 2019). The LCST and UCST behavior rises from the ability of the polymers to balance the hydrophobic and hydrophilic properties of the polymers chain. Adding the hydrophilic monomers in the statistical copolymer of LCST can help to increase the hydrogen bond formation (Sponchioni et al., 2019).

15.2.3.2 Type of thermoresponsive polymers There are several categories of thermoresponsive polymers that commonly present for tissue engineering use such as poly(N-alkyl-substituted acrylamide)s, PNVCL, poly(2-alkyl-2-oxazoline)s, poly(ether)s, poly(N,N-(dimethylamino)ethyl methacrylate), and poly(oligo(ethylene glycone)(methyl ether) (meth)acrylate)s (Fig. 15.5) (Zhao, Ma, et al., 2019).

15.2 Mechanisms

FIGURE 15.5 Type of thermoresponsive polymers (A) Poly (N-alkyl-substituted acrylamide)s, (B) poly(Nvinyl caprelactam), (C) poly(2-alkyl-2-oxazoline)s, (D) poly(ether)s, (E) poly(N,N(Dimethylamino)ethyl methacrylate, and (F) poly(oligo(ethylene glycol)(methyl ether) (mech)acrylate)s. Adapted with permission Zhao, C., Ma, Z., Zhu, X. X. (2019). Rational design of thermoresponsive polymers in aqueous solutions: A thermodynamics map. Progress in Polymer Science [Internet], 90, 269291. Available from: https://doi.org/10.1016/j.progpolymsci.2019.01.001.

15.2.3.2.1 Poly(N-alkyl-substituted acrylamide)s Poly(N-alkyl-substituted acrylamide)s are known as LCST polymers at 32
C and have shown that the water molecules forming the hydrogen bonds producing hydrogen bond bridge to maintain the hydration (Doberenz et al., 2020). The Nsubstitution plays the most crucial role in the LCST behavior. The methyl amino groups in this polymer’s groups will be replaced with the ethyl amino groups. Thus, resulting in the exhibition of LCST transition at temperature range of 23
C32
C (Kong, Guo, Zhang, & Gao, 2017). However, polymer with LCST behavior is usually not suitable for reasonable practices. Therefore we need to stabilize the LCST behavior before officially being used for research.

15.2.3.2.2 Poly(N-vinylcaprolactam) PNVCL is a known thermoresponsive polymer that can undergo transition temperature. PNVCL has a high potential to contribute to medical device application, especially as thermoresponsive abilities that can suit the criteria for the innovation materials for drug delivery and tissue engineering field (Mohammed et al., 2018). PNVCL hydrogels possess several insufficient criteria, such as having poor porosity structure, low mechanical strength, and toxic cross-linkers (Shi et al., 2017). The PNVCL polymer has an LCST behavior. There are several methods of controlling the polymerization of PNVCL, including atom transfer radical polymerization, cobalt mediates radical polymerization, and reversible additionfragmentation chain transfer polymerization (Yu, Yi, & Tang, 2020).

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15.2.3.2.3 Poly(2-alkyl-2-oxazoline)s Poly(2-alkyl-2-oxazoline)s are known as star-shaped macromolecules and have a wide range of application, mainly in the drug delivery field. The poly(2-alkyl-2oxazoline)s have a hydrophobic property structure that can bind to the compounds that have low molar mass, such as drugs (Kirila, Kurlykin, Tenkovtsev, & Filippov, 2020). This thermoresponsive polymer can be controlled through structure and size group modification by combining the hydrophobic chain of the polymers with hydrophilic amide groups (Kirila et al., 2020).

15.2.3.2.4 Poly(ether)s Polyethers are a wide range of polymers that include PEO, PEG, and polypropylene oxide (PPO). At room temperature, PEO is a water-soluble polymer at various concentrations. However, when the temperature starts to increase, the miscibility gap starts to develop. Thus, causing solution-phase separation to become two phases. PEO is a more extended polymer. The LCST for PEO is considered much higher than other polymers due to longer polymer characteristics. However, the PPO polymer tends to have hydrophobic properties compared to PEO that can dissolve in water (Xiaoyan et al., 2009).

15.2.3.2.5 Poly(N,N-(dimethylamino)ethyl methacrylate) Poly(N,N-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a complex thermoresponsive polymers with LCST properties (Flemming, Mu¨ller, Fery, Mu¨nch, & Uhlmann, 2020). PDMAEMA commonly been used in biological activity. It has a weak polybase with temperature-dependent solubility. The LCST of PDMAEMA usually occurred at 50
C in an aqueous state (Id & Nowak, 2019).

15.2.3.2.6 Poly(oligo(ethylene glycol) methyl ether methacrylate)s Poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-based polymers that display LCST properties in the water (Kureha, Hayashi, Ohira, Li, & Shibayama, 2018). The LCST of POEGMA polymer depends on the physiological temperature used. Usually, the POEGMA started to become affected when the temperature reaches 90
C in the aqueous state (Dalgakiran & Tatlipinar, 2019).

15.3 Form of thermoresponsive polymers and polymeric composites The stimuli-responsive polymers exhibit a phase transition in response to environmental changes (stimuli). They can be triggered under various stimuli such as temperature, pH, light, and enzymes. The change of physical properties of these polymers can result in the change of macroscopic behavior of polymers either in a solution, semisolid, or solid phase. This triggered condition will allow the polymerization in a controlled manner under a physiological condition to obtain the

15.3 Form of thermoresponsive polymers and polymeric composites

predetermined physical properties of formulations such as hydrogel, particles, films, and implant, which are favorable for biomedical applications (Doberenz et al., 2020). Specifically, the polymers that capable to change their solubility in response to environmental temperature are known as thermoresponsive polymers. The changes in polymer solubility will lead to conformational changes of the polymer formulations from one phase to another. Notably, the point when the polymer solubility changes occur is determined as CST or transition temperature. Polymers with UCST are water-soluble above this temperature and become insoluble below it (cooling). Polymers that hold LCST behavior are entirely soluble in the water below this temperature, and phase separation occurs when increasing the temperature (heating). The gelation of thermoresponsive polymers is entropy-driven, where the negative free energy of thermoregulation indicates a thermodynamic process, which process is reversible, and the gels can return to solution-phase after removing the stimulus. The thermoresponsive phase transition is often called a coil-to-globule transition. To explain this, hydrophilic subunits can form hydrogen bonds with water molecules and keep the polymer chains in random coilshaped and hydrated form, results in a homogenous phase. When the temperature changes beyond the CST, the conformation is shifted from coil to globule state, causing the hydrophilic subunits of polymers inaccessible to water molecules and thus no formation of hydrogen bonds, which results in the polymer chains to a globule-shaped and dehydrated form to obtain a heterogenous phase (Dastidar & Chakrabarti, 2019). Thermoresponsive polymers hold great potential in tissue engineering and regenerative medicine, and drug delivery systems due to the plethora of “smart” biomaterials that are sensitive to the environmental changes of the surrounding tissues and exhibit reversible physical behavior in response to this factor. Depending on the biomaterials, they can be used as a hydrogel for subcutaneous and injection-based in situ gelation systems, cell sheet engineering, micro- and nanoparticles, 3D printing, and controlled drug delivery systems under physiological conditions. The design and characterization of biomaterial aim to fine-tune the pharmacokinetics and pharmacodynamics of the system toward therapeutic doses, excellent biocompatibility, bioactivity, biomechanical properties, controlled degradation profile, and absent immunogenicity for in vivo and clinical applications.

15.3.1 Hydrogels Hydrogels are three-dimensional (3D) hydrophilic cross-linked polymer networks containing a large amount of water. The thermoresponsive hydrogels can change their conformations under the temperature change of the surrounding environment, contributing to different mechanical and thermal properties depending on the type of polymers and cross-linkers. The gelation of polymers that exhibit LCST behavior can be induced above this environmental temperature to change their solubility, resulting in gel formation. The temperature-dependent phase

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transition can be determined using rheology, differential scanning calorimetry, and spectroscopy analysis (Dastidar & Chakrabarti, 2019). The classical preparation of precast molds for hydrogel preparation has moved forward to an injectable system that allows in situ cross-linking of hydrogel for minimally invasive tissue engineering and controlled drug delivery approaches. In addition, thermoresponsive polymers have been used to fabricate scaffold using a 3D bioprinting system, employing thermogelation for high-precision features of scaffold for tissue engineering application (Hogan & Mikos, 2020). Due to the porosity structure, soft stability, and hydrophilic nature, the hydrogel can mimic the microenvironment of the native ECM, which is feasible to apply for irregular-shaped tissues such as skin. Hydrogel has been well reported for wound dressing application in removing tissue exudates, exhibiting antibacterial activity, maintaining skin humidity, and reducing scar formation and pain. A natural polymer such as chitosan derived from chitin has demonstrated biocompatibility and antibacterial activity against Acinetobacter baumannii in vitro. An in vivo study has revealed that chitosan hydrogel had accelerated wound healing, re-epithelialization, and wound closure and decreased bacterial colony count in the full-thickness excisional wound of a rat model (Ahovan et al., 2020). NIPAM is the most favorable synthetic polymer for tissue engineering applications due to its transition temperature in an aqueous solution is 34
C that is close to body temperature (Isikci Koca, Bozdag, Cayli, Kazan, & Cakir Hatir, 2020). The synthesis of thermoresponsive poly(N-isopropyl-acrylamide)-b-poly (ε-caprolactone)-b-polyethyleneglycol-b-poly(ε-caprolactone)-b-poly(N-isopropylacrylamide)(P NIPAAm-PCEC-PNIPAAm) penta-block hydrogel copolymer has resulted in a 3D structure with interconnected pores that mimicked the ECM, had control degradation rate, maintained cell viability, increased mRNA expression of phenotypic markers of collagen Iα1 and collagen III, and enhanced proliferation of fibroblasts (Oroojalian et al., 2019). Poly(N-isopropylacrylamide)-grafted gelatines (PNIPAM gelatines) hydrogel has demonstrated higher compressive strainstress, cell proliferation, and collagen deposition in vitro (Ohya & Matsuda, 2005).

15.3.2 Nanoparticles For targeted drug delivery, passive targeting is mediated by local heat stimulus, which can be achieved by the different types of nanocarriers such as polymeric nanoparticles using thermoresponsive polymers. The delivery systems are triggered by external heat derived from localized hyperthermia in various pathological conditions such as cancerous and inflammation tissues. Thermoresponsive nanoparticles can be designed for the targeted delivery of chemotherapeutic agents. The idea is to develop thermoresponsive polymers as a delivery system to retain the cargo at systemic circulation (37
C) but then release the cargo within a locally heated environment, for example, tumor tissue that holds temperature range between 40
C and 45
C (Dastidar & Chakrabarti, 2019). The 5-fluorouracil loaded in poly(butylcyanoacrylate) and poly(ε-caprolactone) nanoparticles showed high drug-loading

15.3 Form of thermoresponsive polymers and polymeric composites

efficiency, sustained drug-release profiles, enhanced cytotoxic effect in vitro, and exhibited tumor growth inhibition while increased mice survival rate in a subcutaneous tumor model of immunocompetent mice (Ortiz et al., 2015). The amphiphilic nature of thermoresponsive polymers enables the delivery of a wide variety of hydrophilic and hydrophobic molecules, including drugs and bioactive molecules such as growth factors for drug delivery and tissue engineering application. An injectable hydrogel is an efficient delivery system as it can load cargo owing to its porosity and subsequently release the cargo at the target site. The polymers can be the free-flowing solution at or below ambient temperature; then, a nonflowing gel can be formed when injected into the tissue due to the change in environmental temperature. This way, many hydrophobic drugs can be loaded and administered to produce a sustained drug delivery. For example, poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) was synthesized via sequential polycondensation and free radical polymerization reactions before introducing growth factor of stromal cellderived factor-1 (SDF-1) at the point above polymer LCST. The sustained release of SDF-1 in PPCN hydrogel has promoted endothelial progenitor cell homing and angiogenesis would significantly improve impaired dermal wound healing in a diabetic murine splinted excisional dermal wound model (Zhu et al., 2016). Polymeric nanoparticles are also entrapped in the injectable hydrogel system. A sprayable in situ gelation of injectable hydrogel composed of poly(N-isopropylacrylamide166-co-n-butyl acrylate9)-PEG-poly(N-isopropylacrylamide166-co-nbutyl acrylate9) copolymer denoted as PEP and silver-nanoparticles-decorated reduced graphene oxide nanosheets (Ag@rGO)(AG) exhibited intriguing solution 2 gel irreversibility at low temperatures that have shown biocompatibility, accelerated wound healing and antibacterial ability in Methicillin-resistant S. aureus (MRSA) infected full-thickness round skin wound model of rat, which implies an administration of sprayable hybrid aqueous mixture onto the targeted skin area for a stable wound dressing (Yan et al., 2019).

15.3.3 Micelles Thermoresponsive polymers are attractive for polymeric micellar drug delivery systems as they allow controlled release of the drug based on localized hyperthermia. Thermoresponsive polymers are used to synthesize “smart” micelles that aim to encapsulate hydrophobic drugs and delivery them into an aqueous environment for a controlled release system and protect the drug from metabolism and oxidation in the circulatory system. Polymeric micelles are nanoscopic of selfassembled amphiphilic monomers consist of the hydrophobic inner core that is surrounded by the hydrophilic shell in an aqueous solution that is aggregated and forming a block copolymer when the concentration exceeds critical micelle concentration (Yadav, Dibi, Mohammed, & Emad, 2019). For example, the amphiphilic 4-arm and 6-arm thermoresponsive star-like block copolymers were synthesized through the ring-opening polymerization of

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γ-substituted ε-caprolactone monomers γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxyε-caprolactone and γ-ethoxy-ε-caprolactone have been shown to self-assemble into thermodynamically stable micelles, demonstrated temperature sensitive and exhibit higher drug loading capacity in vitro (Washington et al., 2017). The poly (acrylamide-co-acrylonitrile), a P(AAm-co-AN) copolymer, is another example of a thermoresponsive block in multiblock copolymers that have been used to synthesize thermoresponsive micelles loaded with an anticancer drug. For example, doxorubicin encapsulated in PEGylated P(AAm-co-AN) copolymer micelles has been shown to inhibit tumor growth in vivo (Bordat, Boissenot, Nicolas, & Tsapis, 2019). The thermoresponsive block copolymers with double LCST were reported to precisely control polymerization within the physiological temperature. Thermoresponsive micelles were synthesized using double LCST of poly(3-methylN-vinylcaprolactam) block copolymers. The LCST1 was varied from 19
C to 27
C by copolymerization of N-vinylcaprolactam with a novel hydrophobic monomer, 3methyl-N-vinylcapro-lactam, while the LCST2 at 41
C42
C was obtained by copolymerization of N-vinylcaprolactam with hydrophilic N-vinylpyrrolidone. The idea of LCST1 was to initiate micelle formation and entrapment of anticancer drug doxorubicin, whereas LCST2 was to induce the collapse of the micelle core and release the drug, which may increase the efficacy rate for cancer therapy (Liang, Liu, Kozlovskaya, Palchak, & Kharlampieva, 2015).

15.3.4 Films The materials that can transform energy from the environment into macroscopic deformation by chemical and physical stimuli are known as “smart” mechanical actuators. These materials become attractive for many applications such as artificial muscles and robotic devices due to the lightweight, soft, and excellent thermal and electrical conductor. For example, thermoresponsive aluminum (Al) composite films have been synthesized by casting a solution of poly(acrylic acidco-acrylate) on an Al foil. Both Al and polymer layers were bound via the carboxyl groups to form the films. With a combination of electrical conductivity of Al, these films demonstrated a rapid and reversible thermo-responsive deformation that has good mechanical properties, which can mimic the movement in nature for robotic application (Zhou et al., 2019). Another example, poly(2-phenylethyl methacrylate)-grafted cellulose nanocrystals (MxG-CNC-g-PPMA) are also thermoresponsive polymers that exhibit LCST behavior in imidazoliumbased ionic liquids that have demonstrated mechanically robust thermoresponsive and ion-conductive films, which may imply the application for thermal cut-off safety devices (Kato, Lettow, Patel, & Rowan, 2020). The films can also be fabricated via hydrogel fabrication, in which the thickness of the hydrogel film can be controlled, ranging from nanometer to micrometer to maintain hydrogel permeability. Dopamine was used to coat onto a polyethersulfone membrane surface to introduce double bonds for photo-induced

15.3 Form of thermoresponsive polymers and polymeric composites

surface cross-linking copolymerization of NIPAAm and methacryloxyethyltrimethyl ammonium chloride to form hydrogel film. The quaternary ammonium salts in the hydrogel film were effectively killed bacteria before detaching them by reducing the temperature below LCST of poly(NIPAAm), which the hydrogel film could be potentially used for antibacterial application (Wang et al., 2018). Thermoresponsive submicroporous films in nanometer size were synthesized by blending PNIPAM with polystyrene, which the blends were prepared with polyethylene terephthalate substrate using a spin coater that aims to control pore size. The films have demonstrated higher cell viability and accelerated cell detachment at 20
C conditions. Thus the use of thermoresponsive surfaces allows the harvest of interconnected cells in monolayer culture without enzymatic or physical scrapping, which could be useful for cell sheet technology in tissue engineering applications (Fragal et al., 2019).

15.3.5 Interpenetrating networks Interpenetrating networks are cross-linked hydrogel that consists of two covalently bound polymer networks linked together by physical entrapment instead of covalent bonds. This cross-linked hydrogel requires the polymerization of both networks simultaneously, which results in two interpenetrating networks that can only be segregated by disrupting bonds (Ward & Georgiou, 2011). Sodium alginate (SA)/polyvinyl formal composite with double cross-linking systems was fabricated by blending a PVA gel with SA before cross-linking with formaldehyde (HCHO) and calcium ion (Ca21) to form a hydrophilic and continuous porous structure. The composite has shown an improvement for thermal, mechanical, and hydroexpansivity properties as well as cell attachment, which could be a potential application for surgical filling sponges and wound dressing (Wang, Zheng, et al., 2017). Cryogel is another example of interpenetrating networks that become important for tissue engineering applications due to their interconnected macroporous features. The cross-linking reaction of cryogel is initiated at subzero temperatures ranging between 25
C and 220
C. At this point, the solvent such as water begins to freeze, while hydrogel precursors become highly concentrated in the unfrozen liquid phase. The highly concentrated polymer and cross-linker in the liquid phase allow the cross-linking reaction to occur at these low temperatures. The cryogel is thawed when completing cross-linking reaction, thus obtaining a macroporous network of the cryogel. For example, the DNA-based cryogel network has been fabricated by mixing an aqueous solution of DNA with crosslinker and catalyst tetramethylethylenediamine to initiate chemical reaction of amine groups presented on the nucleobases of DNA strands with the epoxide terminal groups of cross-linker polyethylene glycol diepoxide at 220
C, to form a macroporous network of covalently linked DNA strands. Then, the alginate was introduced to the cryogel construct before being ionically cross-linked with calcium ions to form DNA-based interpenetrating network cryogels. These cryogels were able to sustain large deformations higher than 95% of strain under

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compressive forces, enhance toughness and stiffness, excellent cytocompatibility, and interconnected microporosity, which the cryogel demonstrated a sponge-like pore structure that potentially uses for diverse tissue engineering applications (Basu, Johl, Pacelli, Gehrke, & Paul, 2020). The in situ fabrication of the interpenetrating network of fibrin and hyaluronic acid (HA) was developed by combining enzymatic fibrin cross-linking (thrombin and fibrinogen) with orthogonal disulfide cross-linking of HA (thiol-derivatize and 2-dithiopyridyl-modified HA) to form an injectable hydrogel of two interpenetrating networks. The high hydrophilicity of HA functions to avoid compaction of the fibrin network, while fibrin enables an adhesive niche for in situ cell encapsulation. This fibrin/HA interpenetrating network hydrogel has shown an increase of storage modulus, a lower degradation profile, and enhanced cell proliferation and expression of cell surface receptor CD44, which are promising strategies for tunable extracellular-based biomaterials in tissue engineering and regenerative medicine (Zhang, Heher, Hilborn, Redl, & Ossipov, 2016).

15.3.6 Polymersomes Polymersomes are self-assembled structures based on amphiphilic block copolymers with a hydrophilic interior that allow encapsulation of hydrophilic molecules in their interior protected from the outside environment, which have also been employed for drug and DNA delivery (Ward & Georgiou, 2011). For example, NIPAAm polymers become amphiphilic in an aqueous solution above body temperature (37
C), allowing self-assemble into vesicles that could encapsulate hydrophilic molecules. The block copolymer poly(ethylene oxide)-block-poly(Nisopropyl-acrylamide) forms vesicles, which could be potential systems for drug delivery (Onaca, Enea, Hughes, & Meier, 2009). Dual responsive smart nanocarriers were developed by encapsulating thermoresponsive poly(N-isopropylacrylamide)-doxorubicin into pH-responsive PEG2,4,6-trimethoxy benzylidene pentaerythritol carbonate polymersomes. The system has shown that drug release from polymersomal formulation was pHdependent, inhibiting tumor growth rate in mice models and enhancing drug halflife systemically, suggesting that thermo-pH-dual-stimuli responsive hydrosomal represents an effective strategy for cancer therapy (Oroojalian et al., 2019). Polymersomes were also synthesized using amphiphilic brush block copolymers poly(oligo(ethylene glycol) methyl ether methacrylate)-b-poly(oligo(propylenesulfide) methacrylate) via self-assembly in aqueous solution. The interior of the polymersomes was co-loaded with hydrophilic molecules and water-soluble PNIPAM conjugates that allow it underwent a phase transition to form in situ hydrogels when the temperature above LCST of PNIPAM. The integration of thermoresponsive hydrogel in the polymersomes demonstrated a sequential intracellular release of water-soluble cargos from polymersomes that is stable for the drug delivery system (Du, Bobbala, Yi, & Scott, 2018).

15.4 Applications of thermoresponsive polymers

15.4 Applications of thermoresponsive polymers 15.4.1 Vascular applications Cardiovascular diseases (CVD) are the number one cause of death in the world, bringing in one-third of worldwide deaths. Diseases such as coronary heart disease, peripheral arterial disease, rheumatic and congenital heart diseases, cerebrovascular disease, and venous thromboembolism are all classed as CVD (Stewart, Manmathan, & Wilkinson, 2017). CVD occurs through an accumulation of interactions based on risk factors caused by environmental factors, familial inheritance, or both. CVD occurring through environmental causes involve modifiable risk factors such as unhealthy diets, obesity, smoking, and a sedentary lifestyle. All factors stated contribute to an increase in CVD risk. CVD is a complex disease, whereby obtaining other diseases such as diabetes mellitus, hypertension, and atherosclerosis can contribute to a developmental increased risk of morbidity and mortality. Other environmental factors to which an individual has little control over include tobacco smoke, pesticides, vaporized chemicals, and pollution (Cosselman, Navas-Acien, & Kaufman, 2015). Another factor that determines the risk of an individual obtaining CVD is genetics. Various mutations in genes involved in the regular function of the cardiovascular system can have an adverse effect on an individual depending on the mutation severity and/or in combination with environmental risk factors. Some examples of genetic mutations affecting the cardiovascular system include mutations in sarcomere and structural genes causing hypertrophic and dilated cardiomyopathy, abnormalities in desmosomal genes leading to arrhythmogenic cardiomyopathy, and heart arrhythmias inherited from mutations in the transmembrane ion channel genes, to name a few (Cirino et al., 2017). Often, the damage on the cardiovascular system from CVD is irreversible, leaving the heart muscles with fibrotic scarring and narrowing of the arterial vessel. There are various treatments and preventative care currently available in the market. Medical treatment for CVD preventative care includes lipid-lowering therapy, antihypertensive therapy, and antiplatelet therapy. Besides that, surgical operations such as coronary bypass surgery and valve repair/replacement surgery are also available. In lipid-lowering therapy, it is a form of CVD prevention therapy to reduce lipid levels in the body to lower CVD risk of occurrence. There are two types of cholesterol found in the human body, which are low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). LDL-C is considered the “bad” cholesterol, contributing to an increase in CVD risk (Stewart et al., 2017). Several studies have established that excess LDL-C in the human body leads to the formation of atherosclerosis plaques, leading to an increased risk of other diseases such as ischemic stroke and coronary heart diseases (Soran, Dent, & Durrington, 2017). HDL-C, on the other hand, is the “good” cholesterol, attributing to cardioprotective properties. However, this statement has yet to be reproducible.

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This is because some clinical studies only stated that low levels of HDL-C are linked to an increased CVD risk, whereas other studies contradict this information as an increase in HDL-C levels does not reduce CVD risk, and that HDL-C associated genetic polymorphisms does not correlate with CVD risk as well (Ronsein & Heinecke, 2017). Hence, statin treatments aimed at the reduction of LDL-C levels with a slight elevation in HDL-C levels are prescribed to reduce CVD risk. Besides that, antihypertensive therapy looks into controlling hypertension as it contributes as a risk factor for CVD. Hypertension is diagnosed through having blood pressure of more than 115/75 mmHg consistently, thus increasing the risk of CVD. Hence, hypertension intervention functions to reduce an individual’s blood pressure to reduce CVD risk. However, it was found that there was no lower limit of blood pressure that benefits the reduction in CVD risk, with statistical limitations that further blood pressure reduction is damaging. These studies present a flaw in hypertension treatment, and hence limiting methods of CVD risk reduction as well (Stewart et al., 2017). Antiplatelet therapy, as the name suggests, utilizes antiplatelet and/or anticoagulant drugs to reduce thrombosis and blood aggregation risks, hence reducing CVD risks as well. While these medications are widely used for clinical treatment, it brings on a high risk of excessive bleeding due to a reduction in platelets and coagulating factors, thus possibly increasing the mortality of an individual (Metharom, Berndt Michael, Baker Ross, Andrews, & Robert, 2015). Various surgical procedures are also available in effort of reducing CVD risks. Coronary artery bypass graft (CABG) surgery is a surgical procedure that redirects blood supply to the heart due to the presence of a blocked artery fragment in the vessel (Cho et al., 2017). While effectively reducing CVD risks such as a heart attack, various complications and risks may be presented, one of which is atrial fibrillation. Postoperative atrial fibrillation is the common complication presented following CABG surgery, occurring in between 20% and 40% of patients, which in turn increases hospitalization, other cardiac event risks, and mortality of an individual. Due to several limitations stated in previous CVD treatments and surgeries available, an alternative method should be presented, which can effectively reduce CVD risk. Many research methods look into the possibility of using thermoresponsive polymers to repair or replace damaged blood vessels. One study utilized a 3D-printable glucose-sensitive and thermoresponsive hydrogel to act as a sacrificial material in the construction of vascular-like channels. The researchers used a boronate ester hydrogel as the sacrificial gel, whereas the fabrication of the nonsacrificial gel consists of copolymerization of N-isopropylacrylamine for thermoresponsiveness together with pentafluorophenyl acrylate for postmodification and poly(vinyl alcohol) for gel formation. Interconnecting multichannels within the nonsacrificial gel was successfully achieved, and vascular endothelial cells could proliferate and attach to the multichannels (Tsai, Theato, & Huang, 2020). Another study demonstrated the use of a parrafin-based phase changing material incorporated with acrylic polymeric samples through micromachining in the creation of vascular channels. The results showed that vascularization was enhanced heat transmission of the phase change material in the acrylic composite,

15.4 Applications of thermoresponsive polymers

where increasing the channel width twofold also doubly increases heat transfer in effective areas (Mutua, Balapour, & Farnam, 2020). One other study fabricated nanothin coculture membrane with adjustable pore architecture and thermoresponsive functionality. This membrane allowed for an effective coculture of mesenchymal stem cells and cardiomyoblast cells, with thermoresponsive properties aiding in the viable, ECM-preserved, printable transfer sheets of cardiomyogenically differentiated cells (Ryu et al., 2015).

15.4.2 Gene delivery It is a common fact that genetics play a major role in determining human characteristics. However, when genes are associated with the cause of complex or rare diseases, they are labeled as genetic diseases. Genetic diseases are often due to mutagenic errors present in the DNA structure, either occurring spontaneously or inherited (Sinden & Wells, 1992). This leads to the genetic mutations phenotypically expressed, leading to various diseases such as cancers, blood disorders, and heart diseases (Wray & Maier, 2014). While there is no cure currently available for people with genetic diseases, there are various treatments available to alleviate or reduce symptoms, one of which is gene therapy. Gene therapy is defined as the delivery of nucleic acid polymers aimed at cells to treat genetic diseases (Campa, Gallenga, Bolletta, & Perri, 2017). Gene therapy is essentially able to deliver therapeutic sitespecific modifications to genomes, which allows for normal DNA transcription and translation, producing cellular proteins of nondiseased phenotypes (Gonc¸alves & Paiva, 2017). However, several issues may arise in the delivery of nucleic acids as they are negatively charged and unstable molecules, which may affect the intracellular delivery process into the body (Lostale´-Seijo & Montenegro, 2018). One technique commonly used in the delivery of therapeutic genetic treatment involves recombinant DNA technology. This method involves the use of a segment of a selected healthy gene incorporated into a vector, usually of viral or plasmidial derivation. The vector with genetic material is then inserted into targeted cells for the delivery of therapeutic genes, hence utilized by the cells for the production of normal proteins (Gonc¸alves & Paiva, 2017). Vectors are commonly used for the delivery of genetic material as it can provide protection against genetic material degradation as well as improve biodistribution and uptake (Lostale´-Seijo & Montenegro, 2018). In the context of this chapter, there are two types of thermoresponsive polymers involved in gene delivery, which are single and hybrid thermoresponsive polymers. While single thermoresponsive polymers are largely available, it exhibits many drawbacks such as high gelation concentration and temperature, low mechanical strength, and presents cytotoxicity. Hence, hybridizing thermoresponsive polymers largely resolves these issues in accordance with targeted delivery systems (Sarwan, Kumar, Choonara, & Pillay, 2020). Thermoresponsive polymers can be used in gene delivery experiments. A common issue largely faced in gene delivery is that as previously mentioned, the genetic material present in the vector is negatively charged and a hydrophilic molecule, which makes its delivery into

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the nucleus of cells impossible as the nucleus membrane is negatively charged and hydrophobic (Gandhi et al., 2015). Therefore thermoresponsive polymers’ temperature can be altered to increase transfection efficiency. One study utilized polyethyleneimine, a synthetic polycation, to form compact DNA into small particles together with grafted neutral responsive PNIPAM chains. These compacted DNA particles have structural variations formed according to the graft polymer LCST and were assessed. The results concluded that alterations in temperature-induced phase transitions of thermoresponsive polycation vector configurations increase transgene expression, hence enhance gene delivery (Lavigne et al., 2007). Another study utilized lipoplexes in the delivery of short interfering ribonucleic acids (siRNA), whereby the thermoresponsive polymer N-isopropylacrylamide-co-N,N0 -dimethylaminopropylacrylamide (P(NIPAAm-co-DMAPAAm))modified liposome allowed for more efficient intracellular delivery as compared to commercial transfection agent Lipofectamine RNAiMAX and nonmodified or PEGylated liposomes. This is due to P(NIPAAm-co-DMAPAAm) LCST characteristics, hence altering its hydrophilic to hydrophobic nature in temperatures above the LCST (Wang, Ayano, Maitani, & Kanazawa, 2017).

15.4.3 Drug delivery Thermoresponsive polymer can be used as a drug delivery due to its properties being stimuli-responsive polymers. They are also famously known as “intelligent” or “smart” polymers. Temperature, pH, redox potential, light, ionic strength, electric or magnetic fields, and the presence of enzymes or particular ligands are all examples of environmental conditions that these polymers react to (Alexander, 2006). The ability to alter a system’s physicochemical properties in response to a particular stimulus makes smart polymers appealing in a variety of applications, such as promoting protein and cell adhesion/detachment in cell culture engineering and bioseparation (Okano, Yamada, Okuhara, Sakai, & Sakurai, 1995; Wischerhoff et al., 2008). Temperature changes influenced the changing of its conformation, hydrophobic/hydrophilic balance, and solubility in modern drug delivery applications (Shao, Wang, Wang, Li, & Zhang, 2011). There are two different behaviors: LCST and UCST. LCST polymers include PNIPAM, poly(N,N-diethylacrylamide), poly(N-ethylmethacrylamide), poly (methyl vinyl ether), poly(2-ethoxyethyl vinyl ether), poly(N-vinylisobutyramide), poly(N-vinyl-n-butyramide), PNVCL, polyphosphazene derivatives, and poly(N(2-hydroxypropyl) methacrylamide mono/di-lactate). While UCST polymers are poly(acrylic acid) and polyacrylamide or poly(acrylamide-co-butylmethacrylate), poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (commercially known as Pluronics, Poloxamer). The difference between these two is polymer that is initially soluble but becomes insoluble when heated, resulting in an LCST, and polymer that is initially insoluble but becomes soluble when heated, resulting in an UCST (Klouda & Mikos, 2008).

15.4 Applications of thermoresponsive polymers

FIGURE 15.6 Released drug concentration over time. The solid lines indicate the toxic and minimum effective levels of the drug. The desirable, controlled drug release is indicated by black dotted lines while, shown in gray dotted lines, two cases of problematic drug release indicate drug release ending too soon or, on some occasions, being below the minimum effective level or higher than the toxic level. Note that it is desirable, after a small initial amount of time, that the released drug concentration is constant and between the toxic and the minimum effective level (Ward & Georgiou, 2011).

To achieve a successful drug delivery was never an easy task. Many obstacles need to be under consideration, which include enzymatic or hydrolytic degradation, solubility, toxicity, fast clearance rate, and the ability to not pass through the biological barriers (Goodman, Ng, & Pun, 2008; Juillerat-Jeanneret, 2008). Based on Fig. 15.6, the concentration of the drug released at the target-specific area is either too high or too low, or maybe not delivered at all at a certain time point, which causes the problems in drug delivery. Alternatively, a thermoresponsive polymer, which may respond to external stimulus as mentioned earlier, can be a beneficial and advanced way in modern drug delivery. An expansion of polymer chains as a result of temperature rising to that end may enable drugs to diffuse out and be released from the carrier (Hatefi & Amsden, 2002). As a whole, preloaded polymer networks for drug release at body temperature have almost become standard practice, with more recent research concentrating on polymer networks as a 3D structure for tissue engineering and cell culture.

15.4.4 Wound healing 15.4.4.1 Wound healing phases Skin is the largest organ that plays essentials roles in the human body and acts as a barrier to pathogens from the environment (Jeong, Park, & Lee, 2017). The largest surface area of the human skin exposed to the environment tends to have a high risk of skin injury. A skin injury can occur due to acute and chronic wounds that can be differentiated based on the severity of the wounds and the duration of wound healing progress (Masri & Fauzi, 2021; Salleh & Fauzi, 2021). Human skin tissue consisted of three different main layers; epidermal, dermal, and

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Sweat Duct

Hair

Stratum Corneum

Epidermal

Stratum Granulosum

Stratum Spinosum

Dermal

Sebaceous Gland

Stratum Basale

Keranocytes

(A) Epidermis region

Blood Vessel Hypodermal

Lymph Duct Adipocytes

(B) Skin ssue components

FIGURE 15.7 Normal skin anatomical structure (A) epidermis region, and (B) skin tissue components. Adapted with permission Masri, S., & Fauzi, M. (2021). Current insight of printability quality improvement strategies in natural-based bioinks for skin regeneration and wound healing. Polymers, 13, 2021.

hypodermal. The epidermis layer is known as the skin’s outermost layer that protects against harm caused by the external environment (Xu, Han, Gu, & Wu, 2020). The dermis layer is usually composed of ECM, human dermal fibroblasts, glands, and blood vessels (Xu et al., 2020). Last, the hypodermis layer riches in adipose tissue that aimed to control temperature regulation and provides mechanical properties to humans (Xu et al., 2020). Fig. 15.7 describes the anatomical structure for normal skin layers. Wound healing is a complex process involving several essential phases, including hemostasis, inflammation, proliferation, and skin remodeling. Fig. 15.8 describes the overview for acute and chronic wound structure.

15.4.4.1.1 Hemostasis Initially, the wound healing process begins with the vasoconstriction by the blood vessels following by platelet aggregation to stop the bleeding, followed by neutrophil recruitments to perform phagocytosis after the wound exposed to the outside environment (Dong et al., 2020). The hemostasis phase will immediately start after the wound occurred. Fig. 15.9 describes the hemostasis phases where the exudate containing clotting factor starts to coagulate at the wound site.

15.4.4.1.2 Inflammation Next, the wound healing process continues with the wound inflammation phase. The inflammation phases aimed to remove the pathogens that enter from the outside. This phase also important to prevent the wound from getting infections.

15.4 Applications of thermoresponsive polymers

FIGURE 15.8 Difference between acute and chronic wound structure (A) acute wound, and (B) chronic wound (Larouche, Sheoran, Maruyama, & Martino, 2018).

FIGURE 15.9 Wound healing stages (Xu et al., 2020).

However, delays in the inflammation phase result in the formation of chronic wounds (Singh, Young, & McNaught, 2017).

15.4.4.1.3 Proliferation The proliferation phase in wound healing usually involved days and weeks. New formation of blood vessels will be developed by the fibroblasts and epithelial cells

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(Rajendran, Kumar, Houreld, & Abrahamse, 2018). In the angiogenesis process, the platelets will release the transforming growth factor (TGF-β), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to trigger the angiogenesis. The VEGF will be released in response to hypoxia to trigger neovascularization and tissue repair (Singh et al., 2017).

15.4.4.1.4 Tissue remodeling During the remodeling phase, the protein of ECM, including fibrous protein, collagen, polysaccharides, fibronectin, and proteoglycans, will be synthesized (Rajendran et al., 2018). Tissue remodeling is the final stage for the wound healing process, which sometimes takes up to several years to develop mature tissue. The collagen and protein will be deposited at this phase to allow the new tissue formation and enhance wound organization (Singh et al., 2017).

15.4.4.2 Application of thermoresponsive polymers in wound healing In skin tissue engineering, the scaffold must possess several characteristics, including biocompatible, biodegradable properties, and mimic our native ECM. Thermoresponsive polymer is a tremendous interest for wound healing application and scaffolding due to their unique properties to change the temperature. The changes in the temperature can develop phase transformation toward the polymer. Thus it will release the antiinflammatory, antimicrobial, and wound care drug (Castillo-Henrı´quez, Castro-Alpı´zar, Lopretti-Correa, & Vega-Baudrit, 2021). Several forms of a scaffold, including hydrogel, films, and sponge, have been employed as wound healing materials. However, hydrogels become the most commonly excellent delivery systems to manage acute and chronic wounds (Abbasi et al., 2020). Thermoresponsive polymers show good performance for the wound healing process by changing the temperature. The changes in the temperature lead to the release of a loaded antiinflammatory, antimicrobial, and wound care drug (Castillo-Henrı´quez et al., 2021). Thermoresponsive polymers have good application for wound healing because most of the thermoresponsive polymers show excellent biodegradability and accelerate wound healing progress by increasing the vascularization at the injury site (Zhu et al., 2016). Therefore thermoresponsive polymers show efficiency in wound healing application.

15.5 Future perspectives Thermoresponsive polymers have great potential in the development of novel in vivo drug delivery, tissue engineering, and regenerative medicine, therefore many studies are shedding the light on various natural and synthetic polymers that are beneficial for such applications and which can be utilized for novel therapeutic platforms. However, natural polymers experience some limitations and disadvantages when in requirements of high mechanical strength and rapid gelation process compare to

15.5 Future perspectives

synthetic polymers. Meanwhile, the limitation of the synthetic polymers is that it is frequently found to be nonbiocompatible for cells adhesion and cell bioactivity, hence this drawback is to be modulated to overcome other complications whereas to ensure effective in vitro, in vivo, and clinical studies and future applications. There are several successful trials and studies that have been done to achieve the desired properties of thermoresponsive polymers, one of the methods is chemically modifying and co-polymerizing two or more polymers to enhance its mechanical and biocompatibility properties. Moreover, engineered thermoresponsive polymers and state-of-the-art process techniques have successfully demonstrated sustained released drug delivery with great biocompatibility thus enable these types of polymers to be utilized in cell sheets and 3D-printing (3DP) for tissue engineering and regenerative medicine purposes. The thermal gelation of materials such as elastin-like peptides (ELPs) and pluronic is great for the exploitation of 3DP as they have a high amount of ECM hence making them a great biomimetic material of the ECM tissues. Other than that, gelatin a biomaterial is also highly utilized for the 3DP method, as it can overcome the extrusion of printing and is highly compatible toward cells with an outcome of nonadhesive soft hydrogels as its end product. At present, polymers that can sustain their gel form at physiological temperatures are in need for bioprinting, to assist encapsulation of cells to accelerate cell viability for the duration throughout the printing process. These 3DP-encapsulated gels have already been applied in various application, for example, APLIGRAF, a hydrogel encapsulated fibroblast to enhance diabetic wound healing, which has been approved by the FDA in the market; however, the main focus remains on optimizing these 3DP gels for the analysis of clinical translation. Other than that, stimuli-responsive polymers, also known as “smart” polymers, are in the favor of many novel studies especially in the in vivo medical applications as they can give better control on drug release for therapeutic processes. These stimuli-responsive gels give further control by combining them with pH-responsive, thermoresponsive, photo responsive, or even magnetic responsive moieties or materials to generate energy, hence creating thermoresponsive polymers. However, these materials come with limitations thus the building blocks of these gels are required to stabilize these complex hybrids right from the construction of the gels fabrications as to biomimic the ECM environment. Thermoresponsive polymers are indeed a great benefit for the tissue engineering study; however, a thorough understanding is needed to fully control the mechanism of the polymers itself and to fully utilize the potential of these materials. Various novel studies have highlighted the advantages of these materials such as ELPs, gelatin, and pluronic, which are highly biocompatible and can mimic the ECM, hence clinical approaches are vital in this process to establish a firm foundation for these polymers to enter the pharmaceutical and biomedical market. With these bioengineered polymers, we will able to overcome the burden of chronic wounds, cancer diseases, and chronic disease with the combination of advanced technologies and great expertise.

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15.6 Conclusion In conclusion, thermoresponsive polymers and its polymeric composites hold great potential in medical applications. Thermoresponsive polymers are reactive toward varying degrees of temperature, altering its physical structures accordingly based on its LCST and UCST. These polymers have biodegradability, biocompatibility, and cellular growthenhancing properties, while also having moldability and is customizable as needed in accordance to the chosen application. The mechanisms of thermoresponsive polymers heavily rely on protein adsorption, cell adhesion and attachment, and its polymeric behavior. These polymers can also be presented in various forms including hydrogels, micelles, films, nanoparticles, interpenetrating networks, and polymersomes. Thermoresponsive polymers in various forms can then be applied into the human body for treatment, such as the vascular system, drug delivery, wound healing, cell carrier material, and gene delivery.

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Further reading

Zhang, Q., Weber, C., Schubert, U. S., & Hoogenboom, R. (2017). Thermoresponsive polymers with lower critical solution temperature: From fundamental aspects and measuring techniques to recommended turbidimetry conditions. Materials Horizons, 4, 109116. Zhang, X., Chen, L., Lim, K. H., Gonuguntla, S., Lim, K. W., Pranantyo, D., et al. (2019). The pathway to intelligence: Using stimuli-responsive materials as building blocks for constructing smart and functional systems. Advanced Materials, 1804540, 148. Zhang, Y., Heher, P., Hilborn, J., Redl, H., & Ossipov, D. A. (2016). Hyaluronic acidfibrin interpenetrating double network hydrogel prepared in situ by orthogonal disulfide cross-linking reaction for biomedical applications. Acta Biomaterialia, 38, 2332. Zhao, C., Ma, Z., & Zhu, X. X. (2019). Rational design of thermoresponsive polymers in aqueous solutions: A thermodynamics map. Progress in Polymer Science [Internet], 90, 269291. Available from: https://doi.org/10.1016/j.progpolymsci.2019.01.001. Zhao, C., Dolmans, L., & Zhu, X. X. (2019). Thermoresponsive behavior of poly(acrylic acid- co-acrylonitrile) with a UCST. Macromolecules, 52, 44414446. Available from https://doi.org/10.1021/acs.macromol.9b00794. Zhou, S., Cun, F., Zhang, Y., Zhang, L., Yan, Q., Sun, Y., & Huang, W. (2019). Thermoresponsive aluminum-based polymer composite films with controllable deformation. Journal of Materials Chemistry C, 7(25), 76097617. Zhu, Y., Hoshi, R., Chen, S., Yi, J., Duan, C., Galiano, R. D., . . . Ameer, G. A. (2016). Sustained release of stromal cell derived factor-1 from an antioxidant thermoresponsive hydrogel enhances dermal wound healing in diabetes. Journal of Controlled Release, 238, 114122. Available from https://doi.org/10.1016/j.jconrel.2016.07.043.

Further reading Chen, S., Wang, K., & Zhang, W. (2017b). A new thermoresponsive polymer of poly (N-acryloylsarcosine methyl ester) with a tunable LCST. Polymer Chemistry, 8(20), 30903101. Available from https://doi.org/10.1039/c7py00274b.

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16

Ceramic particle dispersed polymer composites

Bhabatosh Biswas1, Gurudas Mandal2, Apurba Das3, Abhijit Majumdar1 and Arijit Sinha2 1

Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, Delhi, India 2 Department of Metallurgical Engineering, Kazi Nazrul University, Asansol, West Bengal, India 3 Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India

16.1 Introduction Composites are generally produced by the combination of two separate phases or materials, one of which is called the matrix and the other is dispersed phase. Performances of the composites are governed by their individual phases as well as their relative quantity, shape, size, orientation, and distribution. Polymer composites can now be used in various applications involving challenging environment owing to the advance of new high temperature stable polymers. Component weight, cost, and material wastage during production can be reduced by using polymer composites over traditional metals. The manufacturing of a wide variety of polymeric composites opens the opportunity for utilization of a large range of thermosets, thermoplastics, and biodegradable polymers as matrices with incorporation of various types of fillers. These types of polymer composites are suitable for various packaging and structural applications. Fillers come in a variety of forms, ranging from powder to textiles. Fibers are essential elements for fabrication of a vast array of composite materials in the form of short and discontinuous fiber as well as woven or non-woven textiles. It is also worth noting that with the right mechanical and chemical properties, fibers can be employed as particles or whickers. Chemical treatments for the same are also available. Varieties of mechanical elements, such as brakes, cams, gears, wheels, and seals are manufactured by fiber-reinforced polymer composites (FRPCs) for better performance. FRPCs possess low density and helpful to maintain specified design alteration and the sequences of stacking to impart excellent quality and stiffness (Chang, 1983). The glass fiber based polymeric composites have low wear protection and a high grating owing to their limited scheme of the fiber strengthening. This has prompted many experts to use strands/fillers in their polymer castings. It may be noted that the studies are also being undertaken to Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00020-6 © 2023 Elsevier Inc. All rights reserved.

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expand the range of possible applications to benefits producers and consumers. Tribological characteristics of FRPCs were widely studied where synthesis techniques, various reinforcement shapes, sizes, and patterns are evaluated by different researchers (Bijwe, Tewari, & Vasudevan, 1989; Sommers, 2010; Suresha, Chandramohan, Samapthkumaran, Seetharamu, & Vynatheya, 2006; Tripaty & Furey, 1993; Viswanth, Verma, & Rao, 1991). Particulate fillers are another essential form of filler. The major advantage of particulate filler is that it eases the processing. These particles can range in size from microns to nanometers. Metal, nonmetal, and other types of particle fillers may be used. Ceramic particles are one of the most important nonmetal fillers which could be found in nature or generated from natural resources. ZrO2, Al2O3, SiO2, and TiO2, are very commonly used fillers. Energy, aerospace, and automobile sectors are widely using polymer composites due to their several advantages. Polyether ether ketone (PEEK), polysulfone, polyesters, phenolic, vinyl ester, epoxies, and polyamides can be employed in a variety of blends along with various filaments to provide bearing materials for better wear resistance and a low coefficient of rubbing (Rowntree, 1985). Search for possible applications are ongoing to provide cost-effective application. Polymers, viz., vinyl ester polyester and epoxy have been studied for their contact and wear properties by many experts. High thermal gradient, low wear rate, enhanced fatigue life, and low frictional power have become commonplace with the arrival of the space age (Karthik, Raja, Prabu, & Ganesh, 2015; Senthil Kumar, Karthik, & Raja, 2015). The addition of TiO2 and Al2O3 to polymers increases their dynamic mechanical characteristics (Alam & Chowdhury, 2020; Rostam, Mustafa, & Aziz, 2021; Yunus & Alsoufi, 2018). In the context of both stiffness and maximal effort, it was also found that the presence of SiO2, TiO2, and Al2O3 can improve the same (Lascoup, Aboura, Khellil, & Benzeggagh, 2010; Nayak, Dash, & Ray, 2014; Yadav, Purohit, & Kothari, 2019). The usage of thermoplastic materials in the aviation industry has become increasingly important in recent years, as they offer high stiffness. This material has a high tolerance for damage management and a long shelf-life, making it one of the most appealing and practical materials in the context of structural integrity (Krawczak & Maffezzoli, 2020; Weiss, 1991). Green composite materials now exhibit intriguing properties that entice researchers. Fatigue is a key component in this process, as it aids in the discovery of a link between biological usefulness and its application in the biomedical business (Askadsky, Ushkov, Smirnov, & Voronin, 2016; Bendigeri & Jwalesh, 2016; Kumar, Singh, & Hashmi, 2020; Laachachi et al., 2006; Rajesh et al., 2014; Sims & Broughton, 2000). Mechanical properties such as hardness, strength, stiffness, and impact strength are superior for the composite material. Influences of metal oxides on heat-cured polymethyl methacrylate (PMMA) were evaluated by various researchers (Asar, Albayrak, Korkmaz, & Turkyilmaz, 2013; Jeyapragash, Srinivasan, & Sathiyamurthy, 2020; Madhusudhan & Kumar, 2017). Furthermore, the action of particles such as SiO2, TiO2, and Al2O3 has significantly improved mechanical properties (Aljafery, 2018; Farhan & Hussein, 2020). The impact of

16.2 Matrices used in ceramic particle dispersed polymer composites

reinforcements on bio-composite polymer matrix has been considerable in terms of biodegradability, low-cost manufacture, environmentally friendly durability, and high strength. In this technique, polylactic acid (PLA) and high-density polyethylene (HDPE) efficiently meet modern-day structural industrial demands, and the growth of the materials were observed in the atomic force microscopy (AFM) procedure (Deshmukh et al., 2017; Quitadamo, Massardier, & Valente, 2019). Composite can be used as replacement of light materials like aluminum alloys for the aviation domain. Rest of the article is divided into several sections where Section 16.2 describes the matrices used in ceramic particle dispersed polymer composites. Section 16.3 presents the reinforcements used in ceramic particle reinforced composites followed by fabrication of ceramic particulate dispersed composites in Section 16.4. Room-temperature and elevated temperature curing of composite material is described in Section 16.5. Different types of ceramic particle dispersed composites are described in the Section 16.6. Different mechanical characterizations are illustrated in Section 16.7 and finally the concluding remarks are outlined.

16.2 Matrices used in ceramic particle dispersed polymer composites 16.2.1 Biodegradable matrices Recently polymers were widely considered for biomedical field especially in the bone tissue engineering (Athanasiou, Niederauer, & Agrawal, 1996; Prestwich & Matthew, 2002). Two types of biodegradable polymeric materials are mainly natural and synthetic polymers. A biological protein (Lee, Singla, & Lee, 2001) termed as collagen and gelatin (Young, Wong, Tabata, & Mikos, 2005) (made from collagen) are naturally derived polymers with issues such as volatility, incompatibility, immunogenicity, and poor biodegradability. However, because most synthetic polymers like poly lactic-co-glycolic acid (PLGA) and polyurethanes (PURs) have changeable characteristics (Nair & Laurencin, 2007; Puppi, Chiellini, Piras, & Chiellini, 2010; Rezwan, Chen, Blaker, & Boccaccini, 2006). Injured bone healing, orthopedic and dental implant, and tissue engineering are all possible application areas for these biodegradable polymers. In modern medicine, both synthetic and natural polymers serve critical roles (Lo´pez-Noriega, Quinlan, Celikkin, & O’brien, 2015; Przekora, Palka, & Ginalska, 2016; Qin, Zhong, & Ma, 2016; Sultana et al., 2014). Polyesters have received more attention among synthetic polymers than other forms of polymers. PLA, PLGA, PEEK, poly-L-lactic acid (PLLA), and PMMA are widely used polymers for medical applications (Eppley, 2005; Niemela, 2005). PLA can be treated as best suitable materials for several biomedical applications because of its biocompatible nature, easy to manufacture, hydrophobic quality,

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and biodegradable properties (Ambrose & Clanton, 2004; Baro, Sa´nchez, Delgado, Perera, & E´vora, 2002; Lampin, Warocquier-Cle´rout, Legris, Degrange, & Sigot-Luizard, 1997; Li & Yao, 2008; Narayanan, Vernekar, Kuyinu, & Laurencin, 2016). Renewable resources (corn starch and sugar cane) are mainly used to manufacture a biodegradable thermoplastic polymer PLA. Screws, rods, pins, and plates are made from various forms of poly-D-lactic acid, PLLA, and poly-DL-lactic acid. PLA is a semicrystalline polymer where slow crystallization rate is observed (Lasprilla, Martinez, Lunelli, Jardini, & Filho, 2012; Shah, Tatara, Souza, Mikos, & Kasper, 2013). The functions of injured portions in the human body are dependent on soft as well as hard tissues (Crecelius, 2013; Currey, 1999; Migliaresi, De Lollis, Fambri, & Cohn, 1991). PLGA (synthetic biopolymer) has been catching interests of the researchers in a significant manner. The arbitrary ring-opening copolymerization of PLA poly (glycolic acid) (PGA) is known as PLGA. PLGA is a biodegradable polymer and can be used for potential applications due to good mechanical properties, high cell adherence, and regulated degradation rate (Holzapfel, 2001). Herein way, the rate of degradation of PLGA products may be controlled by fluctuation of its constituent’s ratio. As a result, PLGA is favored over PGA due to advantages of controlled rate of degradation. Polymers can be employed in skin, muscle, and other soft tissues and in medicinal applications like as joints and cancer treatment distribution systems (Ulery, Nair, & Laurencin, 2011; Van de Velde & Kiekens, 2002). The rate of bone repair and growth has been shown to be much accelerated when implants are constructed of PLGA (Haghighat & Ravandi, 2014; Kleinschmidt, Marden, Kent, Quigley, & Hollinger, 1993; Sadat Tabatabaei Mirakabad et al., 2014). Collagen and gelatin come from animal sources, while chitin from marine sources is processed into chitosan. However, scientists are focusing their attention on the other two feedstock regions, which are regarded to be the best favorable for coming improvement and expansion. PLA and polyhydroxy alkanoates (PHA) can be produced via microbial biopolymer feedstocks. The biopolymer group of polymers is divided into two groups: hydrocolloids and lipids and fats. Starch is a hydrocolloid biopolymer and presents in agricultural feedstock (like corn, wheat, beans, rice, and potatoes) (Lucena-Martı´n, Gonza´lez-Lo´pez, & de Mondelo, 2001; Salmoral, Gonzalez, & Mariscal, 2000). The most common type of starch is granules, which are made up of one linear and one branched polymer (Chandra & Rustgi, 1998). Amylopectin, a branched polymer, makes up around 80% of total starch weight, while Amylose, a linear polymer, makes up the rest. Natural filler materials are a faster biodegradable component and can be used in synthetic plastic. To speed up the breakdown of polyethylenes, granular starch is typically used. The gelatinized form of starch can also be employed (Verhoogt et al., 1995). As starch is heated with water during inoculation or extrusion molding, a thermoplastic material is created, which can be distorted during mixing. After that, the starchbased product is mixed with synthetic or natural components. Starch’s molecular structure is broken when heated above the glass transition temperature, allowing

16.2 Matrices used in ceramic particle dispersed polymer composites

for more bonding (Kolybaba et al., 2006). Glycerol is commonly used in starch blends as a plasticizer to promote smoothness and malleability. Plasticized starches are made from starch granules by plasticizing with water and glycerol (Ernst, Martin, Stuff, & Willershausen, 2001). Injection molded, extruded, blown, and compression molded plastic materials made from starch-based mixes are available. Fibers (flax and hemp fibers) used as reinforcing fillers are also found in agricultural feedstocks for the biopolymer sector (Bismarck et al., 2002). Low-cost and biodegradable natural cellulose fibers are mechanically robust material. Cellulose fibers are the most often utilized natural filler in plastic goods because of their qualities (Hornsby, Hinrichsen, & Tarverdi, 1997). Results shows that tensile modulus increases considerably by introduction of 25% wt.% cellulose fibers in a polypropylene (PP) matrix. The molecular chain of cellulose is extremely lengthy, making it insoluble and infusible in most vigorous solvent (Chandra & Rustgi, 1998). Hence, it is frequently transformed into derivatives increasing the solubility; as a result matrix adherence also increases. Flax fibers are still the most popular choice since they are mechanically robust and widely available. The surface properties of the fibers are modified by chemical treatment (acetylation) without affecting the fiber structure or shape (Frisoni et al., 2001). These changes slow down the onset of fiber breakdown and improve adhesion at the fiber matrix interface. Fibers with higher moisture content are adequately dried prior to mix with the matrix for better adherence (Bledzki & Gassan, 1999). Because it is extremely polar and biodegradable, polyvinyl alcohol has been found to be an excellent polymer for use as a matrix in natural fiber reinforced composites (Chiellini, Cinelli, Imam, & Mao, 2001). Microbial biopolymer feedstocks use microbial fermentation to create biological polymers (Zhou, Li, & Chau, 2010). The materials are biodegradable and environmentally beneficial alternatives to synthetic plastics. When nutrient deficits arise, a few bacteria store PHAs as internal carbon reserves. Carbon-to-nitrogen (C:N) ratio enhancement improves the specific polymer yield (i.e., increased PHA synthesis) and are mainly for wastewater treatment in chemical plant (Biddiss & Chau, 2006). Researchers have known for a long time that chemical or biological treatments may convert almost any sort of biomass into sugars. The sugars can then be converted into PHAs by certain microbes. Researchers from University of Hawaii have transformed food waste into PHAs (Petkewich, 2003). The expensive cost of creating new material is increasing due to widespread usage in industries and modern civilization need. When used alone, PHAs are brittle and expensive; therefore researchers combine them with less expensive polymers that offer complementing properties. Microbial fermentation produces PLA, the second most prevalent biopolymer. Lactic acid obtained from fermentation processes is condensed to make those (Kolybaba et al., 2006). A thermoformed natural plastic container made from corn was prepared by Wilkinson Manufacturing Co, United States. Plant starches store carbon which is primarily transformed into natural plant sugars. Both biodegradable PHA and PLA (Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003) do not exist

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in nature, and hence, are regarded as synthetic polymers. Biopolymer researchers have looked at and altered a variety of other biological materials. Wheat includes starch and gluten, which are both used in biopolymer production. Canola compounds have polymer and plasticizer potential (Crawford, Couper, & Lamias, 2001). Chitosan (produced from deacetylation) is insoluble in water and needs to dissolve in acidic solutions prior to use as biodegradable polymer films (Park, Gorte, & Vohs, 2001). Soy proteins have good structural characteristics to use as reinforced materials in industrial sector (Park, Kim, Shin, & Jeong, 2000). Therefore it can be concluded that lot of natural entities (both plant and animal) are available which can be used as biopolymers.

16.2.1.1 Modification or recycling polymer matrices This type of thermosetting unsaturated polyester resin (UPR) can be degraded by lichen. If lichens are introduced to the UPR manufacturing process, it becomes a biodegradable UPR (Gowsika & Nanthini, 2014; Okkerse & Bekkum, 1999; Tang, Moyori, & Takasu, 2013). Epoxidation of waste vegetable oil with hydrogen peroxide in acetic acid was utilized to make novel biobased epoxy resins. Deoxidized waste vegetable oil was mixed with acid hardeners (like citric acid, tartaric acid, succinic anhydride, and sebacic acid) to make biobased epoxy resins. The inherent viscosities ranged from 0.06 to 0.70 (dL/g) and were thermally stable up to 350 C. Except for B4, solution cast thin films are translucent. Using bacterial granules, the resin films were completely biodegraded in 14 135 days (made up of bacterial consortia). This study focused on a cottonseed oil based thermosetting polymer. Under regulated reaction conditions, the eco-friendly oligomeric fumarate resin was generated by in situ hydroxylation followed by fumaration of cottonseed oil. Changing the proportions of the comonomers (methyl methacrylate and butyl methacrylate) three new polymeric materials were developed. The new polymeric materials, which spanned from soft rubbers to ductile plastics to highly brittle plastics, showed tensile stress strain behavior. Differential thermal analysis and thermogravimetric analysis (TGA) were used to characterize the synthesized polymers, as well as mechanical characteristics including tensile strength, % elongation, and hardness. The biodegradability of the produced polymer was evaluated using hydrolytic testing, chemical resistance tests, and soil mechanics studies.

16.2.2 Nonbiodegradable matrices 16.2.2.1 Thermoplastics HDPE, Low-density polyethylene, Polystyrene, PP, polyvinyl chloride (PVC), Nylon (different variant), thermoplastic polyurethanes, and Teflon are just some of the thermoplastic synthetic polymers that can be used as polychlorotrifluoroethylene (PCTFE or PTFCE).

16.3 Reinforcements used in ceramic particle reinforced composites

16.2.2.2 Thermosetting Unsaturated polyester (UP) resins, epoxy (epoxide) resins, phenolic (PF) resins, bis-maleimide resins, benzoxazine resins, polyimides, PUR resins, cyanate ester resins, silicones, and vinyl esters are the most common thermosetting polymers used in ceramic particle reinforced composites.

16.3 Reinforcements used in ceramic particle reinforced composites 16.3.1 Reinforcement from natural resources Clay and silica, which are abundant in nature, were the first ceramics. Blast furnace waste slag, Fly ash, sludge, glass waste, polished tile waste, rice husk ash, eggshell, and other natural resources can be employed as filler particles in polymer matrix composites.

16.3.2 Reinforcements from synthetic resources There are a variety of ceramic particles that may be employed in polymer matrices to improve certain properties while also introducing some new properties in the final composites. Following is an overview of several notable ceramic materials. Ferroelectricity is demonstrated by barium titanate (frequently combined with strontium titanate). Body armor is made of boron oxide. Carbon, which is employed as an abrasive, is structurally isoelectronic to boron nitride. Domestic pottery such as plates and mugs are made of earthenware. Ferrite is utilized in electrical transformer magnetic cores and magnetic core memory. Porcelain is utilized in an extensive variety of products in both the home and the workplace. Sialon is a high-strength, thermally shock-resistant, chemically, and wear-resistant material with a low density. Silicon carbide (SiC) is a refractory substance and abrasive in nature. It is used as a susceptor in microwave furnace. As an abrasive powder, silicon nitride (Si3N4) is utilized. Electrical insulators are made of steatite (magnesium silicates). Titanium carbide is a kind of titanium. Re-entry shields for space shuttles and scratchproof watches are made of this material. Varistors are made of zinc oxide (ZnO), a semiconductor that is employed in their manufacturing. Zirconium dioxide is a kind of zirconium (zirconia), which undergoes multiple phase changes between ambient temperature and realistic sintering temperatures in its pure form, and can be chemically “stabilised” in several ways. It has high oxygen ion conductivity, making it perfect for use in vehicle fuel cells and oxygen sensors. On the other hand, metastable structures can provide transformation toughening for mechanical purposes and used in most ceramic knife blades. Like ceramic polysilazane is also used for metal forming tools, valves and

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liners, abrasive slurries, culinary knives, and bearings sensitive to high abrasion (Garvie, Hannink, & Pascoe, 1975; Wachtman, 1999).

16.4 Fabrication of ceramic particulate dispersed composites 16.4.1 Methods of composite fabrication 16.4.1.1 Methods for thermoplastics 16.4.1.1.1 Low-pressure processing techniques In a preconsolidated laminate a variety of processing techniques under lowpressure condition are effectively developed based on matrix distribution and intimate fiber. Lower pressures mean less money spent on forming equipment and tooling, which is a key consideration for smaller production runs.

16.4.1.1.2 Thermoplastic composites considering vacuum forming Vacuum forming technique can be used for thermoplastic and thermoset composites. Pressure constraints, degradation due to heat skin core morphological effects, and stresses between the epidermis and the center of the body are mainly caused by heat. Flow restriction also causes certain practical constraints. Cooling rate is considered as log(dT/dt) 5 6.6, where T is temperature and t represents time. Hand lay-up type procedure is considered for manufacturing thermoplastic 219 composites. Variety of materials can be made using the vacuum bag method but minimum temperature of 200 C should be maintained for PP-based composites. A preshaped environment friendly silicone rubber bag or a flexible nylon vacuum bag (50 75 m) is fitted on the surface of the tool, depending on manufacturing volumes. Consumable costs and processing time can be reduced by using environment friendly silicon membranes. A male tool can be used with preformed silicon rubber forms. For full impregnation, the material and tool are heated under vacuum for 10 20 min (depending on the fabric fiber and matrix scattering) to temperature range of 185 C 190 C, with a vacuum of up to 1 bar applied during the dispensing cycle. The tool is effectively cooled under 70 C (to prevent alteration and simplify handling) in vacuum chamber; after that vacuum bag is withdrawn and the part is freed.

16.4.1.1.3 Autoclave forming of thermoplastic composites Autoclaves is a type of vacuum molding process that can be used for heating the mold, vacuum bag, and textile composite, and it has capacity to add external pressure (3.5 7 bar). In this process tighter thickness is achieved by reducing void content but production rate is lower. The autoclave high temperature is raised to directly above the matrix melting temperture (Tm), the autoclave pressure is increased to induce forming, and the autoclave is subsequently cooled at merging

16.4 Fabrication of ceramic particulate dispersed composites

pressure. Hydrostatic pressure is applied during forming to distort the diaphragm stretching reaction. It is possible to use molds with thin sections and low thermal mass. Autoclave processing of thermoplastic composites can be achieved by double diaphragm shaping (Sadighi, Rabizadeh, & Kermansaravi, 2008).

16.4.1.1.4 Diaphragm forming Laminates production and sandwiching laminates in thin plastically flexible sheets can be performed by diaphragm forming. Here, biaxial strain on the laminate during clamped around the mold edge is maintained. Design and manufacturing of wrinkle-free and split-free textile composites are tricky things where the whole mold, laminate, and diaphragms are heated to a temperatures above the laminate polymer Tm over a period of 30 min or longer. Air pressure above the diaphragm is removed to reduce the diaphragm force for the complicated laminate locations for creation of minute feature. Compressed air or an autoclave can be used to make the diaphragm. A single diaphragm applied over the mold can produce a forming which is equivalent to those produced with two diaphragms. The top section of the lungs’ diaphragm applying pressure to the clamp pressure tool that is both cool and hot vacuum part that has been molded composite material that has been heated.

16.4.1.1.5 Bladder inflation molding The bladder inflation molding (BIM) process allows for the manufacturing of hollow composite components with great geometrical complexity and intrinsic stiffness. To specify the shape of the part, BIM employs an outside mold and an interior bladder. Low process pressures (less than 10 bar) allow for easy tooling, resulting in a method for creating thin hollow composite objects. BIM has been proven in the past for tiny generic components, automotive parts, sports equipment, pressure vessels, and suspension arms. The BIM technique has two process variants: (1) a composite mold temperature which is above the composite matrix Tm, and (2) a nonisothermal process where preheating is done above the matrix Tm outside of the mold. The item was made using a hydraulic press to seal the tool and a rapid pneumatic connecting mechanism to apply core pressure to the bladder. Mold cycle durations were also reduced to 140 s for a typical pressure vessel component with an 85 mm diameter and 2 mm thickness.

16.4.1.1.6 Resin Transfer Moulding (RTM) Resin Transfer Moulding (RTM) procedures employed for thermosetting resins can also be used for reactive thermoplastic resins. Generic plates and portions of a car floor pan can be made using thermoplastic resin transfer moulding (TPRTM). Tanks, gear pumps, and pipes (two numbers each) make up the injection unit, which transports the activator and monomer from the storage tank to the mixer unit. Temperature of the system is required to maintain at 180 C throughout the process to keep the monomer in molted condition.

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16.4.1.1.7 Injection-compression technique By managing crystallization-based shrinkage and concurrent void formation, injection-compression methods have also been used to reduce porosity levels (10% to 1%). Shear edge tool is used for processing of injection-compression where 55 bar average pressure is applied on the surface during the cooling phase. Cyclics 56 polymerizes cyclic butylene terephthalate oligomers to generate a polybutylene terephthalate (PBT) material system, which is also suitable for TP-RTM. When handled between 180 C and 200 C, the prepolymers melt, polymerize, and solidify without the need to cycle the mold temperature. With a prepolymer melting point of 150 C, a mold temperature of 180 C 190 C is stated to provide a fair compromise between improved polymerization and crystallization speed. Cooling is necessary for mold before component release as melting temperature of polymerized PBT has is 220 C. CBTTM resins have been proven in compression molding, vacuum bagging, reaction injection molding (RIM), structural RIM, resin film infusion, RTM, and pultrusion processes for textile composites, according to Cyclics Corporation 56.

16.4.1.1.8 High-pressure processing High-pressure processing needs high forming pressures due to the restricted duration in the tool where separate preheating of the composite is required. This process significantly reduces costs and cycle times in mass production. Alternative (isothermal) tool temperature regime is less appropriate for mass production as time and operating cost are higher due to significant energy transfers. Fully and partly soaked, and poorly consolidated material shapes are selected depending on a mix of economics (avoiding preconsolidation costs vs possibly quicker stamping times), mechanical characteristics (setup times vs void ratio), and type of automation needed. Preconsolidation can help to mix layers of material properly. Preheating-induced deconsolidation can be prevented for fully preconsolidated materials during final stamping stage for accurate mold shape. Increasing preconsolidation levels can improve laminate qualities and lower porosities for poorly consolidated or partially preconsolidated case where the effect being particularly strong at lowers forming temperatures. Following a brief review of common preheating and blank holding methods, the processing techniques are needed for almost all stamp-forming variations.

16.4.1.1.9 Preheating technology for stamp-forming processes Infrared, hot air, and air impingement are commonly used preheating techniques. Apart from that microwave, radio frequency, contact heating, and resistance heating are also currently being researched (Beera, 1996). The heating using infrared has various advantages due to several oven design (Paul, 2018) that possesess better stability and better energy efficiency. Circulated hot air furnaces have shorter reaction times than infrared, but they prevent excessive flux levels to get uniform temperature over the surface. Uses of inert gases can prevent deterioration of the

16.4 Fabrication of ceramic particulate dispersed composites

surface properties. Hot air at high velocity is delivered in air impingement ovens where peak temperature is controlled for the composite blank. In the infrared region electromagnetic radiation is emitted by infrared oven emitters, which is reflected, absorbed, or transmitted by the laminate. The random molecular energy increases as radiation is absorbed, due to heat transfer. Carbon black pigmented PP has limited transmittance across the range of wavelength of 0.8 3 m73, with 3% reflection and 97& absorption. Fiber architecture dependent material is deconsolidated during heating due to the release of strain energy in the reinforcing fibers. The composite block is required shield during heating due to its laminate structure to avoid debonding and delamination. Higher heating temperature is needed to achieve higher matrix viscosities. However, matrix breakdown occurs with excessive and prolonged heating.

16.4.1.1.10 Blank-holders and membrane forces Blank-holders are a major component of the stamp-forming techniques. Matrix phase of thermoplastic composite shrinks during preheating, and a blank-holder is required retain the material in place. In order to maintain accurate fiber alignments in the finished item, an automated system is also necessary to move the heated and hence easily adjustable material. Transfer of material from oven to the tool in manual mode is not recommended due to very high temperature. Blank holds put the material in place during preheating and enable for rapid heating.

16.4.1.1.11 Continuous compression molding In continuous compression molding process (Isayev, 2000), materials are heated in a preheat oven before keeping in an automated intermittent press. During compression of the tool the material is fed and the material is drawn when the press is open. This process can help to join flat sections, “U” sections, “I” sections with closed hollow sections. It is indeed feasible to make forms that have a fixed circumference. The composite sheet that feeds the procedure can contain a variety of lay-ups, such as cross feed and angle feed. Twenty-five bar pressure is usually maintained, while 30 80 m/h feed rates of are considered. Sandwich structures with a 30 mm thickness may be made with pieces as thin as 5 mm. Modern thermoplastic composite manufacturing pathways are schematically presented. Cocompression is used to compress textile molds with a foamed core. It is seen that incorporating composite layers to form a structure is challenging task and good coordination is needed in terms of volume fraction of ceramic and fibers with operating temperature. Flow-molding core can maintain overall volume fraction of matrix and fiber; otherwise flexural stiffness will be slightly affected.

16.4.1.2 Methods for thermosetting The most prevalent methods for producing thermosetting composite matrices are manual lay-up and compression molding. Some of the other techniques are sprayup, resin transfer molding, vacuum bag molding, and pultrusion. There are two types of molding processes: open mold and closed mold.

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16.4.1.2.1 Open molding Hand lay-up. Simplest and widely used composites construction technique is hand lay-up (Kozlowski, Wladyka-przybylak, Helwig, & Kurzydloski, 2004). When compared to other molding or fabrication procedures, it requires a low-cost setup. The ingredients are fed into an open mold and left there until they reach an appropriate, stable shape. The amount of time required is determined by the polymer’s properties. Fabrication of polymer composites at room temperature for 24 h is required (Yahaya, Sapuan, Jawaid, Leman, & Zainudin, 2015). This is a common fabrication method for thermosetting matrices. It is utilized to make a variety of automotive components (Kim, Shim, Sturtevant, Kim, & Song, 2014). Spray-up method. The method of fabrication is the source of the process’ name. A spray cannon is utilized to place both the fiber and the resin into the mold in this production procedure. Rolling the prelayed resin and fiber preform is performed to remove the air bubble entrapment inside the resin. The curing procedure, which is normally done at room temperature, is determined by the type of resin employed. For composites, this technique of production is currently limited.

16.4.1.2.2 Closed molding Pultrusion. Uniform cross-sectional composite parts can be manufactured using a composite synthesis technique, called pultrusion (Yuhazri, Phongsakorn, & Sihombing, 2010). In this technique, the fibers are manufactured from a heated die. As the resin travels through the die, a constant pressure is applied, forcing it to melt and saturate the fiber reinforcement. The quality of the finished items is affected by the manner of preheating, the temperature of the die, and the speed at which the fiber travels through. Resin transfer molding. In resin transfer molding process a closed die is used where resin is forced under pressure through the die. This approach may be used to create complicated things with a smooth finish all the way around (Hollaway, 2010). Compression molding. Compression molding is a well-known process for UPRbased composites. Initially the reinforcing and thermosetting resins are leveraged inside flat die/molds (male and female), then up to 2000 psi pressure and up to 200 C temperature are applied to fix the material for a predefined time. This procedure also includes room-temperature curing. The composition, form, size, and thickness of the resin dictate the kind of curing needed. Once the item has hardened, it is taken out from the mold. For easy removal of the composite from the die a releasing agent is added prior to molding (Asaithambi, Ganesan, & Kumar, 2014; Vijayakumar, Nilavarasan, Usharani, & Karunamoorthy, 2014). Vacuum bag molding. Vacuum bag molding, a version of hand lay-up, is a technique for compressing a composite during the curing process. This process is important for air removal (Deo, 2010; Khalil et al., 2016; Kikuchi, Tani, Takai, Goto, & Hamada, 2014; Pearce, Summerscales, & Guild, 2000; Quan et al., 2016; Sathishkumar, Naveen, & Satheeshkumar, 2014; Thori, Sharma, & Bhargava, 2013; Wiedmer, 2006).

16.5 Curing of the composites

16.5 Curing of the composites 16.5.1 Room-temperature curing Due to their increased chemical and mechanical characteristics, epoxy resins are widely used as adhesives, varnishes, civil engineering applications, and cultural heritage conservation. Practical and financial concerns aspect is major thrust for curing of those structures in room temperature condition. The method of producing composites durable is referred to as “curing.” Curing takes a considerable amount at room temperature. This technique normally includes catalyst, crosslinking reagent, and addition to the resin can accelerate the curing process. Methyl ethyl ketone peroxide is commonly used a catalyst while cobalt naphthenate or cobalt octoate considered as accelerator. On the other hand, styrene is used as the most common cross-linking agent. When concentrations excee 30% 40% cross-linking agent can be used, while the catalyst and accelerator can be used at low concentration levels as 2% 3%. Ambient temperature curing usually needs a 24-h cure period. After that, the composite can be taken from the mold and shipped to be processed further, such as postcuring at higher temperatures (de la Caba, Guerrero, Eceiza, & Mondragon, 1996).

16.5.2 High-temperature curing High-temperature curing is generally performed between 100 C and 150 C. Several heat-cure epoxy adhesives have been developed for plastic bonding or bonding delicate electronics or sensors where greater temperatures could distort or harm parts. Imidazoles are an anionic polymerizing curing agent for epoxy resin, similar to tertiary amines as 2,4,6-tris(dimethylaminomethyl)phenol (DMP30) and benzyl dimethylamine (BDMA). Due to higher lifespan imidazoles have the capacity to cure resin by short duration thermal heating (80 C 120 C) improving the flowability. Imidazole carboxylate, metal salt imidazole complex compounds, epoxy imidazole adducts, and imidazole are the commonly used curing chemicals. Imidazole is the better choice than other secondary amines due to prolonged pot lifespan, higher curing speed, and greater heat resistance of the cured material. The BF3 amine complex is utilized in tiny concentrations in resins as it is a catalyzed curing agent (1% 5%). Cured substances with heat distortion temperature (HDT) 230 C and provides an excellent electrical, chemical property. Carbon hydroxide has completely replaced the active hydrogen in tertiary and secondary amines, resulting in a polymerization catalyst rather than an additional reaction with epoxy resin. As a result, depending on the type of curing agent utilized, the loading changes. Quality of the cured resin largely depends on curing temperature, speed, and heat generation. As a result, the amine is rarely used alone because the heat created causes the properties of the center and exterior areas of large castings to diverge. It is used in a variety of products, including paints and adhesives. Although not as successful as a curing agent, tertiary amine

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is an important accelerator for acid anhydrides and can be employed as a cocuring intermediary for polyamine and polyamide curing reagents. Curing agents are used to enhance flowability for rapid curing at a temperature range from 100 C to 180 C. Imidazoles outperform other tertiary amines as it offers longer pot life, a quicker curing rate, and a higher heat resistance. Polymercaptan is a lowtemperature curing agent that cures between 0 C and 220 C. However, polymercaptan has pot life of 2 10 min at normal temperature, but cures fast and practical strength achieved within 10 30 min. Although polysulfide resin possesses mercaptan groups at its terminals, it lacks the low-temperature and fastdrying properties of liquid polymercaptan. This curing agent also serves as a flexibility enhancer. When combined with a tertiary amine or polyamine curing agent, the resin serves as a room-temperature curing agent. The polysulfide loading ranges from 50 to 100 wt.%. With increase in the polysulfide loading, the cured resin gains flexibility, shock resistance, and permittivity, while shrinkage reduces. As cationic polymerization catalysts, Lewis acids including ZnCl2, BF3, FeCl3, SnCl4, and AlCl3 have been utilized (Three Bond 2285B, 2287B). At normal temperature, the Lewis acids react quickly with resins. These Lewis acids are the complexes of boron trifluoride-amine. The characteristics of the complexes vary depending on the kind of amine (melting point, reactivity, etc.). The BF3 amine complex is utilized in tiny concentrations in resins because it is a catalyzed curing agent (1% 5%). The high HDT ranges of 150 C 170 C give great electrical capabilities, but their chemical stability is limited. Multiplexes are using electrical isolating laminates and carbon fiber reinforced plastics (Crawford, 1987; Huang & Leu, 1993) as inactive nature of cured resins.

16.6 Different types of ceramic particle dispersed composites 16.6.1 Particulate-reinforced composites Micron and nanometer-sized aluminum particles were used by Singh, Zhang, and Chan (2002) as a dispersion phase on theoretically brittle thermosetting UP resin to enhance the fracture toughness. The fracture behavior composites were studied by systematically changing particle size and volume percentage. The fracture surface of polyester resin reinforced particle reinforced composites is shown in Fig. 16.1. In general fracture toughness rises continuously with volume percent of aluminum particles for a given particle size. Smaller particles improved fracture toughness more than larger particles for a given volume percentage of the particle. According to the study (Kim et al., 2012), fracture front trapping is the primary extrinsic toughening process. They investigated the effects of surface modification on the tensile, fracture, and tribological characteristics of Al/epoxy composites. As per the observations, strength of silane-treated Al/epoxy composites was higher than untreated Al/epoxy composites. The mechanical

16.6 Different types of ceramic particle dispersed composites

FIGURE 16.1 Fractographic image of a polyester resin reinforced Al particles composite (Singh et al., 2002).

properties of commercial epoxy resin were investigated using varying weight percentages of copper particles (Mohammed Altaweel, Ranganathaiah, Kothandaraman, Raj, & Chandrashekara, 2011). At room temperature, the effect of the weight fraction of copper powder within the epoxy resin on mechanical properties was evaluated, and it was observed that enhancing the relative density of copper powder leads to an increase in modulus of elasticity, modulus of rigidity, tensile yield stress, impact strength, and fracture energy. Conradi looked into the impacts of adding SiO2 fillers to a polymer matrix (PVC) and observed that the matrix’s modulus and elasticity were greatly increased, improving the overall quality of the composite. Because SiO2 particles tend to clump together, a perfect homogeneous dispersion of the fillers was predicted to be necessary. This might have a big influence on the traits you get. As a result, SiO2 fillers must have adequately modified surfaces in order to maintain a strong interface contact between the inorganic materials as well as the polymer matrices (Conradi, Zorko, Jerman, et al., 2013). Chakraborty, Sinha, Mukherjee, Ray, and Chattopadhyay (2013) examined the scratch and indentation hardness of ZnO nanoparticle reinforced ZnO/ PMMA composites. At low indentation depths, a satisfactory correlation between scratch hardness and indentation hardness was established, with a linear relationship

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between hardness and ZnO nanoparticle reinforcing content. The impact of graphite integration (2, 4, 6, and 8wt.%) inside an epoxy matrix produced by compression molding was investigated. The level of filler particle dispersion, particle size, and aggregate structure all play a role in the mechanical characteristics of composites, according to the study. Elastic modulus, flexural modulus, and impact strength are increased after addition of filler materials. It was also shown that as the filler content in composites increases, the functional group decreases due to fast gelation. The filler percentage in the composite sheet was kept to an absolute minimum of 8% since any more than that might cause gelation, which raises the viscosity of the mixture (Bhagat & Verma, 2013). The tensile, fracture, and tribological characteristics of Al/epoxy following surface modification with 3-aminopropyltriethoxysilane were examined by Kim et al. (2012). Al/epoxy composites with 10% untreated and 10% silane-treated aluminum particles were made by cast molding. All of the matrix’s mechanical properties increase considerably when it is reinforced with silane-treated alumina. In the low particle weight range of 0.3 0.9 wt.%, Marhoon and Hanna (2016) discovered that the K2CO3 weight fraction increased tensile strength and impact energy for made UP/K2CO3 composites, which was greater than the cast neat resin. Higher tensile strength and impact energy were recorded at 0.3 and 0.9 wt.% K2CO3, respectively. Higher weight fraction enhanced the tensile strength but decreased impact energy. Hardness and water absorption percentages rose with filler percentage, peaking at 1.5 and 1.8 wt.%, respectively. For various filler kinds, Marhoon and Hanna (2016) found that the tensile modulus, bending modulus, hardness, and water absorption % increase with filler percentage. The ultimate tensile strength of the UP resin filled with CaCO3 and MgO particles improves as the weight fraction of the filler particles increases, peaking at 3 and 9 wt.% for CaCO3 and MgO, respectively. Both types of fillers have a maximum flexural strength of 9 wt.%. Baskaran et al. used the sol gel process to make polymer composites containing nano alumina. It was found that up to 5% filler content, a fairly uniform dispersion was maintained. The impact strength decreases when filler is added, while the storage modulus, glass transition temperature, and thermal stability enhance (Baskaran, Vijayakumar, & Mohan, 2011). On the basis of UP, Adhikari et al. (2017) created jute-reinforced Al2O3 and ZrO2-filled composites. The microhardness and thermal stability of the composites rose as the filler content inside the matrix increased, with filler amounts of 20 wt.% giving the best results. The UP matrix increases the mechanical and physical bonding between particles and fibers, boosting the composite’s properties, according to Fourier transform infrared (FTIR) analysis. According to DSC tests, adding hard metal oxides has no influence on the glass transition temperature (Tg) of the composites formed. UP/jute/ZrO2 composites exhibit the highest hydrophobicity compared to the other composites for all the testing conditions (sea water conditions, boiling, acidic, alkali). The enlargement of the composite samples was also confirmed by a little decrease in microhardness values. Evora and Shukla (2003) study a production method that employs direct ultrasonification to produce polyester/TiO2 nanocomposites with good particle dispersion throughout the matrix, as measured by transmission electron microscopes. With the inclusion of the nanoparticle, the fracture toughness rises considerably due to crack pinning and crack

16.6 Different types of ceramic particle dispersed composites

trapping processes. Both compression strength and dynamic fracture toughness skyrocket. According to Ray, Easteal, Cooney, and Edmonds (2009), clay-based nanocomposites have better properties than other mineral-based filler reinforced composites. These composites also have better thermal stability and a good barrier performance, making them suitable for a wide range of applications. The purpose of this work is to present an up-to-date overview of a novel type of polymer composite. Pappu et al. (2015) investigated whether coal combustion residue could be used as a recyclable waste in a polymer matrix and discovered that it considerably improved mechanical properties including tensile and flexural strength. In addition to this, using sisal fiber into composites increases tensile strength while decreasing density. Amorphous materials are studied by Karthik and Meenakshi (2015) where the performance of epoxy polyamide (EP) and amorphous Al2O3-ZrO2 nanoparticles in epoxy polyamide resin (NEP)-coated samples was carefully studied. Thermodynamic conductivity, hardness, and elastic modulus have been significantly improved. As demonstrated in SPM and AFM microstructure images, the NEP-coated sample retains its original surface feature with current passive layer growth, but the EP-coated sample deteriorates and deforms. The surface roughness of EP and NEP-coated samples were evaluated for nanofiller in the epoxy matrix. It has been observed that Al2O3-ZrO2 nanofiller may be used to enhance the enviable type of the substrate epoxy matrix. Ahmad and Mamat (2011) synthesized ZrO2-SiO2 sand nanoparticles reinforced composites and investigated their physical and mechanical characteristics in relation to the microstructure. ZrO2-based SiO2 nanoparticles composites of 20, 15, 10, and 5 wt.% were created using a powder processing technique and sintered at 1500 C for 2 h, resulting in an increase in sintered density. Adding 20 wt.% SiO2 nanoparticles exhibits the hardness value of 12.45 GPa. Microstructures of SiO2 nanoparticle show the diffusion has occurred into the pore sites of the composites.

16.6.2 Hybrid composites Hossen et al. (2015) showed the tensile properties of chemically treated jute fiber reinforced PE/clay nanocomposites are higher than nontreated one. To make the raw jute fibers more compatible with the PE matrix, they were chemically treated with benzene diazonium salt. At an ideal fiber content of around 15 wt.%, the treated composites confirmed an enhanced strength. Chitrambalam (2009) investigated how alkali and sodium lauryl sulfate treatments improved the mechanical properties of woven hybrid composites and banana/kenef hybrid composites. They discovered that SLS surface modification enhanced mechanical performance more than alkali treatment because of better fiber matrix interface. Idicula et al. (2006) determined the diffusivity, specific heat, and thermal conductivity (Fig. 16.2), of polyester/banana/sisal composites for various fiber surface treatments with different filler content. The inclusion of banana/sisal fiber lowers the neat thermal conductivity of the composite. It was concluded that the heat conductivity of the polymeric matrix is vital than the banana/sisal fiber. The composite of sisal fiber, red mud, and glass fiber

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FIGURE 16.2 A Schematic representation of thermal conductivity model for fiber-based composites (Idicula et al., 2006).

laminate was calculated. To treat fibers and red dirt, a hydrophobic chelate based on Ti and Si is utilized. Two types of laminates were evaluated: sisal/glass fiber/polyester resin and sisal/glass fiber/red mud with polyester resin. When 40% sisal-coir fiber reinforced composites are subjected to moisture at severe temperatures, moisture absorption increases by 33%, followed by a significant loss of mechanical properties owing to degradation of the fiber matrix interface. The tensile and impact strength of the hybrid composite, which comprises 40% sisal-coir-glass fibers and 60% resin, are excellent. Pujari, Scheres, Marcelis, and Zuilhof (2014) examined the properties of jute and banana fiber composites and physical, mechanical, and chemical properties were evaluated. In high-performance appliances, such as recreational and athletic products, marine industries, and aeroplanes; for example, lower-cost items can be used. As a result, given this context, it is plausible to infer that the composite is the most wanted technology in today’s quickly evolving global trend.

16.7 Characterization 16.7.1 Structural properties 16.7.1.1 Scanning electron microscope (SEM) and field emission scanning electron microscope (FESEM) analysis To evaluate spontaneous crack propagation in the composite, surface properties of fracture samples were efficiently frozen in liquid nitrogen before taking SEM photography. The fractured surface of 30 and 130-nm silica/epoxy composites

16.7 Characterization

and plain epoxy is shown in Fig. 16.1. When comparing the smooth surface of pure epoxy to the greater roughness of such composite’s worn surfaces, the presence of silica fillers in the epoxy matrix is established (represented in Fig. 16.3). When compared to pure epoxy, both silica/epoxy composites shatter in sharp fractured patterns and discrete stages with such a fish-skin-like microstructure (Fig. 16.3D), showing enhanced brittleness. On the other hand, when particle size fell, the roughness of the fracture surface reduced dramatically. Individual particles seemed to be hidden on none of the composite’s surfaces, which is notable. As a result, distinguishing whether the fractures propagated in the matrix, through the particles, or at the particle-polymer matrix contact from SEM images is difficult. To understand more about the crack propagation route, the fracture surfaces were subjected to a Raman spectroscopic examination. The fracture surfaces of each specimen were assessed 10 times on different sections of the fracture surface for all cases. The usual Raman spectra of the materials show that the epoxide ring and the SieOeSi link exhibit separate peaks in the epoxy and silica spectrums,

FIGURE 16.3 Fractographic images of pure epoxy-based composite with 0.5 vol.% SiO2 of size (A) 30 and (B) 130 nm, and (C) fracture surface showing fish-skin-like microstructure in SiO2/ epoxy composite and (D) enlarged view of the encircled portion in (C) (Conradi, Zorko, Kocijan, et al., 2013).

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respectively (Conradi, Zorko, Kocijan, et al., 2013). However, we only identified spikes in the spectrum both of composites that have been characteristic of epoxy and had no silica contribution. The silica particles were not visible on the fractured surface, as shown by SEM photography, and the fractures in the composites progressed into the matrix resin, bypassing the particles.

16.7.2 Charpy impact strength test Impact strength of composites can be enhanced at the expense of ductility and addition of micro-cracks that allow for higher energy absorption. SEM images are used to examine the top surface of the fractured samples adjacent to the location where the impactor impacted the sample (Fig. 16.4). Fig. 16.4 shows material congregation in the path of impact crack propagation, as well as distinctive steps with increased roughness for all cases (A), (B), and (C), indicating that the nature of brittle in the Charpy test differs for pure epoxy and composites. Pure epoxy has a flat surface (Fig. 16.4A), with even steps and little delamination. In comparison to pure epoxy samples, we found increased roughness and

FIGURE 16.4 The area close to the damaged surface after hitting of the sample by the impactor (Conradi, Zorko, Kocijan, et al., 2013). The Figures (A-C) show the SEM surfaces along with crack direction for epoxy resin and the figure (D) is the schematic top view of the crack direction for epoxy reinforced composites.

16.7 Characterization

sharp steps in 30 nm and 130 nm silica/epoxy composites (Fig. 16.4B and C), as well as excess material fragments and more evident delamination, which boosts the materials’ energy absorption capabilities.

16.7.3 Atomic force microscopy A biodegradable thermoplastic matrix consisting of starch and ethylene-vinyl alcohol was used to create the composites (SEVA-C, Novamont, Italy). A Ca-P layer entirely covered the surface after 128 h of immersion in SEVA-C supplemented with hydroxyapatite particles (see Fig. 16.5). These findings emphasize the relevance of the biological fillers in this sort of material, as the filler offers two main purposes: it strengthens the composite and provides bioactivity (Leonor, Ito, Onuma, Kanzaki, & Reis, 2003).

16.7.3.1 Fourier transform infrared (FTIR) analysis Fourier transform infrared (FTIR) spectroscopy studies were carried out to examine if the powder filler fully incorporated in the composite structure. FTIR spectra for basalt powder and composites are shown in Fig. 16.6. The FITR data were captured for both before and after silanization.

FIGURE 16.5 The generation of Ca-P layer on the surface of SEVA-C and 30% hydroxyapatite composite as shown from the in situ atomic force microscopy images (Leonor et al., 2003).

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FIGURE 16.6 FTIR spectra of the basalt powder and composites (Matykiewicz & Barczewski, 2020).

Peaks in the spectra of the composites studied indicated the presence of polymer matrices in the shape of epoxy resin. The aromatic ring was responsible for 800 850/cm, the C O C stretching band was accountable for 1000 1100/ cm, the verified C C O C stretching band was important for 1150 1300/ cm, and the Ar C double bond was involved for 1500 1600/cm. At 2800 3000/ cm, the CH2 and CH3 symmetric stretching bands, as well as the C H stretching band, are stretched. Because SiO2 is the main component of basalt powder, a broad absorption band correlating to the Si O Si connection was found in the region of 1000 1200/cm. As seen by the drop in intensity peak at 3400/cm, which corresponds to the O H group (Jamali, Rezvani, Khosravi, & Tohidlou, 2018; Matykiewicz & Barczewski, 2020), the silane coupling agent can interact with hydroxyl groups on the surface of basalt powder.

16.7.3.2 Tensile testing A tensile test was performed on the electrospun membranes. The stress strain plot for each case is represented in Fig. 16.7A. The tensile test data were used to compute breaking strain, elastic modulus and strength. For S50 sample tensile

16.7 Characterization

FIGURE 16.7 (A) The stress strain curves for the electrospun membranes, (B) tensile modulus, (C) maximum strain value at break, and (D) ultimate tensile strength (Castro et al., 2018).

modulus of 13.5 6 1.27 MPa was noticed, while for the S0 sample it was observed as 9.5 6 1.75 MPa. However, for higher concentrations of Si-NPs (S75), the tensile modulus decreased to 8.9 6 1.32 MPa. The strain at break was similarly affected by the addition of Si-NPs. Fig. 16.3C depicts that with higher silica mixture, the strain at breakpoint reduced compared to pure poly(ε-caprolactone) membranes. S25 and S50 show a distinct enhancement of tensile strength (5.8 6 0.19 MPa for S50, 6.5 6 1.55 MPa for S25, and 2.9 6 0.31 MPa for S0). S75, on the other hand, had the same tensile strength as S0 membranes (3.7 6 0.99 MPa) (Castro et al., 2018).

16.7.3.3 Flexural testing Al2O3/epoxy composites having various Al2O3 concentrations were tested for different sintering temperatures to determine flexural strength and the result is shown in Fig. 16.8. All the composites were found to have typical brittle fracture characteristics. Comparative flexural strength values of the sintered porous Al2O3 ceramic skeletons, degreased green body, and pure epoxy are also plotted in Fig. 16.8. Flexural strengths of Al2O3/epoxy composites and sintered porous Al2O3 ceramic were increased rapidly

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FIGURE 16.8 Flexural strength of the Al2O3/epoxy composites with respect to the content of Al2O3 (Hu et al., 2016).

FIGURE 16.9 Variation of Izod impact strength for ZnO-filled composites with ZnO content (Gull et al., 2015).

16.7 Characterization

with augmentation of Al2O3 content. For example, flexural strength rises from 121 to 305 MPa for the 55% 70% increased of Al2O3 content. This can be attributed due to the fact that intergranular bond in the Al2O3 ceramic grows rapidly with rise in sintering temperature. The degreased green body, on the other hand, exhibited a flexural strength of only 1.57 MPa (Hu, Du, & Chen, 2016).

16.7.3.4 Izod impact test It is a well-known fact that the weight fraction, orientation of fiber, and filler strength play a significant role for the Izod impact strength. The orientation of glass fiber in all samples was kept constant, and the variation of impact strength was measured with different content of filler in this investigation. Fig. 16.9 shows that increasing the filler loading to 5 wt.% results in a noteworthy betterment in the value of impact strength up to around 40%. The adhesion force of matrix and inorganic filler, fiber pullout, and matrix fracture all affect the composite’s impact strength. The value of impact energy in the case with glass fiber reinforced polymer (GFRP) composites enhances with the accumulation of filler due to improvement of bonding strength between inorganic filler, matrix as well as fiber. In this

FIGURE 16.10 Thermogravimetric analysis of polyester composites filled with ZnO (Gull et al., 2015).

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regard, it is to be noted that GFRP composites filled with ZnO may absorb more energy and have better fracture strength than empty GFRP composites; hence crack propagation is restricted. The results obtained from the Izod impact test substantiate the fact that ZnO will be a promising candidate as filler material in the fabrication of future composites (Gull et al., 2015).

16.7.3.5 Thermogravimetric analysis Fig. 16.10 presents the TGA behavior of GFRP composites with and without ZnO addition. Thermal stability can be determined from the TGA behavior. The weight of the composites was reduced during TGA owing to breakdown or volatilization, whereas the weight was increased due to chemical reaction and gas absorption. The profile of heat degradation in glass fiber integrated polyester composites loaded with ZnO shows weight loss in discrete phases. The progressive removal of moisture and bound water from composites causes weight loss in the temperature range of 30 C 130 C. In the second phase, weak bonds are broken and highly strained crosslinks are unzipped, resulting in the creation of straight chains with the maximum disintegration rate. At a higher temperature, the linear backbone chain of the polymer disintegrated into minute fragments. After completion polyester matrix burning at 800 C, the residue of glass fiber and ZnO filler remains. This investigation revealed that using 3 wt.% ZnO resulted in a weight loss of 47.71%, which was raised to 49.79% when using 4 wt.% ZnO (Gull et al., 2015).

16.8 Summary The particulate-reinforced composites, particularly ceramic particle reinforced composites, can greatly enhance different properties of the polymer matrix composites as elucidated in this study. Despite the fact that particles have a lesser reinforcing capacity than unidimensional fibers, the simplicity with which the former may be processed highlights its benefit. Besides, the enhanced stiffness and wear resistance of particulate-reinforced composites play decisive roles in many applications. The nature of the matrix, the reinforcement orientation, and the curing method influence how the polymer composites are made. Many studies have been conducted throughout the world, particularly on natural fiber based polymer matrix composites incorporated with hard ceramic particles. This compendium reinforces the fact that the ceramic particle dispersed polymer composites are inexpensive and possess various outstanding characteristics and therefore, can be the potential candidate for various cutting-edge technologies.

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CHAPTER

Electrospinning for biomedical applications

17

Srividya Hanuman1,2, , Steffi Zimran3, , Manasa Nune1,2 and Goutam Thakur3 1

Manipal Institute of Regenerative Medicine, Bangalore, Karnataka, India 2 Manipal Academy of Higher Education, Manipal, Karnataka, India 3 Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India

17.1 Introduction 17.1.1 Theory of electrospinning Electrospinning, a term derived from the words “electrostatic spinning,” is a fabrication methodology used for the synthesis of fibers in the micro to nano scale using the expulsion of polymers under the appropriate electrostatic force. Here is an insight into the foundation and evolution of this technique. Electrospinning has its origins from the work of Rayleigh in 1897. Looking at its potential over the other technique to produce nano-fibers at the time, the technique was further investigated in 1914 by Zeleny (1914). Due to its competition with the mechanical drawing process that was commercially evolved by then, electrospinning was not adopted or further looked into till 1934. Antonin Formhals published the first ever patent on electrospinning in 1934, where he fabricated cellulose acetate (as yarn) using the solvents, acetone and monomethyl ether of ethylene glycol (Formhals, 1934). Formhals rolled out a series of patents in 1938, 1939, and 1940 to establish the experimental set up required for the making of the polymer fibers utilizing electrospinning (Huang et al., 2003). The prototype used by Formals for the electrospinning mimicked the conventional spinning drum where threads were collected in their stretched form on a movable collecting device. It was in the 1960s, that Taylor identified and studied the conelike formation of polymer droplet when an electric field was applied (Taylor, 1969). The lead to the integral terminology “Taylor’s cone” that is extensively used in all literatures related to electrospinning. In the subsequent year several researchers worked towards refining the fabrication technique to produce uniform, reproducible polymer fibers with extensive utility in several sectors spread across



Authors contributed equally.

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00013-9 © 2023 Elsevier Inc. All rights reserved.

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Table 17.1 Key studies that served as milestone in the electrospinning technology. Year

Event

References

1934

Formhals patented the process of electrospinning cellulose acetate for textiles Streams with diameter 0.1 mm was fabricated by Vonnegut and Neubauer from electrified homogenous droplets Patent for apparatus to fabricate nonwoven fibers using electrospinning Created electrospinning unit that produced fibers in the diameter range 0.05 1.1 μm Conducted a series of studies that aim at production of fibers from polymer melts

Formhals (1934)

1952

1966 1971 1981

1996

Established that electrospinning could be done using various polymer solutions

Vonnegut and Neubauer (1952) Simon (1966) Baumgarten (1971) Larrondo and St. John Manley (1981a, 1981b, 1981c) Reneker and Chun (1996)

multiple industries. The Table 17.1 discusses some key work that were stepping stones in defining the electrospinning process as known today. The past decade has seen a substantial increase in the utility for nano-sized solutions in healthcare therapeutics. The versatility and ease provided by electrospinning make it a key player in creation ideal solutions for several biomedical and regenerative medicine based research across the globe.

17.1.2 Principle of electrospinning The electrospinning technique is similar in functionality to the electrostatic spraying process, with the only point of difference being the production of uniform polymer droplets in the electrospraying process and uniform nonwoven fibers in the case of electrospinning. Over the years, electrospinning has been identified as versatile and efficient technique can be executed with a simple apparatus in the laboratory set up and can be effortlessly up scaled for industrial production (Stankus et al., 2006). This section discusses the fundamental aspects of electrospinning alongside its principle of functioning. The electrospinning apparatus used today has three main components—the spinneret (needle/pipette of small diameter), a collecting plate, and a voltage supplier capable of producing high DC voltage. The components are conventionally arranged either in a vertical or horizontal set up (Kidoaki, Kwon, & Matsuda, 2005). The Fig. 17.1 showcases the schematics of the different set ups of electrospinning. Electrospinning functions on the fundamental theory that stronger repulsive forces surpass the weaker force of surface tension (of electrospinning solution) that has been charged. During fabrication, the polymer solution fixated at the tip

17.1 Introduction

FIGURE 17.1 Schematic of (A) Horizontal and (B) Vertical apparatus set-up for electrospinning.

of the spinneret is exposed to the established electric field thus inducing a charge on its surface. On reaching the critical value, the repulsive forces generated overcomes the polymer’s surface tension leading the ejection of polymer fibers from the pinnacle of the Taylor’s cone (Chew et al., 2005). The fibers are accumulated on the collecting surface because of the unsteady and rapid expulsion of the solution in the gap between the spinneret and collecting surface coupled with the evaporation of the solvent. A plethora of models were used to depict the process of electrospinning and its concluded that all electrospinning set ups can be divided into four regions based on the function executed in that zone. The four regions are: 1. 2. 3. 4.

Base region—nozzle end where the surface of the solution is charged Jet region—expulsion of polymer solution in a streamlined fashion Splay region—jet streams cleave into nanofibers Collector region—formed nanofibers accumulate in this zone.

The work of Hamzeh, Miraftab, and Yoosefinejad (2014) provided detailed explanation of the fiber formation using the electrostatic theory. Fig. 17.2 exhibits the various phases of fiber synthesis in the electrospinning process. He concluded that solution, when subjected to an electric field of high voltage and there is an accumulation of positive charges at the tip of the nozzle, the solution is forced out of the spinneret. He theorized that Taylor’s cone is an outcome of this attractive force that is enveloped by the polymer’s surface tension. The ejected polymer solution cleaves into fine strands as the surface tension gradually proceeds to its critical limit. The repulsive forces developed between these strands leads to their splitting into nano-sized fibers that are then collected on the negatively charged collector surface.

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FIGURE 17.2 Schematic representing the different stages of electrospinning.

17.2 Parameters influencing fiber production Electrospun fibers would be most desirable when all fibers have uniform diameters; the surface of the fibers does not have any undesirable flaws and its easy for collecting continuous and long fibers from the collector. The working parameters of electrospinning play a key role in modifying the fiber morphology in accordance to the application. Working parameters are categorized as either system parameters or solution parameters. System parameters, as the term suggests, are parameters that are modifiable on the electrospinning apparatus while the solution parameters are the parameters to modify the solution before the fabrication process. This section discusses the various system and solution parameters and how the fiber morphology is influenced.

17.2 Parameters influencing fiber production

17.2.1 System parameters 17.2.1.1 Applied voltage In the electrospinning activity, applied voltage is responsible for setting up the electric field followed by inducing the necessary charges on the polymer solution and so initiating the procedure. This makes applied voltage a crucial factor amongst the process parameters. Baumgarten concluded in 1971 that the configuration of the preliminary drop alters based on the spinning conditions, that is, voltage, viscosity and a feed rate. The specific contribution of voltage to the nanofiber morphology has been a matter of dispute amongst researchers. Reneker and Chun (1996) came up with the hypothesis that electric field (created by applied voltage) does not have a critical impact on the diameter. Later research has suggested that higher voltages contribute to more solution release thus producing fibers with higher diameters (Demir et al., 2002). This is conflicted with the findings of several other research who provided evidence that increasing the intensity of the electric field leads to an increased repulsive force on the jet formed from the polymer solution, which ultimately aids the narrowing of fiber circumference; alongside swift evaporation of solvent from the fibers (Deitzel et al., 2001; Megelski et al., 2002). This theory was further supported by the work of Larrondo and Manley (Larrondo & St. John Manley, 1981a,b,c) who proved that there was a reduction in fiber diameter by almost half on doubling the applied voltage. Some also found that there was higher bead formation at higher voltages.

17.2.1.2 Flow rate The speed at which the solution is ejected from the nozzle is termed at the flowrate of the electrospinning process. Flowrate of the solution governs the transfer rate of the material involved and the jet velocity. Primarily, a lower flowrate is advisable as this is essential for uniform fiber production and provide the appropriate time for good evaporation of the solvents. It was observed by many researchers that elevated flow rates lead to the formation of fibers with higher diameter and larger pore size. Studies by Megelski et al. (2002) and Zong et al. (2002) investigated the relationship between fiber morphology and flow rate. Higher flow rates often resulted in beaded fibers as a result of reduced drying time before settling on the collector

17.2.1.3 Tip to collector distance The distance from the tip to collector is an essential factor that determines the drying time proved to the produced fibers before they reach the collector. In the case of the tip to collector distance, identifying the optimal distance is key as both lesser or more distance do not produce the desirable fibers (Geng, Kwon, & Jang, 2005). This distance was not a significant factor in term of modulation of fiber morphology when experimented with gelatin (Ki et al., 2005), chitosan and

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PVA (Zhang, 2005). It was observed that shorter distance produced flatter fibers while increased distance produced rounder fibers in electrospun silk-like polymer with additional fibronectin functionality (Buchko et al., 1999). A closer distance caused tinier fibers in polysulfone fibers.

17.2.1.4 Collector types The collecting platform is the conductive substrate on which the nanofiber settles post formation. The widely used material for this purpose is aluminium foil, but the difficulty posed in transferring the fibers post spinning lead to investigation of other alternatives. Over the years other collectors like, pin (Sundaray et al., 2004), parallel bar, grided bar (Li, Wang, & Xia, 2004), rotating wheel/rod (Xu et al., 2004), wire screen or liquid baths (Ki et al., 2007) have been used as substitutes. A comparative study between aluminium and wire screen as collectors showed that aluminium were better collectors as they have more surface area in comparison to the wire screen that had more beaded firms. Though the wire screens provided easier transfer of the fibers for further application, the surface area provided for collection was less (Wang et al., 2005). The type of collector and rotating speed are determinants for fiber alignment (Laurencin et al., 2008). Several research groups have identified that the use of a rotating drum or metal frame leads to the formation of aligned fibers, which when optimized are almost parallel to each other (Fong, 2002). Another alternative collector to gather aligned fibers are split electrodes with a void in the center to collect the aligned fibers (Jalili, Morshed, & Ravandi, 2006).

17.2.2 Solution parameters 17.2.2.1 Concentration A direct corelation exists between solution viscosity and concentration at which the solution is prepared. It has been experimentally proven that lower concentration produces fiber with higher diameters and increased number of beads. As concentration is gradually increased, one can observe the transition of the beads into spindle and then into elongated and continuous fibers (Bhardwaj & Kundu, 2010). Once this threshold is exceeded there is a difficulty in maintaining a continuous flow of the solution and so discontinuous fibers are formed. Therefore identification of the appropriate concentration is key for desired fiber fabrication (Deitzel et al., 2001). Fong et al. studies the relation between solution concentration and fiber morphology of PEO nanofibers, where they fabricated nanofibers using a varied concentration between 1 and 4 wt.%. It was found that with increased concentration, fibers with lesser beads were produced (Fong & Reneker, 1999; Sukigara et al., 2003). Liu et al. (2008) conducted studies by comparing PBD nanofibers formed at seven different concentrations, from 11% to 17%. The nanofibers produced lesser

17.2 Parameters influencing fiber production

beads as the concentration increased and no beads were found at 17%. This study explained that bead formation was attributed to low surface tension in lower solution concentrations which increased with the concentration, thus eliminating the beads at optimal concentrations. Demir et al. provided experimental evidence that the diameter of the nanofibers formed were proportional to the cube of polymer concentration (Demir et al., 2002).

17.2.2.2 Surface tension Surface tension of solutions differ based on the solvents used to prepare the solution. Reducing the surface tension leads to easier formation of fibers without beads as high surface tension contributes to the instability of the jets and in turn is responsible for the sprayed polymer particles (Hohman et al., 2001). Surface tension of the solution does not impact the morphology of the fibers but play a key role for their optimal production. There is no directive that lower surface tension leads to ideal electrospinning conditions, but it helps fix the range that would be most suitable for the purpose (Haghi & Akbari, 2007; Pham, Sharma, & Mikos, 2006).

17.2.2.3 Molecular weight Molecular weight of the polymer influences the electrical and physicochemical properties of the solution including surface tension, conductivity, dielectric strength, and viscosity (Haghi & Akbari, 2007). It is very evident from the above information that molecular weight is essential for altering the morphology of the electrospun fibers. In general, it can be said that a higher molecular weight is desirable for electrospinning as it increases the viscosity of the solution. Molecular weights lower than optimum are observed to produce more beads, while over a certain threshold, leads to the synthesis of fibers with higher diameters. Gupta et al. (2005), fabricated PMMA fibers using solution with molecular weights varying from 12.47 to 365.7 kDa, to comprehend the impact of molecular weight on fiber morphology. The study concluded that increase in molecular beads reduced the beads and droplets in the resultant fibers (Gupta et al., 2005).

17.2.2.4 Conductivity/surface charge density The conductivity of the solution plays key role in the jet formation of the electrospinning process. Most polymers are conductive in nature except for certain dielectric materials. The type of polymer, solvent utilized, and the presence of ionizable salts are the factors that affect a solution’s conductivity. Several studies have provided conclusive results that higher electrical conductivity causes significant decrease in the nanofiber diameter. On the other hand, less conductive solutions resulted in deficient elongation of the jet, leading to production of nonuniform fibers and increased beads. However, Hayati, Bailey, and Tadros (1987), showed when high conductivity was coupled with strong electric fields, a dramatic bending instability occurred and nanofibers were formed in a wide range

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of diameters. A hypothesis was established after a lot of research that radius of jet varies inversely with the cube of root of the solution conductivity (Huang et al., 2001; Jiang et al., 2004). Zong et al. demonstrated the ability of ionic salts to impact the fiber diameter a morphology by the addition of ionic salts like NaCl, KH2PO4, and NaH2PO4. They found that the addition of ionic salts resulted in the formation of fibers with the diameter of 200 1000 nm (Zong et al., 2002). This technique was further investigated in polymers like, PAA (Kim et al., 2005), PVA, PEO, and polyamide-6 (Mit-Uppatham, Nithitanakul, & Supaphol, 2004b) amongst several others. The addition of salts aided the homogenous production of fibers and reduced bead generation.

17.2.3 Ambient parameters Electrospinning has a higher potential compared to other fiber fabrication techniques due to its simple apparatus requirement, reproducibility and best of all, the ability to be done at atmospheric conditions. While the atmospheric conditions aid the formation of fibers, certain factors like temperature and humidity could be optimized for an optimized fiber outcome. The studies conducted by production of polyamide-6 fibers at various temperatures, from 25 C to 60 C, showed that increase in temperature produced thinner fibers. This is because of the reduction of solution viscosity with the rise in temperature (Mit-uppatham, Nithitanakul, & Supaphol, 2004a). The effect of humidity was investigated by Casper et al., where polystyrene solutions were fibers fabricated at varied humidity levels. Appearance of tiny pore was seen on the surface of the fibers which after a level lead to the pores fusing. Lower humidity let to a high rate of evaporation of the solvent, so rapid that the solvent started clogging the spinneret within a few minutes of initiating the process (Casper et al., 2004). It has therefore been suggested to identify and maintain a high humidity that aids the discharge of the nanofibers while not creating undesirable pores on the surface.

17.3 Polymers for fabrication of electrospun fibers 17.3.1 Synthetic polymers For decades, numerous polymers have been used as scaffolds for diverse applications, which are covered in this chapter.

17.3.1.1 Poly L-lactic-co-glycolic acid PLGA is physically reliable and relatively biocompatible, and it has been extensively researched as a drug, protein, and other macromolecule delivery agent. It is the most widely used biodegradable polymer due to its considerable clinical work, superior degradation properties, and possibility for extended drug delivery.

17.3 Polymers for fabrication of electrospun fibers

For electro spinnability and antibacterial properties of PLGA against methicillin-resistant Staphylococcus aureus, researchers investigated the influence of solvent system on polymer. According to the researchers, HFIP and DCM: DMF are the best solvent systems for PLGA-linezolid and regulated drug release (Eren Boncu, Ozdemir, & Uskudar Guclu, 2020). It was also revealed that administering the linezolid-loaded nanofiber locally reduced linezolid side effects, resulting in enhanced therapeutic response. This chapter suggests a strategy for boosting h-ADSC adherence and proliferation on the scaffold by encapsulating ordered poly PLGA (Seyedebrahimi, Razavi, Varshosaz, Vatankhah, & Kazemi, 2020). Following the assessment of being functionalized biologically PLGA, chitosan nanoparticles (CSNPs) were encapsulated in “brainderived neurotropic factor (BDNF)” along with nanoparticles of gold (AuNPs), were mixed with solution that is derived from laminin, and added on the over-floor of the scaffolds. Bradford assay and inductive paired plasma optical emission spectrometry was utilized to investigate the release of BDNF and AuNPs from the scaffold. After that, immunocytochemical labeling and gene expression studies were used to assess the test group’s ability to develop into Schwann cells. According to the Cytotoxicity assay, h-ADSC proliferation was substantially greater on the laminin-functionalized scaffold than on the PLGA scaffold. Furthermore, the expression of SCs markers was considerably greater when BDNF and AuNPs were present on the scaffold surface. In this work electrospinning was used to create platelet-rich plasma (PRP) loaded PLGA nanofibers (Torabi et al., 2020). An electron microscope was utilized to evaluate the surface of the modified scaffold. Uniform with interconnected pores on fibers was identified. The synthesized scaffolds were characterized for binding, cellular growth, and biocompatibility as well as cell adhesion and proliferation using human iPSCs. According to tests, produced PLGA nanofibers scaffold is more biocompatible than standard cell culture dishes (TCPS). The ability of human iPSCs to differentiate into cardiomyocytes (CMs) was assessed at the gene and protein levels on a variety of substrates. PRP integrated scaffolds exhibited significant improvements in biocompatibility and differentiated iPSCs in this group showed strong activation of cardiac genes, notably MLC2A, ANF, and MLC2V.

17.3.1.2 PLLA-polylactic acid PLLA nanofibers were electrospun with a single nozzle approach (Derakhshan et al., 2016). To examine the surface morphology and fiber diameter, techniques such as SEM imaging was used. Primary human bladder smooth muscle cells were seeded on the electrospun fibers to test the material’s potential for bladder repair. The viability and proliferation were studied using one of the simplistic Almar blue assays, and hBSMCs were viable and showed cells’ native structures on both simple random and thick random fibers. This study depicted the alteration of cells on the electrospun nanofibers. In other words, hBSMCs might form in their original shape, on both random as well as aligned fibers. PLLA electrospun nanofibers were used, which are hydrophobic in nature, and were surface modified by plasma treatment to generate a COOH group on the

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surface, onto which cationized gelatin was grafted using carbodiimide as a coupling agent to increase cell adhesion (Chen & Su, 2011). Even after day 7, the PLLA nanofibers exhibited a considerable increase in cell proliferation. Collagen and sGAG production were analyzed up to day 30. A 30-fold increase was seen for modified PLLA. In cell viability assay, dead cells were seen only on unmodified nanofibers whereas dead cells were seen barely on modified fiber after 14 days of culture, and also, chondrocytes on CG-PLLA NFM showed better morphology and infiltered inside the meshes along with better ECM synthesis. Polymer breakdown resulted in a considerable reduction in membrane size, whereas the cartilaginous construct developed in vivo had a yellowish-white tint. Showed the formation of sub microfibers of PLLA using lower voltages such as 6 kV by adding a small amount of sodium lauryl ether sulfate along with chloroform as a solvent system for electrospinning and studied its use in tissue engineering and cell viability (dos Santos, Duarte, Pezzin, da Silva, & Domingues, 2020). Three-dimensional and highly porous fibers were obtained using the larger distance between the tip to the collector. PLLA is effective in its use due to its biocompatibility with human adipose stem cells. It was proven that water, acts as a nonsolvent in the electrospinning system, facilitating fiber formation with high porosity, which is optimal for cell growth. In this study, PLLA was used to create a scaffold that replicated the heart’s nave ECM exactly, with the addition of proteins on the surface (Muniyandi et al., 2020). The improved surface compatibility and potential for cell viability were studied using human cardiac fibroblasts. Because it mimicked the ECM environment, this alteration helped to attach and produce greater outcomes. Proteome profiling was used to investigate the types of proteins produced by cells, and the results revealed that substrate surface topography stimulates proteins that have a beneficial function in cellular proteome expression.

17.3.1.3 Polycaprolactone PCL is a partially crystalline aliphatic biodegradable polyester with a melting temperature of 55 C. Its crystallinity decreases with increasing molecular weight. It has good tensile strength and is miscible with a wide range of other polymers (Guarino, Gentile, Sorrentino, & Ambrosio, 2017). And due to its hydrophobic nature, it takes a long time to degrade, therefore, widely employed for various applications, and also the possible load-bearing mechanical property is very intriguing for soft tissue engineering (Kurniawan, Nor, Lee, & Lim, 2011). However, since the PCL surface is hydrophobic in nature it limits its application to colonize cells (Kuppan, Sethuraman, & Krishnan, 2013). As a result, various modifications are made in reference with the application of scaffold. Electrospun PCL along with bioactive plant extract is obtained from Inula graveolens (L.) (Wasan et al., 2020). The solution was prepared with a ratio of 7: 3 from 8% PCL and 5% plant extract in 1:2 acetic acid/formic acid solvents. Using SEM, FTIR, and XRD physiochemical characterization of fibers is done. Contact angle measurement reduced from 118.4 6 2.0 to 51.4 6 2.0 after adding

17.3 Polymers for fabrication of electrospun fibers

plant extract to PCL. MTT assay done using fibroblast cells at different time points showed that I. graveolens/PCL scaffold showed higher viability when compared to only PCL and toxic effects were observed on cells. Surface modification of electrospun polycaprolactone (PCL) scaffold was accomplished in a gas setting (Kudryavtseva et al., 2018). The impact of plasma treatment on the structure was explored, and was found to be unaffected by plasma treatment, although it did promote PCL scaffold biocompatibility and hydrophilicity. Finally, this modification method offers the path for the generation of scaffolds that are more biocompatible to tissue. Hyaluronic acid and short peptides, as well as PCL, were used to create a scaffold for bone tissue regeneration (Rachmiel et al., 2021). The processability of hyaluronic acid (HA) is low in electrospinning, hence it must be combined with another polymer. As a consequence, a composite scaffold consisting of a PCL polymer core/shell, HA, and a synthetic self-assembling peptide was synthesized. Through molecular recognition, the peptide increases cellular adhesion by following the arginine-glycine-aspartic acid (RGD) sequence. The preosteoblast cell line adhered to the scaffold effectively and proliferated, demonstrating osteogenic differentiation and calcium deposition. The response of liver cells to structural properties on PCL fibers was studied (Gao & Callanan, 2021). A solvent nonsolvent system was used to construct scaffolds with bigger dimensioned surface of depression, minor surface of depression, and without surface depression. For up to 14 days, HepG2 cells were seeded on the scaffolds. The minor surface depression group demonstrated higher cell viability and DNA content than the other groups. Furthermore, despite the fact that the cell growth rate was varied, the scaffolds increased albumin gene expression in identical quantities in all situations. These findings suggest that HepG2 cells prefer small depressions over smooth and large depression fibers, suggesting that liver cell responses might be tuned. The goal of one of the other studies was to develop and test a combination of nanofibrous PCL/collagen scaffold that can be used as a new skin treatment strategy (Sharif et al., 2018). The behavior of human endometrial stem cells (hEnSCs) was studied on this scaffold. The hEnSCs have been characterized and capable of uterine repair due to their significant capacity to generate new capillaries. Using the electrospinning technique, a PCL 3D scaffold was created. Plasma was used to treat the PCL, which was then grafted with collagen. The mechanical and structural qualities of the structures were assessed. Stem cells were seeded on scaffolds to evaluate cytocompatibility and differentiation. The PCL/collagen scaffold had a higher wettability and better mechanical and structural properties. On the PCL/ collagen scaffold, cell adhesion and growth rates were greater than on PCL.

17.3.1.4 Polyurethane PU’s are a class of synthetic organic polymers formed by the reaction of diisocyanates with any other difunctional compounds like glycols. Because of its efficient features like biocompatibility, nontoxicity, good barrier properties, gas

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permeability, and high efficacy in the control of wound moisture is widely used for biomedical applications. An electrospun PU nanofibrous mat along with Nigella sativa oil (NSO) was ¨ zer, Go¨ktalay, Saat, & tested as a viable wound dressing material (Aras, Tu¨may O Karaca, 2021). The surface morphological properties like fiber uniformity, bead formation, and fiber continuity, pore size, porosity, fiber diameter, and wettability of the NSO-loaded PU nanofibrous scaffolds were studied using a microscope imaging and imaging software. Those randomly oriented fibers were smooth, continuous, and beadless. The total immersion method was used for determining the release characteristics of NSO from PU/NSO nanofibrous mat and the absorption spectra were detected with a UV-Vis spectrophotometer. A significant release of NSO from the PU fibers was observed. In vitro cytotoxicity studies were also done on the HUVEC line to check the viability of the cells. The colorimetric WST-1 assay which relies on the mitochondrial dehydrogenase enzyme within viable cells was used for this. No cytotoxic effects were observed, and the cells were found to be viable. The PU/NSO nanofibrous mats also showed antibacterial properties when investigated against E. coli and S. aureus bacteria. The in vivo wound recovering efficiency of PU/NSO nanofibrous mat was studied on rats by creating a wound model. Significant wound healing and tissue regeneration were observed. All of the findings indicate that NSO-loaded PU nanofibrous mats could be applied as wound dressing materials. Li et al. (2020) developed a novel solvent system by using Trichloromethane (TCM)/2,2,2-trifluoroethanol (TFE) for the fabrication of electrospun thermoplastic polyurethane (TPU) nanofibers. An even morphology and uniformly distributed TPU nanofibers were obtained. The molecular level information of the electrospun nanofibers was characterized by using a FTIR spectrometer. It showed that the TCM/TFE solvent system did not affect the chemical structure of the TPU. The crystal structure information of the electrospun nanofibers were obtained through differential scanning calorimetry analysis. X-ray that is diffrated was used to study the impact of a varied TCM/TFE volume ratio on the crystal structure electrospun nanofibers’ properties which was examined through analysis. CCK-8 assay was used to check cell viability. TPU nanofibers were investigated in vitro for cytotoxicity using rat bone marrow mesenchymal stem cells (rMSCs). No signs of cytotoxicity were seen. All of these findings indicate that the electrospun TPU nanofibers produced with the unique TCM/TFE solvent combination were highly biocompatible.

17.3.2 Natural polymers 17.3.2.1 Gelatin Gelatin is a “denatured protein” that is obtained via the processing of collagen with acid as well as alkaline. The alkaline phase has an effect on glutamine amide as well as asparagine groups, causing them to hydrolyze and form carboxyl

17.3 Polymers for fabrication of electrospun fibers

groups. The acidic intervention has minimal effect on the amide groups since they are acidic in nature. As a result, the acidic treatment produces gelatin that is electrically distinct from “alkaline-processed gelatin” (Xiao et al., 2019). Due to the various treatment parameters for gelatin, adaptability may be achieved with either positively or negatively charged cells and biomolecules by using a gelatin scaffold, hydrogel or carrier. Due to the above-mentioned characteristics of gelatin, it has been deployed in the engineering of different tissues as well as drug delivery systems. Several of these uses are presented in the following section. Lee, Ko, Cho, Park, and Kwon (2017) investigated the changes in properties of crosslinked gelatin electrospun fibers caused by electron beam irradiation. TFE was used to make gelatin sheets, and nanofibers were fabricated using an electrospinning method and crosslinked with glutaraldehyde before being irradiated with an electron beam at various dosages. Surface morphology, pore size and porosity, crosslinking degree, biodegradation, and water absorption were studied for both crosslinked and uncrosslinked gelatin sheets. Using fibroblasts on nanofibrous scaffolds, a cell proliferation assay and histological investigation were performed. In crosslinked gelatin sheets irradiated at higher doses, pore size, porosity, and crosslinking degree, weight loss, and in vitro biodegradation were all increased, whereas water adsorption was decreased. Cell growth was shown to be greater in irradiated samples compared to TCPS, and histological pictures demonstrated fibroblast proliferation and migration on scaffolds after 4 weeks of culture, suggesting that the scaffold was suitable for soft tissue engineering. In this study gelatin nanofibers were synthesized via glutaraldehyde crosslinking and growth factors were bound to the surface via avidin-biotin covalent binding and avidin conjugation (Lee, Lim, Birajdar, Lee, & Park, 2016). To describe the attachment of growth factors, SEM, atomic force microscopy, and confocal microscopy were utilized, and adipose-derived stem cells were used to assess cell survival, cytotoxicity, and proliferation. The fluorescence intensity measurements showed biotin binding to avidin-modified gelatin scaffolds, confocal imaging predicted cell morphology, and the viability of cells was determined using a live/ dead technique. Elsayed, Lekakou, Labeed, and Tomlins (2016) showed “fabrication of novel electrospun multilayer gelatin fiber substrates with controlled fiber layer alignment and gelatin crosslinking optimization for adhesion similar to coronary artery tunica medium. With human smooth muscle cells, the scaffolds were used to assess binding, growth, and migration. In both axial and circumferential directions, the manufactured scaffolds exhibited tensile strength similar to tunica media of coronary artery and better suture retention strength. Cell viability experiments showed a greater rate of cell migration, adhesion, and proliferation on manufactured scaffolds, suggesting that they could be utilized as vascular grafts.” In the study conducted by Dias et al. (2017), at 37 C, gelatin was crosslinked with 1,4-butanediol diglycidyl ether (BDDGE) at varying concentrations and for different amounts of time. The physicochemical and biological characteristics of BDDGE-crosslinked electrospun gelatin meshes were investigated. Meshes with

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well-defined structure and random deposition were created using electrospun gelatin fibers crosslinked with BDDGE. The degree of crosslinking may be adjusted by changing the crosslinker’s value or the incubation time, allowing for more precise control of fiber diameter and mechanical characteristics. The proliferation experiment demonstrated that fibroblasts were capable of attaching and growing within the nanofiber meshes, generating an extracellular matrix, while the cytotoxicity data showed no harm. Because of its nontoxicity and potential to change gelatin’s mechanical and physical properties, BDDGE could be utilized as a gelatin crosslinker, according to this study. Li et al. (2018) synthesized gelatin/genipin nanofibrous scaffolds. To strengthen the scaffolds, gelatin was used as a substrate for nanofibers, and genipin was used as a crosslinking agent. hEnSCs cells and other cells were used to study the cell viability and biocompatibility. Solutions with varying solvent concentrations were prepared, and it was observed that as the amount of gelatin in solution increased, the surface tension and conductivity of the solution gradually reduced. The diameter of the nanofibers increased as the amount of crosslinking agent was increased. The synthesized gelatin/genipin films were found to have good water resistance and mechanical qualities, suggesting that they might be used as a patch.

17.3.2.2 Chitosan Chitin is a naturally occurring polymer that may be seen in the insect’s cuticles, crustacean’s shells, as well as fungal cells. Chitosan can be referred to as a deacetylated version of chitin 71 that is either partly or completely deacetylated. The proportion of deacetylation in chitosan is typically between 70% and 85%, as well as the “molecular weight” is between 10 and 100 kDa, depending on the species. Its range of applications in the areas of tissue engineering such as bone, skin, cartilage, cell culture substrates as well as vascular grafts. Chitosan’s biofunctional characteristics, which include being biocompatible, nontoxic, biologically renewable, nonantigenic as well as biodegradable properties distinguish it as a biologically functional and beneficial biocompatible material. Furthermore, the amino and hydroxyl groups of chitosan may be chemically changed to offer a great degree of chemical variety. It also possesses bio adhesive qualities, which is a bonus. Rajendran et al. (2017) aimed to develop a nanofibrous model for studying 3-D liver model consisting of hepatocytes and fibroblast coculture for maintaining the liver function long term. Electrospun chitosan nanofiber scaffolds were employed. These fibers combine to create a microfiber mat with a lot of porosity, strength characteristics, and surface area. Because of its structure, which is similar to components of the liver ECM, chitosan could be an appropriate scaffold material for hepatocyte cultivation because it is critical to mimic the microenvironment in the liver. Hepatocyte spreading and flattening were observed to be improved by fibronectin more than type I or type IV collagen, and even more than laminin. They employed fibronectin coating on electrospun chitosan

17.3 Polymers for fabrication of electrospun fibers

scaffolds to improve cell adhesion, proliferation, and spreading in this research. They came to the conclusion that cell adhesion molecules like fibronectin on chitosan biomaterials are critical for adhesion, development, and integrin interaction. Under mild conditions, electrospun chitosan was examined (Lee et al., 2021). The surface was then chemically treated with succinyl-beta-cyclodextrin. This increased the hydrophobicity compared to electrospun chitosan without surface modification and also as a result of a reduction in the amount of residual amine on the surface. Physiochemical characterization was done and thermogravimetric analysis (TGA) verified the thermal stability of polymeric composite. FTIR confirmed the surface chemistry. In both the characterization we can see better result in the modified chitosan nanofibers compared to nonmodified chitosan. Next, they analyzed the drug release using hydrophobic drug using a clinical antiinflammatory drug glucocorticoids and DEX, a lipophilic substance to verify surface modified chitosan. They confirmed that the modified chitosan had larger amount of DEX compared to the nonmodified chitosan. Later they characterized the biocompatibility using F-Actin staining, live/dead assay and CCK-8 assay. FActin staining was used to visualize the cellular adhesion and morphology. Both scaffolds provide a favorable environment for cell proliferation.

17.3.2.3 Silk Silk is a protein biopolymer that is generated by a variety of organisms, including silkworms, spiders, flies, scorpions as well as mites. Spider silk is a fascinating biopolymer that is lightweight, flexible, and durable, and it has mechanical characteristics that are similar to the best fibers produced by modern science in terms of strength and elasticity. Additionally, it is an ecologically friendly and biodegradable substance. Since there is a limited supply of spider silk, silk fibroin (SF), a naturally occurred polymer generated by silkworms, is an excellent substitute. Fibroin, as well as sericin, are the two most important elements of this substance. Fibroin, a fibrous protein that contributes to the formation of the silk core, is made up of three chains: the heavy fibroin chain, the light fibroin chain, including the fibrohexamerin. Due to its superior mechanical characteristics, gradual degradation rate as well as biocompatibility, this substance is very promising. Also, due to its transparency possibilities, silk has subsequently been exploited as a biocompatible material in “corneal tissue engineering procedures” (Palchesko, Carrasquilla, & Feinberg, 2018). Nour, Imani, Chaudhry, and Sharifi (2021) created a porous scaffold consisting of rudimentary “FGF-immobilized silk fibroin” and other components. On “bFGF-immobilized silk fibroin scaffolds,” the growth, as well as the proliferation of L929 cells were significantly enhanced. In vivo experiments included the scaffold implantation into a rodent dermal defect, which demonstrated substantial skin regeneration as well as re-epithelialization over some time (Nour et al., 2021). After a four-week treatment period, the growth of new vasculature and the layering of collagen shows the possibility of these scaffolds in tissue regeneration as well as angiogenesis.

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This research looked into the impact of electrospun silk nanofibers on mankind which emerged pluripotent stem cells to distinguish into emitting cells (Enderami et al., 2020). The fibroin proteins are electrospun into nanofibrous silk scaffolds. They cultured human iPSCs (hIPSCs) onto these silk scaffolds and differentiated them into insulin-producing cells (IPCs). Using microscope, the morphological properties of nanofibrous scaffolds with seeded hIPSCs were investigated. The scaffolds were tested for biocompatibility and nontoxicity using the MTT assay. hIPSCs were found to be viable. Cell survival and cell viability were found to be greater on the scaffolds than that of the control groups. Realtime PCR (qRT-PCR) method was utilized for analysis by total RNA extraction of the cell samples. Immunofluorescence assay was carried out to track the differentiation of hIPSCs into IPCs and insulin expression was checked. Strong expression was observed in the silk scaffolds. Flow cytometry was used for analyzing the IPC-specific marker proteins in the differentiated hIPSCs. Insulin, glucagon, and Pdx-1 proteins were detected in the silk scaffolds with differentiated hIPSCs while their levels were low in the control groups. Glucose-simulated insulin and C-peptide secretion was estimated by using the ultrasensitive ELISA process. Cells on the silk scaffolds produced significant amounts of both insulin and Cpeptide with respect to the control groups. Mehraz, Nouri, and Namazi (2020), studied electrospun SF/b-cyclodextrin citrate nanofibers for usage in controlled drug release. b-cyclodextrin was first esterified with citric acid to form the compound b-cyclodextrin citrate or b-CDCA. bCDCA was further esterified with SF to produce the SF/b-CDCA. The SF/bCDCA was electrospun to fabricate nanofibrous scaffolds onto which ciprofloxacin was loaded as the drug molecule and the drug release mechanism was investigated. Morphological characterization of the drug-loaded SF/b-CDCA nanofibrous scaffolds was done by using a scanning electron microscope (SEM). It was discovered that as b-CDCA concentrations increased, the fiber diameter decreased. The morphology of the electrospun nanofibers was also directly altered by the solution’s viscosity. The immersion approach was used to load the drugs onto the scaffolds, and the absorbance was evaluated with a UV-visible spectrophotometer. It was observed that cyclodextrin could trap the drug molecules within the pores of the fibrous scaffolds. The amount of ciprofloxacin reduced was raised, while the drug release time rose. FTIR and H-NMR spectrometric measurements confirmed the production of citrate. According to the Higuchi mathematical model, drug release is mostly controlled by diffusion. Li, Zhang, Luo, Yan, and You (2019) investigated the influence of “biofunctionalized silk fibroin nanofibers on directional and long neurite outgrowth.” They did this by electrospinning aligned SF nanofibers and immobilizing laminin onto them. Their effects were investigated using PC12 cells as a model system. The produced nanofibers and cells were morphologically characterized. For the surface functionalization process, physical adsorption and chemical immobilization methods were used for the modification of SF nanofibers by laminin. Immunofluorescence assay was done by using antilaminin antibody staining. It confirmed that chemical immobilization had procured

17.3 Polymers for fabrication of electrospun fibers

more laminin on covalently bound silk nanofibers than that of physically adsorbed ones. CCK-8 assay kit was used for determining the cell proliferation and viability. Laminin-immobilized nanofibers were observed to support greater proliferation and cell adhesion and thereby, greater viability. These findings demonstrate that functionalized nanofibers provided morphological signals that enhanced neurite development and guided axonal growth over the fibrous conduit in natural neurons. These discoveries could be used to develop functional nerve transplants and regenerative medicines. In a rat sciatic nerve damage model, the effect of electrospun silk-polyaniline conduits on functional nerve regeneration was examined (Das et al., 2017). They made nanofibrous nerve conduits and implanted them in a rat for peripheral nerve regeneration. They conducted cytotoxicity studies for scaffolds with Schwann PC12 cells. Then using SEM, MTT-assay and live-dead assay were studied. Viable cells with normal morphology were observed. MALDI, SEM, and FTIR were used to characterize scaffolds, which revealed fibers of average diameter. The porosity and swelling ratio of cell-seeded and unseeded nerve conduits were determined, and the conduits were characterized by calculating their porosity and swelling ratio. There was no erythema or edoema production in an in vivo intracutaneous toxicity study. The nerve conduits were surgically implanted, and electrophysiological testing, walking track analysis methods, and the calculation of the sciatic function index were used to assess the functional status of the regenerated sciatic nerve. Transmission electron microscopy was used to examine the morphology and internal tissue architecture of the regenerated sciatic nerve. Within the conduit, the formation of a myelin sheath over the axons in the regenerated tissue was confirmed.

17.3.3 Composite and hybrid Single polymer nanofibers can have any of the qualities needed for tissue engineering, but they cannot cover the multicomponent properties found in ECM. As a result, composite electrospun fibers are of tremendous interest in order to overcome limitations and difficulties caused by single component and to develop biomimetic scaffolds. It also addresses both the mechanical and functional requirements of cells (Xize, Shuyan, Zhang, Liu, & Wu, 2019). For example, due to the restricted spinnability of sodium alginate, PVA is added and electrospun by altering the solution and process conditions (Jadbabaei, Kolahdoozan, Naeimi, & Ebadi-Dehaghani, 2021). Spinnability was quite good at 6.5 PVA. To obtain that further, process parameters are adjusted and the scaffolds that were generated under these circumstances have outstanding physical, chemical, mechanical, and biological properties. After 48 hours of incubation, the L929 cell lines had a high vitality. A nanocomposite scaffold for bone tissue regeneration was fabricated by electrospinning a natural polymer gelatin, a synthetic polymer PCL, and also nanohydroxyapatite (Gautam et al., 2021). SEM examination was used to determine the nanocomposite scaffold’s fiber diameter and shape. The quality of nHAp

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Table 17.2 Applications of composite electrospun fiber. Electrospun polymer composite

Tissue engineering application

1

PCL/PVP

2

PVA/Chitosan/PCL

3

PCL/Gelatin/ Hyaluronic acid Cellulose acetate/ Gelatin CFTRIAXONE/PVA

Bone tissue engineering Any diseased or damaged organ Brain tissue engineering Wound healing

Sohrabi, Abbasi, Ansar, and Soltani Tehrani (2021) Sajeev, Anand, Menon, and Nair (2008) Unal et al. (2020)

Wound healing

Fatahian, Mirjalili, Khajavi, Rahimi, and Nasirizadeh (2020) Mani, Jaganathan, Faudzi, and Sunar (2019) Semnani et al. (2017)

4 5 6 7

Polyurethane/ Titanium oxide PCL/Chitosan

8

Chitosan/PVA/CNT

9

Sodium alginate/ PVA Chitosan/PVA/Silk

10

Cardiac tissue engineering Liver tissue engineering Cardiac tissue engineering Liver tissue engineering Skin tissue engineering

Author name and year of publication

Samadian et al. (2020)

Mombini et al. (2019) Manikandan, Yuvashree, Sangeetha, Bhuvana, and Nayak (2020) Fathi et al. (2020)

deposition was also investigated utilizing a variety of characterization methods. Human osteoblasts were seeded on the “Gelatin-PCL-nHAp nanocomposite scaffold, and demonstrated great viability and significant proliferation. Furthermore, over the Gelatin-PCL-nHAp nanocomposite scaffold, cell-scaffold structures revealed excellent cellular adhesion and equally dispersed cells, as well as the usual polygonal morphology of osteoblasts. The Gelatin-PCL-nHAp scaffold was shown to be a promising alternative as a result of the in vitro analysis of electrospun nanocomposite scaffold.” Some of the other combinations of polymers used for tissue engineering are given in the Table 17.2.

17.4 Applications of electro-spun fibers in tissue engineering applications 17.4.1 Use of electro-spun polymers in neural tissue engineering In Fig. 17.3, Chen et al. (2018) attempted to synthesize silica nanofiber (SNF2) by electrospinning using a 1:1; they also synthesized laminin modified SNF2 to

17.4 Applications of electro-spun fibers

FIGURE 17.3 Schematic of the surface modifications of the three SNF2 substrates. (A) Silica nanofibers (SNF2) electrospun onto coverslip; (B) physically laminin-adsorbed SNF2 (SNF2/L); (C) chemically laminin-bonded SNF2 (SNF2-AP-S-L); (D) the chemical reactions for the preparation of SNF2-AP-S-L. Courtesy: Chen, W. S., Guo, L. Y., Tang, C. C., Tsai, C. K., Huang, H. H., Chin, T. Y., Yang, M. L., & ChenYang, Y. W. (2018). The effect of laminin surface modification of electrospun silica nanofiber substrate on neuronal tissue engineering. Nanomaterials, 8(3), 165. (open access).

connect it to the surface of Laminin. The SNF2-AP-S-L, physical and functional experiments demonstrated that it was nontoxic, bioactive, and had sufficient integrity to be classified as a biodegradable scaffold. When the cells named PC12 were seeded onto its surface, the fibers provided good support and helped in cell differentiation, and also the neurite extensions were found to be longer on SNF2AP-S-L, compared to those on unmodified SNF and SNF. These findings suggest substrate might be a suitable biocompatible substrate for tissue engineering applications, as it can support prolonged neuronal development. Neuronal cells derived from bone rMSCs were introduced on engineered scaffolds by M.P Prabhakaran et al. in 2009. Various characterization techniques were used to characterize these nanofibers. MSCs were differentiated into neural cells, and cell proliferation was measured, which revealed that cells grown on

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combined scaffolds had a greater proliferation rate and longer neuronal morphology and neurofilaments, indicating that they might be used in nerve regeneration procedures. PVA electrospun fibers were also used for effective neuronal tissue engineering, Babaie et al. (2020) fabricated and optimized conductive scaffolds that could induce intercellular connections through electrical signals. In this study, electro-spun conductive scaffolds were fabricated out of polyvinyl alcohol and poly(3,4-ethylene dioxythiophene). MTT assay and cell adhesive assay were used to examine their biocompatibility and biological interactions. The results revealed that PEDOT substrates had better physicochemical properties and cell survival. Neural cells derived from rMSCs were analyzed using RT-PCR, and were induced with these scaffolds and subjected with and without electrical stimulations. This study found that combining PVA/PEDOT conductive scaffolds, trying to recreate the function’s neural tissue can increase cellular responsiveness and neural development. Sadeghi et al. (2019) fabricated electrospun nanofibers containing poly (ε-caprolactone) (PCL), chitosan, and polypyrrole (PPy) with those of chitosan and PPy to study and combine the advantages of these electrospun nanofibers. Surface topography and physicochemical properties were analyzed for various compositions of the PCL/chitosan/PPy polymeric scaffolds and the results showed that chitosan in the scaffolds showed improved hydrophilicity. In vitro studies with PC12 cells revealed that the PCL/chitosan/PPy nanofibrous scaffold supported cell adhesion, increased cell propagation, and neurite extension, implying that the PCL/chitosan/PPy nanofiber scaffolds promote cell adhesion, propagate, and multiplication, indicating that they might represent as "convincing neural tissue substitutes.” Heidari et al., in 2019 studied the applications of graphene-doped electro-spun scaffolds in neural tissue engineering. The aim was to evaluate graphene biological properties along with PCL/gelatin nanofibrous mats. SEM analysis and the cellular proliferation of PC12 showed better with presence of graphene. In addition, a drug release study found that the interactions of graphene and TCH led to a controlled release of TCH from electrospun PCL/gelatin/graphene nanofibrous mats. In addition to these properties, improved hydrophilicity and viable biodegradability made these nanofibers potential option for being used as neural tissue engineering scaffolds. Fesharaki et al., conducted a study in 2018, where they studied the neurogenic property of isolated and cultured human scalp adipose derived mesenchymal stem cells (SAD) and evaluate its use in nerve tissue engineering. SADS cells after treatment showed a neurogenic differentiation and the cells were seeded and the impact of electrospun Poly (-caprolactone) (PCL) and PCL/gelatin nanofibrous substrates treated with PRP was also investigated. Their findings revealed that after SADS cells were treated, they exhibited neural cell markers and the CL/gelatin/PRP scaffolds are a promising substrate in in vivo experiments. Naseri-Nosar et al., in 2017 designed a study to develop a drug-loaded scaffold using coaxial wet electrospinning technique, including the fibrils with a

17.4 Applications of electro-spun fibers

core-shell structure. The core was composed of wet-electrospun polylactic acid, and fibril’s coating was made of cellulose acetate, and the scaffold was made of citalopram-loaded gelatin nanocarriers (CGNs). The fibrils created a nonwoven structure, as observed in SEM. The CGN coating rendered the scaffold particularly hydrophilic. It also increased cell viability in rat Schwann cells, according to cytocompatibility tests. The citalopram-containing scaffolds was formed into a neural guiding conduit and implanted in Wistar rats’ sciatic nerves, suggesting that it could help with sciatic nerve repair.

17.4.1.1 Use of electro-spun fibers in cardiac tissue engineering The utilization of nanofibrous scaffolds made of poly (lactic acid) (PLA) and poly (glycerol sebacate) (PGS) elastomers was investigated (Flaig et al., 2020). PGS elastomer was combined with PLA to increase functionalization. The scaffolds were electrospun and fixed to create crosslinking, and tests on them included cytocompatibility, chemical, and mechanical characteristics. The presence of PGS increased material-cell contact. CMs were seeded on these scaffolds, and it was discovered that they were able to replicate the morphology of native tissue with neo-vascularization, and when these scaffolds were grafted on mice hearts, they showed no inflammatory, demonstrating that CMs implanted on PLA: PGS scaffolds increased biocompatibility and cardiomyocyte support, making them a viable biomaterial for cardiac applications. Suh, Amanah, and Gluck (2020) studied how iPSC derived CMs when seeded on electrospun scaffolds can act as a potential tool towards cardiac tissue engineering applications, where the responses are studied to device tissue engineered implants for cardiac repair. As shown in the Fig. 17.4, the use of poly(1,8-octanediol-co-citrate) (POC), which may also be utilized as cardiac patches for tissue healing when combined with poly(-caprolactone) (PCL) and electrospun and it is crosslinked thermally (Vogt & Boccaccini, 2021). Random and aligned PCL/POC fiber mats were examined for their mechanical, physicochemical, and in vitro cytocompatibility characteristics. These PCL/POC fibers were shown to be appropriate for cardiac tissue engineering based on mechanical characteristics and cross-linking. PU/Chitosan/carbon nanotubes composite nanofibrous scaffolds were produced to replicate the extracellular matrix by electrospinning nanofibers (Ahmadi, Nazeri, Derakhshan, & Ghanbari, 2021) and evaluated using a variety of chemical and mechanical characterization methods, as well as cell viability studies. Composite nanofibrous scaffolds were electro-conductive, and aligned nanofibers can potentially be used as scaffolds for regeneration of myocardium after infarction. Mashayekhiyan et al. (2021) showed the use of electrospun nanofibers produced from cardiac extracellular matrix (ECM) that replicate the physicochemical and structural features of cardiac tissue. Polyvinyl pyrrolidone (PVP), gelatin (Gel), and PCL were electrospun into the acellular calf heart ECM in this work. Glutaraldehyde was used in crosslinking the scaffolds, and the resultant fibers were aligned, homogeneous, and bead free. The inclusion of the calf heart ECM

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FIGURE 17.4 Electrospinning of aligned fibers using combination of poly (1,8-octanediol-co-citrate) (POC) and poly(-caprolactone) (PCL). Reproduced with permission from Vogt, L., & Boccaccini, A. R. (2021). Random and aligned electrospun poly (ε-caprolactone)(PCL)/poly (1, 8-octanediol-co-citrate)(POC) fiber mats for cardiac tissue engineering using benign solvents. European Polymer Journal, 160, 110772.

improved the scaffold’s flexibility and hydrophilicity, according to the findings. Furthermore, the ECM’s cell-binding sites improved the viability of cells grown on scaffolds. These findings show that the acellular calf heart ECM, when combined with gelatin, PVP, and PCL, is a suitable biomaterial for creating electrospun scaffolds. Polypyrrole (PPy) scaffolds were used to influence the functioning of synthetic heart tissue (Liang, Mitriashkin, Lim, & Goh, 2021). PPy was electrospun with varying concentrations of SF solution to produce conductive PPy-encapsulated SF nanofibers. The myocardial fibrils were better mimicked by PPy with a smaller fiber diameter. Conductive mats with 7% SF had the best mechanical qualities, resembling genuine myocardium, and had electrical conductivity for CMs. The hybrid mats sustained CM contraction and had elongated and directed sarcomere striations, according to in vitro experiments. In conclusion, these results suggest that the PPy-encapsulated SF electrospun mats could be valuable in various tissue engineering. Mousa et al. (2020) studied the development of a tri-layered nanofiber patch that is compatible and adequately provides mechanical properties and mimics the physiological cardiac tissues. The produced patches were made up of a hydrophilic center layer made up of layer-by-layer electrospun silk fibrin (SF) and polyvinyl alcohol (PVA) composite, with upper and bottom layers made up of PCL polymer and polylactic acid (PLA). The patch’s shape, physiochemistry,

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biodegradability, and mechanical stability were investigated. In vitro tests on these manufactured patches employing a human endothelial cell line (EA. hy926) revealed that they possessed great endothelial cell biocompatibility, mechanical elasticity, and a unique nanofiber structure, contributing to a novel application of cardiac tissue engineering. Electrospinning is used to create random patches of nanofibers to test cardiac tissue repair strategies (Cesur et al., 2020). A cytotoxic, physiochemical, and other mechanical property tests were conducted. The resultant nanofiber patches had smooth, bead-free morphologies. When compared to random nanofiber patches of PLA, PLA/PEG, and PLA/PEG/COL, mechanical characterization findings revealed that aligned nanofiber patches had the highest tensile strength. The aligned patch showed the greatest degradation ratio in an in vitro degradation test. As a result of their research, the newly generated patches demonstrated good tissue alignment in cardiomyocyte cell morphology investigations, indicating that they might be used as a tissue-like cardiac patch as a healing method for myocardial infarction.

17.5 Conclusion In conclusion it can be stated that electrospinning is the technique of nanofiberbased substrate production by the application of a high voltage and under an electric field. A range of process and solution parameters help modify the composition, morphology, porosity, and hence the surface area of the fibers is based on the application they are being fabricated for. Over the years, electrospinning of several natural and synthetic polymers has been extensively investigated to gain an understanding of their characteristics. As the nanofibrous structure of electrospun fibers closely mimic the extracellular matrix prevalent in all eukaryotic cells and tissues, the electrospinning technique is exceptionally desirable in the field for tissue engineering. Several studies have been conducted to characterize a plethora of electrospun fiber as substrates for electrospinning in the last 20 years. Despite the prominent success of the products of electrospinning, the technique still poses limitation in terms of inadequate cell infiltration, heterogeneous pore size and difficulty in optimizing the multiple affecting parameters. The future investigation in this field would be aimed at enhancing the cell migration and design of the fibers to produce ideal tissue engineered substrates.

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Further reading

extracellular matrix. Materials, 13(11), 2661. Available from https://doi.org/10.3390/ ma13112661, PMID: 32545241; PMCID: PMC7321639. Vogt, L., & Boccaccini, A. R. (2021). Random and aligned electrospun poly (ε-caprolactone)(PCL)/poly (1, 8-octanediol-co-citrate)(POC) fiber mats for cardiac tissue engineering using benign solvents. European Polymer Journal, 160, 110772. Vonnegut, B., & Neubauer, R. L. (1952). Production of monodisperse liquid particles by electrical atomization. Journal of Colloid Science, 7(6), 616 622. Available from https://doi.org/10.1016/0095-8522(52)90043-3, Elsevier BV. Wang, X., et al. (2005). Formation of water-resistant hyaluronic acid nanofibers by blowingassisted electro-spinning and non-toxic post treatments. Polymer, 46(13), 4853 4867. Available from https://doi.org/10.1016/j.polymer.2005.03.058, Elsevier BV. Xiao, S., Zhao, T., Wang, J., Wang, C., Du, J., Ying, L., Lin, J., Zhang, C., Hu, W., Wang, L., & Xu, K. (2019). Gelatin methacrylate (GelMA)-based hydrogels for cell transplantation: An effective strategy for tissue engineering. Stem Cell Reviews and Reports, 15 (5), 664 679. Available from https://doi.org/10.1007/s12015-019-09893-4. Xize, G., Shuyan, H., Zhang, R., Liu, G., & Wu, J. (2019). Progress on electrospun composite nanofibers: Composition, performance and applications for tissue engineering. Journal of Materials Chemistry B, 7. Available from https://doi.org/10.1039/ C9TB01730E. Xu, C. Y., et al. (2004). Aligned biodegradable nanofibrous structure: A potential scaffold for blood vessel engineering. Biomaterials, 25(5), 877 886. Available from https://doi. org/10.1016/s0142-9612(03)00593-3, Elsevier BV. Zeleny, J. (1914). The electrical discharge from liquid points, and a hydrostatic method of measuring the electric intensity at their surfaces. Physics Review, 3(2), 69 91. Available from https://doi.org/10.1103/PhysRev.3.69, American Physical Society. Zhang, C., et al. (2005). Study on morphology of electrospun poly (vinyl alcohol) mats. European Polymer Journal, 41, 423 432. Zong, X., et al. (2002). Structure and process relationship of electrospun bioabsorbable nanofiber membranes. Polymer, 43(16), 4403 4412. Available from https://doi.org/ 10.1016/S0032-3861(02)00275-6.

Further reading Al-Kaabi, W. J., Albukhaty, S., Al-Fartosy, A. J. M., Al-Karagoly, H. K., Al-Musawi, S., Sulaiman, G. M., & Soliman, D. A. (2021). Development of Inula graveolens (L.) plant extract electrospun/polycaprolactone nanofibers: A novel material for biomedical application. Applied Sciences, 11(2), 828. Available from https://doi.org/10.3390/app11020828. Amores de Sousa, M. C., Rodrigues, C., Ferreira, I., Diogo, M. M., Linhardt, R. J., Cabral, J., & Ferreira, F. C. (2020). Functionalization of electrospun nanofibers and fiber alignment enhance neural stem cell proliferation and neuronal differentiation. Frontiers in Bioengineering and Biotechnology, 8, 580135. Available from https://doi.org/10.3389/ fbioe.2020.580135. Brigham, C. J. (2017). Chitin and chitosan: Sustainable, medically relevant biomaterials. International Journal of Biotechnology for Wellness Industries, 6(2), 41 47.

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Ginestra, P. (2019). Manufacturing of polycaprolactone—Graphene fibers for nerve tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials, 100, 103387. Available from https://doi.org/10.1016/j.jmbbm.2019.103387. Karimi, A., Karbasi, S., Razavi, S., & Zargar, E. N. (2018). Poly(hydroxybutyrate)/chitosan aligned electrospun scaffold as a novel substrate for nerve tissue engineering. Advanced Biomedical Research, 7, 44. Available from https://doi.org/10.4103/abr. abr_277_16. Schaub, N. J., Johnson, C. D., Cooper, B., & Gilbert, R. J. (2016). Electrospun fibers for spinal cord injury research and regeneration. Journal of Neurotrauma, 33(15), 1405 1415. Available from https://doi.org/10.1089/neu.2015.4165. Xuan, H., Li, B., Xiong, F., Wu, S., Zhang, Z., Yang, Y., & Yuan, H. (2021). Tailoring nano-porous surface of aligned electrospun poly (L-Lactic acid) fibers for nerve tissue engineering. International Journal of Molecular Sciences, 22(7), 3536. Available from https://doi.org/10.3390/ijms22073536. Ziv-Polat, O., Margel, S., & Shahar, A. (2015). Application of iron oxide nanoparticles in neuronal tissue engineering. Neural Regeneration Research, 10(2), 189 191. Available from https://doi.org/10.4103/1673-5374.152364.

CHAPTER

Advances in biomedical polymers and composites: Drug delivery systems

18

Aalok Basu1 and Amit Kumar Nayak2 1

Department of Pharmaceutics, Dr. BC Roy College of Pharmacy and Allied Health Sciences, Durgapur, West Bengal, India 2 Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Odisha, India

18.1 Introduction Polymers have been present in nature since the advent of life (Namazi, 2017). Different polymers, including polysaccharides, nucleic acids (DNA and RNA), and proteins, have played roles in the biological world (Li, Lee, & Dziubla, 2015; Schnitzler & Herrmann, 2012; Zhang, Sun, & Jiang, 2018). Nowadays, starting from daily use to earth’s most cutting-edge operations, polymers have invaded all the possible corners of applications due to their unique molecular arrangements and macromolecular chemistry (George, Sanjay, Srisuk, Parameswaranpillai, & Siengchin, 2020; Sahana & Rekha, 2018; Yang & Kopeˇcek, 2014). The polymer industry is, hence, the fastest developing industry and brings around an invention every other day. Advancements in material sciences over the past decades have led to an exponential development in functional polymers for use in biomedical technology (Kandar, Hasnain, & Nayak, 2021; Maity, Hasnain, Nayak, & Aminabavi, 2021; Nayak, Hasnain, Tabis, & Aminabhavi, 2021; Sahana & Rekha, 2018). Since the last few decades, various composite materials have extensively been applied in diverse fields, including drug delivery, tissue engineering, foods, cosmetics, agriculture, textiles, optoelectronics, automobiles, and aerospace engineering (Hasnain & Nayak, 2019a; Hasnain et al., 2016; Hasnain, Ahmad, Chaudhary, Minhaj, & Nayak, 2019b; Hasnain, Ahmad, Minhaj, Ara, & Nayak, 2019; Mazumder, Nayak, Ara, & Hasnain, 2019; Nayak, Hasnain, Nanda, & Yi, 2019; Nunes, Coimbra, & Ferreira, 2018; Zagho, Hussein, & Elzatahry, 2018; Zhao et al., 2015; Reis, de Moura, & Samborski, 2020). Composites and nanocomposites are quite popular among various materials and biomaterials researchers due to their compelling features, unique “green” designs, ease of preparation, and costeffectiveness (Zagho et al., 2018; Zhao et al., 2015). Composites are essentially multiphase materials, containing one material (in several forms, such as particles, fibers or sheets) embedded in another phase (Hasnain & Nayak, 2018a; Hasnain, Ahmad, Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00009-7 © 2023 Elsevier Inc. All rights reserved.

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Chaudhary, Hoda, & Nayak, 2019a; Mohanty, Vivekanandhan, Pin, & Misra, 2018). The incorporation of particles, fibers, or sheets into a matrix of materials (such as polymers, ceramics, or metals) further enhances their mechanical strength, optical features, water or gas permeability, and electrothermal conductivity. Among various composites, polymer-based composites appear as promising category of biomaterial in the biomedical field as they offer a wide scope for tailoring polymers with specific functional groups to meet very specific requirements (Hasnain & Nayak, 2018a; Hasnain & Nayak, 2019a; Hasnain et al., 2016; Hasnain et al., 2019a; Hasnain, Ahmad, Minhaj, et al., 2019; Nayak et al., 2019; Zagho et al., 2018; Zhao et al., 2015; Hasnain et al., 2019b). Polymer nanocomposites generally comprise a polymer matrix inside which inorganic nanomaterials (such as nanoparticles, nanofibers, nanowires, nanoclays, and nanotubes) are dispersed (Nayak, Alkahtani, & Hasnain, 2021). These polymer composites and nanocomposites often exhibit superior optical, thermal, and mechanical properties. This is due to a synergism of properties of dispersed materials (such as high surface reactivity, large surface area, and high thermomechanical stability) and those of the matrix-forming polymers (such as flexibility, low weight, and high processability). Such properties are further guided by certain aspects, including the type of dispersed nanomaterials within the polymer matrices, their concentrations, sizes, morphologies, and interactions at the interfaces (Kumar et al., 2018). Multidisciplinary research in drug delivery is gradually transforming into industrial developments and many polymer composite based products have been investigated as drug delivery carriers that are ready to dominate the market (Chowdhury, Chakraborty, Maity, Hasnain, & Nayak, 2020; Sharma et al., 2021). This chapter gives an overview of the advancements in the synthesis of polymer composites and their applications related to drug delivery.

18.2 Synthesis of polymer composites The application of polymer composites in drug delivery generally relies on the procedure used to synthesize the desired structures. Different methods have been successful in producing composites with tailored microstructures, unique properties, and controlled drug releasing capacities (Fig. 18.1). Some of these methods are summarized in the following sections.

18.2.1 Hydrothermal method The hydrothermal method involves high temperature (up to 180 C) and high vapor pressure (about 1 atm) conditions. The solvent characteristics of various compounds are prone to drastic changes at such extreme conditions, leading to in situ growth of composites in tunable and controlled morphology. Ramadas and his coworkers developed hydroxyapatite nanorods on graphene oxides sheets through simple hydrothermal process (Ramadas, Bharath, Ponpandian,

18.2 Synthesis of polymer composites

FIGURE 18.1 Classification of conventional methods for synthesis of polymer composites.

& Ballamurugan, 2017). This binary nanocomposite system exhibited superior biocompatibility and was intended for applications in drug delivery, orthopedic and dentistry applications. Nanocrystalline hydroxyapatite dispersed in poly(lactide-co-glycolide) (PLGA) composite system was also prepared via hydrothermal route and was designed for prolonged delivery of bone morphogenetic protein in osteoporosis patients (Liu & Webster, 2010). This design boasted of dual-phase drug release profile as opposed to conventional drug carriers. Certain workers used combination routes, such as coprecipitationhydrothermal method for synthesis of drug-loaded organic/inorganic nanocomposites. The temperature requirement for the modified method is significantly reduced and allows loading of various drugs, including pirenoxine sodium and dexamethasone disodium phosphate (Wang, Zhou, Fang, & Cao, 2020; Xu, Xu, Gu, Fang, & Cao, 2018). The hydrothermal method is nevertheless energy-consuming and often causes the deformation of imprinted cavities at high temperature.

18.2.2 In situ polymerization During in situ polymerization the fillers and monomers are well mixed. In general, the monomers intercalate within the layers of filler matrices. The intercalated

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monomers are then polymerized either by surface functionalization at the matrix surface or through addition of certain agents which catalyze the process. A stimuli-responsive system for delivery of doxorubicin (an anticancer drug) was fabricated by Morfin-Gutierrez et al., through polymerization technique (MorfinGutierrez, Sa´nchez-Orozco, Garcı´a-Cerda, Puente-Urbina, & Mele´ndez-Ortiz, 2020). This system comprised magnetite nanoparticles grafted with poly(N-vinylcaprolactam) and demonstrated excellent cancer cell targeting ability.

18.2.3 Electrospinning method Electrospinning is an attractive method for designing of polymer fiber based composites for drug delivery applications. In this approach, polymer solution or melt is introduced into an electric field (Fig. 18.2) (Luraghi, Peri, & Moroni, 2021). High voltage causes surface charge repulsion of the melt, which forms the spherical drop of viscoelastic solution on the needle into a cone known as the Tylor’s Cone. Once ejected, the charged jet solution evaporates to form fibers of desired dimensions and is collected on the collector. Electrospinning is performed in normal conditions of temperature and pressure. It does not require any expensive setup. The ability to apply on natural and synthetic polymers makes electrospinning a versatile process for the synthesis of polymer composites and nanocomposites (Aruchamy, Mahto, & Nataraj, 2018). The fibrous texture of the electrospun nanocomposites offers site-specific drug delivery features through diffusion or degradation mechanisms. Many drugs,

FIGURE 18.2 A scheme of electrospinning process (Luraghi et al., 2021). From Luraghi, A., Peri, F., Moroni, L. (2021). Electrospinning for drug delivery applications: A review. Journal of Controlled Release: Official Journal of the Controlled Release Society, 334, 463 484., with permission, Copyright © 2021 The Author(s), published by Elsevier B.V.

18.2 Synthesis of polymer composites

including antibiotics, proteins, and DNA, have been loaded into these delivery devices. Spadaro et al. fabricated nanofibrous composites to design dual-sensitive delivery systems for cancer therapy. PEGylated PLGA nanofibrous composites containing gold and iron oxide nanoparticles and silibinin were developed which could allow the clinicians to control the rate of drug release at the target site through the applications of light and magnetic fields (Spadaro et al., 2018). In another work, modified coaxial electrospinning technique allowed Yang et al. to produce a new kind of structural nanocomposite in which polyvinylpyrrolidone and diclofenac sodium core was coated by shellac (Yang et al., 2018). A nanocoat over amorphous hydrophilic composite material was formed and the design offered a colon-targeted pulsatile drug release profile. A porous composite nanofiber based controlled-release system containing a core of PEG and salicylic acid blend and a sheath of polylactic acid was prepared (Nguyen, Ghosh, Hwang, Chanunpanich, & Park, 2012). Porous and nonporous systems were configured through variation in solvent content ratio. Evaluations revealed that the salicylic acid release was higher from the porous nanofiber based composite and followed one-dimensional Fickian mechanism. Electrospinning process involves a large number of parameters with drugcomposite system, structural morphologies, and release rates for designing the top-notch in vivo drug delivery devices (Rogina, 2014). Although this method has been used on several polymer systems for drug delivery applications, a deeper understanding on the processing parameters is still necessary.

18.2.4 Three-dimensional printing technology Three-dimensional (3-D) printing or additive manufacturing process has been used to fabricate 3-D objects from a virtual model by depositing layer-by-layer of printed materials in a confined volume (Hossain, Chowdhury, Shuvho, Kashem, & Kchaou, 2021; Jime´nez, Romero, Domı´nguez, Espinosa, & Domı´nguez, 2019). This allows the composition to be varied throughout the 3-D microstructure, thus providing a degree of control, which are not possible using traditional fabrication techniques. 3-D printing has been used to fabricate microstructures and nanostructures for drug delivery (Curry, Henoun, Miller, & Nguyen, 2017; Qi & Craig, 2016). The final form usually contains a number of bioactives and structural components. The major challenge is the selection of suitable hosts and materials that can be processed using this technique (Giannatsis & Dedoussis, 2009). Very accurate quantities of therapeutics and scaffolding materials can be deposited through printing. Concentration gradients and spatial patterns of drug deposition can further be incorporated into the polymer matrix design. Rattanakit et al. applied a novel extrusion printing technology to produce a delivery device wherein dexamethasone-21-phosphate disodium salt was loaded into biodegradable composites of PLGA/polyvinyl alcohol (PVA). The release of drug from the device could be controlled through the spatial distribution of drug within the printed polymer matrix (Rattanakit, Moulton, Santiago, Liawruangrath,

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& Wallace, 2012). Recently, Reddy Dumpa et al. applied hot-melt extrusionpaired fused deposition modeling 3D printing technology for creating the shells of a novel gastro-retentive pulsatile drug delivery device (Reddy Dumpa, Bandari, Repka, & Novel, 2020). The shell material was composed of hydroxypropyl cellulose- and ethylcellulose-based filaments, while the core was made using a compressed theophylline tablet. This system demonstrated high residence time in the stomach as well as caused pulsatile delivery of theophylline. A polymer-magnetic nanocomposite was designed by Bozuyuk et al. (2018), which could deliver doxorubicin on demand. The work described the fabrication of magnetically controlled micro-swimmers through two-photon direct laser writing technology. Modified chitosan and biocompatible superparamagnetic iron oxide nanoparticles were used to build the matrix and azide-modified doxorubicin was considered as the model bioactive for light-stimulated drug release. It is obvious that the revolutionary 3-D printing technique can be used to engineered composite scaffolds, which offer the desired characteristics through precise manipulation of the assembly process for a given material composition. Reduction in requirements for raw materials, labor costs, faster manufacturing time, and ability to customize according to geometric complexities are some advantages of 3-D printing technique over the traditional methods. Although 3-D printing is continued to be used for the development of materials and complex structures, the application of this technology for the designing of drug delivery systems with sophisticated functionalities is in its early stages (Bekas, Hou, Liu, & Panesar, 2019; Moulton & Wallace, 2014).

18.3 Characterization and drug release properties Understanding of the various physicochemical features of composites (such as surface morphology, stability, or solubility) is necessary for their effective uses in drug delivery applications. Analyses of these features and interactions (in vitro and in vivo) are essential for better identification of their target ability and behavior inside the body at molecular levels. Several analytical methods have been routinely used for the determination of properties, such as size, shape, composition, surface functionalities, purity, and stability of the composites. The methods include Fourier transform infrared (FTIR), dynamic light scattering, zeta potential, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy, and thermal analysis for elucidation of morphological and structural features of composites and nanocomposites.

18.3.1 X-ray diffraction XRD is a multipurpose and nondestructive tool that provides details of the chemical composition and structural information of the specimen studied. XRD vividly

18.3 Characterization and drug release properties

describes the crystallinity and crystal size of samples along with their d-spacing patterns and phase transition. When X-ray hits the atoms in a crystal lattice, they cause scattering phenomenon. Diffraction occurs when the scattered rays from one atomic plane are in phase with the rays from another plane. The interplanar distances can be calculated from Bragg’s equation. XRD analysis of composites reveals the kinds of interactions between the filler and polymer. The mechanism of cargo loading and intercalation of molecules can also be understood from a combination of d-spacing and 2θ values. XRD has been used structural analysis of liquid crystalline hydrogels impregnated with silver nanoparticles, characterization of a novel hydrogel graphene nanoparticle composite loaded with methotrexate, and obtaining the microstructural parameters of PVA-coated magnetic nanocomposites for target-specific delivery of metformin (Ghosh et al., 2021; Lali Raveendran, Kumar Sasidharan, & Devaki, 2017; Saeednia, Yao, Berndt, Cluff, & Asmatulu, 2017). The core shell structure of cellulose and silver nanocomposite was well identified through XRD analysis (Karuppusamy, Pratheepkumar, Dhandapani, Maruthamuthu, & Kulandainathan, 2015). Results confirmed that the slow release of colloidal silver could be pH triggered for enhanced wound healing applications.

18.3.2 Fourier transform infrared spectroscopy FTIR is another useful tool for characterization of composites. The infrared energy is a part of the electromagnetic spectrum and consists of three regions: far-IR (40 400 cm21), mid-IR (400 4000 cm21), and near-IR (4000 14,000 cm21). This technique reveals the structural information as well as molecular interactions, as it relies on the mode of vibration of molecules. The principle of IR spectroscopy rests on the fact that the energy absorption of an irradiated sample is the result of the transitions between the molecular levels of rotational and vibrational energies. These transitions are very sensitive to the structure of the molecules and can be used for identification of interactions responsible for drug loading and release (Basu et al., 2017; Onnainty & Granero, 2019). Most of the works relate the formation of composites through chemical conjugation of the individual materials. Anirudhan and Chithra Sekhar (2020) have identified the surface interactions of doxorubicin and chitosan with layer double hydroxide, which cause successful encapsulation of the drug in the nanocomposite. Application of FTIR for detection of impurity in nanocomposite has been reported by Gopala Kumari et al. (Gopala Kumari, Manikandan, Pakshirajan, & Pugazhenthi, 2020). They showed that the loading of norfloxacin into the double hydroxide layers did not contaminate the polymer nanocomposite with carboxylates. Interactions between synthesized nanoparticles and polymer matrix can be well perceived through FTIR spectroscopy. These interactions contribute to the mechanical stability of nanocomposites and also in their drug release profile (Raja Namasivayam, Venkatachalam, & Arvind Bharani, 2020).

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18.3.3 Thermal analysis Thermal analysis includes a number of techniques that determine the subtle alterations in the physical and chemical natures of the sample as a function of temperature. Generally, a combination of the differential scanning calorimetry (DSC) and the thermogravimetric analysis (TGA) is useful for understanding the temperature effects on solid composites. DSC analysis has been used for polymer characterization, which provides information related to melting point, glass transition, crystallization, and changes in heat capacity. It is fast, reasonably accurate and is widely used for thermal analysis. TGA is another tool used for the determination of weight changes of a particular sample during heating and associated reactions. Though it has been used for studying degradation kinetics of polymer composites, it can also be applied for quantification of impurities, additives, and residual solvents. TGA is routinely used to understand the effect of nano-additives impregnation on the thermal stability of polymer matrices (Pielichowski & Pielichowska, 2018). However, certain workers, such as Shah et al., have applied both DSC and TGA analyses for obtaining various information about drug-loaded nanocomposites (Shah, Ashames, Buabeid, & Murtaza, 2020). This information included polymer polymer miscibility, certain evidence of interpolymer hydrogen bonding, composite degradation profile, and distribution pattern of drugs within the composites.

18.3.4 Scanning electron microscopy SEM is a powerful surface imaging technique that provides detailed information regarding structural properties, surface morphology, particle size, and crystalline nature of composite materials. Due to such high accuracy and resolution capacity, in-depth characterization of various kinds of composites is possible. SEM offers 3-D surface information of the analyzed sample. The technique uses a punctual beam of electrons under vacuum to generate secondary electrons at the surface of the sample. The secondary electrons are amplified through a photomultiplier and directed toward a tube on whose screen the image is formed. Drying of the samples is critical to maintain optimum structural stability. The dried samples are neatly coated through gold or platinum sputtering technique, which is performed under vacuum. The notable feature of SEM is the high depth of field that generates sharp image of large structures (Sarabandi, Gharehbeglou, & Jafari, 2020). Orsu and Matta (2020) reported that the porous nature of carboxymethyl guar gum nanocomposite played an important role in release of ciprofloxacin. Sometimes, a degree of porosity is necessary to facilitate water penetration inside polymer matrix, leading to higher water absorption capacity. This property is essential in case of nanocomposite hydrogels capable of drug releasing under hydrated conditions (Singh, Kumar, & Dhaliwal, 2020). Certain proof of polymerization and chemical reactions during the synthesis of nanocomposites are also available from SEM studies (Anirudhan, Chithra Sekhar, & Athira, 2020).

18.3 Characterization and drug release properties

18.3.5 Determination of drug loading into composites Drugs or bioactives are sometimes incorporated within the matrix during composite preparation. They may also be attached covalently onto the material surface or to the polymer before composite assembly. There are many instances when drug molecules have been attached onto the surface or inside the cavities of the preformed composites. The amount of drug entrapped or encapsulated within the composites generally depends on the solvent to dissolve or disperse it, as well as on the sorption ability of the polymer matrix. The property of drug loading can be expressed by two parameters: drug loading capacity (DLC) and entrapment efficiency (EE) (Yaneva & Georgieva, 2018). EE can be described as the quantity of bioactives encapsulated or incorporated within the composite matrix divided by its total quantity in the formulation. It is expressed in form of the following equation (Eq. 18.1): %EE 5

Total amount of drug 2 amount of drug present in residual solvent 3 100 (18.1) Total amount of drug

DLC represents the mass of drug present in the composite divided by the total mass of the composite. DLC can be expressed using the following equation (Eq. 18.2): %DLC 5

Total amount of drug 2 amount of drug present in residual solvent 3 100 (18.2) Weight of composite

There are often several challenges faced during accurate quantification of these parameters. A combination of various analytical techniques must, therefore, be judiciously chosen.

18.3.6 Estimation of drug release from composites The rate and amount of drug released from composites depends on various factors (AlShaafi, 2017; Aydin, 2020). They include the molecular arrangement of the composite matrix, mechanism of drug loading into the system, crystalline state of the drug postentrapment, and the nature of release medium. There are a number of approaches undertaken to facilitate drug release from the composites into the dissolution medium and some of them have been described next. 1. Dialysis bag approach: During dialysis a diffusion of small molecules (in this case, drugs) occurs from a higher concentrated solution to a lower concentrated solution across a semipermeable membrane (Fig. 18.3A). This technique is generally used for in vitro drug release estimation from nanobased systems. The procedure involves placing the buffer-dispersed nanocomposite system inside a dialysis bag and submerging the bag into the dissolution medium. The system should be kept under stirring or shaking conditions at 37 C 6 0.5 C. Then aliquots of the solution are withdrawn at certain intervals from the dissolution medium and must be replaced with same volume of fresh buffer to maintain sink condition.

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FIGURE 18.3 Drug release studies using (A) dialysis bag, (B) paddle method, and (C) Franz diffusion cell.

2. Paddle method: Release of drug from composites can be stimulated using a paddle rotated at an optimum speed. Composites should be immersed into the dissolution medium at 37 C 6 0.5 C (Fig. 18.3B). Aliquots of the medium are taken at predetermined time intervals and replaced with fresh fluids. The samples are filtered using a 0.45-μm membrane filter and are analyzed through appropriate analytical techniques. 3. Franz diffusion cell apparatus: Drug release from composites intended for transdermal applications are evaluated using Franz diffusion cell (Fig. 18.3C). This approach consists of phosphate buffer and a dialysis membrane (or animal skin) placed between the donor and receptor compartments. The receptor compartment is maintained under stirring conditions using a magnetic stirrer and the temperature of the medium is well controlled through a jacketed water-circulation system. Aliquots of the sample are withdrawn at specific intervals and would be replenished accordingly.

18.3.7 Mathematical treatment of drug release kinetics Most of the works related to drug delivery systems are evaluated and analyzed by fitting with different kinetic models to explain the drug release mechanisms from dosage forms and composites. The data generated from the release study are fitted into kinetic equations to understand the mechanism of drug release from the composites. The kinetic models generally applied are zero-order (cumulative percent drug release vs time), first-order (log percent drug cumulative remaining vs time), Higuchi (cumulative percent drug release vs square root of time), Hixson Crowell (cube root of

18.3 Characterization and drug release properties

cumulative percent drug remaining vs time), and Korsmeyer Peppas models (Hasnain et al., 2016; Yaneva & Georgieva, 2018). These models are discussed in detail in the following section. 1. Zero-order release kinetic model: When the release of the drug from the composite is independent of the drug concentration, the drug release pattern may be said to be following zero-order kinetics. This phenomenon can be represented by the following equation (Eq. 18.3). Qo 5 Qt 1 K0 t

(18.3)

where Qt is the percent of drug released at time t, and Q0 is initial percent of drug in dissolution medium. K0 is the zero-order rate constant and can be expressed as concentration/time. For the kinetics studies the results obtained from in vitro drug release experiments are plotted as cumulative amount of drug release versus time. The value of K0 is thus derived from the slope of the linear plot. 2. First-order release kinetic model: Concentration-dependent drug release from composite can be related through first-order kinetics model and is expressed using the following equation (Eq. 18.4). logCt 5 logC0 2 Kt =2:303

(18.4)

where Ct is the concentration of drug released at time t, C0 is the initial concentration of drug encapsulated in the composite, Kt is the first-order rate constant, and t is time. The results obtained from the in vitro studies are plotted as log cumulative percent of drug remaining versus time which would give a straight line with slope 2 Kt/2.303. This model can explain the drug dissolution in composite, which contain water-soluble drugs in porous matrix. 3. Higuchi model: The drug released from an insoluble polymer matrix through diffusion can be expressed by the Higuchi equation. The Higuchi model considers that: (1) the initial concentration of drug in the composite is higher than drug solubility, (2) the drug diffusion occurs in one dimension only, (3) drug particles are much smaller compared to composite thickness, (4) molecular diffusivity remains constant throughout, and (5) sink condition is maintained in the dissolution medium throughout the experiment. Higuchi model can be expressed by the following equation (Eq. 18.5). Qt 5 KH x t0:5

(18.5)

where KH is Higuchi’s constant and can be derived from the slope of the linear plot of cumulative percent drug release versus square root of time. 4. Hixon Crowell model: According to this model, it is assumed that the composite’s regular area of composites is proportional to the cube root of its volume. The drug release pattern from the composite that follows first-order kinetics may be expressed using the equation (Eq. 18.6). 1=3

W0

1=3

2 Wt

5 κt

(18.6)

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where W0 is the initial quantity of drug entrapped within the composite; Wt is the remaining quantity of drug inside the formulation at time t. The surface volume relation constant is denoted by κ (kappa). This equation has been applied to certain systems, where drug release takes place in planes perpendicular to the drug surface. 5. Korsmeyer Peppas model: This model is used to describe the mechanism of drug release from a polymeric matrix. To study the mechanism, first 60% of drug release data are fitted into the Korsmeyer Peppas equation which is described in the following (Eq. 18.7). Mt 5 Ktn MN

(18.7)

where Mt/MN is the ratio of drug released at time t, n is the release exponent, and K is rate constant. The equation can be rewritten in logarithmic form (Eq. 18.8). logQt 5 logK 1 nxlog t

(18.8)

In this model the value of the release exponent or n depicts the drug release mechanism. The part of the release curve, where Mt/MN is less than 0.6, is applied to determine the n value. To study the drug release kinetics, the data obtained from the dissolution experiments are plotted as log percent cumulative release versus log time. There are other models used for studying drug release mechanism from polymeric systems. Some of them include the Baker Lonsdale model for determination of drug release from spherical diffusion rate-limiting matrix (Huang, Tsui, Tang, & Gu, 2018) and the Hopfenberg model applicable for surface eroding polymer devices (Geraili & Mequanint, 2020). However, these models are very specific to certain composite systems and lack adaptability.

18.3.8 Mechanisms for controlling drug release from composites Drug release profiles of composite-based drug delivery carrier systems are governed by various factors, such as drug solubility, molecular diffusion across polymer matrix, desorption of surface-bound drug molecules, polymer porosity, degradation, and erosion (Hasnain et al., 2016; Son, Lee, & Cho, 2017). The drug release mechanism can thus be precisely guided through interplay of all these factors. It is well known that the cargo molecules attached to the surface are rapidly released by desorption. The initial burst release is observed since the encapsulated drug is adsorbed or weakly associated to the relatively larger surface of the polymer matrix and can be released through diffusion (Cheng et al., 2010). The drugs, which have a higher affinity toward the matrix, are, however, released quite slowly through diffusion, and sometimes erosion (Raut, Gahane, Joshi, Kalthur, & Mutalik, 2019).

18.4 Applications in drug delivery

Drug release from carrier systems can also be facilitated through the application of internal and external stimuli. Such stimuli enable release of drugs in a spatiotemporal manner and these “stimuli-responsive” polymer nanocomposites have attracted considerable interests in the field of drug delivery due to their significant control over cargo release from the nanosurface (Lavrador, Esteves, Gaspar, & Mano, 2021; McCarthy, Zhang, & Abebe, 2021). Internal stimuli-mediated drug release depends on innate physiological states or biological processes, such as redox potential, pH change, and enzymatic activity (Hasnain & Nayak, 2019b; Raza et al., 2019). In pH-triggered and redox potential dependent release, the acidic pH of endosomes or tumor microenvironment initiates the drug release (Das et al., 2020; Hasnain & Nayak, 2019b). Moreover, the enzymes overexpressed in tumor microenvironment or those produced within the subcellular compartments can also facilitate the drug releasing. On the contrary, external actuation offers even greater control using external forces, such as ultra-violet (UV) and visible light irradiations, photothermal therapy, magnetic fields, and ultrasounds (Hasnain & Nayak, 2018b; Hasnain & Nayak, 2019b; Raza et al., 2019). For example, nanocomposites can be guided, and drug delivery can be triggered as well as monitored through use of an external magnetic field (Wang et al., 2018; Zhu, Li, Peng, Hosseini Nassab, & Smith, 2019). Ultrasounds and photothermal therapy involve the use of acoustic perturbation or near-infrared light, respectively, to increase the local temperature of a particular tissue causing cell death or to initiate drug release from the composite devices (Tan et al., 2020; Wang et al., 2019). These systems aim to reduce the side effects, increase efficacy, and can be tuned specifically according to patient’s changing needs. Though externally triggered drug delivery is an essential step toward “personalized medicines,” there are certain issues related to clinical translation, regulatory aspects, and instruments required for the drug release activation (Said, Campbell, & Hoare, 2019) (Fig. 18.4).

18.4 Applications in drug delivery Inclusion of polymer composites in drug delivery applications has drawn significant attention due to multiple functionalities (Sharma et al., 2021; Zhao et al., 2015): (1) protection against early release of drugs, (2) efficient facile drug loading, (3) ability to overcome various intra- and extracellular barriers in the physiological system. Certain features such as zero toxicity, excellent bioavailability, high water solubility, and minimum antigenic effects make polymer nanocomposites one of the preferred choices for drug delivery applications (4). Some of the significant applications of polymer composites in drug delivery have been discussed in the following sections.

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FIGURE 18.4 Different stimuli that trigger drug release at the target site.

18.4.1 Tumor-targeted drug therapy Cancer has caused over 10 million deaths worldwide in 2020 (according to World Health Organization) and is acknowledged as one of the biggest challenges in medical sciences. In the recent years, polymer composites have been routinely tested for treatment as well as detection of tumor cells, and controlled irradiation of specific tumor sites (Cheng et al., 2018). These strategies usually involve the use of near-infrared light to raise the local temperature (i.e., hyperthermia) at the target sites. Such a device may also include chemotherapeutic cargo, which can be released with the help of an external trigger. Polymer composites are thus playing a major role in designing of novel photo-chemotherapeutic strategies, which combine the individual merits of the respective therapies and present a more efficient anticancer response with delivery of lower therapeutic dose (Wen et al., 2021). Most of the reported nanomaterials depend on the enhanced permeation and retention effect for passive entry into the tumors and, consequently, lead to insufficient accumulation of the drug at the tumor site. However, tumor cells display certain features that offer various opportunities for cancer therapy, including higher temperature within the cells, acidic microenvironment, and higher glutathione concentration than that found in normal cells (Niu et al., 2018; Xiao et al., 2019; Yang et al., 2019). These findings encouraged researchers to design composite systems based on pH-, temperature-, or glutathione-responsive polymers for control releases of anticancer drugs (Chen et al., 2020; Mao et al., 2018;

18.4 Applications in drug delivery

Wang et al., 2014). These delivery strategies can be applied to a variety of tumor types and overcome the difficulties associated with conventional chemotherapy. Some recently investigated polymer composites for tumor-targeted drug therapy are listed in Table 18.1.

18.4.2 Ophthalmic drug delivery Ocular drug delivery is another challenge for formulation scientists and clinicians who are involved in the mitigation of ophthalmic conditions, such as dry eyes, corneal neovascularization, glaucoma, and bacterial infections (Gote, Sikder, Sicotte, & Pal, 2019; Kang-Mieler, Rudeen, Liu, & Mieler, 2020). There are numerous anatomical and physiological barriers, such as nasolacrimal drainage, tear turnover, reflex blinking, and conjunctival absorption, which limit the delivery of drugs to the ocular tissues (Gote et al., 2019). Polymer nanocomposites, including micelles, nanosuspension, and hydrogels, have been used to improve ocular bioavailability either through enhanced corneal penetration or increasing the drug retention period on the ocular surface (Almeida, Amaral, Loba˜o, & Lobo, 2014; Xu et al., 2018). Hydrogel composites have been an interesting platform for enhancement of drug retention period due to high viscosity and mucoadhesive capacity (Gorantla et al., 2019). These hydrogels often comprise polymers and copolymers that can extend noncovalent bonds with the mucin present over the corneal surface and they may further impart sustained release characters to the system. Drug release from ocular nanocomposite can also be triggered by the use of an external stimulus (Huu et al., 2015). However, it is worth noting that use of light and UV radiation as stimuli can raise concerns of side effects in the cornea and retina. Thermoresponsive hydrogels can offer sustained release profiles through sol gel transitions without compromising the safety of corneal epithelial cells. Copolymer-based in situ gelling agents provide better precorneal retention by converting the liquid eye drops into a gel, hence improving ocular bioavailability of the drug (Chou, Luo, & Lai, 2017). Advanced nanocomposites employing a combination of stimuli-responsive and in situ gelling strategies hold great potentials for patient compliance and enhanced therapeutic efficacy. Some recently investigated polymer composites for ocular drug delivery are listed in Table 18.2.

18.4.3 Buccal drug delivery Delivery of drugs through the buccal route has attracted a certain deal of interest as it offers a direct access of drugs to the blood circulation by the jugular vein, thereby eliminating the chances of hepatic metabolism (Hasnain et al., 2020; Smart, 2005). However, there are certain aspects, such as low permeability and small absorptive area, which limit the delivery of drug through the buccal cavity. Since last few years many, composite-based polymeric films and hydrogels with mucoadhesive properties have been experimented to circumvent the

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Table 18.1 Some recently investigated polymer composites for tumortargeted drug therapy. Polymer composites for tumortargeted drug therapy Poly(D,L-lactide-co-glycolide) chitosan composite particles Multifunctional PEG chitosan iron oxide nanocomposites Polyoxalates cross-linked chitosan nanocomposites Hyaluronidase enzyme corechitosan PEG gelatin polymer nanocomposites Magnetic nanocomposites of folatemodified chitosan/carboxymethyl surface and bovine serum albumin nanoparticles Hybrid nanocomposite consists of a pH-responsive chitosan, a thermosensitive poly(Nvinylcaprolactam), and a functionalized cell-penetrating peptide Chitosan overlaid Fe3O4/reduced graphene oxide nanocomposite Chitosan-based nanocomposite with legumain sensitive properties Magnetic chitosan-cis-aconitic anhydride doxorubicin nanocomposite Electrospun poly(ε-caprolactonegelatin) @ polydopamine composite nanofibers Inhalable lactoferrin/chondroitin sulfate monoolein nanocomposites Inhalable lactoferrin/chondroitin sulfate nanocomposites Inhalable phospholipid enveloped lipid core nanocomposites Inhalable boronic acid decorated albumin nanocomposites Nanocomposite based on hyaluronic acid ceramide and Soluplus

Drugs

References

Paclitaxel followed by topotecan Methotrexate

Karthika et al. (2020)

Cisplatin

Rajan, Murugan, Ponnamma, Sadasivuni, and Munusamy (2016) Rajan, Raj, Al-Arfaj, and Murugan (2013)

5-Fluorouracil

Lin et al. (2015)

Paclitaxel

Bano, Afzal, Waraich, Alamgir, and Nazir (2016)

Doxorubicin and oleanolic acid

Chen et al. (2020)

Doxorubicin

Karthika et al. (2020)

Doxorubicin

Luo et al. (2018)

Doxorubicin

Xu, Wu, and Jiang (2015)

Doxorubicin

Cen et al. (2020)

Resveratrol

Abdelaziz et al. (2020)

Doxorubicin and ellagic acid Rapamycin and berberine Etoposide and berberine Resveratrol

Abd Elwakil et al. (2018)

Kabary et al. (2018) Elgohary et al. (2018) Lee et al. (2016)

18.4 Applications in drug delivery

Table 18.2 Some recently investigated polymer composites for ocular drug delivery. Polymer composites for ocular drug delivery

Drugs

References

Composite films of hyaluronic acid and hydroxypropyl methylcellulose

Timolol maleate

Pentablock copolymer-based composite nanosystems made of polycaprolactone, polyethylene glycol, and polylactic acid Bilayered films of hydroxypropyl methylcellulose and Eudragit Intercalated nanocomposites with chitosan glutathione glycylsarcosine and layered double hydroxides Composite of poly (lactide-coglycolide) nanoparticles and chemically cross-linked hyaluronan hydrogel Ocular insert made of composite containing polymethyl methacrylate casting with polyethylene oxide Composite lipoidal chitosan poly (ε-caprolactone) nanosystem Nanocomposites made of liposome, glycylsarcosine-anchored layered double hydroxides Nanocomposites-based systems composed of hyaluronan nanogels and Pluronic F68 micellar solutions Thermosensitive nanocomposite hydrogel-based delivery systems

Lysozyme (B14.5 kDa), IgG-Fab (B50 kDa), and IgG (B150 kDa)

Tighsazzadeh, Mitchell, and Boateng (2019) Patel, Vaishya, Pal, and Mitra (2015)

Chloramphenicol Pirenoxine sodium

Boateng and Popescu (2016) Xu et al. (2018)

Bovine serum albumin

Hsu, Wu, Hsieh, and Huang (2021)

Betaxolol HCl

Gevariya and Patel (2013)

Indomethacin

Du Toit et al. (2013) Gu et al. (2019)

Dexamethasone disodium phosphate Ferulic acid

Grimaudo et al. (2020)

Cefuroxime

Sapino et al. (2019)

disadvantages associated buccal administration of drugs (Laffleur, 2014). They guard the drug molecules from degradation at oral pH and release them toward the mucosa in controlled manner. High mechanical stability and mucoadhesivity can be achieved using cationic and anionic polymers (Laffleur, 2014; SalamatMiller, Chittchang, & Johnston, 2005; Shinkar, Dhake, & Setty, 2012). While anionic polymers, such as alginate and carboxymethylcellulose, bind to mucin protein through hydrogen bonds, cationic polymers, such as chitosan, form thiol or sulfide bonds with mucin (Salamat-Miller et al., 2005). These polymers have been blended with inorganic clays, carbon nanotubes, metal, solid lipid, and polymeric nanoparticles to form functional nanocomposites suitable for buccal delivery of drugs.

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Hydrogel composites have been investigated as a versatile platform for encapsulation and release of drugs, including chlorhexidine, calcitonin, ornidazole, curcumin, doxorubicin, and several others into the buccal cavity (Morantes et al., 2017). This is due to the fact that hydrogels can minimize interactions between the polymer matrix and active site, improve drug encapsulation efficiency, and extend the residence time in the oral mucosa. Nanoparticle-impregnated polymer films are also a popular means of delivery of therapeutics through the buccal route. This approach has been successful in improving the bioavailability of nicotine, certain flavonoids, antiretrovirals, antihypertensives, etc. (Macedo et al., 2020; Mahmoud, Ali, Raafat, Badawy, & Elshahawy, 2018; Okafor, Ngoepe, Noundou, & Mac¸edo Krause, 2019). Some recently investigated polymer composites for buccal drug delivery are listed in Table 18.3.

Table 18.3 Some recently investigated polymer composites for buccal drug delivery. Polymer composites for buccal drug delivery

Drugs

References

Thermoresponsive composites of Pluronic F127 and polyethylene oxide Nanofibrous composite buccal films of chitosan of poly(vinyl alcohol) Chitosan/poly(lactide-co-glycolide) nanoparticles nanocomposite buccal films Composite of in situ gellan gum gel and amino methacrylate copolymer microparticles Composite hydroxypropyl methylcellulose and sodium alginate Modified glutinous-rice starchchitosan composite films

Paclitaxel/dimethylβ-cyclodextrin

Choi et al. (2014)

Terbinafine HCl

Szabó, Daróczi, Tóth, and Zelkó (2016) Santos et al. (2017)

Composite dosage form of minitablet matrix-bilayer mucoadhesive film

Desmopressin acetate

Freeze-dried composite wafers made of metolose carrageenan and metolose chitosan Chitosan/gelatin/keratin composite mucoadhesive buccal patch

Aspirin

C-glycosyl flavonoid enriched fraction of Cecropia glaziovii Aceclofenac

Nicotine Lidocaine HCl

Hydrocortisone sodium succinate

Elmowafy, Cespi, Bonacucina, and Soliman (2019) Okeke and Boateng (2016) Soe, Pongjanyakul, Limpongsa, and Jaipakdee (2020) Kottke, Burckhardt, Knaab, Breitkreutz, and Fischer (2021) Farias and Boateng (2020) Davoudi et al. (2018)

18.4 Applications in drug delivery

18.4.4 Drug delivery for bone tissue regeneration Bone healing or regeneration is a complicated process and polymer composites have been tested as newer class of materials to deal with traumatic injuries and bone disorders (Hasnain et al., 2019a; Nayak, Mohanta, Hasnain, Hoda, & Tripathi, 2020). The basic requirements for all such materials are (1) to facilitate tissue regeneration at the defective sites and (2) to eventually get reabsorbed into the newly formed bone tissues. These requirements can be addressed using 3-D nanocomposite scaffolds that are based on various natural and synthetic biopolymers and a range of nanoparticles available to the research community. Existing biopolymers used for bone healing include cellulose, starch, chitosan, alginate, collagen, fibrin, gelatin, polycaprolactone, PVA, and poly(lactide-co-glycolide) (Hasnain et al., 2019a). Nanohydroxyapatite, mesoporous silica nanoparticles, nanoceramics, and metal nanoparticles are some examples of nanoparticles incorporated as fillers within the polymer matrix to produce the desired functional effects (Hasnain & Nayak, 2019a; Nayak, Maity, et al., 2021). Improvement of some essential physicochemical properties of materials, such as surface area, mechanical strength, optimum cell adhesion, proliferation, and differentiation, are observed (Bharadwaz & Jayasuriya, 2020). Moreover, molecular interactions between nanoparticles and polymers can be engineered to modulate the release of drugs and bone tissue inductive factors from the nanocomposites. In recent years, many multifunctional composites and nanocomposites are being used as carriers for localized delivery of therapeutics to accelerate bone tissue regeneration and treat local defects (Hasnain & Nayak, 2019a; Hasnain et al., 2019a; Nayak et al., 2021). Local administration of drugs ensures improved bioavailability and reduces chances of unwanted immune responses (Saltzman & Olbricht, 2002). The presence of antimicrobials in the cargo prevents the integration of the scaffold with the bacterial cells in the vicinity, which might result in biofilm formation postimplantation (Hetrick & Schoenfisch, 2006; Zilberman & Elsner, 2008). Several antimicrobials, including the common ones, such as amoxicillin, gentamicin, tetracycline, and vancomycin, have been loaded in 3-D nanocomposites to support cell infiltration and proliferation for bone tissue regeneration and healing process (Ray, Hasnain, Koley, & Nayak, 2019). It should be, however, noted that the initial burst release is unwanted according to the requirements of bone regeneration therapy and may lead to serious side effects, in vivo. Therefore polymer swelling and degradation are the preferred mechanisms for the release of drugs and other bioactives entrapped in the nanocomposites. An ideal nanocomposite for bone regeneration should thus be capable of releasing multiple bioactives in sequential manner, which can be tuned according to the degradation profile of each polymer layer (Mourin˜o, 2016). Some recently investigated polymer composites for drug delivery for bone tissue regeneration are listed in Table 18.4.

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Table 18.4 Some recently investigated polymer composites for drug delivery for bone tissue regeneration. Polymer composites for drug delivery for bone tissue regeneration

Drugs

References

Chitosan/cellulose nanocrystal composite scaffolds

Tetracycline

Composite scaffold of chitosan gelatin hydrogel/poly(L-lactide-co-glycolide)

Transforming growth factor β1 and bone morphogenetic protein-2 Rosuvastatin

Patel, Dutta, Ganguly, and Lim (2021) Han et al. (2015)

Bioerodible composite sponges made of Carbopol, polycarbophil, sodium alginate, xanthan gum, and chitosan Collagen/chitosan microspheres composite scaffold Composite made of oxidized hyaluronic acid/collagen (type I) hydrogel and tricalcium phosphate Composite containing poly(lactide-coglycolide) capsule and collagen Hydrogel composite scaffolds made of gelatin microparticles and embedded within the oligo[poly(ethylene glycol) fumarate] hydrogel Novel composite nanoparticles made of gelatin and glycidyl methacrylate derivatized dextrans Nanocomposite fibrous scaffold containing silica-coated nanohydroxyapatite-gelatin reinforced with poly-L-lactic acid yarns Polycaprolactone-gelatin composite electrospun nanofibers Composite nanofibers of poly (ε-caprolactone), poly(glycerol sebacate), and hydroxyapatite

Recombinant human bone morphogenetic protein-2 Tetracycline

Vancomycin Insulin-like growth factor-1 and bone morphogenetic protein-2 Bone morphogenetic protein Vancomycin

Basic fibroblast growth factor Simvastatin

Ibrahim and Fahmy (2016) Hou et al. (2012) Wei, Chang, Liu, and Chung (2018) Ueng et al. (2011) Lu et al. (2014)

Chen, Ma, Dong, and Wu (2009) Huang et al. (2015)

Qiu et al. (2016) Rezk, Kim, and Kim (2020)

18.5 Conclusion and future perspectives With the ever-rising need for advanced and effective medicines, the application of polymer composites in drug delivery continues to attract significant interests of the scientific community. The design and synthesis of these composites are now possible through robust and reproducible means. Manufacturing processes can be further tuned to design composites with suitable properties for the desired functions.

References

In other words, unwanted properties and biological effects can occur through slightest variations in the polymer matrix and nanoparticles. Therefore valid protocols for detecting the toxicity associated with these nanocomposites must be in place to estimate and counter possible threats. Until now, the considerations of the risks which nano-therapy may bring onto a patient were neglected, and the knowledge in this field is lacking. Thorough characterization using advanced analytical techniques is slowly paving the way toward a better understanding of the nano bio interactions in vivo. However, before adequate research is done and unless there is satisfactory data, polymer composite based products will continue to face recurring challenges related to their commercialization and toxicity testing.

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Natural gums of plant and microbial origin for tissue engineering applications

19

Kunal Pal1, Deepti Bharti1, Goutam Thakur2 and Doman Kim3 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India 3 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

19.1 Introduction Natural gums (NGs) are polysaccharides/hydrocolloids that are the naturally occurring polymers that are obtained from plants (e.g., gum arabic, gum tragacanth, guar gum (GuG), and gum ghatti), bacteria [e.g., gellan gum (GG)], and seaweed polysaccharides (e.g., carrageenan, and agar) (Ahmad, Ahmad, Manzoor, Purwar, & Ikram, 2019). The classification of the NGs has been summarized in Fig. 19.1. Many of the NGs used in different industries are the exudates that are produced as the pathological products of plants (Prajapati, Jani, Moradiya, & Randeria, 2013). NGs are considered high-molecular-weight hydrophilic materials, which are composed of polysaccharides, proteins, or minerals (Bektas, Gurel Pekozer, Ko¨k, & Torun Kose, 2021). The NGs are also biocompatible and biodegradable. In addition, these polysaccharides do not elicit immunological reactions when implanted within the human body (Mohammadinejad et al., 2020). Accordingly, they have found a special interest in the biomedical industry. The monomeric units (i.e., monosaccharides) are conjugated with each other through the glycosidic linkages (Koyyada & Orsu, 2021). Most of the NGs can be readily solubilized in water. The solubilization of NGs within the aqueous solutions greatly enhances their viscosity. Like any other polysaccharides, the NGs appear in different chemistries, which allow the researchers to tailor the properties of the NGs chemically. Further, NGs have been blended with ceramics and other synthetic polymers to enhance the properties of the polymer matrices (Mohammadinejad et al., 2020; Muthukumar, Song, & Khang, 2019). Accordingly, these polysaccharides have been explored to design different types of polymer matrices, keeping biomedical applications in mind. NG-based matrices have been explored in various areas of biomedical research, including drug delivery, tissue engineering, and Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00029-2 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 19.1 Classification of the natural gums. Reproduced from Mohammadinejad, R., Kumar, A., Ranjbar-Mohammadi, M., Ashrafizadeh, M., Han, S.S., Khang, G., & Roveimiab, Z. (2020). Recent advances in natural gum-based biomaterials for tissue engineering and regenerative medicine: A review. Polymers, 12(1), 176 under Creative Commons License.

wound healing (Azarniya, Tamjid, Eslahi, & Simchi, 2019; Deep et al., 2019; George, Shah, & Shrivastav, 2019; Khillar & Amit Kumar, 2021; Lustosa et al., 2017; Rahman & Arafat, 2021; Sagbas & Sahiner, 2018). Among the different biomedical applications, tissue engineering is one such area wherein NGs have been explored with utmost success. Tissue engineering is a field of study wherein attempts are made to regenerate damaged tissues and organs using a scaffold (Taghavizadeh Yazdi et al., 2021; Tariq, Bhawani, & Alotaibi, 2021). A scaffold is regarded as the threee-dimensional (3D) porous polymer matrix. These polymeric matrices are meant to be used as artificial extracellular matrices that can support mammalian cell adhesion, growth, and, lastly, cell differentiation (Mohammadinejad et al., 2020). The selection of materials for designing implants and scaffolds for biomedical applications is important for the success of the treatment of the patients. The materials that are usually used to develop scaffolds include polymers (both natural and synthetic) and ceramics. Among the various types of polymers, NGs have gained much importance in designing scaffolds for tissue engineering applications in the last decade. The surface properties of the scaffold matrices can also be tailored using gums. In the current study a scientometric analysis was performed to identify the gums of plant and microbial origin that have been mostly used for tissue engineering applications. The data for the analysis were obtained from the Web of

19.2 Scientometric analysis

Science (WoS) database. The properties of the three most commonly used gums will be discussed in brief. Further, the research work of the most highly cited papers will be discussed.

19.2 Scientometric analysis The WoS database was searched with the keywords “gum ” and “tissue engineering” on November 06, 2021. The search returned with a total of 451 research articles, which were written in the English language. The evolution of the research articles over the years for the aforesaid keywords has been depicted in Fig. 19.2. The figure suggests that the first publication in this area was during the year 2005. Thereafter, there has been a consistent increase in the number of publications in the said field of research. The density plot of the keywords that relate to the association of tissue engineering and the NGs has been given in Fig. 19.3. The visual inspection of the density plot suggests that GG, xanthan gum (XG), and GuG are the three most commonly explored NGs for tissue engineering

FIGURE 19.2 Evolution of the manuscript on the applications of gums in tissue engineering over the years.

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FIGURE 19.3 Density plot of the keywords showing the association of tissue engineering and NGs.

applications. Accordingly, in the subsequent section, an attempt will be made to discuss the properties of these NGs.

19.3 Natural gums In the last decade the use of the naturally derived polymers has received much attention in the food, pharmaceutical, and biomedical industries. Such systems have been explored as binders, thickeners, protective colloids, gelling agents, and scaffold formers. The main advantage of these polymers is that they can be used to develop polymeric matrices of different types, including matrix systems, films, coating agents, microparticles, and nanoparticles. Most of these polymers are available at low costs, which have been ascribed to their abundance in nature. Since these polymers are readily available in mother nature, they are hydrophilic polymers. Due to this reason, the polymer matrices of the NGs can easily absorb water. Also, because of their natural origin, they are inherently biocompatible and are safe for human use. Examples of these commonly used plant- and microbe-derived polymers include GuG, XG, GG, gum tragacanth, honey locust gum, and Sesbania gum. The NGs are generally classified as polysaccharides, which are a type of complex carbohydrates, and are formed by condensation polymerization. In the previous section, we have seen that the NGs, namely, GG, XG, and GuG, have been extensively explored for tissue engineering applications. In this section, we will discuss their properties in brief.

19.3 Natural gums

19.3.1 Gellan gum GG is an exopolysaccharide synthesized by the microbe Sphingomonas elodea (family: sphingans) during the fermentation process. It is an anionic linear polysaccharide and hence exhibits acidic properties. Chemically, GG is composed of the tetrameric repeating units of [D-Glc(β1-3)D-GlcA(β1-4)D-Glc(β1-4)LRha(α1-3)]n (1,3-β-D-glucose (Glc), 1,4-β-D-glucuronic acid (GlcA), 1,4-β-Dglucose (Glc), and 1,4-α-l-rhamnose (Rha)) (Muthukumar et al., 2019; Palumbo, Federico, Pitarresi, Fiorica, & Giammona, 2020). 1,3-β-D-glucose (Glc) consists of an L-glyceryl substituent and an acetyl group. The acetyl group is present in some of the 1,3-β-D-glucose (Glc) units. Such GG is regarded as acetylated GG. On the other hand, the acetyl group can be removed from the polysaccharide by the process of deacetylation. Such polysaccharide is regarded as deacetylated GG. The chemical structures of both the GGs are shown in Fig. 19.4. The deacetylated form of GG is usually marketed as “Gellan Gum” (Palumbo et al., 2020). The polymer has been used for various applications like cosmetics, pharmaceuticals,

FIGURE 19.4 Chemical structure of gellan gum. Reproduced from Muthukumar, T., Song, J.E., & Khang, G. (2019). Biological role of gellan gum in improving scaffold drug delivery, cell adhesion properties for tissue engineering applications. Molecules (Basel, Switzerland), 24(24), 4514 under Creative Commons License.

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tissue engineering, and food. Mainly, the gum has been used in the aforesaid industries as the gelling agent, viscosity modifier, and stabilizing agent for emulsions and suspensions. This natural polymer exhibits a high degree of biocompatibility and has been approved by the Food and Drug Administration of the United States (US-FDA) for food and biomedical applications. Some of the common biomedical applications of GG have been summarized in Fig. 19.5. Another important property of GG is its heat-resistance property.

19.3.1.1 Applications The use of poly(vinyl) alcohol (PVA)/GG-based nanofibrous scaffolds has been proposed for tissue engineering applications (Aadil, Nathani, Sharma, Lenka, & Gupta, 2019). The electrospinning method was employed for the development of the scaffolds. Fourier Transform Infrared (FTIR) spectroscopic analysis of the scaffolds suggested that the interaction between the PVA and the GG was

FIGURE 19.5 Some biomedical applications of GG. Reproduced from Muthukumar, T., Song, J.E., & Khang, G. (2019). Biological role of gellan gum in improving scaffold drug delivery, cell adhesion properties for tissue engineering applications. Molecules (Basel, Switzerland), 24(24), 4514 under Creative Commons License.

19.3 Natural gums

mediated through intermolecular hydrogen bonding. In vitro cell culture studies with murine embryonic stem cells indicated the nontoxic and biocompatible nature of the developed scaffolds. The researchers concluded that the developed nanofibrous scaffolds have a great potential to be explored as tissue engineering matrices. Nanocomposite films of GG and titanium dioxide nanotubes have been developed by Ismail, Mat Amin, and Razali (2018) for skin tissue engineering applications (Ismail et al., 2018). The cross-linking of the films was achieved by calcium chloride via ionic cross-linking. The films were synthesized by the solution casting method. Analysis of the films by X-ray diffraction (XRD) suggested that the films were predominantly amorphous. It was also found that the nanotubes of titanium dioxide were present on the surface of the films. In vitro cell proliferation study of 3T3 mouse fibroblast cells over the nanocomposite films confirmed the nontoxic and biocompatible nature of the developed films. The authors finally concluded that the developed nanocomposite films were suitable for skin tissue engineering applications. Vieira et al. (2019) has reported the synthesis of calcium-enriched methacrylated GG beads for bone tissue engineering applications (Vieira et al., 2019). Preparation of the gels was achieved by the ionic gelation method using calcium chloride solution. The developed gels were capable of self-mineralization, which was promoted by the ion-binding capability of the gels. It was found that a bonelike apatite layer was formed over the gel beads when they were immersed within the physiological fluids. Employing in vitro studies, the researchers found that the developed gels were biocompatible and that there were negligible chances of disproportionate proinflammatory reaction if the gels were to be implanted within the human body. The biological compatibility of the gels was further confirmed by subcutaneously implanting the gel beads in CD1 male mice for up to 8 weeks. The in vivo study suggested complete calcification of the developed gels when they were implanted into the mice. Also, the implanted gels did not elicit inflammatory reactions within the mice. The authors concluded that the developed gel beads of methacrylated GG could be explored for bone tissue engineering applications. Composite hydrogels of GG and demineralized bone powder have been proposed for bone tissue applications by Cho et al. (2021). Spongy composite hydrogels of GG and hydroxyapatite have also been found to be useful for bone tissue applications (Manda et al., 2018). The development of the spongy composite scaffolds helped the researchers mimic the organic and inorganic phases of the bone tissue. GG mimicked the organic part while the hydroxyapatite could mimic the inorganic phase. Choi et al. (2020) has proposed the development of a bilayered hydrogel of GG. The hydrogels primarily consisted of GG and demineralized bone particles. The prepared bilayered hydrogels were developed for osteochondral tissue engineering applications. The polymer matrices for osteochondral tissue engineering applications are designed such that they can help to repair/regenerate both damaged bones and cartilages. The hydrogel layer that was meant to repair the

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cartilage tissue was developed using pristine GG. On the other hand, the hydrogel layer meant for bone tissue regeneration consisted of a mixture of GG and demineralized bone particles. The amount of demineralized bone particles within the composite hydrogel layer was varied in the range of 0.5% and 2.0% (w/v). The in vitro biocompatibility of the bilayer hydrogel was established using NIH/3T3 mouse embryo fibroblast cells. In vivo biocompatibility studies were performed by implanting the hydrogels within the subcutaneous parts of Sprague Dawley rats for a period of 8 weeks. The hydrogels did not induce inflammatory reactions. In addition, the developed polymeric architectures were able to induce regeneration of osteochondral defects.

19.3.2 Xanthan gum Xanthan gum (XG) is categorized as a long-chain anionic polysaccharide with a bacterial origin (Riaz, Iqbal, Jiang, & Chen, 2021). In other words, XG is a bacterial polysaccharide. It is synthesized by the bacterium Xanthomonas campestris (Hidalgo, Armendariz, Wagner, & Risso, 2016). XG is a high-molecular-weight polysaccharide. In general, the molecular weight of XG ranges from 1 to 10 3 106 Da (Dadou et al., 2017). The polymer is widely used in various industries, including food, textile, and biomedical industries. It is a highly watersoluble polysaccharide and has often been used as a viscosity modifier. Though XG is a viscosity modifier and not a gelling agent, several researchers have reported XG as the gelling agent (Hidalgo et al., 2016; Quinn, Hatakeyama, Takahashi, & Hatakeyama, 1994; Yoshida, Takahashi, Hatakeyama, & Hatakeyama, 1998). Chemically, XG consists of a linear (1 4)-β-D glucose backbone. The alternate glucose residue consists of a charged trisaccharide side chain (Hidalgo et al., 2016). The trisaccharide side chain consists of two D-mannose units and one D-glucuronic acid (Dadou et al., 2017). D-Glucuronic acid residue, which has a carboxylic group, makes XG an anionic polyelectrolyte. The chemical structure of XG is depicted in Fig. 19.6.

19.3.2.1 Applications Synthesis of novel self-healing XG hydrogels has been proposed in Hua, Gao, Zhang, Ma, and Huang (2020). The developed hydrogel was reported to be tough and electrically conductive. Such a hydrogel could be developed by exploring the synergistic interactions among different components like montmorillonite, poly (acrylamide-co-acrylonitrile), XG, and ferric ion. The unique property of the hydrogels that were prepared herein was their ability to recover shape even after several loading/unloading cycles. The hydrogels were also exhibited excellent resistance to fatigue and pH-dependent swelling behavior. They were also found to be biocompatible with L929 mouse fibroblast cells. It was concluded by the authors that the developed authors could be explored as matrices for tissue engineering applications.

19.3 Natural gums

FIGURE 19.6 Chemical structure of gellan gum. Reproduced from Dadou, S.M., El-Barghouthi, M.I., Alabdallah, S.K., Badwan, A.A., Antonijevic, M.D., & Chowdhry, B.Z. (2017). Effect of protonation state and N-acetylation of chitosan on its interaction with xanthan gum: A molecular dynamics simulation study. Marine Drugs, 15(10), 298 under Creative Commons License.

Byram et al. (2020) have reported the synthesis of biomimetic silk fibroin and XG-based blend hydrogels (Byram et al., 2020). The synthesized hydrogels were explored for connective tissue regeneration applications. The blend hydrogels were prepared by the physical cross-linking method. The interconnected polymeric network and the porous structure of the developed hydrogels were established by scanning electron microscopy and micro-computed tomography imaging techniques. In vitro cell proliferation study was performed using L929 fibroblast cells. The viability of the cells was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which is a colorimetric analysis. MTT assay helps the researchers to have an estimate of the metabolic activity of the cells. It was found that the developed hydrogels were cytocompatible in nature. Further, the authors found that the prepared hydrogels could mimic the extracellular matrices of the cartilage tissue. By analyzing the results the authors came to the conclusion that the developed hydrogels could be explored as support matrices for soft tissue engineering applications. Silk fibroin/XG-based injectable hydrogels have been proposed by Zhang et al. (2020). The prepared hydrogels exhibited self-healing properties. Synthesis of the hydrogels was achieved by the ionic cross-linking method using sodium trimetaphosphate as the cross-linker. The hydrogels were then 3D printed into self-supporting polymeric architectures, wherein the hydrogels were present as fibers. The developed polymeric architectures were porous in nature, suggesting the formation of scaffolds.

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The developed scaffolds were found to be suitable for tissue engineering applications. Chitosan/XG-based mechanically enhanced scaffolds were developed by Bombaldi de Souza et al. (2019). The developed scaffolds were proposed for the regeneration of soft tissues. The porosity of the scaffolds was tailored using the surfactant Kolliphor P 188. An increase in the concentration of the surfactant increased the porosity of the scaffolds. Simultaneously, silicone rubber was also added to the polymeric blend so as to compromise the loss of the mechanical stability of the scaffolds. The addition of the silicone rubber improved the elastic moduli of the scaffolds. The developed scaffolds were reported to exhibit viscoelastic properties. It was also found that the presence of silicone rubber did not affect the biodegradability and the biocompatibility of the developed scaffolds. The developed scaffolds had sufficient properties to be used as scaffolds for soft tissue regeneration. Nanocomposite scaffolds of sodium alginate/XG have been proposed for bone tissue engineering applications (Kumar, Rao, & Han, 2017). The blend polymer matrices were reinforced with cellulose nanocrystals and halloysite nanotubes. The scaffolds were prepared by the freeze-casting/drying method. Both the reinforcing agents were uniformly dispersed within the blend polymer matrices. It was observed that there was a good interfacial interaction between the polymer matrices and the nanofillers. The addition of the nanofillers compromised the water uptake capacity of the scaffolds. Also, there was a reduction in the porosity of the scaffolds as the nanofillers were added. However, the nanofillers could considerably improve the mechanical properties of the biopolymeric blend scaffolds. The nanocomposite scaffolds could promote the proliferation of the MC3T3-E1 osteoblastic cells, which suggested the utility of the prepared scaffolds for bone tissue engineering.

19.3.3 Guar gum GuG is a plant-derived high-molecular-weight galactomannan polysaccharide. The gum is obtained from the seeds of the plant Cyamopsis tetragonolobus that belongs to the family of Leguminosae (Palumbo, Berent, Proniewicz, & Bana´s, 2019). Since the polysaccharide is a naturally occurring polymer, it is inherently biodegradable. Further, the polymer is a biocompatible and environment-friendly polymer. Chemically, the polysaccharide is composed of D-mannopyranose (M) and α-D-galactopyranose (G) monomer units. The M units are attached to each other by β-(1-4) linkage, which forms the backbone of the polysaccharide. At every alternate M unit, the G units are attached by α-(1-6) linkages to the M unit. The chemical structure of GuG is represented in Fig. 19.7. It is a viscositymodulating polysaccharide and is usually used as a stabilizer and/or thickening agent. The polysaccharide has found applications in various fields, including food, cosmetics, and biomedical applications. GuG-based polymeric architectures

19.3 Natural gums

FIGURE 19.7 Chemical structure of guar gum. ´ J. (2019). Guar gum as an eco-friendly Reproduced from Palumbo, G., Berent, K., Proniewicz, E., & Banas, corrosion inhibitor for pure aluminium in 1-M HCl Solution. Materials, 12(16), 2620 under Creative Commons License.

have been greatly explored for regenerative and tissue engineering applications, which are included in biomedical applications.

19.3.3.1 Applications Das et al. (2021) has synthesized a novel GuG indole acetate ester (Das et al., 2021). The ester with a high degree of substitution was further used to develop films for tissue engineering applications. The modified GuG was then crosslinked with hydrolyzed keratin. Hydrolyzed keratin was obtained from chicken feather waste, which can be used as a sustainable resource. Since the films developed with GuG ester and the hydrolyzed keratin are developed with bio-based polymers, the films were found to be nontoxic and biocompatible. Further, the films exhibited antimicrobial properties both against Gram 1 ve and Gram 2 ve bacterias. The authors concluded that the developed novel films could be used for skin tissue engineering applications. In another study, polymer blend scaffolds of GuG/gelatin have been proposed for soft tissue engineering (Indurkar, Bangde, Gore, Agrawal, et al., 2020). The scaffolds were developed by the freeze-drying of the blend hydrogels. The authors found that the optimized scaffold had sufficient pore interconnectivity and porosity such that the scaffolds could allow an easy mass transfer. This is of utmost importance because the mass transfer property help in the efficient transport of the nutrients to the cells and, at the same

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time, can remove the cellular waste products out of the scaffolds. The biocompatibility test with human keratinocytes suggested the nontoxic nature of the developed scaffolds. The authors reported that the optimized scaffold could be used for soft tissue engineering applications. Recently, GuG/gelatin bioink has been proposed by authors (Indurkar, Bangde, Gore, Reddy, et al., 2020). It was proposed that the developed bioink can be used for the 3D bioprinting of mammalian cells. In the study the authors used the bioink for the 3D printing of human keratinocyte (HaCaT) cells. The 3D structures were cross-linked with borax. In the recent past, films of carboxymethyl GuG have been proposed for wound healing applications. In a similar study, novel chitosan/GuG bioinks were developed by Cleymand et al. (2021). The developed inks were supposed to be used for extrusion-based 3D bioprinting. The properties of the hydrogel scaffolds of silk fibroin were improved by reinforcing with oxidized GuG, which contained curcumin-loaded Ze in nanoparticles (Nezhad-Mokhtari, Ghorbani, & Abdyazdani, 2021). The developed hydrogel was used for wound healing applications. An increase in the oxidized GuG could improve the degradation properties of the hydrogels. In vitro cell culture analysis using NIH-3T3 mouse embryonic fibroblast cells in the presence of the hydrogels suggested their nontoxic properties. Further, the hydrogels were found to exhibit antimicrobial properties against Bacillus and Escherichia coli bacteria. It was found that the developed hydrogels were suitable for wound healing applications. In another study, arabinoxylan/GuG hydrogels have been proposed for skin wound healing applications (Khan et al., 2020). Even though the proposed hydrogels were cross-linked with tetraethyl orthosilicate, they were biodegradable. The associative interactions among the arabinoxylan and GuG molecules were confirmed by FTIR spectroscopy. Microstructural analysis of the hydrogels suggested the presence of a homogenous porous structure throughout the hydrogel matrices. The antimicrobial properties of the hydrogels were confirmed by performing antimicrobial studies against Pseudomonas aeruginosa (Gram 2 ve) and Staphylococcus aureus (Gram 1 ve) bacteria. As the hydrogels were designed using natural polymers, they were nontoxic and biocompatible.

19.4 Conclusion NGs are polysaccharide-based biopolymers that are obtained from several natural resources, which allow sustainable development. Due to this reason, they have found enormous interest in various fields of applications, including food, cosmetics, pharmaceutical, and tissue engineering. This can be reasoned to the fact that NGs are inherently biocompatible, nontoxic, and biodegradable. Further, the NGs can help in overcoming some of the limitations of the synthetic polymers when they are blended with the synthetic polymers. In this chapter, we have discussed the three most commonly used NGs of plant and microbial origin that

References

have been explored for tissue engineering applications. The identification of the commonly studied NGs was performed by scientometric analysis. It has been found that the NGs are promising candidates for developing scaffolds and bioinks for tissue engineering applications. The development of the scaffolds has been primarily made via the freeze-drying method or electrospinning method. Many researchers have proposed the reinforcement of the NG-based polymeric architectures for improving their properties. The NG-based biopolymeric architectures have been explored to develop structures for stem cell culture, soft tissue engineering, bone tissue engineering, skin tissue engineering, and wound healing. The use of NG-based bioinks for tissue engineering applications is in its nascent stage. Extrusion-based 3D printing has been mostly used while using the NG bioinks. The ongoing research on bioinks has opened new dimensions in the field of tissue engineering.

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CHAPTER

Polymers and nanomaterials as gene delivery systems

20

Sundara Ganeasan M1, Amulya Vijay2, M. Kaviya1, Anandan Balakrishnan2 and T.M. Sridhar1 1

Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, Chennai, Tamil Nadu, India

2

20.1 Introduction Treatment of diseases has always been a challenge to mankind and with pandemics such as plague and COVID-19; the loss of life is not under the physician’s control. In case of traditional diseases such as cardiac, cancer, and viral infections where the diagnosis and treatment is a challenge, gene therapy offers a new ray of hope. Its primary aim is to give the genetic information to produce the therapeutic proteins in order to modulate genetic diseases. The challenge with the efficacy of this technology is the introduction of the gene. Therapeutic genes are used to restore the proteins that have been dysfunctional or missing from the nucleus with the help of vectors. Similarly, in case of RNA, there is a need to change or dictate the existing genes at the nucleus as changes that cannot be made directly to the call are not translated into action. The stability of the foreign gene molecule dictates the stability of the host cell and this determines the success of the therapy (Mali, 2013). In 1980s virus-based vectors served as a tool to fulfill the major expectation from transgene expression (Moss, Smith, Gerin, & Purcell, 1984). In early reports, to protect the hepatitis B attack on chimpanzees, vaccinia virus was used as a vaccine vector. A few years back Sung and Kim have reviewed the use of nonviral gene delivery (nVGD) systems. They reported the modifications that occurred in cell phenotype on acquaintance to DNA in the body (Sung & Kim, 2019). The main criterion for designing a gene delivery system is the prediction of the specific communication among the gene delivery vehicle with the target cell. The three main systems that control the gene delivery system are (1) a gene is a therapeutic protein, (2) direct gene function to the gene expression of target cells, and (3) control the gene delivery system and deliver gene expression to a specific target location. The development of gene delivery systems is mainly divided into liposomes, microvesicles, and viral and nonviral structures (Je, Cho, & Kim, 2006). Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00025-5 © 2023 Elsevier Inc. All rights reserved.

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Due to changes in adenovirus, adeno-associated virus (AAV), lentivirus (LV), and retrovirus (RV), the delivery of viral genes is possible, depending on their virions and their interaction with host cells. The most commonly used gene delivery system is naked DNA, which uses vascular endothelial growth factor (VEGF) to have a major impact on myocardial ischemia, angiogenesis, and intratumor injection. The main limitation of naked DNA is due to the presence of phosphate groups and its hydrophilicity, its large size makes it difficult to enter cells (Manjila et al., 2013). Although gene delivery has so many advantages, it has been limited to practical applications due to its replication risk, cumbersome sterilization, DNA insertion, and high immunogenicity. These shortcomings that are present in gene delivery, polymers, nanoparticles, bioceramics, and other factors can be overcome by combining them into the gene delivery system to enhance its characteristics. In particular, polymers incorporated into gene delivery systems have several significant advantages, such as their ability to form nanoscale complexes with nucleic acids (size from 50 to 150 nm). The surface properties permit the targeting of biomolecules of various sizes, such as bacteria, normal cells, and neuronal cells are 0.15 2 μm, 10 30 μm, and 4 100 μm according to their sizes. According to Therapeutic Goods Administration standards, (synthetic) polymers are safer, cheaper and have higher production capacity than viral vectors (VVs). In spite of several clinical advantages, these polymers in comparison with conventional vectors have some limitations which include the presence of higher levels of toxicity along with poor transfection effectiveness that do not meet the conditions for clinical gene therapy applications. In designing and elucidating functional gene delivery systems with transfection efficiency and biocompatibility, some focused efforts have been made to eliminate these problems (Shi et al., 2017). There are many nonviral gene vectors available, one of which is nanoparticle as an ideal vector with all the characteristics that are needed for gene-based delivery vehicles. The main advantage with nanoparticles is that their size, measured in nanometer, allows vehicles to penetrate deep into the target location. It also contributes to polymer diversity and unlimited DNA packaging. The addition of nanoparticles as well as polymers and ceramics is an excellent platform for delivering genes directly to the target location (Lin, Zhang, & Huang, 2015). This chapter mainly discusses about the gene types, techniques, and the applications of gene delivery systems which mainly employs the polymer and its composites.

20.2 Types of gene delivery The concept of gene therapy is to introduce an external matter into the host cells in order to achieve a desired treatment to enable the patient to lead a normal life. Gene therapy was initially concentrated toward the treatment of specific genetic disorder-based diseases but with the acquired knowledge it has expanded to the

20.2 Types of gene delivery

treatment of a family of complex diseases. Gene therapy can be classified into two types, mainly the germinal line and the other involving somatic cells. Human gene therapy has been restricted to somatic cells since in the case of germinal line it is ethically forbidden though it has resounding potential. The development and stabilization of the process of introduction of the gene determines the efficacy of the therapy. Since the genetic materials introduced directly into the cells are not generally transcribed, therapeutic genes used in recovery of the missing or damaged proteins or RNA deployed in therapy to modify the transcription of existing genes are delivered into the nucleus. A complete knowledge of the interaction between the target cell and the gene delivery system is necessary to bring out in an effective gene delivery system. A gene delivery process is built on three gears, namely, a plasmid-based system where the functioning of the gene and its expression is controlled by the target cell. The next one involves selection of a particular protein used in therapy and encoding its gene followed by design of a control system to transport a gene expression plasmid at precise locations in the human body (Han, Mahato, Sung, & Kim, 2000). In the past three decades, there has been immense work on the development of viral gene delivery and nVGD systems. Despite several strides, an ideal delivery system for gene therapy applications that can be applied to various types of cells in vitro and also in vivo without any restrictions and toxic effects is needed. Gene delivery systems for germline are stem cells, sperm cells egg cells, embryonic nuclear, microinjection, nuclei, and in case of somatic cells, it is by viral dependence (pox virus, adenoviral, hybrid adenoviral systems, adeno association, herpes simplex, retroviral helper-dependent adenoviral systems, LV, and Epstein Barr virus) and nonviral systems (physical: naked DNA, DNA bombardment, electroporation, hydrodynamic, ultrasound, magnetism) and (chemical: Cationic lipids, different cationic polymer, lipid polymers) (Nayerossadat, Maedeh, & Ali, 2012).

20.2.1 Germline gene therapy This gene therapy technology is a very simple way of manipulating the germline cells as genetic abnormalities that can be corrected without targeting. It has two advantages where the treated subject is cured, but the partial distribution can also carry the corrected genotype to the next offspring as it is hereditary. During this treatment the gene responsible for the therapy is relocated to the germ cell that is egg or sperm. Although it has almost never been used in humans, various diverse transgenic methodologies have been experimented in additional species which include nucleus engaged from the somatic cells at metaphase for gene delivery; in vitro changes in eggs following in vitro impregnation; pronuclear microinjection of exogenous DNA solution using a glass needle; management of mouse embryonic stem cells by inducing the desired changes during in vitro culture with the help of various types of gene delivery systems; gential system modification involves either direct or indirect injection of the sperm cell to the testis by

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transgenic delivery. This therapy involving germline gene is precisely more thought-provoking and there is an essential need for consistent in vitro fertilization. Along with this preimplantation genetic testing (PGT) accompanied by several protocols such as genome editing is required (Tang & Xu, 2020).

20.2.2 Somatic gene therapy This is a special technique where the genetic contents would not be transferred to the next generation. The therapy involves supplementing of the genes into diploid cells without involving either the egg or the sperm which form the normal reproductive cells. Therapeutic applications using somatic cells is ideally preferred as it is not inherited since only the target cells present in the diseased patient are attacked which also increases its safety levels. The impact of somatic therapy is its short life span as it is designed such that the tissue cells will ultimately die and on replacement it would regenerate. In this therapy, the new genes are successfully unified into the genome which is the first hindrance to somatic gene therapy. Further, during unification of the modified gene if the erroneous part of the DNA occurs then this could encourage a new defect instead of preventing the disease. However, despite these shortcomings, somatic gene therapy is tolerable as it is able to cure many diseases in patients with acceptable recovery levels. The present research is concentrated on developing protocols to precisely heal the genetic defects in somatic cells. Somatic gene therapy can be broadly classified into three types:

20.2.2.1 Ex vivo delivery The aim of this delivery system is to treat a defect in a specified region of the body or a particular organ instead of treating the entire human as a whole. In this technique, the genetic information is systematically engineered in the cells outside the body leading to the development of organ or system specific targeted delivery system. (Gregory-Evans, Emran Bashar, & Tan, 2012). This ex vivo genetic therapy is a promising technique as it provides an exclusive advantage to verify the expression and health of the transfected cellular gene before it is administered to the subject affected by the disease. This helps the physician to treat complicated cases as they can plan the treatment which allows them to select the compounds from the culture plate itself and evaluate its specific expression before it is released (Moirano, Emborg, Kaplitt, & During, 2006). This ex vivo plan can be deployed in situations where corrected cells can be readily obtained as in the case of bone marrow. It can also be used in subjects with rectification of hematopoietic stem cells (HSC) for severe combined immunodeficiency, when the amended cells have a superior benefit in treatment. Hereditary diseases can be effectively treated with ex vivo therapy which involves the utilization of the amended as a source of secreted proteins. This is applied in factor VIII, F8, gene transfer to autologous fibroblasts in hemophilia A. The therapeutic process used can result in obstructions such as transfer of a suicide gene

20.3 Methods and techniques used in gene delivery

to T lymphocytes in order to regulate the graft refusal in nonautologous bone marrow transplants and can be overcome with ex vivo plan (Dwivedi et al., 2018).

20.2.2.2 In situ delivery Direct administration of genetic material to target tissue is referred to as in situ delivery. The presently deployed delivery system does not require effective orientation; this method is appropriate. This technique has found wide application in the delivery of cystic fibrosis which refers to the disease condition of the airway epithelium. This disease condition can be treated delivering CFTR gene with the aid of lipid and adenoviral vectors by targeting the site explicitly in the respiratory tract. This method can be explored for cancer gene therapy as well. The disadvantage with cancer treatment is that a single malignant cell is sufficient to renew the tumor for which researchers are working toward a solution (Nayerossadat et al., 2012).

20.2.2.3 In vivo delivery Systemic administration of modified copies of genes by injection is a very effective way to transfer transgenes into patients. The foremost issue with the in vivo approach is its inefficient targeting. In this method, a viral or non-VV (nVV) is used as a carrier to transfer the genetic substance. This is further transmitted to the target tissue to incite an immune response. The immune system further responds to the vector results in its elimination and transient expression of the transgene at best. The presence of neutralizing antibody further prevents the second injection of the vector. The reduction of neutralizing antibodies is a promising area of research that would lead to the improvement of the delivery systems using gene therapy vectors. This strategy has two requirements: one the target cell is easily accessible to infuse or inject the virus and second, the transfer vector is easy to infect which is followed by targeted integration. This accompanied by the therapeutic gene (transgene) which is expressed in the target cell whereas the surrounding cells are not affected for prolonged time periods (Selkirk, 2004). This delivery system is presently ranked the least advanced strategies deployed which are found to be insufficient in the selection of targeting vectors but in future they may help treat tissue sites in specific diseases.

20.3 Methods and techniques used in gene delivery 20.3.1 Nanoparticle gene delivery systems Nowadays, nanoparticles are one of the prominently used transfection agents used to deliver genes into the specific cell location. The different types of gene delivery systems are represented in the Figure 20.1. Inorganic particles can be deployed in the gene delivery systems as they can be modified at its surface in a

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FIGURE 20.1 The representation of gene delivery systems.

straight forward way, which have a very low cytotoxicity and also have a very long shelf life compared to other systems. There are a number of inorganic particles which are being used in gene delivery applications such as calcium carbonate, calcium phosphate, mesoporous silica, and metal nanoparticles such as gold, etc. (Conde et al., 2012). They also show a number of promising applications in drug delivery, which are governed by size reduction, their capability to interact with different types of biomolecules with volume-to-surface ratio which makes a platform for targeting a location and drug or gene delivery. The major setback of the nanoparticles is its time-consuming approach and also a low conventional method over the years (Wang et al., 2010). One such nanoparticle is layered double hydroxide (LDH) which is possessed to have very low cytotoxicity, excellent biocompatibility, very good anion exchange capacity, systematic degradation in the cellular environment, along with the very high drug/gene loading capacity. LDHs are generally clay materials of anionic class which contain mono, di, and trivalent metal ions, namely, Li1, Ca21, Mg21, Al3 1 , Co31, Fe31, etc., along with anions that are located at the interlayer gallery to restore equilibrium with the positive charge present on layered LDH (Senapati, Sarkar, Das, & Maiti, 2019). Nowadays, the core shells present in nanoparticles containing lipids are gradually gaining attention as a vehicle to transmit genes in nVGD systems. The major

20.3 Methods and techniques used in gene delivery

advantages are the easily available preparation techniques, handling, and also due to their least immunogenic nature which helps in delivering a large piece of DNA. The core shell-type nanoparticles are made up of two main components, namely, inner and outer blocks. The inner blocks comprise DNA condensed with polycations, namely, poly-L-lysine, calcium phosphate, etc., which acts a overall base providing specific size distribution, controlling its morphology which automatically lowers the toxicity of the lipid during the encapsulation. The outer blocks are made up of lipid that safeguards the DNA and also improves the stability in the body fluids. With these two blocks combined makes the core shell-type nanoparticle a potential vehicle for gene delivery (Pozzi et al., 2013).

20.3.1.1 Mesoporous silica nanoparticles For gene delivery systems, inorganic nanoparticles have numerous benefits over polymeric ones. Many inorganic nanoparticles can now be easily produced as a result of advances in nanotechnology. Moreover, nanoparticles with minimal harmful effects to the cells can also be designed. The majority of them have excellent long-term storage stability. Many different types of inorganic nanoparticles, including gold, quantum dots, calcium phosphate, carbon nanotubes, and silica, have been utilized for gene transfer till date. Nucleases may readily destroy deoxyribonucleic acids that have been bound to the nanoparticle exterior wall. As a result, DNAs are best packed in a proper area to prevent deterioration until they reach the point that is ideal for their delivery into living cells. Silica nanoparticle (SiNP) is an interesting nanoparticle for these applications for its host of benefits, which include the presence of required porous surface, simplicity of exterior nanoparticle modification, biological acceptance, and the ability to produce large quantities economically. SiNPs with an optimal pore size ranging from 2.5 to 2.8 nm are currently being used to transport plasmids. Monodispersed Mesoporous Silica Nanoparticles (MMSN-23) have a number of benefits, including ease of synthesis and modification, large-scale manufacturing at a low cost, and drug and gene encapsulation. It is worth noting that, despite significant pDNA loading, strongly aminated MMSN-23 enter cells leaving behind the polymers or lipids. The modification of the surface of MMSN-23 with amine functional groups helps it to exhibit effective cellular absorption. Furthermore, silica surface on the MMSN-23 can be modified through bioconjugation with primary amines. This may be readily changed to integrate functionalities to target specific cells or anticancer drugs. MMSN-23, which have a wide surface area, might be used to transport both tiny medicines and therapeutic biomacromolecules at the same time, resulting in a synergetic effect. MMSN-23 can be explored for various biological applications in vitro and in vivo, assembly of critically complex enzyme sensors in addition to protein-based delivery systems (Kim et al., 2011).

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20.3.2 Liposome gene delivery systems Liposomes are a collection of lipid molecules that assemble to form vesicular structures. Liposomes are made up of hydrophilic and hydrophobic head and tail respectively. This can be altered by exposing the hydrophilic head into aqueous systems to increase the efficiency of gene transferring capacity. It promotes membrane fusion upon acidification. Lipid vesicles offer a wide range of advantages as they come in a choice of shapes and compositions. They have the capacity to wrap and protect therapeutic biomolecules, come at an economical cost, and are muted toward immunogenic reaction. These properties have enabled their use in analysis of biomolecules, drug transport system, facial, and food products’ gene delivery (Balazs & Godbey, 2010). The binding properties of the liposomes to endosomes are achieved by acidification as it is sensitive to pH. The lipoproteins interact with DNA are known as lipoplexes which can be revised to anionic, cationic, neutral, or the presence of the three together. Genetic molecules are mainly loaded in vitro using lipofection via liposomes or lipoplexes. In this technique, the encasing of pDNA, siRNA, or microRNA in spherical structures can possess a hydrophilic polar head group and a hydrophobic tail, as in the case of a typical cell membrane structure (Torchilin, 2005). Cationic-based lipids were the first and most widespread used lipofection systems such as N-[1-(2,3-dioleyloxy) propyl]N,N, N-trimethylammonium chloride (DOTMA). The major challenges in this process at the starting stages came from the joining of nonspecific proteins but with new developments due to iterations such as neutral helper lipids, there has been a reduction in cytotoxicity and, at the same time, efficacy of transfection has improved (Dabkowska et al., 2012). It has been found that cationic lipids have been further active in transfection because they can capture the plasmid in neutral conditions. Lipid emulsions are another class of superior systems used for genetic delivery on comparison with delivery of liposomes (Hashida, Kawakami, & Yamashita, 2005). In the presence of bulky nucleic acid constructs, these systems instantly form complexes. Their attractive features include their superior efficacy in vitro delivery, degradation of the material in vivo, and the option of functionalizing them as per requirements. The efficacy of in vivo transfection is restricted by lipid-based methods, as there are challenges in developing formulations required for mass production (Gantenbein et al., 2020). The basic requirement for liposome gene-based delivery vehicle is the formation of a lipid film by hydration along with DNA. This along with heavymolecular-weight DNA is enclosed in egg phosphatidylcholine liposomes. Following this, the metaphase chromosomes are impregnated in the egg phosphatidylcholine-cholesterol in the ratio of 7:2 mol/mol of liposomes. Alternatively, a gene delivery system with high efficacy levels can be prepared using a cationic lipid that is mixed with a monomer or an alternative hydrophobic plasmid DNA micelle can be combined to acquire a DNA-cationic lipid composite of hydrophobic plasmid. The current research strides in liposomes are an

20.3 Methods and techniques used in gene delivery

increase in the time period of liposomes by reducing the response of phagocytic cells present in reticuloendothelial structure on the established order. Delivery of target cells are realized by coupling the surface of liposomes using either covalent or noncovalent approaches and by further mixing them with monoclonal antibodies. Fusogenic liposomes are being investigated and are in the advance stages of implementation for intracellular delivery. Recently, the use of nanoparticle liposome-based cancer chemotherapy is another promising application along with several others being investigated (Manjila et al., 2013). Several candidates for medicines with very strong and low therapeutic indicators can point to the affected sites required using liposomes-based drug delivery techniques. Encapsulated drugs in liposomes may have pharmacokinetics that changes significantly. The effectiveness of the formulations prepared with liposomal depends on its capability to select drug molecules to the target area for a prolonged time frame. This should be accompanied by reduction in the toxicity of the drug. Normally, in the formulation of liposomal drug delivery systems, the drug which is implanted in a phospholipid bilayer should disseminate at a controlled low rate from the dual layer. Several factors should be considered while developing this system, such as concentration of the drug used, the mixing ratios of drug to lipid, entrapment efficacy, along with in vivo drug release. The advancements in the use of deformable liposomes and ethosomes technology along with the delivery system of the liposomes containing preloaded drugs using alternative routes of administration such as inhalation and optical routes are the future health-care routes. Reducing the toxicity of several very powerful medications can concurrently bring progress in pharmacokinetics and therapeutic effects using liposome methods (Kalepu, Sunilkumar, Betha, & Mohanvarma, 2013).

20.3.3 Microbubble gene delivery systems Gramiak and Shah reported the detection of multiplication in signal strength after an intracardiac saline infection. Later on, they attributed it to the ultrasound reflection arising out of its interaction with tiny bubbles in saline solution (Gramiak & Shah, 1968). After this report, the role of microbubbles was investigated as budding therapeutic agents and also for diagnostic imaging (Sieswerda, Kamp, & Vier, 1998). Microbubble enabled the development of a noninvasive approach for ultrasound-based intracellular drug and gene delivery system that can be deployed for clinical applications. The controlled space-time application of ultrasonic energy combined with microbubbles makes it conceivable to transport site explicit therapeutic drugs to the areas of interest in the diseased sites with negligible systemic side effects. Ultrasound is also used for increasing the penetrability of cell membranes also known as sonic pores to remain temporarily. This newly designed channel would enable impermeable drugs to go into the cell with the introduction of either rapid expansion or contraction along with the breakdown of microbubbles. Microbubbles designed under ultrasound exposure can be deployed

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to induce sonoporation and thereby intensify the penetrability of cell membranes with the desired drugs or gene molecules (Fan, Kumon, & Deng, 2014). The application of ultrasound to treat diseases has opened new domains of research which include delivery of drugs to the tumor sites resulting in the breakdown of the blood brain barrier momentarily and transfection of nonviral genes in vitro and in vivo. Gene-based therapeutic uses can be realized with the use of ultrasound contrast agents to pick out the diseased sites. The use of ultrasoundguided technique is used to accomplish the delivery of nonspecific areas of adenovirus and plasmid reporter genes. In certain specific cases, the adenoviral vectors or their plasmids have remained integrated into albumin-type ultrasound contrast agents and they were further released into the myocardium using ultrasound, thereby destroying the microbubbles that are present in the selected area of the disease (Bekeredjian, Chen, Frenkel, Grayburn, & Shohet, 2003). VEGFencoding plasmids can be carried by microbubbles leading to induce angiogenesis in rat myocardium with subsequent application of ultrasound energy. However, traditional microspheres are negatively charged and their transfection efficiency for cells with negatively charged RNA and DNA molecule is low. The development of DNA and use of microbubbles can cause the DNA to fuse with the outer shell, thereby facilitating coinjection. Previous reports have indicated that after intravenous injection of albumin microbubbles and combining plasmid DNA with ultrasound, the gene can be delivered to the heart muscle. Subsequent research advanced a technique to introduce DNA into the lipid microbubble layers leading to localized transfection following intravenous injection and use of ultrasound. The success rate of transfection by intravenous injection has been in practice for some time but data associating intravenous and intraarterial injection of microbubble-loaded plasmids revealed that the effectiveness of intraarterial injection by at least more than 200% in accomplishing local tissue transfection (Christiansen, French, Klibanov, Kaul, & Lindner, 2003). Maximum gene expression with microbubble-based gene transfer system has proven to be 700 times progressive when compared with ultrasound. This technique successfully transfected RNA into tumor-affected areas and prevented angiogenesis resulting in reduction of tumor development (Song, Shen, Chen, Brayman, & Miao, 2011), and the genes were successfully transferred to the rat myocardium. Microbubbles have been attempted to transfer genes directly to the damaged liver thereby resulting in the delay and further developing the growth of liver fibrosis by modifying the appearance of foreign genes. This technology was also used to accomplish viral DNA vectors in patients suffering from brain parenchyma disease. But, this was earlier impractical as blood brain barrier was impeding its progress and now microbubbles technology has opened up the possibility of gene therapy for genetic diseases treatment of the central nervous system. No clinically appropriate microbubble levels have been inoculated intravenously to establish beneficial levels of drug or genes to treat a disease. Development of microbubble agents and their dose optimization to introduce the microbubbles for future translational experiments would result in gene transfection.

20.3 Methods and techniques used in gene delivery

Further, a drug or gene uptake capacity of the microbubbles and the concentration of these bubbles required during the application of ultrasound need to be investigated. In case of interventional artery or muscle therapy, microbubbles are injected in large quantities to release the drug or genes into the system. In a few clinical trials, microbubble inoculation into the muscular region along with plasmids resulted in local transfection (Ferrara, Pollard, & Borden, 2007). Further research needs to be carried out to evaluate the efficacy of gene delivery optimize the variations in results, understanding about the role of ultrasound in inducing the therapy in terms of tis transport and the biochemical changes it can induce in the human body. Ultrasound-based gene delivery systems with their fine action of microbubbles offer a capable and striking approach for in vivo applications, the inexpensive and universal availability of ultrasound technology.

20.3.4 Viral and nonviral gene delivery systems Gene transfer systems practice numerous methods to allow the amalgamation of the designated gene to the selected cells. Apart from liposome-mediated and microbubble-mediated systems, they are also classified as viral-based and nonviral-based systems. The genetic administration system can be mediated by virus which is altered to carry and release DNA for modified results. The commonly used viral genetic vectors are adenovirus, LV, and RV and they have been successfully used in the development of corona virus vaccines as well within a short span of time. The delivery systems based on nonviral genes were presented to decrease the dependence on virus-built systems. These vectors have resulted in several; successful transfection. These nonviral systems are categorized based on their chemical and physical properties and a few methods based on them are gene gun, electroporation, electrophoresis, microinjection, ultrasound, and hydrodynamic applications (Cevher, Sezer, & C ¸ a˘glar, 2012).

20.3.4.1 Viral gene delivery systems Clinical trials results with VVs have shown that they are an efficient tool for genetic material delivery. VVs have shown their efficacy in transfecting cells and in the long run they have been able to meet the needs of the required therapeutic target. The VVs (Fig. 20.2) are obtained from the main virus during the process of infection, eliminating or substituted with the pathogen gene leading to the production of nonpathogen virus. Virus gene delivery systems display numerous action formulas with different molecular weight of polymers, changing length of the peptides and similarly with lipids. The role of nanoparticles and use of minor biomolecules are being investigated. There are several categories of viruses, but the indispensable compounds are similar. The completely assembled infectious virus known as virion is composed of two or three fundamental parts. The first part is RNA or DNA genetic material that constitutes the virus, which takes information from some types of coded

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FIGURE 20.2 Structure and properties of viral vectors.

proteins. The second part consists of a defensive shell that guards the viral gene from nuclease enzyme and is a capsid coated with proteins that acts to identify the receptors of the cell surface in which the virions are present during the infection. For a few cases, the final part is a wrapping of lipid molecules neighboring the capsid. This is further programmed by a gene which permits the virion to be directed by filtration through the target cell membrane (Wang et al., 2021). In terms of clinical trials, adenovirus is the utmost extensively deployed VV for gene transfer. Adenoviruses have the advantage of delivering an elevated transduction efficacy when it comes across a variety of dividing and nondividing cells. The tropism of adenoviruses is primarily obtained by the capsid protein, hexanone, and fibers that are present in the fibrous knot domains available for the collaboration of the Coxsackie virus and the adenovirus receptor (CAR). Adenovirus serotype 5 (Ad5) is one of the most common carriers in clinical trials; however, due to the high preexisting immunity to Ad.33, it accumulates in the liver and shows strong immunogenicity. AAV has developed as a capable vector that offers little immunogenicity, but in the long run it is stable which has resulted in its approval for commercialization treatments under the brands as Luxturna and Zolgensma. The chances of large-scale production are offered by the LV which belongs to the family of RV. LV possess the intrinsic capability to infect both the dividing and nondividing cells, thereby resulting in incorporation into the host’s genetic material (Kaygisiz & Synatschke, 2020). Over the years, VV therapy alone appears to have reached the limit of therapeutic effect. Recent research on the amalgamation of viruses with biomaterials has revealed a talented future pathway that may allow this therapeutic technology to eventually reach its full potential in a clinical environment. This can be achieved by selecting the proper polymeric and nanoparticle-based biomaterials that would address the reduction in undesirable virus release, harmful products that cause toxicity, the immune reactions with the host, along with incompetent

20.3 Methods and techniques used in gene delivery

gene delivery. These biomaterial and nanomaterials are expected to deliver effectual functionality coupled with consistent gene expression that provides adjustable control and adaptability to the local environment. A series of challenges for these bionanomaterials include confining viral elements close to the carrier biomaterial which could aid the local gene transference and on the other hand the spread of the carrier with potentially dangerous material would become remote. The gene release profile can be preprogrammed to regulate the level and period of transgene expression. In addition, these systems can regulate the immune reply to viral gene transfer, generally leading to reduced cellular and antibody responses that which can meaningfully intensify the accomplishment rate of viral gene delivery. Furthermore, when a modified biomaterial is loaded viral elements, it restricts the viral tropism. These altered systems modified biomaterial with viral elements have been proven to be effective means to improve the transduction efficacy of controlled cell populations (Wang et al., 2021).

20.3.4.2 Nonviral gene delivery system The replacement of chemicals in the place of viruses with their biochemical composition is referred to as nVV. These nVVs are mainly composed of organic and inorganic molecules and compounds. The organic compound base vectors include lipid containing vehicles, polymers derived from natural resources, peptide containing vehicles in addition to synthetic polymers. The combination of lipofectamine 2000 and polyethyleneimine (PEI) is the most established nVV termed as the golden standard that provides the required transfection efficacy. The stepwise changes and action of nVGD in the cells are yet to be ascertained. Among the several proposed mechanisms they can be broadly classified into five stages as the vector consignment has to cross several biological hindrances that include the extracellular atmosphere, cell casing, endolysomal organization, nuclear cell, along with interruptions due to transcription/translation (Wu et al., 2018). The biological safety of the nVVs serves as a foremost advantage for their preference. But its poor transfer efficacy has caused a low transient appearance of its transgenes which has hindered the use of nonviral gene transmission. nVVs have attracted a great deal of attention due to their decreased immunotoxicity. In the past decade, the use of nVVs in clinical trials has tremendously increased compared to VVs which have declined significantly. Progression in efficacy, precise point of delivery, and period of gene expression accompanied by its safety due to decreased immunotoxicity have resulted in a surge in the quantity of nVV products going for clinical trials (Ramamoorth & Narvekar, 2015). nVVs are clinically employed to deliver the different types of nucleic acids which include minor DNA like oligodeoxy nucleotides which are obtained synthetically Major DNA molecules that include pDNA also known as plasmid DNA, ribozymes, and Si RNA- and mRNA-based RNA with the aid of different gene delivery systems (Fig. 20.3). The advancements in gene delivery technology have resulted in the development of additional purposeful nVVs. These vectors have been modified with functions to detect and monitor their progress of the

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FIGURE 20.3 Outline of different nonviral vector delivery methods.

gene delivery by fluorescence imaging. This would result in understanding the mechanism of specific gene deliver along with the process of biodegradation and its excretion out of the biological system (Su, Wu, Wang, & Yeh, 2012).

20.4 Polymers and bioceramics for gene delivery Natural and synthetic biocompatible polymers have been explored for gene delivery applications.

20.4 Polymers and bioceramics for gene delivery

20.4.1 Natural polymer chitosan Chitosan is made up of random β-(1 4)-linked D-glucosamines representing deacetylated components and N-acetyl-D-glucosamines representing the acetylated units which constitutes 50% of the chain length. Water treatment, food processing, and agriculture are just few of the sectors that utilize chitosan. In the medical field, chitosan has been widely used in treatment of diseases as drugs and gene delivery systems owing to their biocompatible and nontoxic nature. Chitosan has a pKa value of 6.5. It is soluble dilute acids because the amount of protonated amine groups in the material increases as the pH of the solvent goes below 6.5. Chitosan is used as a cationic nonviral gene transfer vector as it can be easily obtained from natural and synthetic sources and its ease of solubility. It has the capability to shorten DNA that gives it the capability to prevent nuclease degradation. The chain length of chitosan determines the degree of deacetylation and its molecular weight which in turn influences its physical and chemical behavior. Low-molecular-weight chitosan with deacetylation levels of above 80% results in increased transfection efficacy with minimal toxicity for gene delivery applications. Chitosan may be broken down by enzymes including lysozyme, collagenase, glucosidase, N-acetyl glucosaminidase, and human chitinase. The biodegradability of chitosan may help in the release of pDNA that is embedded to chitosan or pDNA polyplexes under simulated body fluid conditions. Combining chitosan along with different types of cationic-based gene vectors and inducing modifications to chitosan structure have shown to improve transfection effectiveness. Pure chitosan is nontoxic, which is general information. The LD50 of chitosan in mice was reported to be more than 16 g/kg of the body weight when it was delivered through the oral route (Wongrakpanich, 2015). The utilization of on-demand induced disintegration of polymer nanoparticles is used in a wide range of delivery systems that include medication distribution, fragrance release, and nanoreactors. The degradation of polymer side groups can be affected by changing the pH from acidic or basic regions, bifurcation of chemical linkers using enzymes and magnetic and electric fields are the most common ways for disassembly.

20.4.2 Synthetic polymers The cationic poly (2-(dimethylamino) ethyl acrylate) (PDMAEA) has a potential to self-catalyze into poly-acrylic acid (PAA). The degree of PDMAEA breakdown in aqueous solution water was found to be self-determining for both the pH variations examined (5.5 to 10.1). PDMAEA’s cation nature enabled for robust bonding with an oligo DNA which is used as a typical molecule for siRNA and after 24 h, it is released owing to DMAEA hydrolysis for harmless PAA. The degradation of harmless PAA offers the potential for multidose applications, which is very significant in medication administration. In less than 4 h, 80% of cells took

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up the PDMAEA/oligo DNA combination, allowing enough time for the oligo DNA to be released into the cytosol. PDMAEA’s self-degradation process in polymer nanoparticles has been used to study the biochemical pathways to transport which would release siRNA. An arbitrary additional second block of P (N(3-(1 Himidazol-1-yl) propyl) acrylamide) (PImPAA) and poly (butyl acrylate) was connected to a first block of PDMAEA (PBA). The first PDMAEA block is coupled to siRNA resulting in transfection of cells, whereas the second block (P (ImPAA-co-BA)) remained intended to promote endosome membrane fusion besides allowing the siRNA/polymer complex to discharge into the cytosol after 17 h. The results of in vitro studies revealed hand picking and effective knockdown of two very discrete cell lines at a N/P of about 80% corresponding to the nitrogen present on the polymer to phosphorus on the oligonucleotide. A thermoresponsive di-block copolymer that produced micelles may be used to regulate the delivery timing of oligo DNA or siRNA which is part of the polymer complex.

20.4.2.1 Thermoresponsive polymers Thermoresponsive polymers and their related copolymers are widely used in the biomedical application such as cell immobilization, development of drug delivery systems, in situ implants with gels, stem cell and tissue engineering, and many more. Because of the thermoresponsive PNIPAM’s unique properties, it has been used in the preparation and advancement of the gelation process to produce injectable hydrogels by varying the temperature above the polymer’s low critical solution temperature which could result in cytotoxicity. The physical junction formed by hydrophobic contact flanked by warped polymer globules and growing polymer chains results in setting of the hydrogel formation. However, these hydrogels could produce a persistent inflammatory response due to their non degradable property limiting their usage in vivo. Hydrogels with biodegradable properties such as poly (N-iso propyl acrylamide) PNIPAM are prepared by combining polymers which can be broken down or using cross-linkers with PNIPAM. Cross-linkers can restrict the injectability of hydrogels, whereas the breakdown of the hydrogel necessitates a requirement of triggers to initiate the process like enzymes. The inability to maintain the similar environment between cell lines were stimuli is induced and the tissues were transplanted in vitro to vary the rates of deterioration and inconsistency in results between in vitro and in vivo studies is a major drawback. This at certain times restricts the application of thermoresponsive degradable hydrogels for gene delivery. Biodegradation rates and its progress in PNIPAM-based copolymers have been studied by changing the gel formation temperature before and after degradation to understand the degradation process. Chemicals containing hydrolysable side groups were copolymerized with PNIPAM polymer in these degradable hydrogel structures. Polymer solutions demonstrated the formation of gel at temperatures below 37 C, allowing them to be injected before gelation. When the

20.4 Polymers and bioceramics for gene delivery

temperature is raised to 37 C, the polymer solution transforms into a gel. The hydrogel recovers to a sol state when the side groups are degraded, allowing the disintegrated products to liquify in biological solutions and be removed from the human body, though rates of deterioration of these hydrogels cannot be regulated (Tran Thi Dat, 2015). Poly-(L-lysine) (PLL), polyethyleneimine (PEI), and poly-amido amine dendrimers (PAMAM) are the examples of cationic polymers that are commonly employed. These polymers have a broad range of structures, extending from straight lines to complicated branched structures, which affect nucleic acid complex formation and transfection effectiveness. PLL (poly-(L-lysine)), a polycation, was the first to be identified as a possible nonviral gene delivery polymeric vector and accepted for its functions. Reports on their capacity to transport genes both in vitro and in vivo are available. The presence of linear polypeptides induces the biodegradable property along with the amino acid lysine which is present as a repeating unit. The typical number of polylysine molecules within a particular solution rather than a precise amount of lysine molecules per polylysine molecule present in polylysine comes with a variety of sizes. Researchers often carry out synthesis of these polymers with the help of a solid substrate utilizing the sequence to protect/deprotect the reaction processes to avoid this heterogeneity problem.

20.4.2.1.1 Polyethylenimine Polyethylenimine is a polymer that has been utilized in a variety of operations for a long time, including paper manufacturing, water filtration, and shampoo creation. This material has aroused curiosity among researchers to study its applicability as a potential gene delivery system. PEI is a widely used chemicals for transfecting cells during the culture. It is found to be highly effective nonviral substance in a rapidly expanding customer requirement aided by the need for human and animal genome sequencing. PEIs come in a variety of topologies, including linear and branching, and commercially available in the molecular weights ranging from 700 to 800 kDa. Polymerization of aziridine monomers by acid catalysis results in branched products polymers, whereas in case of linear form it is prepared at a lower temperature following the same technique. These two types of PEI have different structures. These polymers have a high-water solubility due to the ethylene amine repeating unit. Each third atom contains an amino nitrogen which is potentially protonable along with high cationic charge which is yet another essential property of these molecules. These are a few details for preferring PEI as an active cationic polymer for gene transport. The branched PEI is given in Fig. 20.4. Polymers made from polyesters poly (lactic acid) (PLA) and poly (D,L-lactideco-glycolide) (PLGA) are biocompatible and biodegradable. These polymers have been cleared by the FDA for specific clinical applications in humans due to their biocompatibility. When these polymers are implanted into the body, they undergo hydrolysis, generating physiologically acceptable and metabolically identifiable

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FIGURE 20.4 Structure of branched PEI. PEI, polyethyleneimine.

units such as lactic acid and glycolic acid which are subsequently eliminated from the physiological system using citric acid cycle. The slow pace of formation of biodegradable polymer particles has little effect on normal cell activity. Because of their biodegradability and submicron size, biodegradable nanoparticle systems have numerous benefits in gene transfer. Overall these polymeric systems can be modified chemically to attack the diseased target cells and tissues to enable the genes and drugs to be loaded for the success of the therapy. It can be applied to increase the bioavailability of the drug in oral environment, have a controlled release of the gene with vector to attack the target tissue, and bring about improvements in genes to overcome the stability in case of enzymes (nucleases and proteases) degradation particularly for peptides, proteins, and nucleic acid systems. These delivery methods at the nanoscale ranges provide a number of specific benefits for gene and medication delivery. Nanoparticles may infiltrate inside the tissues making use of the space and pores in the capillaries, traveling through fenestration present in epithelial linings of the organs such as spleen, liver, etc., and they are absorbed by cells which are at submicron size. This enables the effective administration of medicinal substances to specific bodily locations. Also, by controlling the amount of gene or drug release entrapped in the nanoparticles, one may optimize the therapeutic level required to be present in target tissue needed to obtain the maximum therapeutic efficiency. In addition to this, nanoparticles can be specified to target distant areas of the disease cells with localized release

20.5 Applications of gene delivery

utilizing the method founded on catheter, which is minimally invasive. They can be mixed with ligand molecules that are bispecific in order to reach the diseased cell precisely. PLGA poly (D, L-lactic-co-glycolic acid) consists of several hydroxy acid monomers, namely, D-lactic, L-lactic, or glycolic acids. It may be produced to be extremely crystalline [e.g., poly-(L-lactic acid)] or completely amorphous PLGA. This is achieved by altering the ratio of monomer in order to obtain them in any polymeric shapes and sizes up to 200 nm with molecules of varying sizes. By altering the amount of lactic acid to glycolic acid ratio, polymer molecular weight changes resulting in the formation of PLGA nanoparticles thus enabling the degradation period of PLGA to be changed from days to years (Junping, 2007).

20.5 Applications of gene delivery 20.5.1 Cancer Cancer occurs because the normal process of cell propagation and apoptosis is disrupted. Though several treatment protocols for cancer treatment are available, all of them are accompanied by their own demerits. Figure 20.5 represents the applications of gene delivery. Advancements in cancer treatment require a new type of therapeutic mediators with a new mechanism of action, multiple cell death mechanisms and interaction with conventional treatments. A variety of gene therapy methods for cancer treatment have been developed which include suicide gene therapy, siRNA/miRNA targeted therapy, antiangiogenesis gene therapy, oncolytic virus therapy, immunotherapy, proapoptotic gene therapy, gene directed-enzyme pro drug therapy (Belete, 2021), naked nucleic acid-based therapy, cell-mediated gene therapy, and CRISPR/Cas9-based therapy (Yahya & Alqadhi, 2021). The extended circulating time arising out of changes to the surface of the molecules enables the attack on interstitial cell populations with the targeted gene

FIGURE 20.5 Applications of gene delivery.

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delivery tools. It has been reported that the use of lipid complexes coated with antitransferrin receptor monoclonal antibodies can successfully target DNA and siRNA delivery to tumor cells to bind to the overexpressed transferrin receptor. Gene transfer involving cancer therapeutic genes, whether using enzyme-directed prodrugs or tumor suppressors, or through siRNA-based gene deletion techniques have been discovered as a conceivable treatment involving cancer gene therapy. Further, treatment of nervous system diseases along with brain tumors are treated with gene treatment involving monoclonal antibody facilitated transcytosis of lipid complex-based brain gene delivery (Al-Dosari & Gao, 2009). Physical gene delivery using nonviral systems are prevalent in cancer treatment. One of the technologies is needles and jet injection. Jet injection-based gene transfer has tremendous potential for in vivo gene transfer where DNA-based vaccines are developed for topical immunization drive. This therapy is presently under phase I clinical experimental testing to study its efficacy of skin metastases of breast cancer and malignant melanoma. Further, this technique can be deployed to directly transfect skin cancer cells that would pave the way for normal chemotherapy. Studies have shown that the short hairpin RNA expression vector targeting the multidrug resistance gene 1 (MDR1) can completely reverse the MDR1 phenotype after metastasis in the tumor in vivo. This in turn increases the efficiency of chemotherapy by preventing tumor growth (Stein et al., 2008). In recent decades, researchers have shifted their focus toward the delivery of nucleic acid materials by gene therapy using nonvirals. Advances in immunotherapy, genomics, and the discovery of somatic cell reprogramming into induced pluripotent stem cells have created a new medical model, leading to the reintroduction of gene therapy as a potential device for treating a host of diseases ranging from cancer to viral inflammation. Further, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing system has drawn the involvement of experts due to its capability to edit genome built on RNA-guided nuclease (Mohammadinejad et al., 2020).

20.5.2 Cardiovascular Cardiovascular disease (CVD) has become a serious threat to human life and health as it leads the cause for being the number one killer disease. Patients who have recovered from the corona virus pandemic have also lost their lives during post corona virus treatment to cardiac arrest. Although there are many drugs available in the market that work through different mechanisms of action using orthodox compositions for the dealing with CVDs, their performances are under rated as there are several issues such as inadequate water dissolution, least bioefficiency, nonspecific targeting, and inactiveness to the drugs on prolonged use. Nanotechnology-based nano-drug delivery system (NDDS) provides a new horizon to delivery drugs and genes on various platforms for the recovery of CVDs and offers great advantages in solving the problems mentioned above. However, cytotoxicity in NDDS remains a key issue to be addressed. The future is directed

20.5 Applications of gene delivery

toward the encapsulation and deliver of genes using suitable vectors built on nanocarriers platforms to treat CVDs. In angiogenesis treatment, gene therapy is being explored to deliver the growth factor genes or delivery of nonangiogenic genes for myocardial rescue. This technique is currently evolving and would redefine the treatment of CVDs. In case of virus involved gene transfer system, the safety issues are dominating its efficacy in clinical applications and this needs to be addressed. Several nonviral transmission methods have been established to enable the release of transgenes to disease specific cells deprived of apprehension for insertional mutations, but due to possible cytotoxicity, dose optimization of nVVs is required. In recent years, studies are underway to improve delivery methods by increasing local circulation and decreasing the systemic movement of chemical and drug carriers. Naked DNA is another viable option to transfer DNA to cardiovascular ischemic tissues due to its ease of delivery, efficacy, and safety and clinical trials have to be established (Su et al., 2012). In CVD atherosclerosis is inflated due to the undoubted involvement of monocytes/macrophages (Woollard & Geissmann, 2010). After endothelial cells are injured, monocytes are engaged at the place by discharging chemokines and glycoprotein CD36 is essential in this process. CD36 is an affiliate of the class B knockout receptor and is widely seen on macrophages/monocytes, platelets and endothelial cells. Its reputation in atherosclerosis has been confirmed by the study of ApoE-deficient mice, proving that the inactivation of CD36 leads to a significant reduction in the size of the lesion (Demers et al., 2004). Research on liposomes for atherosclerotic lesions has tested their potential in contrast agents for imaging and antiinflammatory drugs for the design of treatments. Adding on to this, multiple studies have exposed that liposome administration of dexamethasone, cyclopentenone prostaglandin, and serum amyloid A peptide fragments has significant antiatherosclerotic properties in the body (Kelly, Jefferies, & Cryan, 2011). The technology involving cardiovascular gene therapy lies in its continuous and measured appearance of the required protein in the affected cell type or tissue bed to enhance homeostasis and induce the potential for cell regeneration. These combinations can consider the use of protein and gene transfection of ischemic tissue beds or injured blood vessels (Brewster, Brey, & Greisler, 2006).

20.5.3 Kidney The anatomical location and organization of the kidney structure offers various options to locally deliver the genes with efficacy different locations: (1) renal artery, the site of delivery is one at glomerulus and another at tubular epithelium; (2) retrograde renal vein delivery is achieved by administering via basolateral domain along the renal tubules. Nucleic acid extravasation is carried out by creating pores which remain for a short duration caused by the upsurge in pressure locally in renal capillaries comparable to the situation in renal arteries. (3) Retrograde ureteral transfer is where the renal tubular epithelium is targeted and

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(4) intraparenchymal drug delivery. The use of oligonucleotides administered by gene therapy and VVs has been reported to cure kidney ailments (Rubin, Nguyen, Allen, Ayasoufi, & Barry, 2019). In a study conducted by Rocca et al., a recombinant adeno connected virus (rAAV) inoculated retrogradely into the renal vein of mice was used to optimize kidney directed gene delivery strategy. Injection of rAAV into the renal vein resulted in greater renal transduction. This study reported for the first-time renal gene transmission with clinical data (Rocca, Ur, Harrison, & Cherqui, 2014). Rubin et al., have shown that the application of several different vector platforms through direct kidney injection can increase the genetic modification of kidney cells. The results further established that introduction of a large number of carriers injected into the kidney leak out of the organs to facilitate widespread transduction of external tissues indicating that vectors containing kidney-specific promoters might be an advantage. In this background, researchers should focus on increasing renal cell transduction and rise carrier retention in organs (Rubin et al., 2019). Distribution of vectors comprising expression cassettes encoding shRNA forerunners, antagomir, CRISPR Cas9, or miRNA mimics has also received much attention. Reports have highlighted that a lentiviral-based shRNA concept directing toward split and hairy related protein-2 that leads the transplant refusal which meaningfully accomplished gene silencing in rat mesangial cells after subsequent perfusion of inaccessible organs (Carto´n-Garcı´a, Saande, Meraviglia-Crivelli, Aldabe, & Pastor, 2021).

20.5.4 Bone Despite bone’s extraordinary capacity to redevelop without the formation of scars, challenges exist with several diseases and disorders that make it difficult for it to mend. Bone regrowth is an exceedingly slow process where new bone cells are developed and calcification takes place. Bone complications accompanied by pain and weakening of the bone followed by bone loss, and necrosis of the bone cells is a major challenge for the elderly people and patients suffering from bone diseases—osteoporosis, rheumatoid arthritis, etc. To fix osteogenesis issues in bones, a variety of growth factors have been discovered as having the capacity to promote bone growth in endochondral and intramembranous ossification. The bone morphogenetic proteins (BMPs) are the well-studied ones of these factors. Clinically, recombinant human BMP-2 and BMP-7 are offered as dynamic constituents in the commercial products Infuse and OP-1 (osteogenic protein-1). Notwithstanding the fact that these proteins have significant osteogenic characteristics in experimental animals, their clinical efficacy is variable. For example, based on the anatomy of the bone, it varies and in this case it is successful fusion of spinal bones, whereas the same is not true for long bones. On the other hand, the requirement is in extraordinarily large dosages of many milligrams and need to administered several times than the amounts found naturally in bone.

20.6 Conclusion

The challenge in fractured bone healing is to sending of cDNAs which encodes the osteogenic proteins to be active at the site of injury. On delivery of the genes at this site activates the osteogenic protein and undergoes transformation with the help of osteoclast and osteoblast to have new bone deposits. On comparison with recombinant materials, they are free from sedentary and misfolded results which could initiate immune responses. Gene transfer improves safety since the synthesis of osteogenic compounds and its proteins rarely lead to cause systemic dissemination of proteins than bolus delivery which involves huge quantities of recombinant protein. Among the several growth factors that have been found to activate new bone deposition a majority of them promote the distinction of progenitor cells inside the chondrocytes or osteoblasts or boost the bone-making capabilities of established osteoblasts. Bone is an exceedingly vascularized tissue; hence, VEGF and their angiogenic mediators are necessary in the case of intramembranous bone development. In case of chondrogenesis, which is the initial phase of endochondral ossification, blood supply is not required. Peng and colleagues utilized gene therapy and demonstrated that VEGF increased while VEGF antagonist (sFlt1) hindered the restoration of cranial lesions in mice, demonstrating the role of angiogenesis in bone healing. As various growth features operate at various phases of osteogenesis, amalgamation of several factors would be more effective when compared with presence of single component alone in promoting bone repair. BMP-2 and BMP-7 gene delivery, BMP-4 and VEGF gene delivery, and BMP-4 and transforming growth factor(TGF-) gene delivery have all been proven in animal models and this needs to be transformed into safe and nontoxic products.

20.6 Conclusion Gene delivery is the process of transferring an engineered material with the help of synthetic or natural materials into host cells to treat disorders and diseased conditions. There are a host of techniques available to deliver genes into the cells and organs which include mechanical devices from injections to electrical pulses. Gene vectors play an essential role as carriers to transfer DNA or RNA nuclear materials into cells as their large size and electrical charge prevents the direct delivery creating the need for gene mediators. As a hopeful delivery structure, nVVs have established significant consideration because of their least cytotoxicity and nonimmunogenicity. On the other hand, antisense oligonucleotides, aptamers and siRNA-based therapies have shown promising clinical outcomes on human subjects. The success of these gene delivery systems is due the advancement and strides made in understanding the molecular biology concepts. The next stage of development is to evaluate their efficacy, safety, and toxicity and correlate with data from long-term clinical trials before introducing it to patients. Recent

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advances with genome manipulation and development of new preparations of nonviral transfection vectors with valid clinical data have brought out the authorization to practice new gene therapies. In addition, further studies will lead to the development of a variety of life-changing gene therapies.

Acknowledgment The authors (MS and TMS) acknowledge DST SERB CRG Project, New Delhi (CRG/2019/ 003921) for providing Financial support. The authors also thank the support from UGC-SAPDrs-I program New Delhi to Department of Analytical Chemistry, University of Madras.

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21

Essential oil-loaded biopolymeric films for wound healing applications

Kunal Pal1, Preetam Sarkar2, Goutam Thakur3 and Doman Kim4 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 3 Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India 4 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

21.1 Introduction In the last few decades, there has been an increase in the number of cases of inflammatory diseases (Mehta et al., 2019). Among the various inflammationrelated diseases, disease conditions associated with the wounds have gained much attention (Silva et al., 2018). A wound surface is considered a biosynthetic environment wherein the human body carries out a large number of synthesis processes to heal the wound opening (Daemi et al., 2019). The biological processes responsible for wound healing are considerably hampered if the patients are suffering from other diseases, including diabetes. The presence of other pathophysiological conditions can improve the complexity of the cure of the wounds (Okonkwo & DiPietro, 2017). Further, the contamination of the wounds with microbes can also interfere with healing. The hampering of the healing process is reasoned to the ability of the microbes to alter the wound environment. Due to the change in the wound environment, there is a suppression of the biosynthesis at the wound surface. Usually, such infective wounds are treated with antibiotics and synthetic antimicrobial agents (Huang et al., 2020). However, there has been an increase in the number of superbugs in recent times. Superbugs are multidrugresistant bacteria, the growth of which cannot be controlled by many of the current-day antibiotics (Zheng et al., 2018). In other words, the current-day antibiotics are not able to neutralize the superbugs. Keeping this concern in mind, various researchers have proposed the use of medicinal herbs and plants and natural antimicrobial agents (Atef, Shanab, Negm, & Abbas, 2019; Farahpour, Pirkhezr, Ashrafian, & Sonboli, 2020; Ghodrati, Farahpour, & Hamishehkar, Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00002-4 © 2023 Elsevier Inc. All rights reserved.

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2019; Solanki, Sultana, & Singh, 2021). Essentially, such natural products have been explored well in various traditional medicines for therapeutic applications. Among the numerous natural antimicrobial agents, essential oils (EOs) are one of the most important classes of natural antimicrobial agents (Radu¨nz et al., 2019; Trong Le et al., 2020). EOs have been found to be antibacterial, antifungal, and antiviral. Apart from the antimicrobial properties, EOs also exhibit antioxidant and antitumor/anticancer properties (Akhbari, Kord, Jafari Nodooshan, & Hamedi, 2019; Jaradat & Al-Maharik, 2019). EOs have also been explored for their ability to promote wound healing in a regulated manner. As mentioned above, the wound may be regarded as a natural bioreactor wherein there is a continuous biosynthesis of various products, which starts instantaneously after the formation of the wounds. The biosynthesis of these products is very important for the wound healing process. EOs have been reported to promote the biosynthesis of the products required for wound healing (Ghodrati et al., 2019; Sayyedrostami, Pournaghi, Vosta-Kalaee, & Zangeneh, 2018). The ready availability of these bioproducts at the wound site helps in the quick recovery of the injured site. Unfortunately, the development of EO-based formulations for the treatment of wounds is challenging due to their volatile nature (Amalraj, Raj, Haponiuk, Thomas, & Gopi, 2020). Accordingly, numerous researchers have proposed different types of formulations of EOs, including emulsions and films, for wound healing applications (Amalraj et al., 2020; Bao, Wu, & Ma, 2020; Guilherme et al., 2021; Pereira dos Santos et al., 2019). In the current chapter, we will initially discuss wound healing physiology in brief. Thereafter, discussions would be made on the different types of EOs and their components that have been explored for wound healing applications. Attempts would also be made to understand the role of EOs in wound healing. Lastly, the different types of EO-loaded biopolymeric films that have been explored for wound healing applications will be discussed.

21.2 Wound healing physiology The physiology of wound healing has been extensively described in Guimara˜es, Baptista-Silva, Pintado, and Oliveira (2021). The readers are advised to go through the literature for in-depth knowledge. In this section, we will discuss wound healing physiology in brief. The healing of wounds is a well-orchestrated biosynthetic process initiated by our human body as soon as there is a lesion (Kanji & Das, 2017). The lesion is defined as the physical discontinuity of the tissues. Wound healing is mainly targeted toward regenerating and repairing damaged tissues (Guillamat-Prats, 2021). This well-orchestrated process, managed by the human body itself, is implemented in several phases wherein several tissues work in a coordinated manner toward the common goal of repairing the damaged site. The process of wound

21.2 Wound healing physiology

healing is categorized into hemostasis, inflammatory, proliferative, and remodeling subprocesses, which occur in a successive manner (Fig. 21.1) (Guimara˜es et al., 2021). A blood clot is formed at the wound site during the hemostasis phase. The activation of the platelets causes the initiation of hemostasis. Platelets release chemokines and growth factors at the wound site, which promote the formation of a fibrin network around the platelets (Guimara˜es et al., 2021). Then, the phagocytes (neutrophils and macrophages) are rushed to the wound surface in response to the inflammatory reactions. The phagocytes help to clean the wound surface by removing dead cells and bacteria. Concurrently, the fibroblasts also migrate at the wound site. They are responsible for depositing collagen fibers, the

FIGURE 21.1 Different phases of wound healing. Reproduced from Guimara˜es, I., Baptista-Silva, S., Pintado, M., & L. Oliveira, A. (2021). Polyphenols: A promising avenue in therapeutic solutions for wound care. Applied Sciences, 11(3), 1230. Retrieved from https://www.mdpi.com/2076-3417/11/3/1230, under Creative Commons License.

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basis for extracellular matrix formation (Frescaline et al., 2020). Synchronously, angiogenesis is also being initiated. Angiogenesis is the process of generation of new blood vessels and is promoted by the vasculo endothelial growth factor (VEGF). The release of VEGF is triggered by macrophages. The formation of new blood vessels helps bring oxygen and nutrients to the regenerating wound site. At the same time, the blood vessels also help remove the metabolites from the wound site. A combination of these allows quick regeneration that results in the formation of granulation tissue (Jiang & Scharffetter-Kochanek, 2020). Granulation tissue is the new tissue that is formed from the clot formed during hemostasis (Wang, Xu, Wang, Li, & Liu, 2020). The formation of the granulation tissue is important for the reepithelialization of the wound site (Rousselle, Montmasson, & Garnier, 2019). During the final stage, type-II collagen that is formed within the granulation tissue is converted to type-I collagen tissue, thereby resulting in the complete remodeling of the extracellular matrix. The remodeling of the extracellular matrix increases the mechanical properties of the healing tissue and subsequently closes the wound fully (Govindaraju, Todd, Shetye, Monslow, & Pure´, 2019). Unfortunately, some cases (e.g., metabolic disorders and infection of the wound site) can significantly affect the coordination among the different subprocesses of the healing process (Fig. 21.2) (Guimara˜es et al., 2021). This lack of coordination consequently hampers the wound healing and its closure. Diabetes mellitus is one of the common metabolic disorders that can disrupt wound healing. Some of the other disorders include obesity, aging, and abuse of tobacco. Sensory neuropathy has also been related to the disruption of wound healing. If the wound is not healed for a prolonged duration due to pathophysiological disorders, the wound may become a chronic wound. Chronic wounds are prone to infection that leads to delayed wound healing (Wu, Cheng, & Cheng, 2019). In other words, chronic wounds may be described as wounds wherein there is an impairment of the healing process. The impairment may either be due to external and/or internal factors (Bazali´nski, Przybek-Mita, Bara´nska, & Wie˛ch, 2017; Mihai et al., 2018). Empirically, the wounds that do not heal even after several months are categorized as chronic wounds. Such wounds considerably increase the morbidity of the patients. If proper care is not taken, then there are chances of life-threatening infections (Mahmoudi & Gould, 2020). In some cases, there might be a need for amputations. One such example is the amputation of the foot due to a diabetic foot ulcer. The major reason for the conversion of the wounds to chronic wounds can be explained by the abnormality in the physiological process (es), including the inflammatory phase (e.g., hampered phagocytosis and apoptosis), reepithelization, and angiogenesis that causes functional disturbances of the cells (Guimara˜es et al., 2021). An abnormal inflammatory phase increases the availability of neutrophils in quite high numbers at the wound site. The increased neutrophils cause a consequent increase in the free radicals and reactive oxygen species (ROS) at the wound site, thereby increasing the oxidative stress within chronic wounds. Further, the synthesis of collagen is also greatly affected. A combination of

21.2 Wound healing physiology

FIGURE 21.2 Different phases of chronic wound healing. Reproduced from Guimara˜es, I., Baptista-Silva, S., Pintado, M., & L. Oliveira, A. (2021). Polyphenols: A promising avenue in therapeutic solutions for wound care. Applied Sciences, 11(3), 1230. Retrieved from https://www.mdpi.com/2076-3417/11/3/1230, under Creative Commons License.

the above factors leads to the inability of the human body to heal wounds. Apart from the aforesaid conditions, which are intrinsic factors, some external factors can also alter the healing process (Guimara˜es et al., 2021). Some of the common examples include poor nutrition, smoking of cigarettes and other tobacco products, consumption of alcohol, etc. The collagen synthesis is greatly affected if the patient does not receive proper nutrition (Haughey & Barbul, 2017). The infection of the wound can also delay the healing process significantly, thereby causing the formation of a chronic wound. In such infected wounds, the

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bioavailability of the antibiotics at the wound surface is greatly required (Rangel et al., 2020). Further, in the case of multidrug-resistant microbial infection of the wounds, the selection of a suitable antimicrobial agent is of utmost importance. In gist, irrespective of the intrinsic or external factors, the main problems with the chronic wound are the inability of the human body to synthesize the extracellular matrix in sufficient quantity and the altered immune response of the host.

21.3 Essential oils Extensive information about the extraction methods of EOs from plants and the functional properties EOs have been provided in (Basavegowda & Baek, 2021). In this section, some aspects of EOs relevant to the chapter will be briefly discussed. EOs are plant-derived volatile hydrophobic liquids, which are majorly a combination of small molecules (85% 99%), also known as odoriferous compounds. The rest fraction of the EOs is nonvolatile compounds. They are synthesized in plants as secondary plant metabolites. The composition of the EOs is quite varied, which depends on the species of the plants, cultivation conditions, and place of cultivation. EOs are composed of numerous compounds, including alcohols, acids, esters, aldehydes, terpenes, terpenoids, and aromatic phenols (Basavegowda & Baek, 2021). However, the main compounds that provide functional capabilities to the EOs can be categorized as terpenes, terpenoids, and aromatic phenols. Fig. 21.3 depicts some of the major chemical entities that can be found in EOs. EOs are inherently antimicrobial and antioxidant in nature. The antimicrobial activity of EOs is due to the compounds such as menthol, geraniol, cinnamaldehyde, thymol, and carvacrol (Negut, Grumezescu, & Grumezescu, 2018). The antimicrobial spectrum of EOs is considered to be broad. They exhibit antimicrobial activity against Gram 1 ve, Gram 2 ve, fungi, and even viruses. The presence of chemical constituents such as carvacrol, thymol, eugenol, cinnamic aldehyde, and p-cymene denature lipids, lipoproteins, and phospholipids in the bacterial cell membranes (Basavegowda & Baek, 2021). This interaction among the EO constituents and the compounds of the bacterial cell membrane leads to damage to the cell membrane, thereby increasing the cell membrane permeability. Consequently, there is a cellular leakage of the bacterial cytoplasm. The susceptibility of Gram 1 ve bacteria is higher than the susceptibility of Gram 2 ve bacteria. This is related to the fact that Gram 2 ve bacteria consist of lipopolysaccharides, which are hydrophilic in nature, in their cell wall. Due to the presence of the lipopolysaccharide layer, the hydrophobic compounds are considerably shielded from the cell wall membrane (Basavegowda & Baek, 2021). The phenolic constituents (e.g., carvacrol, thymol, eugenol, and oregano) have both hydrophilic and hydrophobic parts. The hydrophilic part of the phenolic constituents interacts with the polar groups of the cell membrane. On the other hand, the

21.3 Essential oils

FIGURE 21.3 Chemical structures of some of the commonly found chemical entities in EOs. EOs, essential oils. Reproduced from Basavegowda, N., & Baek, K.-H. (2021). Synergistic antioxidant and antibacterial advantages of essential oils for food packaging applications. Biomolecules, 11(9), 1267. Retrieved from https://www.mdpi.com/2218-273X/11/9/1267, under Creative Commons License.

hydrophobic parts interact with the apolar groups, which are present as the middle layer of the cell membrane (Basavegowda & Baek, 2021). However, it is important to note that EOs may also exhibit their antimicrobial property in other ways. The components of the EOs may also be internalized through the cell membrane. Once the EO gains entry into the bacterial cells, they can cause protein denaturation, ribosomal disassembly, mitochondrial damage, enzyme inactivation, and

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bacterial DNA damage (Basavegowda & Baek, 2021). Certain EOs can induce oxidative stress in the bacterial membrane, thereby increasing its permeability (Yang et al., 2020). Since EOs can elicit antimicrobial action through multiple mechanisms, it is implausible that the microbes could develop resistance against EOs. Fig. 21.4 shows the plausible mechanisms of antimicrobial activity of the EOs. ROS are the free radicals (e.g., hydrogen peroxide, the superoxide anion radical, and the hydroxyl radical) that are often generated at the wound site. The ROS induces oxidative stress in the biological system and has the capability to damage DNA and cell membranes. The harmful effects of ROS can be easily counteracted using antioxidants. EOs are natural antioxidants due to the presence of phenolic and terphenolic compounds. Among the various phenolic constituents of EOs, carvacrol, thymol, eugenol, and linalool have been found to exhibit the most potent antioxidant properties (Basavegowda & Baek, 2021). Some of the other constituents of EOs such as rosmarinic acid, caffeic acid, carnosol, and quercetin have also been reported to exhibit excellent antioxidant properties. These hydrophobic liquids exhibit their antioxidant property via several mechanisms. The antioxidant property of EOs is primarily due to their free radical scavenging activity. They

FIGURE 21.4 Plausible mechanisms of antimicrobial activity exhibited by EOs. EOs, essential oils. Reproduced from Basavegowda, N., & Baek, K.-H. (2021). Synergistic antioxidant and antibacterial advantages of essential oils for food packaging applications. Biomolecules, 11(9), 1267. Retrieved from https://www.mdpi.com/2218-273X/11/9/1267, under Creative Commons License.

21.3 Essential oils

also act as a preventive antioxidant by preventing the free radical chain initiation and chain-breaking antioxidants by working as a reducing agent, peroxide scavenger, and quenching agent of singlet oxygen (Fig. 21.5). Further, the EOs can also bind with metal ion catalysts that promote the formation of free radicals (Basavegowda & Baek, 2021). Fig. 21.6 highlights some of the plausible mechanisms of antioxidant activities of the EOs. The inflammatory response is a protective mechanism adopted by the body for the removal of the damaged cells at the site of injury or infection. During inflammation, the blood leukocytes gain entry into the interstitium due to the increased permeability of the endothelial lining of the blood vessels. Also, the cytokines (interleukins and tumor necrosis factor-α) are released during the inflammation stage (Miguel, 2010). Cytokines are proinflammatory chemicals and potentiate inflammation. EOs and their constituents have been found to inhibit the release of cytokines. Concurrently, there is an increase in the activity of the various enzymes, namely, oxygenases, nitric oxide synthases, and peroxidases. These enzymes are associated with proinflammatory reactions and can potentiate the inflammation process. The constituents of EOs have also been found to inhibit these enzymes. The arachidonic acid metabolism and expression of cellular adhesion molecules also considerably increased during the inflammation response of the human body (Basavegowda & Baek, 2021). Arachidonic acid is synthesized by phospholipase A2, which is present in the cell membrane during inflammation.

FIGURE 21.5 Schematic representation of mechanisms of preventive antioxidation and oxidation reaction chain-break exhibited by EOs. EOs, essential oils. Reproduced from Basavegowda, N., & Baek, K.-H. (2021). Synergistic antioxidant and antibacterial advantages of essential oils for food packaging applications. Biomolecules, 11(9), 1267. Retrieved from https://www.mdpi.com/2218-273X/11/9/1267, under Creative Commons License.

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FIGURE 21.6 Plausible mechanisms of antioxidant activity exhibited by Eos (Spisni et al., 2020). EOs, essential oils. Reproduced from Spisni, E., Petrocelli, G., Imbesi, V., Spigarelli, R., Azzinnari, D., Donati Sarti, M., . . . Valerii, M. C. (2020). Antioxidant, anti-inflammatory, and microbial-modulating activities of essential oils: Implications in colonic pathophysiology. International Journal of Molecular Sciences, 21(11), 4152. Retrieved from https://www.mdpi.com/1422-0067/21/11/4152, under Creative Commons License.

It is metabolized through the cyclooxygenase and lipoxygenase pathways. The metabolism of arachidonic acid leads to the formation of prostaglandins and leukotrienes. However, in platelets, arachidonic acid is metabolized into thromboxane A2. The synthesis of prostaglandins (primarily PGE2) amplifies pain perception, while leukotrienes increase the proinflammatory responses. The various constituents of EOs (e.g., 1,8-cineole, α-pinene, β-caryophyllene, p-cymene, thymol, and eugenol) have been reported to be good inhibitors of the 5lipoxygenase enzyme. Some of the EO constituents (e.g., limonene and α-pinene) can block PGE2 synthesis by inhibiting the cyclooxygenase-2 enzyme. 1,8Cineole, another common EO constituent, can inhibit the synthesis of leukotrienes and prostaglandins. The antiinflammatory effects of EOs and their constituents have been extensively discussed by (Miguel, 2010). Fig. 21.7 summarizes the different mechanisms by which EOs can exhibit antiinflammatory activity.

21.3.1 Mechanisms of promoting wound healing by essential oils In recent years, it has been reported that several EOs (e.g., lavender oil, tea tree oil, thyme oil, and Ocimum oil) can help heal wounds. The topical application of the EOs at the wound site can promote cell growth and cell migration, thereby

21.3 Essential oils

FIGURE 21.7 Plausible mechanisms of antiinflammatory activity exhibited by EOs. EOs, essential oils. Reproduced from Spisni, E., Petrocelli, G., Imbesi, V., Spigarelli, R., Azzinnari, D., Donati Sarti, M., . . . Valerii, M. C. (2020). Antioxidant, anti-inflammatory, and microbial-modulating activities of essential oils: Implications in colonic pathophysiology. International Journal of Molecular Sciences, 21(11), 4152. Retrieved from https://www.mdpi.com/1422-0067/21/11/4152, under Creative Commons License.

promoting biosynthesis activity. The increased cellular activity results in the increased biosynthesis of collagen, the main polymeric architecture of the extracellular matrix. Collagen stabilizes the healing tissue by improving its mechanical properties and tissue integrity. The so-formed extracellular matrix with improved stability improves not only hemostasis but also promotes the epithelization of the wound surface. The epithelization process is further encouraged by the generation of an increased number of hair follicle stem cells, accounted for by the increased corresponding gene expression (Khezri, Farahpour, & Mounesi Rad, 2019). This can explain less scaring of the wound sites that are treated with EOs. EOs can also help in suppressing the inflammatory response of the body. As mentioned in the previous section, the inflammatory phase is one of the important phases in the wound healing process. However, prolonged inflammation of the wound site is detrimental to the healing process. The ability of the EOs to induce antiinflammatory responses also helps in the healing process. In addition to the aforesaid reasons, the antioxidant properties of EOs have also been related to the accelerated healing process. This has been accounted to the fact that EOs help to reduce the oxidative stress at the wound site (Manconi et al., 2018). An increase in oxidative stress hinders cell growth by causing cell death. Fig. 21.8 shows the application of lavender oil-based topical emulsion for wound healing application.

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FIGURE 21.8 Lavender oil-based emulsion for wound healing application (Boukhatem et al., 2021). Reproduced from Boukhatem, M. N., Chader, H., Houche, A., Oudjida, F., Benkebaili, F., & Hakim, Y. (2021). Topical emulsion containing Lavandula stoechas essential oil as a therapeutic agent for cutaneous wound healing. J, 4(3), 288 307. Retrieved from https://www.mdpi.com/2571-8800/4/3/23, under Creative Commons License.

21.3.2 Methods of preparation of essential oil-loaded films As discussed above, EOs have numerous biological effects which are beneficial in the wound healing process. However, the major disadvantage of EOs is their volatile nature. The volatility of the EOs can be restricted to a great extent by entrapping them with the biopolymer matrices. Taking a cue from the ability of the biopolymer matrices to hinder volatilization of the EOs, various researchers have developed EO-loaded films as wound dressing materials. The EO-loaded films for wound healing applications have been developed mainly by two methods. The first is the conventional solution casting method, while the second is the electrospinning method. The conventional solution casting method employs the development of an emulsion of EOs in a biopolymeric aqueous solution. The emulsion, so formed, is regarded as the film-forming emulsion wherein the biopolymeric phase helps to stabilize the EO phase. Further, the biopolymeric phase hinders the evaporation of the EO. Then, the film-forming emulsion is transferred to a container with a flat base and a large surface area. Subsequently, the container is incubated at a specified temperature and relative humidity conditions to remove the water molecules. As the water molecules are evaporated, the biopolymeric layer is solidified.

21.3 Essential oils

The solidified biopolymeric phase helps control the evaporation of EOs from the films. The release rate of EOs from the films can be controlled by tailoring the composition of the biopolymeric phase (Sedlaˇr´ıkova´ et al., 2019). Fig. 21.9 shows the thyme oil-loaded chitosan films prepared by this method. This method of film preparation is the easiest method and is being widely used by researchers across the globe. However, the method has its own disadvantages. The first disadvantage is the slow process. The evaporation of the water molecules takes a long time. In most cases, the drying period for the preparation of the films varies from 24 to 72 h. Moreover, although the aqueous biopolymeric hinders the escape of the EOs, due to the long drying period, a considerable amount of EO is lost. Hence, there is a need to load the film-forming emulsions with a higher amount of EOs to compensate for its loss. The loss of EOs during the preparation stage unnecessarily increases the cost of the developed films. Also, as the drying process is quite long, the preparation of the films is a slow process and is not a commercially viable method. In the electrospinning method, the film-forming emulsion is used to develop films of nanofibers. Conventionally, the electrospinning method was used for the development of nanofibers. However, the method has been used to develop films

FIGURE 21.9 Thyme oil-loaded chitosan films developed by solution casting method. ˇ ´, J., Janalı´kova´, M., Rudolf, O., Pavlackova ˇ ´, J., Egner, P., Peer, P., . . . Krejcı´, ˇ J. Reproduced from Sedları´kova (2019). Chitosan/thyme oil systems as affected by stabilizing agent: Physical and antimicrobial properties. Coatings, 9(3), 165. Retrieved from https://www.mdpi.com/2079-6412/9/3/165, under Creative Commons License.

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in recent times. The films that are developed consist of nanofibers that are arranged in a random fashion. Hence, such films are often regarded as mats. There are two electrospinning methods for the development of films. The first is known as emulsion electrospinning, while the second method is known as the coaxial electrospinning method (Becerril, Nerı´n, & Silva, 2020). In the emulsion electrospinning method, the film-forming emulsion is loaded in a hypodermic syringe. The plunger of the syringe is pushed at a constant rate to extrude the film-forming emulsion. A typical electrospinning machine employs a voltage between the needle and the metallic collector plate in the range of 5 and 50 kV. As the fluid (film-forming emulsion) is extruded out, it comes under the influence of the electrostatic field. The electric field generates heat within the film-forming emulsion, resulting in the evaporation of the water molecules from the biopolymeric phase. This phenomenon causes an instant solidification of the biopolymers, thereby entrapping the EOs (Kesici Gu¨ler, Cengiz C¸allıo˘glu, & Sesli C ¸ etin, 2019). The development of nanofibrous films can be synthesized in another way, wherein the biopolymer solution and the EO are loaded into a coaxial syringe. This method helps eliminate the preparation step of film-forming emulsion and significantly reduces the complexity associated with film preparation. Fig. 21.10 demonstrates the development of films by both the electrospinning methods. Another advanced electrospinning technique is nozzle-less electrospinning method. In this type of electrospinning method, there is no extrusion needle, that is, needleless setup. However, there is a rotating spinneret that helps in the generation of jets from a thin layer of the polymer solution. During the electrospinning process, multiple jets are formed, which consequently reduces the time for the formation of the films (Nagam Hanumantharao & Rao, 2019). Fig. 21.11 demonstrates the schematic diagram of the working arrangement of the nozzle-less electrospinning machine. As compared to the solution casting method, the electrospinning method is very quick and user-friendly. Further, the loss of EOs is also minimal, which makes the process very economical. The industries can adopt this method for the bulk production of films.

21.3.3 Essential oil-loaded biopolymeric films for wound healing applications Various biopolymers (e.g., collagen, alginate, chitosan, and zein) in conjunction with EOs have been used to develop wound dressing materials. Such wound dressings exhibit good antimicrobial and antioxidant properties. In general, EO-loaded films have been observed to promote the formation of granulation tissue. Further, the synthesis of collagen in the presence of EOs has also been reported by various authors. The entrapment of EOs within the biopolymeric matrices results in the controlled release of EOs over the wound site for a prolonged period. The subsequent paragraphs will discuss some of the applications of EO-loaded biopolymeric films as wound dressings.

21.3 Essential oils

FIGURE 21.10 The schematic diagram for the development of EO-loaded biopolymeric films by the electrospinning method. EO, essential oil. Reproduced from Becerril, R., Nerı´n, C., & Silva, F. (2020). Encapsulation systems for antimicrobial food packaging components: An update. Molecules (Basel, Switzerland), 25(5), 1134. Retrieved from https://www. mdpi.com/1420- -3049/25/5/1134, under Creative Commons License.

Collagen has been explored by many researchers as a material for wound healing applications for a long time. Riella et al. (2012) had reported the synthesis of thymol-containing collagen dressings. The EO-containing films showed antiinflammatory activity in rodents. The analysis was concentrated on the paw edema and peritonitis models. It was found that thymol was capable of inhibiting the migration of leukocytes to the wound site. The healing of the wounds when thymol-containing films were used as the wound dressings showed significantly increased wound retraction rates as compared to the other films. Improvement in the wound healing rate was accounted to better granulation reaction and collagen density (due to improved collagen formation) at the wound site during the healing process. The authors had concluded that thymol can be explored as a bioactive agent for antiinflammatory and wound healing applications. In a similar study, borneol-loaded chitosan films were prepared by Barreto et al. (2016). The presence of bicyclic monoterpene alcohol, that is, borneol, within the films promoted

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FIGURE 21.11 Schematic diagram of a linear nozzle-free electrospinning machine setup (Li et al., 2018). Reproduced from Li, T.-T., Yan, M., Xu, W., Shiu, B.-C., Lou, C.-W., & Lin, J.-H. (2018). Mass-production and characterizations of polyvinyl alcohol/sodium alginate/graphene porous nanofiber membranes using needleless dynamic linear electrospinning. Polymers, 10(10), 1167. Retrieved from https://www.mdpi.com/ 2073-4360/10/10/1167, under Creative Commons License.

granulation reaction and collagen synthesis. Due to the ability of the borneolloaded films to promote collagen synthesis, the rate of wound healing was very quick. Hypericum perforatum oil (HPO)-loaded chitosan films have been proposed for wound healing applications (Gunes & Tihminlioglu, 2017). It was found that the developed films exhibited good antimicrobial and antioxidant properties. The results in this study also suggested that the developed films could be explored for wound healing applications. In a related study, HPO was loaded within the chitosan cryogels. The HPO-loaded chitosan cryogels were successfully explored as wound dressing material (Bolgen, Demir, Yalcin, & Ozdemir, 2020). Bionanocomposite-based polymeric architectures have been explored for various biomedical applications, including drug delivery, tissue engineering, and wound healing. In one such example, EO-loaded sodium alginate/copper oxide nanoparticle-based bionanocomposites have been prepared (Mallick et al., 2020). The bionanocomposite films were loaded with neem EO, eucalyptus oil, and clove oil. It was observed that the copper oxide nanoparticles were homogeneously distributed throughout the polymeric matrices. The EO-loaded developed films have been proposed for wound healing applications. In another study, dextran-based bionanocomposite films were prepared for wound healing applications (Singh, Gupta, Sharma, & Gupta, 2018). The films were loaded with clove oil and sandalwood oil. It was observed that the addition of EOs promoted the wound healing process without the formation of scars. Qin et al. (2020) has reported developing in situ films using zein and clove oil. The films were prepared by electrospinning method employing a

21.4 Conclusion

portable electrospinning device. The use of the portable device allowed the researchers to directly prepare the film over the wound surface. In situ synthesis of the films over the wound surface had considerably improved the comfort of the patients. The developed films were porous, which allowed them to immobilize a large amount of wound exudate. Wound dressings are expected to have good gas exchange properties. It was found that the developed films exhibited excellent gas exchange properties. The films were superhydrophilic, biocompatible and also showed excellent antimicrobial properties. In vitro tests in mice wound models suggested that the prepared films could significantly improve the wound healing process. In recent years, researchers have explored layered films as wound dressing materials. In one such study, Gunes, Tamburaci, and Tihminlioglu (2020) have reported the development of bilayer zein/MMT nanocomposite films (Gunes et al. 2020). The developed films were loaded with H. perforatum oil (HPO). The upper layer of the bilayered films consisted of HPO/zein/MMT, while the bottom layer consisted of electrospun films of HPO/zein/MMT. In this study, the authors used both solution casting (upper layer) and electrospinning (bottom layer) methods for the development of wound dressings. The developed films were compatible with NIH3T3 mouse fibroblast and HS2 keratinocyte cells. It was found that the HPO-containing films promoted wound healing by inducing fibroblast migration.

21.4 Conclusion The process of wound healing is a complex process, which can be attributed to the biosynthesis of multiple biologics at the same time. The synthesis of these biologics is affected by slight irregularity in physiology. If proper care is not taken, the wound may transform into a chronic wound. A chronic wound is regarded as a wound that has not healed even after 6 weeks. Yet, the healing process can be greatly influenced by selecting a wound dressing that can promote the healing process. In the last couple of decades, EOs have been found to promote wound healing. The EOs have also been successfully employed to heal chronic wounds. The wound healing capability of the EOs has been mostly related to promoting granular tissue formation and improved synthesis of the stable collagen matrix. Nevertheless, other mechanisms, including antiinflammatory, antimicrobial, and antioxidant properties, of promoting wound healing by EOs have also been reported. The wound surfaces, especially the chronic wound surfaces, are prone to microbial growth. Till the recent past, such infections were treated with antibiotics. Unfortunately, recently there has been an increase in superbugs, which are resistant to existing antibiotics. The EOs provide a better alternative to these antibiotics. Since the EOs exhibit antimicrobial activity through numerous mechanisms, the microbes cannot develop resistance to these naturally occurring antimicrobial agents.

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Lately, researchers have been developing EO-based formulations for wound dressing application with considerable success. Among the various types of wound dressings, EOs-loaded biopolymeric films have gained much attention in recent times. This is due to the fact that such wound dressings help to release the EOs onto the wound surface in a controlled fashion. The biopolymeric matrix also minimizes the volatility of the EOs. The loading of EOs in biopolymeric matrices makes the wound dressings bioactive. Further, such wound dressings are biodegradable due to the natural origin of all the components. In addition, due to the natural origin of the components, the development of the wound dressings would become sustainable. Nevertheless, there is a lack of clinical data on these wound dressings, thereby restricting their clinic application. Accordingly, there is a need for further investigations to gather more information on the safety and regulatory issues.

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Jaradat, N., & Al-Maharik, N. (2019). Fingerprinting, antimicrobial, antioxidant, anticancer, cyclooxygenase and metabolic enzymes inhibitory characteristic evaluations of stachys viticina boiss. Essential oil. Molecules, 24(21), 3880. Jiang, D., & Scharffetter-Kochanek, K. (2020). Mesenchymal stem cells adaptively respond to environmental cues thereby improving granulation tissue formation and wound healing. Frontiers in Cell and Developmental Biology, 8. Kanji, S., & Das, H. (2017). Advances of stem cell therapeutics in cutaneous wound healing and regeneration. Mediators of Inflammation, 2017. Kesici Gu¨ler, H., Cengiz C ¸ allıo˘glu, F., & Sesli C¸etin, E. (2019). Antibacterial PVP/cinnamon essential oil nanofibers by emulsion electrospinning. The Journal of the Textile Institute, 110(2), 302 310. Khezri, K., Farahpour, M. R., & Mounesi Rad, S. (2019). Accelerated infected wound healing by topical application of encapsulated Rosemary essential oil into nanostructured lipid carriers. Artificial cells. Nanomedicine, and Biotechnology, 47(1), 980 988. Li, T.-T., Yan, M., Xu, W., Shiu, B.-C., Lou, C.-W., & Lin, J.-H. (2018). Mass-production and characterizations of polyvinyl alcohol/sodium alginate/graphene porous nanofiber membranes using needleless dynamic linear electrospinning. Polymers, 10(10), 1167. Available from https://www.mdpi.com/2073-4360/10/10/1167. Mahmoudi, M., & Gould, L. J. (2020). Opportunities and challenges of the management of chronic wounds: A multidisciplinary viewpoint. Chronic Wound Care Management and Research, 7, 27. Mallick, N., Pattanayak, D. S., Singh, R. K., Patel, V. K., Naik, U. K., Kumari, M., . . . Pal, D. (2020). Development of bio-nanocomposite film based on sodium alginate-CuO nanoparticles and essential oils towards medical applications. Journal of the Indian Chemical Society, 97(7), 1088 1093. Manconi, M., Petretto, G., D’hallewin, G., Escribano, E., Milia, E., Pinna, R., . . . Usach, I. (2018). Thymus essential oil extraction, characterization and incorporation in phospholipid vesicles for the antioxidant/antibacterial treatment of oral cavity diseases. Colloids and Surfaces B: Biointerfaces, 171, 115 122. Mehta, M., Tewari, D., Gupta, G., Awasthi, R., Singh, H., Pandey, P., . . . Hansbro, P. M. (2019). Oligonucleotide therapy: An emerging focus area for drug delivery in chronic inflammatory respiratory diseases. Chemico-Biological Interactions, 308, 206 215. Miguel, M. G. (2010). Antioxidant and anti-inflammatory activities of essential oils: A short review. Molecules (Basel, Switzerland), 15(12), 9252 9287. Available from https://www.mdpi.com/1420-3049/15/12/9252. Mihai, M. M., Preda, M., Lungu, I., Gestal, M. C., Popa, M. I., & Holban, A. M. (2018). Nanocoatings for chronic wound repair—modulation of microbial colonization and biofilm formation. International Journal of Molecular Sciences, 19(4), 1179. Nagam Hanumantharao, S., & Rao, S. (2019). Multi-functional electrospun nanofibers from polymer blends for scaffold tissue engineering. Fibers, 7(7), 66. Available from https://www.mdpi.com/2079-6439/7/7/66. Negut, I., Grumezescu, V., & Grumezescu, A. M. (2018). Treatment strategies for infected wounds. Molecules (Basel, Switzerland), 23(9), 2392. Available from https://www. mdpi.com/1420-3049/23/9/2392. Okonkwo, U. A., & DiPietro, L. A. (2017). Diabetes and wound angiogenesis. International Journal of Molecular Sciences, 18(7), 1419.

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Wang, D., Xu, P., Wang, S., Li, W., & Liu, W. (2020). Rapidly curable hyaluronic acidcatechol hydrogels inspired by scallops as tissue adhesives for hemostasis and wound healing. European Polymer Journal, 134, 109763. Wu, Y.-K., Cheng, N.-C., & Cheng, C.-M. (2019). Biofilms in chronic wounds: pathogenesis and diagnosis. Trends in Biotechnology, 37(5), 505 517. Yang, S.-K., Yusoff, K., Thomas, W., Akseer, R., Alhosani, M. S., Abushelaibi, A., . . . Lai, K.-S. (2020). Lavender essential oil induces oxidative stress which modifies the bacterial membrane permeability of carbapenemase producing Klebsiella pneumoniae. Scientific Reports, 10(1), 1 14. Zheng, Y., Liu, W., Qin, Z., Chen, Y., Jiang, H., & Wang, X. (2018). Mercaptopyrimidineconjugated gold nanoclusters as nanoantibiotics for combating multidrug-resistant superbugs. Bioconjugate Chemistry, 29(9), 3094 3103.

CHAPTER

Biomedical antifouling polymer nanocomposites

22

Javad B.M. Parambath, Mahreen Arooj and Ahmed A. Mohamed Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates

22.1 Introduction The phenomenon of adhesion and growth of unwanted materials and organisms at the interface between any aqueous source and a solid surface is termed fouling. This development on surfaces is a critical issue that causes defilement, corrosion, and mishap of engineered devices (Dafforn, Lewis, & Johnston, 2011; Delauney, Compe`re, & Lehaitre, 2010; Hellio & Yebra, 2009; Kerr et al., 1998; Kochkodan & Hilal, 2015; Lejars, Margaillan, & Bressy, 2012; Lewandowski & Beyenal, 2003; Martz, Carr, French, & Degrandpre, 2003; Railkin, 2004; She, Wang, Fane, & Tang, 2016; Tang, Chong, & Fane, 2011; Zhang, Wang, & Zhang, 2018). Fouling is a major concern in the biomedical industry, water filtrationdesalination industry, food industry, and marine devices technology. For example, membrane fouling is a challenge in water desalination processes such as reverse osmosis. Fouling blocks the pores of the membranes and gradually decreases the efficiency which eventually degrades the device quality by salt rejection and permeation flux (Tang et al., 2011). This issue is very common in pressure-driven desalination devices, including nanofiltration, ultrafiltration, and reverse osmosis. Similarly, marine fouling on the surface of devices, ships, and oil platforms severely increases the rate of corrosion of the equipment which leads to significant economic loss. Various fouling organisms, including algae, barnacles, bacteria, spores, and diatoms attach to the surfaces (Dafforn et al., 2011). Biological adhesives produced by some of these organisms such as barnacles can corrode the antifouling coatings (Lejars et al., 2012), which significantly affects the roughness and performance of the device surface. The broad classification of the fouling agents (Fig. 22.1A) includes organic, inorganic, biofouling, natural, and anthropogenic (Chan & Wong, 2010; LoVetri, Gawande, Yakandawala, & Madhyastha, 2010; Schulz, Shanov, & Yun, 2009; Shirtliff & Leid, 2009; Vadgama, 2005; Vo-Dinh, 2007). Organic fouling is caused by the amalgamation of organic materials such as proteins, polysaccharides, carbohydrates, and lipids, whereas inorganic fouling arises due to the precipitation of salts and metal oxides. Biofouling occurs due to the colonization of Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00006-1 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 22.1 (A) Broad classification of fouling agents. (B) Illustration showing few susceptible ways of biofouling infection in the human body (Milo et al., 2017). (C) SEM, scanning electron microscopy image of catheter showing encrustation and blockage by Proteus mirabilis. (A and C) uncoated control. (D) Scheme illustrating biofilm formation on medical implantation. (B) Adapted from P. Dirckx Center for Biofilm Engineering. (C) Reprinted with permission from Milo, S., Hathaway, H., Nzakizwanayo, J., Alves, D.R., Esteban, P. P., Jones, B. V., & Jenkins, A. T. A. (2017). Prevention of encrustation and blockage of urinary catheters by Proteus mirabilis via pH-triggered release of bacteriophage. Journal of Materials Chemistry B, 5(27), 5403 5411. https://doi.org/10.1039/ C7TB01302G. (D) Reprinted with permission from Erathodiyil, N., Chan, H., Wu, H., & Ying, J. Y. (2020). Zwitterionic polymers and hydrogels for antibiofouling applications in implantable devices. Materials Today, 38, 84 98. https://doi.org/10.1016/j.mattod.2020.03.024.

22.1 Introduction

microbes and biological substances on the surface of materials. This buildup of living/dead microorganisms, organelles, blood, and body waste shows the category change from the micro to the macro level of fouling. The extent of fouling varies with the type and nature of the agents which affects significantly medical, marine, and industrial applications. Generally, biomedical fouling includes biofilm, whereas marine and industrial biofoulings include a fusion of biofilm, macromolecules, and inorganic particles (Hellio & Yebra, 2009; Lewandowski & Beyenal, 2003; Railkin, 2004). Sensors and detectors used in marine and aquatic applications are considerably affected by fouling agents. Optical properties-based sensors are very common in oceanography monetarization (Kerr et al., 1998; Martz et al., 2003). The quality, lifetime, and sensitivity of these devices are hampered mainly by the presence of biomedical fouling agents (Delauney et al., 2010; Zhang et al., 2018). Biofouling on membranes consumes more energy and reduces their lifetime along with their permeation quality and membrane flux (Kochkodan & Hilal, 2015; She et al., 2016). Biomedical fouling occurs on prosthetics, dental implants, sensors, catheters, and other medical devices. This may cause serious medical conditions such as implant refusal/failure, associated malfunction of the devices and sensors, and metastasize of contagious diseases (Chan & Wong, 2010; Schulz et al., 2009; Shirtliff & Leid, 2009; Vo-Dinh, 2007). The major cause of contracted infections from hospitals is through infected medical devices due to biofouling. Body parts, which are susceptible to biofouling, are represented in Fig. 22.1B. Catheters are mainly infected by these biofilms and are the major cause of the spread of infections (Chan & Wong, 2010; LoVetri et al., 2010). Statistics showed that 10 among 100 patients get infected from the clinical implants, including catheters, and 9 among 100 patients get pneumonia when supported by ventilators due to biomedical fouling (Chan & Wong, 2010; Shirtliff & Leid, 2009; Vadgama, 2005). Furthermore, 8 among 100 orthopedic implants possess entanglement due to biofilms buildup or other forms of biofouling (Shirtliff & Leid, 2009). According to the National Institutes of Health, 80% of the infections due to biofouling/biofilm on the devices and equipment are difficult to treat and may lead to chronic conditions and harbor antibiotic-resistant microorganisms. For example, biofilms that trigger medical conditions such as cystic fibrosis are difficult to treat due to antibiotic resistance (Monroe, 2007). Primary infections may develop due to fouling on catheters, implants, and devices, whereas secondary infections may develop and can seriously affect vital body organs. Biofouling on medical devices such as dialysis units can occur and its presence is detrimental to patients, difficult to clean, and finally leads to inefficiency of the device (Shirtliff & Leid, 2009; Stickler, 2008). Few examples of biomedical fouling are shown in Fig. 22.1C D and summarized in Table 22.1. There are severe complications clinically reported such as removing ventilation tube after 3 months due to foul smelling (El-Baky, 2012), needles with biofilm caused infections (Donlan, 2001), and bacterial infection on pacemakers which leads to their removal (Marrie, Nelligan, & Costerton, 1982). Polymer nanocomposites are superior in their physicochemical properties compared to their unmodified counterparts. They are prepared by mixing the

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Table 22.1 Biomedical susceptible fouling areas and their common problems. Susceptible area

Problems

Respiratory Contact lens Orthopedic implants Catheter Hemodialysis Dental implants Biosensors

Ventilator-associated pneumonia Eye infection Infection-associated inflammation Urinary tract infection Infected blood Periodontal disease Device failure due to adsorption

nanoparticles with the polymer matrixes and fabricated or engineered using various techniques. Their fabrication strategies have attained wide attention due to the possibilities of tailoring both the physical properties and surface modification. Recent research interest has focused on biocompatible polymer nanocomposites to address the challenges reported in biofouling. For this reason, a fundamental understanding of the attachment of the microbes to the surfaces and their mechanisms of action on the molecular and cellular surfaces is highly important. Thus nanotexturing of polymers can lead to the development of surfaces that have a nonfouling or self-cleaning nature. Early antifouling systems were designed against simple metals, namely, Cu, Pb, As, and Hg. However, the increased toxicity and the short lifetime ceased their wide applications. The quest for nontoxic biocides has lead to the use of natural antifouling agents such as wax and oil. Due to the limited commercialization possibility of these natural resources, researchers focused on the development of fouling-release coatings and polymers, for example, silicones and fluoropolymers. These polymer materials are cheap, versatile, efficient, and biocompatible (Rodrı´guez-Herna´ndez, 2016). The interfacial properties can be tuned by doping with additives. Polymer brushes are common examples as they prevent fouling agents from approaching surfaces (Vos, Kleijn, Keizer, Cosgrove, & Stuart, 2010). Still, achieving efficient methods of antifouling remains a challenge in many applications, especially in the field of the biomedical industry due to the lack of biocompatibility, weak mechanical stability, and short-term antifouling properties. The most common method for preventing fouling is through antifouling coating since the major fouling occurs initially as an inevitable surface phenomenon. Surface properties such as charge, energy, wettability, and texture mainly attract the fouling agents toward the substrates. Therefore designing suitable coatings on the surfaces to target the fouling agents by understanding their nature of the action and the mode of interactions is critical. Apart from the coating, biomedical antifouling requires the development of suitable polymer materials with antifouling properties for enhanced efficacy and biocompatibility.

22.2 Mechanism of antifouling

22.2 Mechanism of antifouling 22.2.1 Strategies of antifouling Since there are different routes of fouling, there are a few strategies also followed for antifouling. Fouling-resistance, release, and degradation are the three general strategies for antifouling (Fig. 22.2A B). Preventing the attachment of fouling

FIGURE 22.2 (A) Schematic illustration of the three principal antifouling strategies (Maan, Hofman, Vos, & Kamperman, 2020). (B) Illustration of the common strategies used in biomedical antifouling (Zander & Becker, 2017). (A) Reprinted with permission from Maan, A. M., Hofman, A. H., Vos, W. M., & Kamperman, M. (2020). Recent developments and practical feasibility of polymer-based antifouling coatings. Advanced Functional Materials, 30(32), 2000936. https://doi.org/10.1002/adfm.202000936. (B) Reprinted with permission from Zander, Z. K., & Becker, M. L. (2017). Antimicrobial and antifouling strategies for polymeric medical devices. ACS Macro Letters, 7(1), 16-25. https://doi.org/10.1021/acsmacrolett.7b00879.

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agents such as proteins, microbes, and other organisms on the substrate surface is fouling-resistance since they are often constructed by highly hydrated surfaces and creates a free energy barrier between the substrate and fouling agents (The´rien-Aubin, Chen, & Ober, 2011). Fouling-release is the approach of weak attraction between the substrate and fouling agent, which facilitates easy desorption from the surface by an external force (Damodaran & Murthy, 2016). Fouling-degrading is how the adsorbed fouling agent will be oxidized or killed (Sakala & Reches, 2018). Surface chemistry is the driving force in the design of efficient antifouling agents. There are different strategies on how the fouling agents are prevented from adsorption. Hydrophilic or zwitterionic surfaces prevent fouling by increasing the surface energy and creating a free energy barrier for fouling agents by strong layers of water molecules already adhered to the surface in the case of low surface energy materials where hydrophobic interactions play a major role in preventing adhesion of fouling agents by self-cleaning (Chen, Li, Zhao, & Zheng, 2010). In addition, surface charges can play a major role: charged or neutral has its strategies according to the nature of the fouling agents. The charged surfaces act as biocidal surfaces to kill bacteria and other microbes, whereas neutral surfaces prevent the electrostatic attraction of the fouling agents to the surfaces (The´rien-Aubin et al., 2011). Since proteins and microbes are the major fouling agents on biomedical devices and blocking their adsorption prevents the infection. Bacterial biofilms and cellular growth are generally colonized after receiving a potent platform of protein adsorbed on the surfaces (Costerton, Montanaro, & Arciola, 2005). Protein fouling is a prime concern in developing biomedical devices due to their proinflammatory nature, which leads to thrombosis. There are a few reviews on antifouling strategies for biomedical applications (Bixler & Bhushan, 2012; Bixler, Theiss, Bhushan, & Lee, 2014; Salta et al., 2010). Specifically, the following strategies have been utilized: (1) the use of biocides to kill bacteria or the use of antimicrobial agents coated on surfaces, (2) materials to repel proteins so that cell adhesion can be prevented, and (3) to create self-cleaning surfaces where the organisms or cells cannot adhere. The surfaces are either chemically or physically modified to avoid fouling by the blood.

22.2.2 Natural antifouling Fouling occurs all around nature, but there are many examples of antifouling which may occur around us with a variety of techniques. These are either mimicked or restructured to prevent fouling in artificial systems. Generally, the antifouling mechanisms are through reduced adhesion, wettability, roughness, and chemical secretions, which are observed in different species in the animal and plant kingdom. For example, superhydrophobic self-cleaning lotus plant surface (Barthlott & Neinhuis, 1997) to chemical secreting seaweed (Ulva lactuca) (Rao, Webb, & Kjelleberg, 2005) in flora and eyelid secretions of the human cornea

22.3 Biomedical antifouling

(Menton, 2008) to flexion of scales and mucous of shark (Kesel & Liedert, 2007) in fauna show their different techniques of antifouling. Superhydrophobic surfaces are seen in lotus leaves, broccoli, insect cicada, wings of butterflies, feathers of pigeon, and superoleophobic surfaces in most fish scales, which helps these species to prevent biofouling and resist microbial attacks. Similar topography was constructed artificially to prevent fouling. Carman et al. (2006) reported biomimetic topography similar to the skin of sharks. They measured the wettability of these constructed surfaces and tested the settlement of zoospores and the alignment of porcine cardiovascular endothelial cells. It was suggested that the low adhesion of both cell types was due to the low wettability. There are several alkaloids and terpenoids secreted by living plants, which inhibit bacteria to reduce biofouling, as Holmstro¨m, Steinberg, Christov, Christie, and Kjelleberg (2000) demonstrated using Pseudoalteromonas tunicate into hydrogels and showed the inhibition of barnacle settlement.

22.3 Biomedical antifouling 22.3.1 Nanogel engineering Three-dimensional network polymer nanoparticles can form nanogels of a size below 100 nm. The cross-linked polymer chains formed either by physical forces or chemical bonding or both are of wide interest in biomedical antifouling applications (Sasaki & Akiyoshi, 2010). Nanogels respond rapidly to minute environmental changes such as temperature and pH. Nanogels are obtained using liposomes in an aqueous medium reaction, lipid-coated nanogels, and gold nanoparticles template to form hollow nanogels for various biomedical applications (Fig. 22.3A) (Singh & Lyon, 2007). Nanogels can create highly hydrated surfaces, which indeed promote antifouling properties. Nanogel coatings have been used to prevent protein adhesion (Nolan, Reyes, Debord, Garcı´a, & Lyon, 2005), against macrophages (South, Whitmire, Garcı´a, & Lyon, 2009), biosensor design (Sigolaeva et al., 2018), cellular adhesion (Lynch, Miller, Gallagher, & Dawson, 2006), and antimicrobial (Nystro¨m, Stro¨mstedt, Schmidtchen, & Malmsten, 2018). Nanogel fabrication on the material surface is a facile method that can be carried out through electrostatic interactions. Generally, they are prepared by the pretreatment of both the material and the nanogel surfaces for the required interactions (Schmidt, Hellweg, & Klitzing, 2008). Nanogels have been developed extensively as stimulus-responsive materials since they can display different types of intermolecular interactions. Hydrophobic surfaces are created using polylactic acids, which are water-repellent polymers onto polysaccharides (Nagahama, Mori, Ohya, & Ouchi, 2007). For example, heat-induced association and the dissociation of polysaccharides coated with PNIPAM polymers were reported and are easily soluble in water (Fig. 22.3B) (Akiyoshi et al., 2000).

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FIGURE 22.3 (A) Nanogel preparation by the nanotemplate methods: (a) liposome and (b) gold nanoparticle. (B) Nanogels at critical solution temperature showing heat-induced association and dissociation. (A) Reprinted with permission from Singh, N., & Lyon, L.A. (2007). Au nanoparticle templated synthesis of pNIPAm nanogels. Chemistry of Materials: a Publication of the American Chemical Society, 19(4), 719 726. https://doi.org/10.1021/cm061878d. (B) Reprinted with permission from Akiyoshi, K., Kang, E., Kurumada, S., Sunamoto, J., Principi, T., & Winnik, F.M. (2000). Controlled association of amphiphilic polymers in water: Thermosensitive nanoparticles formed by self-assembly of hydrophobically modified pullulans and poly (N-isopropylacrylamides). Macromolecules, 33(9), 3244 3249. https://doi.org/10.1021/ma991798d.

Karmali et al. (2012) synthesized stealth nanogel by cross-linking 1-chloro2,3-epoxypropane with iron oxide nanoparticles. This material stopped the recognition and clearance by liver macrophages and considered long-stealth nanoparticles. They have also shown that selective interactions of proteins than the total repelling of proteins for long circulation. Hybrid nanogels of polymers and magnetic nanoparticles Fe3O4 were used as antifouling agents for improving the

22.3 Biomedical antifouling

nanoparticles circulation in vivo. The combined nanomaterials and hydrogel properties resulted in the accuracy of reaching the desired site. The effect of gelated magnetic iron nanoparticles was studied by injecting mice with cross-linked and non-cross-linked magnetic nanoworms. Schmidt et al. (2012) prepared crosslinked polymer nanogels using poly(L-lysine) and hyaluronic acid and increased the elastic modulus by doping with gold nanoparticles by more than one order magnitude. These highly swollen polyelectrolyte films have shown immense potential for optimized cell adhesion (Fig. 22.4). They have also shown that the gold nanoparticles are not incorporated inside the polymer bulk rather on the surface. Molla et al. (2014) demonstrated pH-dependent cross-linking of proteins with nanogels. Their findings can be utilized in a broader perspective of protein encapsulation on nanogels and stimulus-sensitive release for biomedical antifouling. The nanogel contains a β-thiopropionate cross-linker, which helps the polymeric composite to respond differently at different pH values. They used this platform to sequester and turn off a lysosomal protein. Similarly, Takahashi, Sawada, and Akiyoshi (2010) synthesized the cyclodextrin-nanocomplex using nanogels and showed its high capacity for adhering proteins through complexation. They termed it as artificial chaperones and have shown significant protein association. Kruss, Erpenbeck, Scho¨n, and Spatz (2012) developed a nanogel microchannel to study cell-surface interactions. They fabricated polyethylene glycol hydrogel with gold nanoparticles and biomolecules to modify surface density and biocompatibility. This was utilized to study the cell adhesion through a microchannel with and without flow.

FIGURE 22.4 Schematic illustration of the film structure and changes of the cellular adhesion; AuNPs (in yellow) bind by complexation with poly(l-lysine) (in green) with the film. Top photographs show changes of the film with surface concentration γ, which vary on AuNPs concentrations. Reprinted with permission from Schmidt, S., Madaboosi, N., Uhlig, K., Ko¨hler, D., Skirtach, A., Duschl, C., . . . Volodkin, D.V. (2012). Control of cell adhesion by mechanical reinforcement of soft polyelectrolyte films with nanoparticles. Langmuir: the ACS Journal of Surfaces and Colloids, 28(18), 7249 7257. https://doi. org/10.1021/la300635z.

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Shen et al. (2011) developed core shell nanogel with PEG (polyethylene glycol) as linear chain and OEG (oligo ethylene glycol) as side chains. This was developed using the RAFT (reversible addition-fragmentation chain transfer) polymerization method with a low polydispersibility index and high thermosensitivity (Fig. 22.5). The nanogels exhibited good stability in different biological conditions and have shown significant biocompatibility with tunable sizes. Zhao et al. (2013) reported the antifouling properties of two types of poly(Nhydroxyethyl acrylamide) (polyHEAA)-based nanogels. PolyTM-g-HEAA nanogel synthesized by two-step polymerization formed a core shell after coating with polyHEAA onto the cationic polyTM core and showed improved stability in fetal bovine serum (FBS). They also found that this material has negligible cytotoxicity with estimated 94% cell viability without protein adhesion. They suggested that polyHEAA-based nanogels are an excellent choice for antifouling biocompatible materials.

FIGURE 22.5 Schematic representation of macro-CTAs and the formation of core shell nanogels. Reprinted with permission from Shen, W., Chang, Y., Liu, G., Wang, H., Cao, A., & An, Z. (2011). Biocompatible, antifouling, and thermosensitive core 2 shell nanogels synthesized by RAFT aqueous dispersion polymerization. Macromolecules, 44(8), 2524 2530. https://doi.org/10.1021/ma200074n.

22.3 Biomedical antifouling

Xia et al. (2015) developed silver nanoparticle-grown polymer nanogel using poly(ethylene glycol) methacrylate (PEGMA) and methacrylic acid (MAA). They have grown silver nanoparticles in situ on the polymer matrix using vitamin C as a reducing agent. This nanogel composite was experimented with to show prolonged blood clotting on the surface and minimum cellular adhesion. Antibacterial studies revealed that this material could kill both Staphylococcus aureus and Escherichia coli bacteria. Their approach of constructing robust antifouling material with appreciable biocompatibility has immense potential in clinical practices. Recently, Keskin et al. (2020) reported surface coating of multifunctional nanogel with quaternary ammonium compounds. These coatings possess antifouling behavior against S. aureus ATCC 12600, Gram-positive bacteria through a highly hydrated surface. This also allows the triclosan intercalation through internal hydrophobic nature due to the presence of aliphatic chains. Triclosanintercalated nanogels showed a 99.99% antibacterial activity compared to nonquaternized nanogel coatings which suggest their impact on the design of suitable clinically applicable coatings. Furthermore, optimizing the design and synthesis of novel nanogel composites for biomedical antifouling is an important strategy to prevent biofilms associated with protein adhesion and microbial growth. There are several more biocompatible nanogel composites used for proteins releasing/encapsulating, stimuliresponsive, and cell adhering studies reported and yet to be explored for antifouling-antimicrobial activity (Bhuchar, Sunasee, Ishihara, Thundat, & Narain, 2011; Chen et al., 2013; Gonza´lez-Toro, Ryu, Chacko, Zhuang, & Thayumanavan, 2012; Leo´n et al., 2016).

22.3.2 Zwitterionic nanomaterials Zwitterionic nanomaterials are a new emerging class of charged materials, which possess positive and negative charges. They are more biocompatible and mimic biological systems such as lipid bilayer of cell membranes. Their promising characteristics provoke the development of antifouling biomaterials with surface engineering. Recently, the increased number of assembling molecular scaffolds containing various functional groups has become a toolkit for tailoring surface properties at the molecular level. Several reports have shown the zwitterionic nanomaterials have a significant role as biocompatible and bioactive antifouling surfaces. There are two types of polyzwitterionic materials generally utilized for antifouling applications. They are: 1. Polybetaines: Polymer composites with positive and negative charged moieties on the same monomer and 2. Polyampholytes: Polymer composites with positive and negative charged moieties at different monomer units.

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Controlling charge distribution uniformity and neutrality are critical parameters for the development of antifouling zwitterionic nanomaterials. In such a way, they can increase the surface hydration and decrease charge particle adhesion, including proteins. Thus hydrophilic and the charge-neutral surface provides an energy barrier to repel nonspecific adhesion (Fig. 22.6) (Chen et al., 2010). Generally, bare nanoparticles are highly prone to association with proteins, lipids, and other biomolecules, leading to fouling (Lundqvist et al., 2011; Walczyk, Bombelli, Monopoli, Lynch, & Dawson, 2010). The binding of these nanoparticles to biomolecules resulted in phagocytosis as a normal immune response and the body tries to clear rapidly from the bloodstream (Owensiii & Peppas, 2006). To achieve biocompatibility, nanoparticles were attached with polyethylene glycol chains (PEGylation) (Karakoti, Das, Thevuthasan, & Seal, 2011). But the drawback of this method to prevent phagocytosis and increasing biocompatibility is the increased size of nanoparticles and lack of efficiency. Thus this new class of nanocomposites with low molecular weight is potentially more versatile for avoiding nonspecific adsorption of biomolecules and microbes. Consequently, the circulatory half-lives and clearance times for zwitterionic nanoparticles were reported relatively very short. Liu et al. (2007) reported the synthesis of different sizes of biocompatible fluorescent cysteine-coated quantum dots. Zwitterionic amino acid capped nanoparticles showed neither aggregation nor any protein adhesion in the presence of serum. Furthermore, these cysteine-coated quantum dots of size 5.5 nm showed renal clearance by excretion. Oxidation of the amino acid capped nanoparticles like this was found as an issue and utilized d-penicillamine to coat nanoparticles found no nonspecific interaction with human monocytic cells (Treuel et al., 2014). Susumu et al. (2011) reported zwitterionic dihydrolipoic acid derivatives

FIGURE 22.6 Schematic representation of the guidelines to design antifouling polyzwitterionic materials. Reprinted with permission from Chen, S., Li, L., Zhao, C., & Zheng, J. (2010). Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials. Polymer, 51(23), 5283 5293. https://doi.org/ 10.1016/j.polymer.2010.08.022.

22.3 Biomedical antifouling

prepared by adding both tertiary amine and carboxyl functional groups to stabilize the quantum dots and gold nanoparticles. These zwitterionic nanocomposites were utilized for biomedical applications and showed high stability in the cytosol and the absence of fluorescence indicating no protein adhesion. Apart from neutral zwitterionic surfaces, Murthy et al. (2013) reported citrate-capped gold nanoparticles that were functionalized by cysteine and lysine with net negative charge also showed no protein adsorption on the surface. It was postulated that the amino acids bind through their side chains such as amino/carboxyl groups forming a protective outer layer that prevents the inner citrate capping agent from interacting with serum proteins. Thus there are more developments in constructing varioussized nanoparticles using amino acids. Recently, Hameed, Ahmady, Han, and Mohamed (2020) reported tyrosine, tryptophan, and cysteine amino acids capped gold nanoparticles and showed negligible cytotoxicity to human fibroblast cell lines. They have also shown that there is no additional capping agent required apart from these zwitterionic amino acids. There are various sulfobetaine derivatives used in the synthesis of zwitterionic nanoparticles. Rouhana, Jaber, and Schlenoff (2007) reported the use of sulfobetaine-based disulfide to stabilize gold nanoparticles of a very small size of 4 nm. The zwitterionic nanoparticles were aggregation-free, stable in solution in the presence of charged proteins, lysozyme, and bovine serum albumin (BSA). Dopamine sulfonate was used by Wei et al. (2011) to develop magnetic iron oxide nanoparticles. They developed 10-nm-sized zwitterionic magnetic nanoparticles that were stable in various pH values and salinity and have shown to be more resistant to the adsorption of serum proteins. Similarly, Estephan, Hariri, and Schlenoff (2013) reported iron nanoparticles using zwitterionic sulfobetaine siloxane derivatives. These zwitterionic nanoparticles were stable in various conditions such as 3 M NaCl and 50% FBS for 24 h and were easily released through renal filtration. The size of these iron nanoparticles was controlled by controlling the concentration of the zwitterionic siloxane. There are also various phospholipids used to synthesize zwitterionic nanoparticles apart from PEGylated phospholipids although they are more widely used. Jin, Liu, Xu, Ji, and Shen (2008) utilized the thioalkylated phosphorylcholine, 11-mercapto undecyl phosphorylcholine (HS-PC) to prepare zwitterionic silver nanoparticles. These neutral composites showed minimal adsorption of DNA, lysozyme, and BSA in solutions. Similarly, Liu, Huang, Jin, and Ji (2011) reported gold nanoparticles using two differently charged phospholipids. They utilized negatively charged 10-mercaptodecanesulfonic acid and positively charged (10-mercaptodecyl)-trimethyl-ammonium bromide with the citrate capped gold nanoparticles to make them zwitterionic composite. This method resulted in increased stability of citrate-capped gold nanoparticles in cell culture medium with FBS and prevents its antifouling nature. It is desirable to develop more biocompatible zwitterionic materials that have strong antifouling properties. Wang, Fei, Xia, and Zuilhof (2018) reported drop-casting zwitterionic polymer colloidal particles with strong protein resistance and self-cleaning capabilities. They were further investigated for antibiofouling properties. Sa¨llstro¨m,

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Capel, Lewis, Engstrøm, and Martin (2020) reported a 3D printable zwitterionic sulfobetaine hydrogel with a nano-clay cross-linker in controllable shapes. They conducted experiments directly and indirectly to show its minimal cytotoxicity. The neural cytotoxicity of the developed system showed a significant difference in cell viability between the different conditions. Erathodiyil et al. (2020) reported robust and biocompatible calcium cross-linked zwitterionic antifouling material. Simple acrylate monomers containing carboxylic, sulfonic, and tert-amine groups were used as ligands and bound with Ca21 later added zwitterionic methacryloyl-l-lysine (MLL). These zwitterionic composite demonstrated strong resistance against the adhesion of cells, proteins, and microbes. Much less collagen formation was observed in the hydrogel G group compared to the PHEMA group.

22.3.3 Superhydrophobic surfaces and wettability Surface factor wettability is crucial in developing antifouling materials. Extreme water-loving and extreme water-repellent surfaces act as antifouling agents. Both prevent microbial colonization, cellular adhesion, and protein adsorption on the surfaces. Wettability is determined by contact angle measurements, whereas angles less than 10 degrees are superhydrophilic and over 150 degrees are superhydrophobic. Self-cleaning, superhydrophobic surfaces promote antifouling due to their very less surface energy. Whereas superhydrophobic surface exhibits high wettability and high surface energy which prevents foulants adsorption. Antifouling using superhydrophilic surfaces forms a uniform strong water layer that has high surface energy and disrupts further adsorption of fouling agents (Hellio & Yebra, 2009). Water layer on nanoscale coatings restricts microbial adhesion, microorganisms settle in areas slightly larger than their size for protection and to have more surface area contact. Notable efforts have been made toward superhydrophobic surfaces, in biomedical antifouling. As mentioned, superhydrophobicity occurs due to surface structure, often at the nanoscale, with low surface energy. Several reports have been published over the last few years on superhydrophobic nanocomposites to minimize bacterial/cell adhesion and thus to prevent biofilm on implants and catheters (Che, Liu, Chang, Wang, & Han, 2015; Hizal et al., 2017; Pechook et al., 2015). The main materials used for medical implants are titanium, stainless steel, cobaltchromium, and some polymers (Eltorai et al., 2016). Hizal et al. (2017) reported different nanostructures on aluminum substrates, including 2D nanoporous and 3D nanopillared surfaces. They have studied the adsorption of S. aureus and E. coli bacteria (Fig. 22.7). These nanostructured surfaces showed significant antifouling properties by reducing colony-forming units. Qian et al. (2017) also published on superhydrophobic multilayer film coated on stainless steel to monitor the antibacterial properties by incubating E. coli and S. aureus. Hierarchical micro/nanostructures were developed using polydopamine (PDA) and silver nanoparticles. There was no cell adhesion on the surface for 3 days and they compared its efficiency with other noncoated surfaces.

22.4 Computational studies

FIGURE 22.7 (Top) FE-SEM images and schematics representing the bacterial adhesion on superhydrophobic nanopillared surfaces and hydrophilic surfaces. (Bottom) FE-SEM images of hydrophobic layers on aluminum substrates: (a) nanoporous and (b) nanopillared. The top right inset in each figure shows a droplet of water on each surface, angles of 115 and 162 degrees, respectively. Reprinted with permission from Hizal, F., Rungraeng, N., Lee, J., Jun, S., Busscher, H.J., Mei, H.C., & Choi, C. (2017). Nanoengineered superhydrophobic surfaces of aluminum with extremely low bacterial adhesivity. ACS Applied Materials & Interfaces, 9(13), 12118 12129. https://doi.org/10.1021/acsami.7b01322.

22.4 Computational studies 22.4.1 Exploring the antifouling properties of polymers using computational methods Along with several experimental studies of antifouling materials, computational techniques such as molecular dynamics (MD) simulations, molecular mechanics (MM),

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Monte Carlo (MC), and quantum chemical methods have been extensively applied to explore various aspects of the structure, dynamics, and interfacial properties of antifouling materials at the atomistic level as summarized below (Liu et al., 2019, 2020; Ma et al., 2020; Nagumo et al., 2012, 2014; Shao & Jiang, 2013, 2014; Shao et al., 2014; Shao, He, White, & Jiang, 2012; Yiapanis, Maclaughlin, Evans, & Yarovsky, 2014).

22.4.2 Effect of surface hydration on antifouling properties Antifouling polymers can be categorized into two groups based on surface hydration, including hydrophilic-based and zwitterionic polymers. Experimental findings specified that hydrophilic polymers have continually exhibited outstanding antifouling properties against unwanted biomolecular adsorption such as detrimental proteins, bacteria, and cells compared to hydrophobic polymers. Consequently, it is commonly understood that the antifouling properties of the hydrophilic polymers are mainly due to the presence of strong surface hydration. These layers of closely bound water molecules will serve as a physical barrier and act as an energy barrier as well against the binding of biomolecules since significantly high energy is required to replace the water molecules surrounding both polymers and biomolecules. Recently, Liu et al. (2019) reported the effect of carbon spacer lengths (CSLs) on the hydration properties of acrylamides (AMs) and their interactions with proteins. The polymerization of acrylamides and their grafting on gold substrates have been used to fabricate polymer brushes for antifouling applications. All-atom MD simulations were used to study the effect of CSLs on the hydration properties of four acrylamides (AMs) including (N-hydroxymethyl acrylamide) (HMAA), (N-hydroxyethyl acrylamide) (HEAA), (N-hydroxypropyl acrylamide) (HPAA), and (N-hydroxypentyl acrylamide) (HPenAA). The interaction of these acrylamides with C12 protein was also investigated. Outcomes of the simulations study showed that shorter CSLs (1 2 2) allowed synchronization of more water molecules which instigated their longer stay thus caused slow diffusion around the AM chains of HEAA and HMAA compared to HPAA and HPenAA with longer CSLs (3,5). Due to the strong hydrophilic nature of AMs, the extended residence time and slow self-diffusion of water molecules near AMs aided in binding between AMs and water molecules via various kinds of interactions, including Van der Waals, electrostatic, and hydrogen bonding interactions. Results from MD calculations revealed that in the presence of protein, all AMs were able to bind strongly with the water molecules located in the close vicinity thus demonstrated strong hydration. As a result of strong hydration, HEAA and HMAA did not show almost any interactions with the protein, while HPenAA comparatively exhibited weak interactions. These weaker or insignificant AM protein contacts and strong AM 2 water interactions validate that the actual protein resistance primarily is due to the interfacial layer of water molecules around AMs. Better awareness of the interfacial behavior of these four AMs regarding hydration and protein resistance will help in the design of new and useful antifouling materials beyond the conventional poly(ethylene glycol) (PEG).

22.4 Computational studies

In another recent study by Liu et al. (2020), the antifouling properties of polyacrylamide (PAMs) brushes were investigated in detail by applying a combination of computational methods, including MM, MD simulations, and MC calculations (Fig. 22.8). The packing structure, lysozyme protein resistance, and surface hydration of four different PAM brushes were evaluated. Findings from this work also showed that PAMs brushes poly(N-hydroxymethyl acrylamide) (pHMAA), poly (N-(2-hydroxyethyl)acrylamide) (pHEAA), and poly(N-(3-hydroxypropyl)acrylamide) (pHPAA) with shorter CSLs (1 3) retained significantly stronger binding capability with large number of water molecules than a poly(N-(5-hydroxypentyl) acrylamide) (pHPenAA) brush with larger CSL of 5. These results indicate the CSL effect with better surface hydration of pHMAA, pHEAA, and pHPAA directed the elevated surface resistance to lysozyme adsorption, compared to its adsorption on pHPenAA PAM. The use of computational methods aided in understanding how slight structural differences can influence the surface hydration and antifouling attributes of antifouling surfaces and materials.

22.4.3 Polyzwitterions Due to their outstanding biocompatibility, polyzwitterions (PZs), including carboxybetaine (CBs), phosphobetaine (PB), and sulfobetaine (SB) polymers, are among the

FIGURE 22.8 Optimum conformation of the lysozyme protein on (A) pHMAA, (B) pHEAA, (C) pHPAA, and (D) pHPenAA brushes. Reprinted with permission from Liu, Y., Zhang, D., Ren, B., Gong, X., Liu, A., Chang, Y., . . . Zheng, J. (2020). Computational investigation of antifouling property of polyacrylamide brushes. Langmuir: the ACS Journal of Surfaces and Colloids, 36(11), 2757 2766. https://doi.org/10.1021/acs.langmuir.0c00165.

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highly studied biomaterials. To divulge the reaction of the body to implanted biomaterials, for instance PZs, it is crucial to obtain a better understanding of the behavior of proteins in the locality of the surface of the biomaterial (Fig. 22.9). A computational study by Nagumo et al. (2014) explored the antifouling properties of PZs via calculation of free energy profile of the protein absorption at the biomaterial surface thus provided thermodynamic insights into the antifouling mechanism. The predicted energy profiles of carboxybetaine showed no energetically stable points, irrespective of the type of residue. While the profiles for polyethylene terephthalate (PET) display some energetically significant minima mainly for the hydrophobic residues. These results imply that hydrophobic residues can be useful as probe molecules for evaluating the protein antifouling mechanisms of polymers. Another MD simulations study examined the effect of CSL on the molecular properties of zwitterionic carboxybetaines (Yiapanis et al., 2014). Five CB molecules with a range of CSLs (0 4) were investigated. Results showed that the hydration-free energy of CB molecules with a CSL , 3 is strongly dependent on the CSL. On the other hand, CB molecules with a CSL $ 3 do not indicate substantial deviation. The evaluation of the radial distribution functions, lifetime, and free energy profile of CB 2 Na1 binding also illustrate a strong reliance on the CSL, asserting its significance. A computational modeling study was performed to investigate the antifouling behavior of polyethylene glycol (PEG) divulged that PEG chains are attached to hard hydrophilic substrates, for instance, silica compared to deformable polymer

FIGURE 22.9 Schematic presentation of the methodology to evaluate antifouling properties of polymeric biomaterials. For simplicity, explicit water molecules are not displayed. Reprinted with permission from Shao, Q., & Jiang, S. (2013). Effect of carbon spacer length on zwitterionic carboxybetaines. The Journal of Physical Chemistry. B, 117(5), 1357 1366. https://doi.org/10.1021/jp3094534.

22.5 Conclusion

FIGURE 22.10 Charge-density matching decides associations between zwitterionic groups in zwitterionic polymers. Reprinted with permission from Shao, Q., Mi, L., Han, X., Bai, T., Liu, S., Li, Y., & Jiang, S. (2014). Differences in cationic and anionic charge densities dictate zwitterionic associations and stimuli responses. The Journal of Physical Chemistry. B, 118(24), 6956 6962. https://doi.org/10.1021/jp503473u.

substrates (Shao et al., 2014). The study concluded that the impact of steric repulsion versus surface hydration on the antifouling capability of surfaces is greatly reliant on the nanoscale structure and deformability of the substrate. Shao et al. (2012) examined the thermal- and salt-responsive behavior of sulfobetaine and carboxybetaine polymers using MD simulations and concluded that the difference in the charge-density among the cationic and anionic moieties regulates the connotations between the zwitterionic groups responsible for diverse stimuli responses of carboxybetaine and sulfobetaine polymers (Fig. 22.10). In another computational study, the effects of carboxybetaine and (ethylene glycol)4 (EG4) solutes on chymotrypsin inhibitor 2 (CI2) protein were investigated using MD simulations (Ma et al., 2020). Because of the shared zwitterionic property of both CB and protein, superhydrophilic CB has a nominal effect on the protein; however, amphiphilic EG4 solute modified the properties of the protein through hydrophobic contacts. All the abovementioned studies affirm the importance of computational methods in delivering enhanced understanding and fundamental insights regarding the antifouling properties and mechanisms of various polymers which may aid in the design of innovative and efficient antifouling materials and surfaces.

22.5 Conclusion Fouling has been the major cause of medical complications in hospitals and implanted medical devices. Several reports described the complications associated with biomedical fouling. To tackle fouling, surface modification has been the

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most studied in the last two decades. Polymers have been the most preferred choice due to their diverse synthesis methods and the ability to synthesize biocompatible classes. Calculation methods have been extremely beneficial in providing the most plausible mechanisms of antifouling over polymers. Both experimental and calculations are complimentary in probing the role of polymers as the most efficient antifouling modifiers for the fabrication of robust and biocompatible surfaces.

Acknowledgments A.A.M. acknowledges the University of Sharjah support of SEED grant VC-GRC-SR83 2015, competitive grants 160 2142-029-P and 150 2142-017-P, Organometallic Research Group grant RISE-046 2016, and Functionalized Nanomaterials Synthesis Lab grant 151 0039.

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Application of antiviral activity of polymer

23

Shradha Sharma, Howa Begam and Ananya Barui Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India

23.1 Introduction Viral infections are responsible for morbidity, mortality, and one of the leading causes for substantial economic losses (Weiss et al., 2020). Viruses are nonliving organisms and exist beyond the boundary between living and nonliving. Baltimore classified viruses depending on the types of genome and replication process as shown in Table 23.1 (Jarach, Dodiuk, & Kenig, 2020). Viruses are very problematic to treat for many reasons. Not every infected person will show symptoms and in case of COVID-19, most infection will present as subclinical. Therefore infected person may show few-to-no symptoms which may not be diagnosed. Human immune system has the ability to stop the infection but it may have negative effect on populations by enabling viral spread of virus (Gentile & Micozzi, 2016). Pathogenesis is the mechanism to produce an injury induced by virus. A virus is called pathogenic when it can cause infection in host and virulent means it is extremely harmful than other strains of viruses. Viral infection depends on the nature of virus, the host, and the environment. There are six major steps of viral replication: attachment, target cell penetration, loss of envelope, replication, infection and assembly of viral protein, and egress (release of new viral particles) (Maus, Strait, & Zhu, 2021). To ensure the infection the following conditions must be satisfied: sufficient number of virus available at infection site and the site should be obtainable and permitted for virus and the absent or ineffective defense system of host. Therefore, for infection, initially a virus crosses the cell of surface of body such as mucosal lining of respiratory, urogenital and alimentary tract, and conjunctival membranes of eye and skin (Manjarrez-Zavala, Rosete-Olvera, Gutie´rrez-Gonza´lez, Ocadiz-Delgado, & Cabello-Gutie´rrez, 2013). The worldwide COVID-19 pandemic has exaggerated global attraction for infections due to frequently used surfaces. Therefore the researchers are exploring surface that can minimize the number of viable pathogens, which can be applied in various sectors such as health-care centers, public transport, and long-term care facilities to lower exposure and spreading of microorganism (Imani et al., 2020). The major problem with infectious microbes is that they have the ability to Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00011-5 © 2023 Elsevier Inc. All rights reserved.

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Table 23.1 Classification of virus (Jarach et al., 2020). Groups

Acronym

Characteristics

Example

Class 1 Class 2

dsDNA ssDNA

Double-stranded DNA virus Single-stranded DNA

Class Class Class Class

dsRNA ssRNA(1) ssRNA(2) ssRNART dsDNART

Double-stranded RNA virus Single-stranded RNA having positive polarity Single-stranded RNA having negative polarity Single-stranded RNA having reverse transcriptase Double-stranded DNA viruses having reverse transcriptase

Smallpox Human papilloma virus Rotavirus Corona virus Measles virus HIV

3 4 5 6

Class 7

Hepatitis B virus

survive on the abovementioned surface and because of that, they cause transmission of disease. Therefore, to prevent the disease transmission, it is necessary to prepare any surface, which will kill the microbes or reduce attachment of microbes on surface (Ribeiro et al., 2017). These can be performed by using surface-bound antimicrobials and biocidal coating or by using pathogen repellent surface developed by using nanomaterials, chemical modification of surface, and by nanostructures.

23.2 Types of antiviral polymers Depending upon the primary structure, antiviral polymers (AVPs) can be categorized into the following categories. The usage of these types depends on the virus we are dealing with and other factors such as type of host cell in question, the severity of disease, and toxicity.

23.2.1 Polysaccharides Polysaccharides are one of the nature-based antimicrobial materials. Ever since their discovery in 1947, they have been extensively studied. Several characteristics of polysaccharides, such as abundance in nature, nontoxicity, biocompatibility, biodegradability, and striking biological activity, make them an ideal candidate for pharmaceutical industry (Balasubramaniam et al., 2021; Song et al., 2020). Pharmaceutical and food industry are one of the many that have been greatly impacted by this. Being used to make intravenous bags, syringes, drug delivery systems (DDSs), and even blood bags, it has become ubiquitous in hospital surroundings (Balasubramaniam et al., 2021; Geyer, Jambeck, & Law, 2017; North & Halden, 2013). Food industry is heavily dependent on plastics too. Because of their low cost and durability, it has become an integral part of the

23.2 Types of antiviral polymers

industry. However, recently, there has been a recent shift of focus from synthetic nonbiodegradable packaging materials to sustainable products that are nontoxic and exhibit same mechanical strength. For this purpose, polysaccharide polymers such as cellulose, starch, agar, starch, and chitosan have been proposed that have shown significant antiviral and antibacterial activity when compounded with other agents (Motelica et al., 2020). Chitosan is the most abundant biopolymer after cellulose. At low pH, chitosan behaves as a polycationic species and is highly soluble in nature. At higher pH, due to the deprotonation of amino acids, it behaves as anionic species and becomes insoluble in nature as shown in Fig. 23.1. At industrial level, it is produced by deacetylation of chitin by using strong inorganic alkali compound at 50
C60
C and then treating with acids to remove inorganic compounds. Hydrochloric acid is used to eliminate salts such as calcium carbonate and calcium phosphate, and sodium hydroxide is used to remove pigments such as melanin and carotenoids and lipids to obtain a colorless compound (Akbar & Shakeel, 2017; Morin-Crini, Lichtfouse, Torri, & Crini, 2019). Its versatile nature has attracted the attention of scientists to study its properties more closely. Chitosan has shown antiviral activity when conjugated with other materials. One such modification is terminating the polymer chain with disialo-oligosaccharide. It was observed that with the increase of Degree of polymerization of chitosan, the influenza inhibition increased. On the other hand, when degree of substitution of disialo-oligosaccharide was increased, due to decrease in steric crowding, the inhibitory effect was also decreased (Umemura et al., 2008; Yutaka et al., 2006). Another use of chitosan that has been reported is in the treatment of human simplex virus (HSV). Acyclovir is used for the treatment of HSV. But its low bioavailability has been a subject of research. A study published in 2008 by Palmberger et al. showed that thiolated chitosan has been effective in improving the muscular uptake of ACV (Palmberger, Hombach, & Bernkop-Schnurch, 2008). Apart from above, chitosan has also been used for noravirus inhibition. But its activity is highly dependent on concentration, incubation time, molecular weight,

FIGURE 23.1 Structure of chitosan at different pH. It is insoluble at high pH while soluble at low pH.

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and degree of acetylation. All of the above factors need to be carefully calibrated to obtain satisfactory results (Su, Zivanovic, & D’souza, 2009).Our oceans are a huge reservoir of flora and fauna. Seaweeds and algae have had the long-standing interest of research community since time immemorial. Due to the harsh environment in which they thrive in, they have developed some extraordinary features and properties that make them unique (Ahmadi, Moghadamtousi, Abubakar, & Zandi, 2015; Almeida et al., 2011). Their antiviral activity was suspected in 1958 when a 0.25% suspension solution of agar gave protection to chicken against Influenza B virus (Gerber, Dutcher, Adams, & Sherman, 1958). Since then, there have been numerous studies regarding effects of algal-derived polysaccharides on viruses. Carrageenan is among the most important polysaccharide obtained from red algae. It’s a naturally occurring anionic sulfated polysaccharide. The presence of sulfate group in its basic structure accounts for the existence of three types of carrageen, namely, λ-, κ-, and ι-carrageenan as shown in Fig. 23.2 (Wang, Wang, & Guan, 2012). Over the years, the antiviral activity of carrageen has been investigated for a range of viruses. It has shown promising results against human papilloma virus, HSV-1 and HSV-2, and human rhinovirus. It inhibits all of these virus at primary stages of virus replication itself (Buck et al., 2006; Carlucci, Scolaro, & Damonte, 2002; Grassauer et al., 2008). Fengxiang et al. in 2011

FIGURE 23.2 Three structures of carrageenan. Image was obtained with permission from Hans, N., Malik, A., & Naik, S. (2021). Antiviral activity of sulfated polysaccharides from marine algae and its application in combating COVID-19: Mini review. Bioresource Technology Reports no. 13.

23.2 Types of antiviral polymers

prepared low-molecular-weight κ-carrageen by microwave-aided hydrolysis, followed by nanofiltration. At 2.18 pH, 310 kDa κ-carrageen were produced in domestic microwave oven that showed similar IR characteristics as native carrageen. These low-molecular-weight carrageens showed antiviral activity against influenza virus (Tang, Chen, & Li, 2013). Carrageen has also been investigated against SARS-CoV-2 virus. Different group of scientists investigated the effect of carrageenan at different concentrations as well as when compounded with any other polysaccharide and compound to study its effect. Bansal et al. showed that Iota-carrageen-based nasal spray at 6 μg/mL concentration inhibitory effects in vitro. Not only this, carrageen when compounded with fucoidan, another sulfated polysaccharide inhibited the entry of the virus by binding to the S-glycoprotein (Hans et al., 2021). Apart from carrageen, other naturally occurring sulfated polysaccharides such as galactan, fucoidan, naviculan, and calcium spirulan are some other sulfated polysaccharides that have been thoroughly investigated for their antiviral properties (Ahmadi et al., 2015). The activity of these sulfated polysaccharides depends on the spatial distribution of sulfate groups in the polysaccharide backbone, degree of sulfation, and its molecular weight (Hardena, Falshawb, Carnachanb, Kerna, & Prichard, 2009). The action mechanism of sulfated polysaccharides is also highly dependent on the type of viral species as shown in Fig. 23.3. A study published in 1988 by Masanori et al. showed that dextran sulfate and heparin, another sulfated polysaccharides, protected MT-4 cells against human immunodeficiency virus (HIV) at the concentration of 25 μg/mL (Masanori, Robert, Rudi, & De, 1988). It was reported that the length of the sulfated polysaccharide and content of sulfate in the polymer chain is directly proportional to their activity. Sulfation of polymers rendered the polymers hydrophobic hence playing role in inhibiting viral entry in host cell (Valliant, 2016).

23.2.2 Antiviral peptide polymer Ever since the discovery of insulin for therapeutics in the 1920s, there has been in surge in the number of research, pertaining to the role of peptides in fighting diseases (Lau & Dunn, 2017). Their small size (100 amino acids), high specificity at nanomolar range, and its compatibility with the body make it an attractive candidate for antiviral therapeutics. To produce these antiviral peptides, three procedures have been reported computational, biological sources and natural sources (Agarwa & Gabrani, 2020). Needless to say, computer has become an integral part of biology. Applying in silico methods for the designing of AVPs has expedited the process of drug discovery. Proteinprotein interactions are a common occurrence in living organisms. It is the basis of all biochemical events. Nevola and Geralt in 2015 published a paper that aimed at step-by-step identification of Proteinprotein interaction. They used in silico molecular docking analysis for predicting regions of maximum affinity (Nevola & Giralt, 2015). Antimicrobial peptides (AMPs)

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FIGURE 23.3 Action mechanism of SP. The stage at which SP will exert antiviral effect, depending on the attacking viral species. (i) The negative charge of sulfated polysaccharides expressed on the cell surface binds to the positive charge of viral surface and hence inhibits its attachment with the host cell. (ii) SPs may also interact with receptors present on surface of the virus and block their penetration. (iii) SPs can bind to the receptor proteins on virus capsid and prevent their uncoating and internalization. (iv) SPs can also interfere with transcription and replication process of the virus. SP, sulfated polysaccharides. Image was obtained with permission from Hans, N., Malik, A., & Naik, S. (2021). Antiviral activity of sulfated polysaccharides from marine algae and its application in combating COVID-19: Mini review. Bioresource Technology Reports no. 13.

can also be obtained from natural sources. They are present in all prokaryotes as well as eukaryotes. The earliest identified AMP is lysozyme. Identified in 1922, its importance was soon overshadowed by the discovery of penicillin. AMPs received the attention during the 1960s with the rise in multidrug-resistant microbial species. AMPs got a newfound interest as substitutes for antibiotics against bacterial infections (Zhang & Gallo, 2016). Recently, in the past few years, they have also been investigated against viral infections as well. AMPs are present in both prokaryotes and eukaryotes as a host defense mechanism. In scientific literature, the nature of AMPs has been described as cationic and amphipathic. Their cationic nature can be attributed to their high arginine and lysine content at physiological pH (Bulet, Sto¨cklin, & Menin, 2004). Due to the high presence of positively charged amino acids, AMPs can bind easily to negatively charged bacterial

23.2 Types of antiviral polymers

membranes and engage in nonenzymatic degradation. Cyclotides, a family of peptides, derived from a plant source has shown activity against HIV, influenza, and dengue virus by initiating binding with virus membrane and then subsequently rupturing it, leading to cell death (Weidmann & Craik, 2016). The last approach to develop peptides is biological approach. In vitro display has been used, with involvement of different methodologies to produce desirable peptides. Phage display, mRNA display, yeast display, bacteria display, and ribosome display are some of the methodologies discussed in this category. EB peptide, a 20 amino acid long signal peptide of human FGF4 protein, showed activity against broad range of viruses. Viruses, such as herpes virus, influenza virus, and vaccinia virus, fall within the spectrum of EB peptide activity (Altmann, Brandt, Jahrling, & Blaney, 2012). As the research regarding usage of peptides as AVPs is still in its infant stage, there are very few drugs of the category that are available in the market. The first one to be approved was enfuvirtide (Enf), a 36 amino acid long peptide, for HIV. Similarly, cyanovirin-N, a protein isolated from Nostoc Ellipsosporum, inhibited the entry of HCV at low concentrations (Teissier, Penin, & Pe´cheur, 2011).

23.2.3 Nucleic acid polymers These are recently developed AVPs. Just like any other AVPs, it was reported that the sulfated nucleic acid polymers (NAPs) showed higher antiviral activity than their nonsulfated counterparts. The sulfated NAPs belong to the class of phosphorothioate oligonucleotides (PS-ONs) where one oxygen molecule in the phosphate group of phosphodiester bond is replaced by a sulfur atom. A study done on hepatocytes infected with hepatitis B virus showed that NAP blocked the secretion of hepatitis B surface antigen. Pharmacologically, PS-ON is a stable compound that are unaffected by nuclease-mediated degradation. They degrade with time and are eventually removed from body via excretory system (Valliant, 2016). The earliest study done in this area was published in 1987, where antiviral activity of phosphorothioate oligonucleotide was observed against HIV (Matsukura et al., 1987).

23.2.4 Polymer-drug conjugates The idea of DDS brought a revolution in the field of medicine. The fact that they provided constant drug release into the system gave them an upper hand over the conventional drug administration techniques. The involvement of polymers in DDS has not only increased the flexibility in structure but also provides opportunity for both hydrophobic and hydrophilic drugs. They have also been found to increase the bioavailability of the drug (Elvira, Gallardo, Roman, & Cifuentes, 2005). Polymer-drug conjugates are a subclass of DDS and were introduced in 1975 by Helmut Ringsdorf. In this of system, a biodegradable backbone is covalently

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FIGURE 23.4 Structure of polymer-drug conjugate.

linked to a drug. The structure is shown in the Fig. 23.4. The water-solubilizing agent is a hydrophilic unit that is incorporated to make the whole macromolecule nontoxic and soluble. The targeting moiety, as the name suggests, is important to navigate the molecule to appropriate location. Some molecules used as targeting moiety are engineered antibodies, peptides, sugars, and folic acid. Lastly, the therapeutic agent is attached to the backbone with the help of a linker. The use of linker here is very important as it controls the release of drug according to external environment like pH, enzymes present, etc. (Larson & Ghandehari, 2012). The role of these polymer-drug conjugates has been suggested to overcome the side effects of chemotherapeutics (cardiotoxicity, neurotoxicity, immunosuppression, mucositis, and myelosuppression) and multidrug resistance for cancer treatment. The physiochemical properties of polymer-drug conjugates, such as their surface charge, biocompatibility, size, conformation, absorption, distribution, and excretion, give them an edge above the traditional existing methods. They have shown successful results against breast cancer as well as lung cancer. A study published in 2018 showed that when paclitaxel, an anticancer drug was conjugated with a polymer, it displayed tumor growth inhibition (TGI) of 95.5%, whereas the free drug particle showed just 16.7% TGI. Similarly, doxorubicin and gemcitabine were also used with hyaluronic acid as backbone for breast cancer (Alven, Nqoro, Buyana, & Aderibigbe, 2020). Some polymers that have been used as backbone are PEG, poly(vinylpyrrolidone) (PVP), polyvinylalcohol (PVA), dextran, chitosan, hyaluronic acid, pullulan, dextrin, poly(aspartamides), PGA, etc. (Pasut & Veronese, 2007). Similarly, these polymer-drug conjugates have also been used against viruses as well. 30 -Azido-30 -deoxythymidine (AZT, zidovudine), a drug used against HIV, was incorporated with sulfated polysaccharides to overcome its side effects of anemia, mitochondrial myopathy, neutropenia, and resistance against HIV. κ-Carrageenan was used as the polymer backbone that inhibited the attachment of virion with cell (Bianculli, Mase, & Schulz, 2020).

23.2 Types of antiviral polymers

23.2.5 Metal containing polymers The peculiar way in which virus attacks host cell raises the problems for antiviral agents to handle it. Virus uses host cellular machinery to survive and thrive in invading environment. Any antiviral agent developed to target virus or affected cell will also affect uninfected cells. It is due to this reason that so many types of antiviral agents have been developed. The polymeric drugs have shown advantage over small molecules, as their structure can be calibrated to target a specific set of cells. They can also be engineered to release the drug according to the surrounding situation. Incorporating metals in the polymers further increases the scope of polymers as antiviral agents. The empty p, d, and f orbitals, of these metals, provide interaction space for biological entities. The antitumor activity of compounds containing metals, such as gold, iron, platinum, copper, titanium, ruthenium, vanadium, lanthanides, and various metal ions, has been investigated (Roner, Carraher, Dhanji, & Barot, 2008). The scope of organotin polymers as antiviral agents has not been investigated a lot and a very little literature is present on the subject. R2SnX2L2, a general representation of organotin complex, showed activity against both HSV-1 and HSV-2 in vitro. Some organotin polyethers, derived from aliphatic diols, have also showed antiviral activity. One such polymer is hydroxyl-capped polyethylene glycol (PEG). It is a watersoluble polymer that is readily available to cytoplasm and hence is very effective against HSV-1 and vaccinia virus. Dibutyltin chloride (Bu2SnCl2) has similar activity against the abovementioned two viruses. Its cytotoxic action is effective in blocking 20% of virus replication at a concentration of 0.15ug/mL (Roner, Charles, Carraher, Shahi, & Barot, 2011). According to another group of researchers, organotin fractions with acyclovir, which is an antiviral drug used for inhibiting HSV-1, HSV-2, EpsteinBarr virus, cytomegalovirus, and varicella zoster virus (VZV). To obtain the product, Diorganotin dihalide (3 mmol) dissolved in 30 mL heptane, and was mixed with 5 mmol acyclovir and sodium hydroxide (6 mmol) solution prepared in 30 mL water. The mixture obtained was thus stirred at 18,500 rpm for 10 s to obtain the final product. Using the above technique, the following products were obtained—dimethyltin dichloride, diethyltin dichloride, di-n-butyltin dichloride, di-n-octyltin dichloride, diphenyltin dichloride, and dicyclohexyltin dibromice. The GI10 of the resultant product was tested on HSV-1 and VZV strains. The order of inhibition was dibutyltin . diphenyltin . diethyltin 5 dioctyltin . dicyclohexyltin . acyclovir (Jr et al., 2006).

23.2.6 Dendrimers These are another type of DDS that are meticulously designed to overcome the toxicity and drug resistance of conventional therapeutics. They have complicated and diverse 3D branched structure. The exterior of such system constitutes of functional group that conjugates with drug and helps in binding. The interior

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structure encapsulates the drug and regulates its release. Their size ranges from 1 to 16 nm diameter and 30 to 200 kDa (Mhlwatika & Aderibigbe, 2018; Sepu´lveda-Crespo et al., 2015). Typical dendrimer structure are represented in Figs. 23.5 and 23.6. Activity of dendrimers has also been reported against HIV. Polyanionic carbosilane dendrimers (PCDs) were synthesized and showed better in vitro and in vivo activity against HIV, HSV-2, and other STDs. Through molecular simulations, it was found that PCD forms stable complexes with gp120 (coat protein of HIV) and CD4. Because of this interaction, the virus entry is inhibited and the infection is curbed. Another strategy involved making PCD and microbicide conjugates. In an experiment, Tenofovir (TFV) microbicide, conjugated with G1-S16, G2-STE16, and G2-S24P variety of PCP, was tested against X4/R5-tropic viruses (R5 strain, predominant in early stages of HIV infection, is found in body fluids such as semen, blood, and cervicovaginal and rectal secretions while X4 evolves at later stages) in TZM.bl cell line. Dual and triple combinations of PCD and

FIGURE 23.5 Structure of dendrimer is divided into 3 layers: core, interior shell (branching units), and a multivalent surface (surface/terminal groups). Image was obtained with permission from Sepu´lveda-Crespo D., Rafael Go´mez, P., Francisco Javier De La Mata, P., & Jose´ Luis Jime´nez, P.M.A´.M.-F.P.M. (2015). Polyanionic carbosilane dendrimer-conjugated antiviral drugs as efficient microbicides: Recent trends and developments in HIV treatment/therapy. Nanomedicine: Nanotechnology, Biology and Medicine, 118.

23.3 Application of antiviral polymers

FIGURE 23.6 Structure of dendrimers—(A) PAMAM, (B) poly(L-lysine), (C) boltorn, (D) phosphorouscontaining dendrimer, (E) polypropylenimine, and (F) carbosilane dendrimer. Image was obtained with permission from Mhlwatika Z., & Aderibigbe, B. A. (2018). Application of dendrimers for the treatment of infectious diseases. Molecules (Basel, Switzerland), 23(9), 2205.

TFV such as G2-STE16/TFV and G2-STE16/G2-S24P/TFV, G2-STE16/G1-S16/ TFV, and G2-S24P/G1-S16/TFV at 2:2:1 ratio were created. Among all, the combination of G2-STE16/G2-S24P/TFV showed considerable decrease in EC50 values and hence was the strongest competent. Apart from TFV, Maraviroc (MRV) is another microbicide that has been used with PDC against HIV (Sepu´lveda-Crespo et al., 2015).

23.3 Application of antiviral polymers 23.3.1 Drug delivery system DDSs are the formulations or devices that have been investigated by researchers to eliminate the constraint of conventional drug therapy. Different types of materials are used in DDSs such as lipids and polymers in the form of films, hydrogels, nanospheres, nanocapsules, etc. Each carrier system has different property so that they can bind, adhere, or absorb drug molecules. For an ideal DDS the following characteristics must be present within it such as nontoxicity, high drug absorption efficiency, sustained and controlled drug release, physiochemical stability within the body, and it must protect the drug from degradation (Ribeiro et al., 2017). Nanoparticles (NPs) are widely used as nanocarriers due to their unique properties such as improved bioavailability, less drug resistance, controlled release

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rate, easy crossing of cell barriers, targeted drug delivery, and protection of drug from degradation (Kazmierski, 2011; Maus et al., 2021). Due to its nanosize and unique surface properties, NPs are generally used for targeted drug delivery. NPs transfer drugs actively or passively to intracellular fluid from extracellular fluid at epithelial junction of the inflamed tissue site. Drug targeting is favored by the cell-specific ligands such as proteins, antibodies, peptides, or other organic molecules coated on surface of NPs. After receiving the NPs by the target cells, channels allow the drug through membranes (Maus et al., 2021). Several synthetic drugs are available which are used to prevent viral infection or replication, for example, ribavirin, adefovir, entecavir, telbivudine, tenofovir, acyclovir, foscarnet, abacavir, lamivudine, tenofovir, enfuvirtide, maraviroc, indinavir, and interferons (siRNA) (Kazmierski, 2011). Several types of NP are generally used for drug delivery, among them polymers are frequently used for nanocarriers. Polymer NPs allow the loading of bioactive molecules and they provide the protection against external agents, show bioavailability of drug, and assure a controlled drug release. Pedroso-Santana et al. (2020) prepared nanocarrier using chitosan and sodium tri-polyphosphate for delivery of bovine serum albumin and recombinant porcine alpha interferon. Their formulation showed maximum protein release within 90 h and in vitro study revealed antiviral activity. Zuwala et al. (2018) explored antiviral activity of polymer by combining the antimicrobial property and drug delivery capability. They analyzed the agglomeration activity of blood, attachment and accumulation of albumin, suppressive reactions on polymerase, cellular toxicity, and antiinfective property of different polyanions. In addition, they prepared ribavirin-loaded polyanions to investigate to drug release kinetics. They observed that all maromolecular prodrugs exhibited substantial release within 2 h (Fig. 23.7A). They reported the influence hydrophobic polymer on drug release kinetics. Li, Yu, Chen, and Oupicky´ (2015) explained the pharmacological activity of polymeric drug having therapeutic benefits. Polymeric drug shows multivalent benefits, which is a characteristic feature of different biological process such as attachment of virus and bacteria to host cell surface, interface between macrophages and antigens, interaction between cells, and binding between transcription factors with DNA. Polymeric drugs hold several ligands that mimic multivalent ligand, which offer substantial therapeutic effects. It can bind with various receptors or binding sites as shown in Fig. 23.8A. Due to the bulky nature of polymers, polymeric drug generates steric stabilization effect to prevent interaction with different pathogens. Antiviral polymeric drugs interfere interaction between virus and host cell. Due to high molecular weight and multivalent binding capacity, AVP shows its antiviral action by means of steric shielding and competitive inhibition of host cellvirus interactions (Fig. 23.8B). Reolon et al. (2019) developed microparticles by spray-dry technique using different polymers, hydroxypropylmethyl cellulose (HPMC), and EudragitRS 100 to encapsulate acyclovir and curcumin. The study revealed satisfactory results in terms of encapsulation efficiency, compatibility, etc. In vitro drug release study revealed that the polymer microparticles showed sustained release of drugs.

23.3 Application of antiviral polymers

FIGURE 23.7 (A) Drug release kinetics of various polymer carrier that revealed substantial drug release within 2 h.PMAA, PAPA, PAEP, and PHPMA exhibited similar kind of release kinetics due to their structural similarity. Hydrophilicity of polymers played an important role on the release kinetics. (B) Filtration performance analysis of PSDT nanofiber, which depicted that filtration efficiency of the nanofiber, was better compared to commercial air filter. (C) Antiviral capacity of the nanofiber was performed against E. coliphage D24291 and it showed biocidal effect within 2 min. Image was obtained with permission from (60). Image (A) was obtained with permission from Zuwala, K., Riber, C. F., Løvschall, K. B., Andersen, A. H. F., Sørensen, L., Gajda, P., Tolstrup, M., & Zelikin, A. N. (2018). Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects. Journal of Controlled Release: Official Journal of the Controlled Release Society, 275, 5366. Image (B) was obtained with permission from Tian, C., Wu, F., Jiao, W., Liu, X., Yin, X., Si, Y., . . . Ding, B. (2021). Antibacterial and antiviral N-halamine nanofibrous membranes with nanonet structure for bioprotective applications. Composites Communications, 24.

The polymer microparticles loaded with these drugs exhibited improved antiviral effect against BoHV1 virus.

23.3.2 Polymers in protective application Infectious microorganisms spread when those microbes settle onto surface and people touch the surface. By using antimicrobial polymer coating on the surface,

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FIGURE 23.8 (A) Multivalent receptorligand interactions enable attachment of microbes to host cell surface and interaction between antigen and macrophages and between cells. Image describes different ways of receptorligand interactions: (a) chelation, (b) sub-site binding, (c) clustering, and (d) statistical rebinding.(B) Antiviral polymeric drug restrict interaction between viruses and host cell. Figure shows inhibition of viral entry into cell by antiviral polymeric drug (a) by blocking host cell receptors and (b) by masking viral epitopes. Image was obtained with permission from Li, J., Yu, F., Chen, Y., & Oupicky´, D.,(2015). Polymeric drugs: Advances in the development of pharmacologically active polymers. Journal of Controlled Release, 219, 369382.

the infection of virus and bacteria can be prohibited. The AVPs act by neutralizing viruses and blocking the virus entry. Studies reported that the ability of transmission of disease of a virus is inhibited by making bond with polymers (Ginsberg, Goebel, & Horsfall, 1947). AVP coating prevents contamination by antifouling and killing of viruses (Su et al., 2019). The current pandemic created by COVID-19 has upstretched worldwide prerequisite for special type materials with wide-spectrum antimicrobial property for personal protective equipment (PPE) and virus detection device. PPE is vital for occupational safety when a person comes in close contact with biological pathogens. Widely used polymeric materials do not have adequate antiviral property and therefore it makes the frontline health-care workers susceptible of being infected (Nguyen et al., 2020).

23.3 Application of antiviral polymers

Song et al. (2021) prepared coating using 1 H,1 H,2 H,2H-perfluorodecyl acrylate (PFDA) and dimethyl amino methyl styrene (DMAMS) having liquidrepelling and antimicrobial activities. They demonstrated that the coated material would combat against transmission of bacteria and viruses and prevent infection by hindering the attachment of pathogens from the contaminated liquid. Zuwala et al. (2018) observed similar findings where they showed negative charge and hydrophobic polymer an effective inhibitor of virus replication. Tian et al. (2021) designed antiviral and antibacterial polymer using polystyrene doped with 5, 5-dimethylhydantoin and trimethylamine (PSDT) and they prepared nanofiber net membrane by using electrospinning method. N-halamine and ammonia salt groups were incorporated within the membranes to improve its biocidal effects. The materials showed superior bactericidal and virucidal efficiency within 2 min. The filtration efficiency was also measured and it showed 96.7% with pressure fall of 95.4 Pa and its bacterial filtration efficacy was higher (99.77%), which is better than commercial air filter (Fig. 23.7B and C). Yin, Zhang, Xu, and Tian (2021) also prepared antiviral N-halamine-based polymer membrane by electrospinning of polyurethane blended with 2,2,6,6-tetramethyl-4-piperidinol grafted PAA (gPAA) which showed excellent antiviral and antibacterial activity. The authors recommended the application of this membrane on protection wound, protective clothing, drug, and food packaging. The antiviral efficiency against norovirus of neoagarohexaose (NA6) was studied by Kim et al. (2020). The authors found that NA6 have to ability to abandon norovirus. The activation of Toll-like receptor 4 of NA6 was due to myeloid differentiation factor 2 and cluster of differentiation 14 and it produce interferon-β and tumor necrosis factor-α. Fig. 23.9 depicts the mechanism of norovirus infection inhibition by the NA6, which is proposed by the authors (Kim et al., 2020). The antiviral efficiency of poly(sodium 4-styrenesulfonates) (PSSNa) against type 1 Feline herpes virus (FHV-1) and feline calicivirus (FCV) was evaluated by Synowiec et al. (2019). The material showed dual activity, it blocked FHV attachment site as by binding the surface of virion and forming protective shell. At the same time, it inhibits the FCV replication by affecting essential steps of viral replication. The anti-HIV activity of β-cyclodextrin- Ebselen with Soluplus was evaluated by Vartak et al. (2020). The film has potential in anti-HIV and antifungal activity and the authors demonstrated that it could use to prevent HIV infection. Ghosh, Mukherjee, Basak, and Haldar (2020) designated coating for clinical surface that will prevent bacteria, fungi, and influenza viruses. They developed coating using quaternary benzophenonebased ester (QBEst) and quaternary benzophenone-based amide (QBAm). It was seen that the coated surface completely killed bacteria and fungi upon contact. The coated surface also exhibited antiviral activity against influenza virus. Neufurth et al. (2020) proposed covering of the surface of epithelium of intrapulmonary airways with polyphosphate (PolyP) to prevent SARS-CoV-2. PolyP is released from blood, which is morphogenetically active, and metabolic energy producing biopolymer. The authors reported that PolyP effectively inhibit binding of spike protein of corona virus with cellular receptor ACE2. Fig. 23.10 demonstrated the consequence of events during the inhibition of virus infection by PolyP in host tissue.

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FIGURE 23.9 Kim et al. proposed process of norovirus contamination inhibition by means of NA6. NA6 activate TLR4, which showed antiviral activity by producing IFN-β and TNF-α that cause upregulation of IRF-1 expression. Image was obtained with permission from Kim, M., Lee, J.-E., Cho, H., Jung, H.-G., Lee, W., Seo, H.Y., Lee, S.-H., Ahn, D.-G., Kim, S.-J., Yu, J.-W., Oh, J.-W. (2020). Antiviral efficacy of orally delivered neoagarohexaose, a nonconventional TLR4 agonist, against norovirus infection in mice. Biomaterials, 263, 120391.

FIGURE 23.10 Steps showing prevention of attachment of virus to host cell by polyphosphate.

23.3 Application of antiviral polymers

23.3.3 Food packaging Foodborne pathogens are serious matter for consumers, industries, and public institutions. Food contaminated by pathogenic microorganisms mostly viruses and bacteria can cause wide range of disease. Therefore these foodborne diseases may cause high risk of morbidity and mortality for vulnerable persons such as immune-deficient patient, children, old people, and pregnant women (Randazzo, Fabra, Falco´, Lo´pez-Rubio, & Sa´nchez, 2018). Public places such as hospitals, restaurants, and community kitchens have high health risk associated with viral outbreaks. Mostly used nonporous surfaces such as aluminum, glass, plastics, and porous surfaces such as cloths, papers, and cottons may harbor viruses. Due to lack of suitable antiviral packaging materials, many viruses can contaminate food. This contaminated food causes viral foodborne outbreaks. Therefore it is an urgent need to restrict and inhibit the pathogen on food by raising the hygienic practices specifically for food industry and food handling services (Boone & Gerba, 2007; WHO, 2015). Viruses can transmit through different surfaces and they can stay on surface for several days. Therefore, to eliminate the pathogen from the surface, it is necessary to prepare antimicrobial coating using polymers. Food packaging films with antimicrobial properties can prevent viruses, bacteria, and fungi. Antiviral polymeric surfaces prevent the health care-related contaminations therefore the transmission viruses like COVID19 can be stopped by using two mechanisms, such as contact killing and surface repelling. Therefore an ideal food packaging material should show their antiviral properties throughout its use and it should nontoxic. Other features should be smooth and nonporous surface, washable, and tolerance to chemical detergents (Randazzo et al., 2018). Active packaging is a pioneering technology to preserve freshness and to provide extended shelf life of food. The commonly used polymers used for food packaging are polystyrene, polyvinylchloride, polyamide, polypropylene, polyethylene, and polyethylene terephthalate (PET) due to their low cost, good mechanical property, etc. These types of materials are not fully biodegradable and have toxic effect and use of these plastics cause ecological issues. Therefore biodegradable and nontoxic materials for food packaging is more preferable compared to the conventional plastics (de Carvalho & Junior, 2020; Sung et al., 2013; Motelica et al., 2020; Al-Tayyar, Youssef, & Al-Hindi, 2020). Martı´nez-Abad, Ocio, Lagaro´n, & Sa´nchez (2013) evaluated the active renewable food packaging material to control the virus. They prepared polylactide films incorporating silver ions using solvent casting method. The antimicrobial activity was measured against Salmonella enterica and a human norovirus FCV. Their result showed that the materials had excellent potential for food packaging application. Wang et al. (2020) prepared film for food packaging applications using bacterial nanocellulose, silver NPs, and PVA. The film showed great antimicrobial activity and mechanical property. These types of biodegradable materials will reduce environmental pollution. Researchers developed biodegradable polymer composite for use of food packaging is listed in Table 23.2.

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Table 23.2 Biodegradable polymer composite for applications in food packaging. Preparation method

References

Solvent casting Casting

Martínez-Abad et al. (2013) Wang et al. (2020)

Montmorillonite Melissa essential oil/ pomegranate peel extract TiO2

Casting Casting

Avella et al. (2005) Pirsa, Sani, Pirouzifard, and Erfani (2020)

Casting

Silver nitrate

Abutalib and Rajeh (2020) Mei et al. (2017)

Polymers

Nanomaterials

Polylactide

Silver ions

Bacterial nanocellulose Potato starch Chitosan

Silver ions

Sodium alginate and polyaniline PMAA Chitosan Chitosan/PVA

TiO2 SiO2

Coating on Zein film Casting Casting

Chitosan/poly (ε-Caprolactone)

essential oil

Electrospinning

Zhang et al. (2017) Yu, Li, Chu, and Zhang (2018) Ardekani-Zadeh and Hosseini (2019)

Haghighi et al. (2019) developed films for food packaging using chitosan and polyvinylalcohol reinforced with ethyl lauroyl arginate. The films were translucent and it exhibited barrier against visible and UV light that may protect food from photooxidation and UV degradation. It was observed that films containing 5% and 10% ethyl lauroyl arginate were efficient to inhibit typical food pathogens. The incorporation of antimicrobial agents into polymers affects its property. If the agents are compatible with the polymer, it will be more effective. Water vapor transmission is a crucial factor during selection of food packaging materials. A good food packaging material should have good water vapor barrier property to prevent excessive water loss from the packaged food as well as to prevent moisture from environment to enter in food. Controversies have been arisen on the safety of synthetic food additives. Therefore researchers are interested on natural antimicrobial food packaging. The limitations of development of natural antiviral and antibacterial food packaging materials are insufficient studies on efficacy of natural films, economic impact, and consumer acceptance. Therefore preparation of antiviral food packaging materials should be simple, low cost, good mechanical property, and good water barrier capacity and should not degrade easily during packaging period (Sung et al., 2013). Biodegradable films of chitosan and PVA are cost effective but they suffer from poor mechanical property. Yu et al. (2018) showed that modification of PVA/chitosan film using silica enhanced mechanical strength as well as improved the barrier for oxygen and water.

23.4 Concluding remarks

23.4 Concluding remarks Like COVID-19 pandemic, the world has faced similar conditions over past 100 years. As the evolution of viruses occurs naturally, it becomes more aggressive for infection. The antiviral drugs and vaccines are effective to fight against viral infection, however, the drug preparation as well as the clinical trial are time consuming processes. Antiviral polymeric material with its broad-spectrum properties is an emerging alternative to control the viral infections. Due to the emergence of new pathogens, antimicrobial therapy is required to prevent the infections. Along with drugs, development of antiviral surface is another way to inhibit infections. Polymers provide better semipermeable barrier against air-borne particles and thus play important role in fight against viral transmission like corona virus. The use of petroleum-based composite materials is alarming because they are nonbiodegradable and nonrenewable. Some widely used synthetic polymers have antiviral property but they exhibit allergic reactions and nonbiodegradability. For example, polyvinylpyrrolidone, a water-soluble synthetic polymer that is frequently applied in biomedical, pharmaceutical, cosmetic, and food industry due to its physicochemical properties. However, it has been claimed that PVP shows allergic reaction, pulmonary vascularization, subcutaneous granulomas, and reticuloendothelial system deposition (Kurakulaa & Raob, 2020). Bionanocomposite is a good option compared to synthetic material. Polymer nanocarriers offer sustained drug release and these overcome limited aqueous solubility and drug resistance. Nanomaterials have the capacity to improve permeability and thermal and mechanical properties which can be utilized in food packaging purposes. The challenges raised by the synthetic polymers can be overcome by nanobiocomposites. Cellulose and chitosan are utmost abundant polymers present in nature having excellent properties. These types of biomaterials can be used in antiviral applications. Recently, nanomaterials are effectively used to develop drugs and vaccines to combat infection of corona virus. Nanomaterials are used in coating of fabrics for making masks and PPE using spray coating. Nanomaterials such as silver NPs are very efficient in controlling the viral infection. However, the challenges still remain in commercialization of these materials. Toxicity, cost, and their antiviral efficacy are important parameters that should be controlled. The main challenge is the preparation of cost-effective, environmentfriendly, or biodegradable antiviral material. This can be achieved by use of biodegradable natural polymers instead of synthetic polymers. We hope the advancement of AVP in future for clinical treatments. In future, to overcome the challenges in developing antiviral strategies such as drug formulations and manufacturing antiviral polymeric surfaces, new approach with broad-spectrum properties are necessary. The virushost interactions and receptor binding affinity can be predicted through computational modeling so that structure of virus can be predicted and fabrication of AVP and their effectiveness can be predicted.

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Biosensor: fundamentals, biomolecular component, and applications

24

Manoj Kumar Tripathi1, C. Nickhil2, Adinath Kate1, Rahul M. Srivastva3, Debabandya Mohapatra1, Rajpal S. Jadam1, Ajay Yadav1 and Bharat Modhera3 1

ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Department of Food Engineering & Technology, Tezpur University, Tezpur, Assam, India 3 Department of Biotechnology, MANIT, Bhopal, Madhya Pradesh, India

2

24.1 Introduction Biosensors are the analytical devices that translate biological or chemical signal of the reaction into its readable formats which are directly or inversely proportional to the actual change taking place with the analytes. The biosensors constitute of two main components—biological and physical. The biological components are the analyte (substance to be detected) and biorecognition elements which capture the analytes. The physical components consist of transducer and electronic elements where transducers capture the change in reaction occurred during binding of analyte to biorecognition reaction, whereas electronics make the raw data into information and convert into readable form (Bhalla, Jolly, Formisano, & Estrela, 2016). The beginning of fabrication of biosensors was started in 1920, first electrode biosensor developed to measure the blood glucose level. In which, the biosensor monitored the level of glucose oxidase (GOx) by change in current (Leva-Bueno, Peyman, & Millner, 2020). Later in 1967 enzyme electrode was established by Updike and Hicks where GOx was immobilized on polyacrylamide gel on surface of oxygen electrode (Yoo & Lee, 2010). In 1975 first microorganism biosensor and an immune-sensor for the detection of ovalbumin on platinum wire electrode were developed (Thakur & Ragavan, 2013). The use of ferrocene and its derivatives as mediators for amperometric biosensor was mentioned by Cass and his coworkers in 1984 for the detection of glucose and the device was called as glucose pen (Cass et al., 1984). In 1990 Pharmacia Company launched Biacore instrument which was surface plasmon resonance (SPR)-based biosensor for monitoring bimolecular interactions. With the advent of nanotechnology, biosensor development found a new pathway which included BioNEMS, nanoparticles, nanowires, and tubes (Vo, Paul, Kumar, Boykin, & Wilson, 2019). In 2018 DNA probe electrode-based biosensor was established for the detection of Cu 1 2 amino acid oxidation (Lee, Ahn, Park, & Park, 2018). Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00026-7 © 2023 Elsevier Inc. All rights reserved.

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A distinctive biosensor is mainly made up of a biorecognition that identifies and interrelates with the analyte to generate a biological indication. The biorecognition component such as enzymes, nucleic acids, and aptamers are specially used in biosensors. Clinical diagnosis is no longer a challenge due to the lots of research and advancements in the field of biosensors as variety of estimation and diagnosis tools (Myszka, 1999). In the most recent field of material science, intensive research are being carried out in order to create a number of biosensor materials that can aid in the preservation of biorecognition operation element, allowing effectively tap into the biological signal (Vopa´lenska´, Va´chova´, & Palkova´, 2015). Metal nanoparticles, conducting polymers, carbon-based nanomaterials, biopolymers, and other materials have all been used. Because of their natural origins and favorable interactions with living systems, biopolymers, for example, are particularly promising. Nowadays, the practice of biosensors is fairly popular in the field of food quality analysis, environment detention, chemical residue estimation, pollution level indicator, etc.

24.2 Fundamentals of biosensor 24.2.1 Principle of biosensor Instruments that sense the occurrence of a biological analyte in the environment, are biomolecule, biological structure, and bacterium, respectively. The working of biosensors is dependent upon its components assigned for particular roles (Bhalla et al., 2016). The basic components of biosensor are: (1) analyte: substance to be detect; (2) receptor: the specific biomolecule which recognizes the analyte; (3) transducer: the key part of biosensor which converts the biochemical/electrical/thermal/optical/chemical reaction into a measurable form of the signal; (4) electronics: the part which converts the signal into a readable form which can be displayed; and (e) display: the output hardware where the result of a particular reaction is displayed (Turner & Newman, 1998). Depending upon the type of analyte, the receptors and transducers can be of different type. For example, if the analyte is DNA, the receptor could be an aptamer, if analyte is some biomolecule, then the receptor could be the enzyme or any reactive species and if an analyte is an antigen, the antibodies could be receptor. However, the binding of receptor to the analyte is vital part of the detection process as it decides reliability of the biosensor. Although the transducers are the components which sense the change in reaction, the transducers are selected and designed as per the specific application of biosensor (Pandey, 2017). Fig. 24.1 shows the biosensor system and various bioselective materials and bio-recognized signals.

24.3 Classification of the biosensors There are different means of the classification of different biosensors (Corcuere & Cavalieri, 2003).

24.3 Classification of the biosensors

FIGURE 24.1 Illustration of working principle and different components of the biosensor system.

Based on different categories of transducer 1. Resonant biosensors: In this category of biosensors, change in frequency of the acoustic wave is measured. Biorecognition element is attached to the surface acoustic wave transducer, so that before and after binding of the analyte, mass transfer takes place which results into change in frequency through which detection is performed. 2. Optical biosensors: Here the change in light is measured as detection method. Any change occurring in the diffraction, refractive index, thickness of the samples, or any of the optical properties is directly corelated with occurrence of biochemical reaction (French & Cardosi, 2006; Leatherbarrow & Edwards, 1999). There are two types of biosensors under optical biosensors: a. SPR biosensor: These types of biosensors are used for the recognition of bacteria where the capture element is coated on the thin gold film, and samples holding bacteria are detected by change in reflection minima in photo-detector array of transducers. b. Piezoelectric biosensor: The sensor detects the presence of analyte with change in resonance and oscillation. The mass of biorecognition element will increase after binding which will decrease the oscillation depicting the occurrence of reaction (French & Cardosi, 2006). 3. Thermal biosensors: These are basically enzyme-based sensors performing endothermic or exothermic reaction which are sensed by change in

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temperature (Amaro, Turkewitz, Martı´n-Gonza´lez, & Gutie´rrez, 2011; Wang, Sipe, Xu, & Lin, 2008). 4. Magnetic biosensors: Magnetic biosensors detect magnetic characteristics changes or magnetically induced effects (French & Cardosi, 2006). These sensors are used in microfluidic channels which work on the principle of magnetoresistance effect and they have high sensitivity and small size. In microfluidic channels these biosensors will identify the magnetic micro- and nanoparticles with high sensitivity (Scognamiglio, Arduini, Palleschi, & Rea, 2014). 5. Electrochemical biosensors: Electrochemical biosensors identify the concentration of the targeted analyte during their reactions by producing the electrical signal. The electrical signal acts relative to the focus of the targeted analyte. In such systems, the biorecognition constituent is immobilized on conducting/semiconducting medium, so whenever the analyte comes in contact with biorecognition element, change occurs in readable form of physical quantity (Pejcic, De Marco, & Parkinson, 2006). Although there are different forms of system under electrochemical biosensor, likewise: a. Conductimetric biosensor: Here the detection is performed on the basis of rate of change in conductance and resistance of a system. b. Amperometric biosensor: Here the detection is based on generation of current by electroactive species conjugated with either analyte or with biorecognition elements. c. Potentiometric biosensors: These systems sense the oxidation or reduction reaction are given by a biochemical or chemical active specie immobilized on the electrodes. At particular voltage, the specific conducting medium given enzymatic or nonenzymatic reactions occurs at the electrodes which are detected by the transducers. The arrangement of the electrochemical biosensors is shown in Fig. 24.2. Based on the type of biological element or bioreceptors, biosensors are classifies as: 1. Enzyme biosensors: This depends on ability of an enzyme to produce a signal that can be sensed and transformed into an electrical response. Depending on the analyte and the physical transducer to be employed, either a singleenzyme or multienzyme system can be used (Lakhanpal, Gupta, Sharma, & Vaidya, 2011). 2. Immunosensor or bioaffinity sensors: In these biosensors, the transducers are used for linking the immunochemical reaction, which are very affinity to ligand-based solid-state devices. Also, the immune response of certain biological species (usually bacteria) to contaminants produces antibodies, which in turn can be measured (Ronkainen, Halsall, & Heineman, 2010). They are divided into two groups, namely, labeled type (involves a labeling agent such as enzymes, nanoparticles and fluorescent or electrochemicalluminescent probe to measure the quantity of antibodies or analyte) and

24.4 Characteristics of the biosensors

FIGURE 24.2 Arrangement of the electrochemical biosensors.

label-free type (detects the analyte and the antibodies on a transducer surface without any label) (Jiang et al., 2008). 3. Microbial sensors: Microbial cells attached to the physical transducer which measures the utilized concentration of a substrate. The metabolic activity of the cells is observed by an electrochemical modification which is always found to proportional to the substrate concentration (D’Souza, 2001). 4. Nucleic acid biosensor: In recent year DNA and RNA have been employed for the recognition of several types of food-borne pathogen (Velusamy et al., 2011). These types of sensors involve in recognition of an exclusive sequence of nucleic acid bases through the process of hybridization. DNA biosensors are constructed by immobilization of the oligonucleotide sequence/single standard DNA (probe) on to a different electrode to measure the hybridization between DNA probe and their complementary DNA strands.

24.4 Characteristics of the biosensors The main attributes for fabrication of efficient biosensing are selectivity, stability, sensitivity, linearity, and reproducibility. Selectivity of the biosensor affirms the specific detection of analyte resulting in less false positive results. Stability of a biosensor decides the capability of a biosensor to give efficient results in long run. This feature counts for the susceptibility of the system for the disturbances created with change in time, temperature, pH, etc. Sensitivity decides the affinity of the biosensor to detect the pathogen/molecule at minimum concentration. The minimal concentration of a substance detected by the biosensor is termed its limit of detection. Linearity of a biosensor plays major role in quantitative detection. Linearity defines the ability of a biosensor to produce results according to the alteration in concentration of analyte. It can be significantly calculated by y 5 mc,

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where y indicates output and m indicates sensitivity if the system and c define the concentration of analyte. Although the reproducibility feature predicts the efficiency of a system to produce similar results when any test is performed in duplicates or triplicates. For better understanding of different types of biosensors and their utility, the fundamentals of biosensors are explored in the following sections.

24.5 Biopolymers for the development of biosensors Biopolymers are biodegradable polymers which are made of several bio-based resources. Biopolymers mainly include proteins and peptides, cellulose and starch, and DNA and RNA. The monomeric units of above biopolymers are sugars, amino acids, and nucleotides. The living organisms develop biopolymers, which are polymeric biomolecules. The list of different biopolymers classes are shown in Fig. 24.3. On the basis of monomeric units, biopolymers are categorized into three types: 1. Polynucleotides are important polymers and entirely composed of nucleotide monomers. (e.g., DNA and RNA). 2. Polypeptides are polymers made up of amino acid. (e.g., hemoglobin) and 3. Polysaccharides are sugars that are polymeric in nature. (e.g., carbohydrates, cellulose) Biopolymer development is the natural process involving enzymes or microorganisms which degrade the source material to elemental entities, making them environment-friendly (Sarma, Vatsyayan, Goswami, & Minteer, 2009). Natural decomposition of these polymers produces a final product that can be reabsorbed into the atmosphere with minimal carbon footprints. Because biopolymers are BIOPOLYMERS

Biodegradable

NonBiodegradable

Based On Repeating Units Polysaccharides

Biobased Polyesters

Polysaccharides

Non-Biobased Polycarbonates

FIGURE 24.3 Different classes of biopolymer divisions.

Proteins

Nucleic Acids

Based On Polymer Backbone Polyamides

Vinyl Polymers

24.5 Biopolymers for the development of biosensors

made from renewable resources, they contain a broad range of chemical and structural biopolymers (e.g., gelatin, cellulose, alginate, acacia gum, pectin, and others) are mainly used in the biosensors development. In terms of biosensor applications, these polymers have a number of advantages. Table 24.1 represents the biopolymer sources and preparation involved.

24.5.1 Biopolymer composites Various types of biopolymer composites pertinent in area of biosensor development are as follows. 1. Nanoparticle-based biopolymer composites An ionic liquid based enzyme biosensor dispersed in ionic liquid, and matrix was cross-linked with glyoxal and epichlorohydrin (1-butyl-3-methyl imidazolium hexafluorophosphate). This ionic liquid matrix with enzyme peroxidase and the biosensor electrode was developed by mixing it with graphite powder. Using square-wave voltammetry, the biosensor positively can be used to judge the quantity of rosmarinic acid in pharmaceutical samples. The bioactive molecules were immobilized on the magnetic iron oxide nanoparticle surfaces. This part of biosensor is of great interest, since Table 24.1 List of some polymers sources and method preparation. Type of polymer Plastics with a hybrid composition

Plastics derived from cellulose

Lactic acid polymer (PLA) Polyethylene derived from plants Polyesters

Source and method Denatured algal biomass is used to fill petroleumbased polymers such as polyurethane and polyethylene Glucose biopolymer

Polymerization of lactic acid Cracking is a chemical reaction that produces ethylene from ethanol Biomass

Bacteria used in synthesis as an example Cladophorales are filamentous green algae

It’s estimated that 30% of the biomass produced following algal oil extraction contains cellulose Algal biomass fermentation by bacteria Algal biomass is fermented by bacteria Bacteria such as Akaligenes europhus, E. coli, and others

References Mohan, Oluwafemi, Kalarikkal, Thomas, and Songca (2016) Mohan et al. (2016)

Karamanlioglu, Preziosi, and Robson (2017) Bardhan, Gupta, Gorman, and Haider (2015) Ojumu, Yu, and Solomon (2004)

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these bioconjugates’ magnetic properties can be used to enable the recovery of biomolecules specially used for applications of biomedical fields (Thakur, Amarnath, & Sawant, 2014). The nanoparticles of the iron oxide can be used in biopolymers for dispersing purposes. Usually some other compound chitosans are used in formation of nanocomposite films which mainly concern to enzyme immobilization in the biosensors (Teles & Fonseca, 2008). 2. Composites made of carbon-based biopolymers The biosensors made of carbon-based nanomaterials show substantial promising test for high investigative concern due to various characteristics (e.g., conductivity, high surface area, and effective electron transfer). The low dispersibity achieved from the other biosensors can be avoided from the use of these sensors specially equipped in biopolymer with carbon-based functional nanomaterials (Elnashar, 2010). These nanocarbon material-based biosensors have sparked tremendous wide variety of applications which can directly enhance the transfer of electrons in the enzyme-catalyzed electrochemical systems. In various biosensing configurations, these carbonbased nanomaterials help in immobilizing the biocatalysts (Mantha, Pedrosa, Olsen, Davis, & Simonian, 2010). Graphene has been commonly used in the production of electrochemical biosensors and they are made of flat 2D monolayer of carbon atoms. These are helps in faster rate of electron transportation, with high surface area and biocompatibility. The carbon-based nanomaterials have another class called carbon nanospheres based on the nature of their homogeneity, stability of chemicals, and the design of pores. The spherical structure is made up of pentagonal and heptagonal carbon rings in combination, this help in forming the waving flakes, which follow the sphere’s curvature (Nieto-Ma´rquez, Romero, Romero, & Valverde, 2011). There are numerous open edges on the surface, since the graphite sheets are not padlocked shells. 3. Oxide-based biopolymer composites Layered double hydroxides (LDHs) are lamellar ionic solids which are positively charged and they contain metallic cations and exchangeable anions which are bounded weakly. These LDHs are specially called as anionic clays. The main application of LDHs in biosensor is to help in immobilization of enzyme by acting as a promising material (Brondani, Zapp, Vieira, Dupont, & Scheeren, 2011). Alternatively, reusability is reduced during storage in dry inorganic clay matrixes with LDH cracks. In recent use, Laponite is considered as clay having high porosity with excellent adsorption properties such as chemical inertness. These types of biosensors also help in immobilization of biomolecular particles (Fan, Shan, Xue, He, & Cosnier, 2007). These sensors when delaminated in water, they take a shape, which are made of colloidal deferment of negatively charged elementary platelets. This phenomenon helps in developing the cationic biopolymers composites such as chitosan (Li, Chen, Tang, & Zhang, 2009). In addition, when they are in

24.6 Biomolecular component of biosensor

aqueous and organic solutions, the sol gel matrices impart more rigidity in physical nature; chemical inertness and swelling are minimal. 4. Aptamer-based biosensors The generation of DNA or RNA molecules (single-stranded) in vitro, the SELEX, is the best method recently followed. Aptamers have the ability to bind to a broad range of target analyte molecules, which includes viruses, bacteria, proteins, whole cells, and some smaller molecules such as amino acids, organic dyes, metal ions, drugs, etc. They are broadly applicable in the progress of biosensors. Due to their high specificity, synthesis and alteration are more favorable and they also enhance chemical stability (Wang, Yan, Cui, & Wan, 2012). The combination of aptamer with nanomaterials (e.g., as graphene, carbon nanotubes (CNTs) metal nanoparticles, and quantum dots) will intensely increase the aptamer-based biosensors’ performance. Biopolymer-coated nanomaterial composites are commonly used to enhance the biocompatibility of nanomaterial composites in terms of biomolecular activity retention (Song, Wang, Li, Fan, & Zhao, 2008). Biopolymers with excellent film-forming ability helps disperse aggregating nanomaterials including CNTs and reduced graphene oxide, which combines due to heavy interaction.

24.6 Biomolecular component of biosensor Biosensors are analytical tools that mainly comprise of several biological materials. These materials are mainly comprised of tissues, enzymes, microorganisms, antibodies, and cell receptors. In some cases biologically derived materials are closely associated with physicochemical transducer or transducing microsystem (Sharma, Sehgal, & Kumar, 2003). The identification method necessitates the immobilization of recognition elements in order to create these layers. Biomolecules are used to identify individual compounds. Several immobilization methods are available in the lab, but they are not always suitable for the production of biosensors. Catalytic and noncatalytic are the two types of biological components in biosensors (Rocchitta et al., 2016). In the typical biosensor, biological materials such as enzymes, multienzyme complexes, tissues, microorganisms, organelles, cell receptors, antibodies, nucleic acids, or whole cells are used (bacterial, fungal, animal, or plant) for the identification of analyte. In this system electrons are regularly required to be transferred to an amplifier or microprocessor from enzyme molecule-based biological components. Many examples of such transmission can be found in natural cells, such as cytochromes, which are hemoproteins with the primary biological function of transporting electrons or hydrogen via iron valency changes. The detection method recognition elements must be immobilized before these layers can be created. There are several methods for immobilizing biomolecules, but not all of them are suitable for the making of biosensors. The most frequently used biomaterial immobilization methods for the

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design and manufacture of distinctive sensors are physical adsorption method, entrapment method, intermolecular cross-linking method, and covalent binding methods (Mantha et al., 2010; Narayanaswamy, 2006) as given below; 1. Adsorption The substrates used to adsorb enzymes are mainly silica gel, cellulose, glass collodion, collagen, and hydroxyapatite. Main sources of binding forces for the substrates are mainly hydrogen bonds, multiple salt linkages, Van der Walls forces, and the formation of electron transfer facilities. 2. Entrapment The entrapment is common method which occurs with stucking of biomolecules within the gel matrix, during the formation of polymeric gel in a solution having biomolecule. Some important sources that can be used to entrap biomolecules are mainly starch, polyacrylamide, nylon and siliastic gel. 3. Cross-linking Various bifunctional or multifunctional reagents used in the biomolecules’ cross-linking process are hexamethylene di-isocyanate, glutaraldehyde, 1, 5difluro 2, 4-dinitrobenzene, and bisdiazobenzidine-2, 20-disulphonic acid. These molecules are proficient in binding of biomolecules to concern solid supports. Enzyme functional groups are used in covalent binding but they are not required for catalysis. Some important biological functional groups mainly occur in amino acid side chains, such as amino, carboxylic, imidazole, thiol, and hydroxyl, which are required commonly for coupling. 4. Biorecognition element Biorecognition element component is the significant unit that mostly interrelates with the target analyte and helps in producing the signals, which are relative to the concentration of analyte. As deliberated in the previous sections, the biorecognition elements such as enzymes, antibodies, nucleotides, and aptamers are widely used in different types of biosensors depending on their type as well as specific use. Among the biomaterials enzymes are most popular for the development of biosensor, for example, glucose sensors utilize glucose oxidase enzyme such as biomaterial. Enzymatic biosensors are used for undergoing any chemical reactions or transformation enzymes are biochemical catalysts which have high specificity for substrates in single or in group. The structure of such enzyme sensors is made of protein or any cofactors or prosthetic groups.

24.7 Recent trends in biosensors Research and development of various types of biosensors has marked its importance in various fields of biotechnology such as various industries such as food industry, environmental care, pollution assessment sectors, pharmaceutical sector, disease diagnosis, health care, etc. From the year 2019 international health

24.8 Recent applications of biosensors

emergency has arrived in the form of COVID-19, where the biosensing technology is playing major role as part of health-care industry. Wearable devices are the communication tools between the COVID-19-positive patients and health-care authorities connected via internet. The data such as medical history of the patients transferred from patients to authorities is saved in the cloud. There are many digital and nondigital wearable devices that monitor and show the heart rate, calories, glucose level, stress level, etc. Earlier smartwatches such as Apple watch, GPS tracking devices, smart headbands such as Withings, fitness band such as Fitbit were available in the market, whereas now studies have shifted their focus on personal sensor patches and devices. Recently, some advanced biosensor devices came in the market such as a Boston-based start-up WHOOP. It has made a device WHOOP Strap 3.0 which can detect the value of abnormal respiratory rates which is considered as one of the symptoms of COVID-19. At the same time, the Fitbit and Garmin devices also claim to monitor respiratory rates. Similar line, Biosensor Patch1AX is another wearable patch device which is enabled with cardiovascular health monitoring technique which assists in the early detection of COVID-19 disease. This patch is held on chest of the patients which record the temperature, respiration rate, ECG trace, and heart rate. The data of patch is automatically sent to a mobile application which can be transferred to centralized cloud data from where the nearby hospitals or health-care authorities can get an alert regarding the patients.

24.8 Recent applications of biosensors There are a lot of applications of the biosensors in various fields where test, analysis, and investigation of specific change/reaction need to be carried out with output in qualitative or quantitative form (Liu et al., 2016). Recently, the biosensors are used in diagnosing COVID-19 which was their latest use during 2020 (Jiang et al., 2020). Most of the applications based on the characteristics, elements, reactions, etc. taking place in the biosensors are as follows: 1. Ammonia sensor Nitrosomonas sp. and Nitrobacter sp. are the special type of nitrifying bacteria deriving all of its energy from ammonia. Ammonia is measured amperometrically using an oxygen electrode along with a microbial sensor with immobilized nitrifying bacteria. 2. Biological oxygen demand sensor BOD (biological oxygen demand) is a well-known and significant indicator of organic pollution. BOD sensors are mostly microbial type consisting of immobilized microorganisms that assimilate the organic compounds during the testing of sample solution. Microorganisms consuming oxygen produced a reduction in dissolved oxygen passing across the membrane. As a consequence, the current flowing through the sensor steadily

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3.

4.

5.

6.

dropped until it reached a steady level. The microbial sensor has long been used to calculate the BOD of waste water treatment plants in food industry. Fish freshness sensor There are numerous biochemical and physicochemical changes that occur in fishes during its catching to final consumption. The marine food industry relies on precise and quick freshness determination. The hypoxanthinespecific enzyme-based sensors have been developed for determining the freshness of fish. During fish storage, there are various biochemical changes occur inside the fishes leading to degradation of protein and stored glycogen into various lower molecular weight compounds that can readily utilized by Altermonas putrefaciens which gives an indication of the freshness of the fishes. Biosensors based on nucleic acid The nucleic acids are considered to be the storage house of all the genetic information of human being as well as other spices. The DNA and RNA are the nucleic acids which are made up of nucleotides. This can be used in recognition of gene, diagnostics of molecular, screening of drugs, and environmental safety. In order to develop the DNA biosensor, the use of definite functional matrix is necessary for efficient immobilization of DNA probe (single-stranded). Hence, for this purpose, composites of biopolymer material are the commonly used. Antibody-based biosensors Antibodies can be defined as proteins that are produced by triggering of immune system in a response to foreign body (antigens) like pathogens. Immunosensors are biosensors that will detect antigens, by using antibodies for biorecognition, since antibody and antigen are more affinated. In immunesensors, biopolymers are frequently used to capture and stabilize the functional composites. These biopolymer composites are used in detection to identify the bacteria, biomarkers, and viruses. Nano-biosensor

A biosensor is a detection system for an analyte in the form of chain of biological components and a physicochemical detector, whereas a nano-biosensor is nanoscale dimensions (Pumera, Sanchez, Ichinose, & Tang, 2007). The applications of nano-biosensor in the field of biology include (Bellan, Wu, & Langer, 2011) the following: • • • • •

genetic monitoring and disease detection; HIV, hepatitis, and other viral disease immune-sensors, drug testing, and environmental monitoring; cell-based sensors, functional sensors, and drug testing; blood, urine, electrolytes, gases, steroids, drugs, hormones, proteins, and other point-of-care sensors; bacteria sensors (Escherichia coli, Streptococcus, and others): applications in the food industry, medicine, the environment, and other fields;

24.8 Recent applications of biosensors

•

diabetes, drug testing, and other applications for enzyme sensors: applications in the environment; • pollution and toxicity detection in the environment; • agricultural surveillance; • screening of groundwater. 7. Applications of biosensors in food a. Determination of food component: They might be used for scanning and determining various food components such as organic acid, vitamins, alcohols, phenols, and amino acids in food. b. Sensory analysis: The two types of biosensors, namely, electronic nose (E-Nose) and electronic tongue (E-tongue) are widely explored for the organoleptic evaluation of various foods. E-Nose instrument is developed with an aim to mimic the human olfactory system and works on the principle of variations in sensor resistance when the sensor is exposed to odors or vapors (Lawrence & Masih, 2011). E-Nose is used in identification of hop varieties and aroma of orange juice, coffee, and whiskey samples; aging process to know cheese maturity, fish freshness, and degradation of cooking oil; detection of contaminant such as diacetyl in orange juice and trichlonoanisole in wines; quality control to recognize acceptable and rejectable samples in raw material and finished product; wine and brewing industries for online measurement of ethanol; dairy industry to classify the various types of cheese and strains of bacteria (Thakur & Kingsly, 2007). E-tongue is chemical array sensor-based system used for analysis of multi components mainly from the liquid solutions in short time frame. E-tongue is used in process monitoring (batch fermentation process of starter cultures for cheese production), foodstuff recognition (to distinguish regular and diet cola drink), freshness evaluation, quality control (to assess bitterness in beer), and quantitative analysis (to assess sourness in red and white wines) (Peris, 2011). c. Contaminant analysis: Different types of the biosensors are used to detect specific pathogens and to determine pesticide and fertilizer residues and heavy metals in different foods (Cock & Verdugo, 2011). d. Biosensors in food packaging: Biosensors are used in intelligent packaging of foods for real-time monitoring of microbial load inside the package. For example, Food Sentinel System had developed a barcode by integrating a biosensor for the detection of microbial growth in food containers. The increase in microbial load in the package leads to more interaction of antibodies immobilized on the biosensor with microbes that resulted in darkening of barcode and make it unreadable during scanning. Toxin guard is also being developed to detect pathogens. e. Monitoring of fish quality: Biogenic amines are produced in meat, fish, cheese, wine, and milk due to microbial decarboxylation of amino acids.

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Biosensors based on direct coupling of amine oxidase and horseradish peroxidase maybe used to detect quality of fish.

24.9 Merits and limitation of biosensors There are several advantages of using biosensor in food industry. Use of biosensors is analyte specific, rapid and does not require extensive sample preparation. Analyte is measured in its natural microenvironment as it is not modified during sample preparation. The instrument is small, compact, portable, versatile, and simple to use and gives reproducible results. Biosensors can be employed in monitoring the process online due to their rapid response and continuous signals. The system can be used for simultaneous multiple assays. Possibly, they would not be very expensive when produced on large scale. Besides several merits of using biosensors in food industry, there are also many constraints. Development of biosensors involves a team of experts from diverse field such as food science and technology, electrochemistry, biochemistry, optics, electronic engineering, microbiologist, etc. Linear response may not be maintained over a range of concentrations of the analyte. Membrane carrying biosensors may pose problems of fouling and biocatalyst which is the least stable part of biosensor would limit the life of biosensor. Also, sometimes the lack of applicability of a single-analyte targeting by the biosensors for food, for example, in taste or smell analysis may limit their use.

References Amaro, F., Turkewitz, A. P., Martı´n-Gonza´lez, A., & Gutie´rrez, J. C. (2011). Whole-cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila. Microbial Biotechnology, 4(4), 513 522. Bardhan, S. K., Gupta, S., Gorman, M. E., & Haider, M. A. (2015). Biorenewable chemicals: Feedstocks, technologies and the conflict with food production. Renewable and Sustainable Energy Reviews, 51, 506 520. Bellan, L. M., Wu, D., & Langer, R. S. (2011). Current trends in nanobiosensor technology. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 3(3), 229 246. Bhalla, N., Jolly, P., Formisano, N., & Estrela, P. (2016). Introduction to biosensors. Essays in Biochemistry, 60, 1 8. Brondani, D., Zapp, E., Vieira, I. C., Dupont, J., & Scheeren, C. W. (2011). Gold nanoparticles in an ionic liquid phase supported in a biopolymeric matrix applied in the development of a rosmarinic acid biosensor. Analyst, 136(12), 2495 2505. Cass, A. E., Davis, G., Francis, G. D., Hill, H. A. O., Aston, W. J., Higgins, I. J., & Turner, A. P. (1984). Ferrocene-mediated enzyme electrode for amperometric determination of glucose. Analytical Chemistry, 56(4), 667 671.

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Cock, L. S., & Verdugo, J. G. P. (2011). Biosensor application in agri-food industry. In Vernon Somerset (Ed.), Environmental biosensor (pp. 43 64). Intech-Open Access Publisher. Corcuere, J. I. R. D., & Cavalieri, R. P. (2003). Biosensors. In R. D. Heldman (Ed.), Encyclopedia of Agricultural, Food, and Biological Engineering (pp. 119 123). Marcel Dekker. D’Souza, S. F. (2001). Microbial biosensors. Biosensors & Bioelectronics, 16, 337 353. Elnashar, M. (2010). Low-cost foods and drugs using immobilized enzymes on biopolymers. IntechOpen. Fan, Q., Shan, D., Xue, H., He, Y., & Cosnier, S. (2007). Amperometric phenol biosensor based on laponite clay chitosan nanocomposite matrix. Biosensors and Bioelectronics, 22(6), 816 821. French, C. E., & Cardosi, M. F. (2006). Biosensors in bioprocess monitoring and control: current trends and future prospects. In E. M. T. EI-Mansi, C. F. A. Bryce, A. L. Demain, & A. R. Allman (Eds.), 2nd. Fermentation microbiology and biotechnology (pp. 363 394). CRC Taylor & Francis. Jiang, S., Shi, Z., Shu, Y., Song, J., Gao, G. F., Tan, W., & Guo, D. (2020). A distinct name is needed for the new coronavirus. Lancet (London, England), 395(10228), 949. Jiang, X., Li, D., Xu, X., Ying, Y., Li, Y., Ye, Z., & Wang, J. (2008). Immunosensors for detection of pesticide residues. Biosensors & Bioelectronics, 23, 1577 1587. Karamanlioglu, M., Preziosi, R., & Robson, G. D. (2017). Abiotic and biotic environmental degradation of the bioplastic polymer poly (lactic acid): a review. Polymer Degradation and stability, 137, 122 130. Lakhanpal, P., Gupta, A., Sharma, S., & Vaidya, D. (2011). Analytical instruments: Application for quality control in food industry. Beverage & Food World, 24 29. Lawrence, S., & Masih, D. (2011). Electronic nose technique for aroma analysis of food and beverages. Beverage & Food World, 19 23. Leatherbarrow, R. J., & Edwards, P. R. (1999). Analysis of molecular recognition using optical biosensors. Current Opinion in Chemical Biology, 3(5), 544 547. Lee, J. Y., Ahn, J. K., Park, K. S., & Park, H. G. (2018). An impedimetric determination of alkaline phosphatase activity based on the oxidation reaction mediated by Cu 2 1 bound to poly-thymine DNA. RSC Advances, 8(20), 11241 11246. Leva-Bueno, J., Peyman, S. A., & Millner, P. A. (2020). A review on impedimetric immunosensors for pathogen and biomarker detection. Medical Microbiology and Immunology, 209(3), 343 362. Li, F., Chen, W., Tang, C., & Zhang, S. (2009). Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol gel process. Talanta, 77(4), 1304 1308. Liu, J., Dai, S., Wang, M., Hu, Z., Wang, H., & Deng, F. (2016). Virus like particle-based vaccines against emerging infectious disease viruses. Virologica Sinica, 31(4), 279 287. Mantha, S., Pedrosa, V. A., Olsen, E. V., Davis, V. A., & Simonian, A. L. (2010). Renewable nanocomposite layer-by-layer assembled catalytic interfaces for biosensing applications. Langmuir: the ACS Journal of Surfaces and Colloids, 26(24), 19114 19119. Mohan, S., Oluwafemi, O. S., Kalarikkal, N., Thomas, S., & Songca, S. P. (2016). Biopolymers application in nanoscience and nanotechnology. Recent Advances in Biopolymers, 1(1), 47 66.

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Myszka, D. G. (1999). Improving biosensor analysis. Journal of Molecular Recognition, 12 (5), 279 284. Narayanaswamy, R. (2006). Optical chemical sensors and biosensors for food safety and security application. Acta Biologica Szegediensis, 50(3 4), 105 108. Nieto-Ma´rquez, A., Romero, R., Romero, A., & Valverde, J. L. (2011). Carbon nanospheres: synthesis, physicochemical properties and applications. Journal of Materials Chemistry, 21(6), 1664 1672. Ojumu, T. V., Yu, J., & Solomon, A. (2004). Production of polyhydroxyalkanoates, a bacterial biodegradable polymers. African Journal of Biotechnology, 3(1), 18 24. Pandey, C. (2017). Biosensors: Fundamentals and applications. Pejcic, B., De Marco, R., & Parkinson, G. (2006). The role of biosensors in the detection of emerging infectious diseases. Analyst, 131(10), 1079 1090. Peris, M. (2011). Electronic tongues and their application in the monitoring of alcoholic fermentation process. Beverage & Food World, 51 53. Pumera, M., Sanchez, S., Ichinose, I., & Tang, J. (2007). Electrochemical nanobiosensors. Sensors and Actuators B: Chemical, 123(2), 1195 1205. Rocchitta, G., Spanu, A., Babudieri, S., Latte, G., Madeddu, G., Galleri, G., & Serra, P. A. (2016). Enzyme biosensors for biomedical applications: Strategies for safeguarding analytical performances in biological fluids. Sensors, 16(6), 780. Ronkainen, N. J., Halsall, H. B., & Heineman, W. R. (2010). Electrochemical biosensors. Chemical Society Reviews, 39(5), 1747 1763. Sarma, A. K., Vatsyayan, P., Goswami, P., & Minteer, S. D. (2009). Recent advances in material science for developing enzyme electrodes. Biosensors and Bioelectronics, 24 (8), 2313 2322. Scognamiglio, V., Arduini, F., Palleschi, G., & Rea, G. (2014). Biosensing technology for sustainable food safety. TrAC Trends in Analytical Chemistry, 62, 1 10. Sharma, S. K., Sehgal, N., & Kumar, A. (2003). Biomolecules for development of biosensors and their applications. Current Applied Physics, 3(2 3), 307 316. Song, S., Wang, L., Li, J., Fan, C., & Zhao, J. (2008). Aptamer-based biosensors. TrAC Trends in Analytical Chemistry, 27(2), 108 117. Teles, F. R. R., & Fonseca, L. P. (2008). Applications of polymers for biomolecule immobilization in electrochemical biosensors. Materials Science and Engineering: C, 28(8), 1530 1543. Thakur, A. K., & Kingsly, A. R. P. (2007). Biosensor and electronic nose: Complement of human perception towards food-quality. Indian Food Industry, 26(2), 50 54. Thakur, B., Amarnath, C. A., & Sawant, S. N. (2014). Pectin coated polyaniline nanoparticles for an amperometric glucose biosensor. RSC Advances, 4(77), 40917 40923. Thakur, M. S., & Ragavan, K. V. (2013). Biosensors in food processing. Journal of Food Science and Technology, 50(4), 625 641. Turner, A. P. F., & Newman, J. D. (1998). An introduction to biosensors. In A. O. Scott (Ed.), Biosensors for food analysis (pp. 13 27). UK: Cambridge, Royal soc. Of chem. Velusamy, V., Arshak, K., Yang, C. F., Yu, L., Korostynska, O., & Adley, C. (2011). Comparison between DNA immobilization techniques on a redox polymer matrix. American Journal of Analytical Chemistry, 2(03), 392. Vo, T., Paul, A., Kumar, A., Boykin, D. W., & Wilson, W. D. (2019). Biosensor-surface plasmon resonance: A strategy to help establish a new generation RNA-specific small molecules. Methods (San Diego, Calif.), 167, 15 27.

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Vopa´lenska´, I., Va´chova´, L., & Palkova´, Z. (2015). New biosensor for detection of copper ions in water based on immobilized genetically modified yeast cells, . Biosensors and Bioelectronics (72, pp. 160 167). . Wang, l, Sipe, D. M., Xu, Y., & Lin, Q. (2008). A MEMS Thermal biosensor for metabolic monitoring applications. Journal of Microelectromechanical Systems, 17(2), 318 327. Wang, W., Yan, T., Cui, S., & Wan, J. (2012). A bioresponsive controlled-release biosensor using Au nanocages capped with an aptamer-based molecular gate and its application in living cells. Chemical Communications, 48(82), 10228 10230. Yoo, E.-H., & Lee, S.-Y. (2010). Glucose biosensors: An overview of use in clinical practice. Sensors (Basel), 10, 4558 4576.

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25

Memoona Akhtar, Muhammad Farrukh Sarfraz, Samra Fatima and Muhammad Atiq Ur Rehman Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan

25.1 Introduction Cell encapsulation requires immobilizing the cells in a semipermeable polymer membrane. This membrane has a special feature that allowed the diffusion of molecules in both directions along with the invasion of nutrients, oxygen, and other essentials for cell metabolism as well as outward diffusion of therapeutic proteins and waste. The advances in microbiology in numerous microorganisms are of industrial importance and in tissue engineering, the main motive is to overcome the graft rejection by the recipient’s immune system. However, cell encapsulation of all living and dead cells through semipermeable membranes reflects a great potential (De Vos, Lazarjani, Poncelet, & Faas, 2014). Alginate, owing to its moderate gelation conditions and low toxicity, is the most extensively used material for cell encapsulation (Gasperini, Mano, & Reis, 2014). Although the membranes must be permeable for molecules, they must also not allow the passage of molecules greater than a certain essential size. Biofilm reactors, microbial fuel cells, bioremediation, phage therapy, cell transplantation, and regenerative medicine are few examples of the usage of cell encapsulation in biotechnology (Gasperini et al., 2014). The encapsulation method must be gentle enough to avoid exposing the cells to excessive osmotic strain and mechanical stress. It also requires the usage of chemicals that do not interfere with cellular activity. Encapsulating viable and usable cells in such a semipermeable biocompatible matrix must promote cell life (Farina, Alexander, Thekkedath, Ferrari, & Grattoni, 2019). For this purpose, the construction of a cytocompatible matrix is required from materials to allow permeability to nutrients, by-products of cell metabolite, and oxygen. Immunoprotecting the allo/ xenotransplanted cells, helping in tissue-engineered structures, and cytoprotection of yeast, animal cells, and bacteria in bioengineering as well as in fermentation products processing are only a few of the matrix properties that strongly depend upon an individual’s activity (Gasperini et al., 2014). The heart of medicinal research to

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00017-6 © 2023 Elsevier Inc. All rights reserved.

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cure illnesses encased in biological cells in a physical polymeric membrane. Cell encapsulation is a procedure in which living cells are encased in a nonliving semipermeable membrane, allowing the cells to continue to expand and express their genes even after the nonliving membrane has been (Liu, Chiu, et al., 2019). The significance of using biocompatible polymer for membrane with semipermeability is that the immune system of the patient is not capable of sensing the presence of encapsulated cells. So, researcher’s prime objective is transplantation of cells into patient’s body without disturbing their body’s normal balance (Vemmer & Patel, 2013). A huge amount of protective cells (phagocytic cells and lymphocytes) are released by the recipient’s body when injected with the donor’s naked cells. The transplanted cells are destroyed by the cytokines and cytotoxic molecules produced by the host’s immune system (Simo´, Ferna´ndez-Ferna´ndez, Vila-Crespo, Ruipe´rez, & Rodrı´guez-Nogales, 2017). The condition worsened in situations where there is less availability of cell donors or where there is difficulty in growing the artificial culture medium of the necessary donor. Cell encapsulation is an effective solution to the challenges that organ donation specialists face, as well as a way to help people who are suffering from severe complications (Rathore, Desai, Liew, Chan, & Heng, 2013). Cell encapsulation materials can imitate the extracellular matrix and be treated in conditions that are compatible with the presence of cells (Yang et al., 2016). Bacterial cell encapsulation is gaining popularity as a way to improve the probiotic’s bacterial viability in acidic foods such as yogurt (Rathore et al., 2013). Encapsulation is the strategy of coating or encasing one material or mixture of materials within another material/system (Park, Lee, & Hyun, 2015). Core content, payload, actives, fill, and internal process are all terms used to describe the material that is coated or entrapped (Liu, Wang, et al., 2019). Encapsulation isolates cells from their surroundings, dramatically reducing cell damage (Lammari, Louaer, Meniai, & Elaissari, 2020). Encapsulation is being used as a technology to secure susceptible cultures of probiotic, improve durability as well as viability in food products, and deliver probiotic cultures to the target location in the gastrointestinal tract (Coiffier, Coradin, Roux, Bouvet, & Livage, 2001). Probiotic bacteria must be encapsulated to withstand human gastric juice in the gut (pH , 2) (Cross, 1990). Encapsulation technology enhanced the viability of Biffidobacterium pseudolongum and Biffidobacterium longum in a simulated gastric fluid environment. Encapsulated bacteria were found to be more resistant to freezing and freeze drying (Huq, Khan, Khan, Riedl, & Lacroix, 2013). The wall material, carrier, membrane, casing, or covering is the material that forms the coating (Baek, Joo, & Toborek, 2019). Fig. 25.1 shows the properties of microcapsules in cell encapsulation (Gasperini et al., 2014). The objective of this chapter is to explore the best approach for encapsulating probiotic bacteria in a biopolymeric system along with their promising applications in biomedical, food, and industry (John, Tyagi, Brar, Surampalli, & Pre´vost, 2011).

25.2 Encapsulation method

FIGURE 25.1 Microcapsule properties for cell encapsulation are depicted schematically. Adapted from Gasperini, L., Mano, J. F., & Reis, R. L. (2014). Natural polymers for the microencapsulation of cells. Journal of the Royal Society, Interface/the Royal Society, 11. https://doi.org/10.1098/rsif.2014.0817; reproduced with the permission from the Royal Society of Interface.

25.2 Encapsulation method Microencapsulation of microbial cells entails the use of relatively mild encapsulation measures to ensure the cell’s viability. In order to support microbial cell growth and long-term culture, microspheres must be mechanically stable. Various microencapsulation methods for microbial cells were being researched for the conservation and enhancement of microorganism viability over the last several years, with differing degrees of performance. Few of the most popular techniques are coacervation, emulsification, spray drying, and extrusion. Each approach has certain distinctive properties, so the use of microspheres specifies which methodology is used (Vemmer & Patel, 2013). To avoid a gritty taste when swallowed, probiotic cells in microspheres, for example, must be less than 100 nm in diameter. In bioreactors, fermenting microbes used, on the other hand, must be physically solid to endure high physical and mechanical stresses such as shear forces, acidic temperatures, and exposures to the solvent and gases produced during fermentation. In contrast, microspheres holding fermenting microorganisms used in bioreactors must be mechanically solid to endure strong physical and mechanical pressures, including shear forces, acidic temperatures, and exposures to

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fermentation solvents and gases. As a result, research strategy must be capable of producing microspheres with desired physicochemical parameters, thus allowing minimal harm to cell’s viability and integrity, and it must be simple to scale up while maintaining appropriate processing costs (Jime´nez-Pranteda et al., 2012).

25.2.1 Nanoprecipitation Using the gelling and solubility properties of polymers, polymeric nanocarriers are obtained by mixing two main phases (solvent and nonsolvent) and immersing precursors (drug, polymer, and stabilizer) in one of them (Barreras-Urbina et al., 2016). Phase separation and thus nanoprecipitation can be achieved by selfassembly of a hydrophobic polymer in a nonsolvent phase. The polymer and drug are dissolved in a volatile or nonvolatile organic solvent, or a mixture of solvents, to accomplish the organic step (Park et al., 2015). It is then dispersed in aqueous form, either with or without a colloidal stabilizer (nonsolvent). The organic phase is applied to the aqueous phase in a volume-controlled fashion to create smaller and more controlled drops (usually with a syringe). The nanoparticles coprecipitate as the solvent is removed by evaporation or agitation whether the solvent is soluble, or through dialysis unless the solvent is not volatile (Cordt, Meckel, Geissler, & Biesalski, 2020). Nanoprecipitation has been used to encapsulate hydrophobic or hydrophilic active principles into polymeric nanoparticles to treat intracellular infections (Barreras-Urbina et al., 2016). Nanoprecipitation process was shown in Fig. 25.2 (Lammari et al., 2020).

25.2.2 Emulsification Two nonmiscible liquids are dispersed using a stabilizing medium that prefers the continuity to the scattered phase is known as an emulsion as shown in Fig. 25.3 (Vinner, Richards, Leppanen, Sagona, & Malik, 2019). Separating these two phase droplets can also be done with the aid of a solidifying agent (Vinner et al., 2019; Zhang et al., 2019). The emulsion is known as water in oil emulsion. If the scattered form is aqueous, use reverse state or an oil in water (O/W) emulsion. In each case, only two steps are needed to create simple emulsions. Adding up a third stage, double emulsions can be produced (Singh, Medronho, Miguel, & Esquena, 2018). These emulsified systems can enhance encapsulated cell safety by using encapsulated probiotics (Vinner et al., 2019). Owing to the hydrophilic nature of bacterial cells, the diffuse aqueous process is preferred (SchwarzSchilling, Aufinger, Mu¨ckl, & Simmel, 2016). By means of xanthan and guar gum in the water-based process and sunflower oil in the lipidic phase, a clear emulsion was used to encapsulate Lactobacillus plantarum 299 v and metronidazole. This encapsulation technique increased the viability of the cell during storage and is shown to be ideal for the controlled release of encapsulated agents (Yaakov et al., 2018).

25.2 Encapsulation method

FIGURE 25.2 Schematic representation of nanoprecipitation method. Adapted from Lammari, N., Louaer, O., Meniai, A. H., & Elaissari, A. (2020). Encapsulation of essential oils via nanoprecipitation process: Overview, progress, challenges and prospects. Pharmaceutics. 12:1 21. https://doi.org/10.3390/pharmaceutics12050431; reproduced with the permission from Elsevier.

25.2.3 Coacervation As in the case of chitosan nanoparticle systems, coacervation occurs when positively charged amino groups penetrate cationic groups, which then interact with anionic groups from tripolyphosphates to form coacervates (da Silva et al., 2019). Depending upon the quantity of macromolecules used, the method can be categorized into simple or complex coacervation (Timilsena, Akanbi, Khalid, Adhikari, & Barrow, 2019). Execute the steps below with constant agitation to separate a macromolecular solution into dense coacervate (Zhao et al., 2020). In order to further precipitate a surface-active hydrocolloid, the active concept must also be dispersed in the solution. Changes in pH, a nonsolvent, temperature, and the use of electrolytes will all influence precipitation (as represented in Fig. 25.4) (Timilsena et al., 2019). In the case of complex coacervation, the addition of other hydrocolloids to the solution allows the formation of a polymer polymer matrix (Bosnea, Moschakis, & Biliaderis, 2014).

25.2.4 Capillary encapsulation method The idea of Eukaryotic cells, which have multiple inner compartments containing nuclei, mitochondria, and lysosomes, is analogous to a revolutionary method for

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FIGURE 25.3 Schematic representation of encapsulation method by emulsification. Adapted from Vinner, G. K., Richards, K., Leppanen, M., Sagona, A. P., & Malik, D. J. (2019). Microencapsulation of enteric bacteriophages in a pH-Responsive solid oral dosage formulation using a scalable membrane emulsification process. Pharmaceutics, 11. https://doi.org/10.3390/ pharmaceutics11090475; reproduced with the permission from Elsevier.

encapsulating many components in a single capsule. A membrane separates these compartments (biopolymer microcapsules) from the host cell, allowing them to perform diverse and distinct functions (Mkam Tsengam et al., 2019). In an aqueous solution, the biopolymers chitosan and alginate, as well as microcapsules, are synthesized, implying multiple stages. Droplets of anionic biopolymer are formed by shearing off the capillary tip with gas pulses, instead of dissolving oppositely charged ions in the solution. The ions are injected into a tank, where they react with multivalent biopolymeric cations to form a shell over each droplet, as shown in the diagrams. The next step is to use the same technique but with a larger capillary to encapsulate multiple capsules. In the inner compartments, colloidal spores, enzymes, and bacteria could be encapsulated without compromising their native functions (Lu, Oh, Terrell, Bentley, & Raghavan, 2017). Fig. 25.5 represents the capillary encapsulation method (Lu et al., 2017).

25.2.5 Electrospinning Electrospinning was used to spin viruses or bacteria trapped in a polymer matrix (Noruzi, 2016). Dilute salt solution or Luria-Broth media are used to spread

25.2 Encapsulation method

FIGURE 25.4 Schematic representation of coacervation. Adapted from Timilsena, Y. P., Akanbi, T. O., Khalid, N., Adhikari, B., & Barrow, C. J. (2019). Complex coacervation: Principles, mechanisms and applications in microencapsulation. International Journal of Biological Macromolecules, 121:1276 1286. https://doi.org/10.1016/j.ijbiomac.2018.10.144; Reproduced with the permission from Elsevier.

viruses or bacteria. In same volumetric ratio, a 14% w/w aqueous solution of vinyl alcohol was mixed with this. The suspension as of 1 mL syringe was electrospun, when we used a hypodermic needle of 0.5 mm internal diameter. The rate of flow was 0.2 0.5 mL/h (Sarioglu, Keskin, Celebioglu, Tekinay, & Uyar, 2017). After the injection of a copper electrode into the polymer matrix, the suspension was spun onto the side of a grounded collector disk. Frequency of the electrostatic field was 1.1 kVcm21 with a distance of 12 cm between both the electrode tip and the disk’s edge (Zussman, 2011). These all experiments were conducted at 25 C with relative humidity level of 50% (as represented in Fig. 25.6) (Sarioglu et al., 2017). As a result, this approach may be a good substitute for lyophilization for storing organisms used for strain collections, retaining commercially valuable genetically engineered bacterial strains, and biosensing systems. Furthermore, electrospinning is indeed a great strategy to encapsulate and orient biological materials (DNA, proteins, medicines, etc.) as well as cells. For the purpose of wound healing, gene, phage therapy and cutaneous fungal infections, and other uses, electrospun nanofiber mats may be used to cover 3D surfaces and free their contents (Salalha, Kuhn, Dror, & Zussman, 2006).

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FIGURE 25.5 Schematic representation of capillary encapsulation method. Adapted from Lu, A. X., Oh, H., Terrell, J. L., Bentley, W. E., & Raghavan, S. R. (2017). A new design for an artificial cell: Polymer microcapsules with addressable inner compartments that can harbor biomolecules, colloids or microbial species. Chemical Science, 8:6893 6903; reproduced with the permission from Royal Society of Chemistry.

25.2.6 Layer-by-layer self-assembly method Due to robustness, reliability, enhanced efficiency, as well as scalable tool, this method is commonly used to design and fabricate different nanostructured films, coatings, and scaffolds having tunable architectures as well as customized physical and chemical properties. Owing to the stability as well as tunability of multilayer shells, layer-by-layer encapsulation method already was developed for a range of biomedical applications, for instance, cell-based biosensors, cellular transplantation, transport of cells or molecules, as well as tissue engineering (as seen in Fig. 25.7A) (Belbekhouche, Bousserrhine, Alphonse, & Carbonnier, 2019). Depending upon the variations in encapsulation approaches, one can differentiate LbL cell encapsulation into two fundamental classes (such as in Fig. 25.7B) (Liu, Wang, et al., 2019). The direct cell encapsulation technique entails subsequent deposition of predetermined polymers forming multilayered nanofilms on an individual cells or cell aggregates to accomplish single cell or cell aggregate encapsulation. The indirect cell encapsulation process involves encasing cells in a biocompatible hydrogel nucleus, accompanied by LbL polymer self-assembly on the hydrogel to form multilayer shells. Primarily focused on how hydrogel center is disposed of, indirect cell encapsulation is divided into two functional configurations: (1) LbL encapsulated bulk hydrogel, in which the hydrogel core is maintained in the final application, and (2) LbL

25.2 Encapsulation method

FIGURE 25.6 (A) Schematic representation of electrospinning process for bacteria encapsulated PVA and PEO webs, and photographs of PVA and PEO webs, (B) representative images for bacteria encapsulated webs including a SEM micrograph and a schematic representation of a bacterial cell inside PVA/PEO fibers. Adapted from Sarioglu, O. F., Keskin, N. O. S., Celebioglu, A., Tekinay, T., & Uyar, T. (2017). Bacteria encapsulated electrospun nanofibrous webs for remediation of methylene blue dye in water. Colloids Surfaces B Biointerfaces, 152:245 251. https://doi.org/10.1016/j.colsurfb.2017.01.034; reproduced with the permission from Elsevier.

encapsulated hollow capsule, in which the hydrogel core is discarded by hydrogel liquefaction, resulting in a hollow core shell configuration after LbL encapsulation of the multilayered polymers on the hydrogel (Liu, Wang, et al., 2019).

25.2.7 Spray drying A way of microencapsulating probiotics involves atomizing a microbial cell suspension in a polymeric solution into hot air, and then quickly evaporating the water (Arepally & Goswami, 2019). The substance is then isolated from the conveying air as a dry powder in a cyclone. Numerous parameters, such as feef rate of the product, air flow, feed temperature, inlet as well as temperature of the air, must be optimized so that to develop distinct well-formed microspheres as shown in Fig. 25.8 (Burgain, Gaiani, Linder, & Scher, 2011). The inlet air temperature

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FIGURE 25.7 (A) The biomedical applications of the LbL self-assembly technique for cell encapsulation and the mechanism of cell encapsulation by the LbL self-assembly technique are depicted in this diagram. (B) Diagrams depicting various methods for cell encapsulation using the LbL self-assembly method. Single cells or cell aggregates can be directly encapsulated using the LbL self-assembly process, or they can be encapsulated inside biocompatible materials (such as hydrogel or inorganic material) and then LbL self-assembled on the material’s surface to form a multilayer shell. Adapted from Liu, T., Wang, Y., Zhong, W., Li, B., Mequanint, K., Luo, G., & Xing, M. (2019). Biomedical applications of layer-by-layer self-assembly for cell encapsulation: current status and future perspectives. Advanced Healthcare Materials, 8:1 16. https://doi.org/10.1002/adhm.201800939; reproduced with the permission from Wiley.

must be adjusted properly since decrease in water evaporation rate due to low temperature of the air resulted in aggregated microspheres having highly dense membranes and weak flow properties, whereas, an extremely high temperature of the air harms viability of the cell (Boza, Barbin, & Scamparini, 2004). Furthermore, adjusting the feed temperature is critical for modifying the polymer solution and, as a result, the ability to be sprayed uniformly (Zhao, Sun, Torley, Wang, & Niu, 2008). The B. lactis Bb-12 was microencapsulated in whey protein and spray dried to increase cell viability when exposed to bile. The probiotic cells’ viability improved after 12 weeks of storage as well (Pe´rez-Chabela, LaraLabastida, Rodriguez-Huezo, & Totosaus, 2013).

25.3 Applications 25.3.1 Intestinal tract health Probiotic usage was shown to be effective to treat multiple forms of diarrhea, particularly antibiotic-associated diarrhea in adults, travelers’ diarrhoea, and

25.3 Applications

FIGURE 25.8 Spray drying process. Adapted from Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic living cells: From laboratory scale to industrial applications. Journal of Food Engineering, 104:467 483. https://doi.org/ 10.1016/j.jfoodeng.2010.12.031; reproduced with the permission from Elsevier.

rotavirus-related diarrhea in infants (Naghmouchi et al., 2019). Lactobacillus GG, Lactobacillus casei, Bifidobacterium bifidum, and Streptococcus thermophilus are the most widely studied probiotic bacteria in such studies (Aisida et al., 2020). Since diarrhea is a leading cause of infant mortality globally but can be debilitating in adults, frequent probiotics’ use may be an effective, noninvasive means of preventing as well as treating such diseases, especially in developed countries (Daly, Riley, Segura, & Burdick, 2020). Inflammatory bowel disorders, irritable bowel syndrome, colitis, and alcoholic liver disease were also linked to probiotic bacteria, which were found to maintain intestinal integrity and mediate their impact. Furthermore, lactic acid bacteria (LAB) has been shown to increase intestinal mobility as well as alleviate constipation (Cacicedo et al., 2016).

25.3.2 Bioavailability and nutrient synthesis Food fermentation with LAB was shown to boost folic acid levels in yoghurt, bifidus milk, and kefir, as well as niacin and riboflavin levels in yogurt, vitamin B12 levels in cottage cheese, and vitamin B6 levels in Cheddar cheese (Naghmouchi et al., 2019). Probiotics can boost the ability of certain dietary nutrients, such as protein and fat, to be digested, in addition to nutrient synthesis (Giessen & Silver, 2016). LAB manufacture simple fatty acids such as butyric

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acid, lactic acid, and propionic acid that might help sustain an adequate pH and protect the colonic mucosa from pathological changes (Tolve et al., 2016).

25.3.3 Probiotics’ antimicrobial potential In recent years, the role of probiotics in human diet has been recognized. This research suggested an enhanced in vitro model for studying antimicrobial action of probiotics toward enteropathogens via attempting to mimic atmospheric growth condition similar to those found in the small intestine in a popular culture medium (Pinilla & Brandelli, 2016). To investigate a culture medium that could accommodate both probiotics as well as infectious agents, a primary experimentation was conducted. This was intended to ensure that the relationship was correctly assessed under joint growth conditions (Shokryazdan et al., 2014). The popular culture medium, brain heart infusion, was chosen for antimicrobial activity assays. The communications between Salmonella 1344 and Lactobacillus rhamnosus and Lactobacillus reuteri were then tested under a variety of pH and oxygen supply circumstances to simulate the small intestinal environment (Cueva et al., 2010). The antimicrobial effect of L. rhamnosus GG ATCC 53103 was the highest, particularly in anaerobic conditions as well as at lower pH levels (Urdaci, Bressollier, & Pinchuk, 2004). Both lactic acid and secreted nonlactic acid molecules were involved in its antagonistic activity (Marianelli, Cifani, & Pasquali, 2010).

25.3.4 Cancer prevention The effects of probiotic intake on cancer tend to be promising in studies. In the Western world, colorectal cancer (CRC) is the major cause of cancer death (Rodrigues, Cedran, Bicas, & Sato, 2020). About 70% of CRC cases are linked to environmental causes, most notably food (Yu & Li, 2016). Probiotic cultures in fermented milk have been shown to guard against CRC. After the consumption of fermented milk or probiotics, interventional trials do seem to have altered intermediate markers of CRC exposure in human subject from a high to low risk sequence. Animal tests consistently indicate that probiotic administration reduces the occurrence of chemically mediated colorectal tumors and the development of aberrant crypts (Wen et al., 2020). In vitro experiments also offer proof of safety and enable researchers to learn more about the active compounds that are involved as well as the pathways behind their anticancer properties (Rafter, 2004). Probiotics have been shown to improve many main intestinal functions, including detoxification, colonic fermentation, transport, and immune state, both are linked to colon cancer growth (Saikali, Picard, Freitas, & Holt, 2004). The bacteria’s LAB or a soluble compound formed by the bacteria can directly interact with tumor cells in culture, inhibiting their development (Uccello et al., 2012). The colon cancer cell line HT-29 was significantly decreased in growth as well as viability in culture after exposure to LAB, while dipeptidyl peptidase IV and

25.3 Applications

brush boundary enzymes were dramatically enhanced, indicating that such cells were undergone a differentiation phase (Hirayama & Rafter, 2000).

25.3.5 Tissue engineering Used as a scaffold for accelerated cell growth in tissue engineering applications. Safe handling and good entrapment of cells are critical when developing biomaterials for tissue engineering to ensure a sufficient number of cells following transplantation (Alazhari, Sharma, Heath, Cooper, & Paine, 2018). Chitosan is a best alternative for tissue engineering in cartilage. There are critical aspects of cell form and materials in cartilage tissue engineering with the aim of delivering growth factors and cells to the injured area. Chondrocytes, fibroblasts, and stem cells are among the cell types that have been researched (Gu et al., 2021). Cartilage mainly comprises of both a sparse web of cells embedded in thick matrix of type II collagen and proteoglycans that are essential for cartilage tissue’s advanced mechanics (Yang, Zhang, Yue, & Khademhosseini, 2017). As a consequence of an aging population, obesity, and a more active adult population, cartilage damage from osteoarthritis, degenerative joint disease, and injury is on the rise, necessitating cartilage repair approaches (Dekosky et al., 2010). Owing to the scarcity of natural chondrocytes, researchers are looking into alternative materials and cell sources which facilitate proliferation as well as chondrogenesis. As a result, chitosan, a natural polymeric substance, is being used in cartilage tissue engineering for cell encapsulation approaches (Zhang et al., 2019). Other research has shown that chitosan-based scaffolds encapsulated with cells can be used for wound treatment, orthopedics, and epithelial cell applications (tracheal tissue) (Gasperini et al., 2014).

25.3.6 Methylene blue dye remediation from water An industrial strain of Pseudomonas aeruginosa with methylene blue (MB) dye remediation capability was selected as the bacteria encapsulated nanofibrous webs’ electrospinning matrices, using polyvinyl alcohol (PVA) and polyethylene oxide (PEO) as the polymer matrices. The bacteria/PVA site and the bacteria/PEO system have a high MB remediation potential. But bacteria/PEO web have demonstrated better removal efficiencies than PVA web bacteria, kept at 4 C for 3 months and found to be potentially storable to hold the bacterial cells encapsulated alive. Overall, it can be used as a beginning inoculum for water systems bioremediation (Sarioglu et al., 2017).

25.3.7 In Agriculture and the food processing Probiotic encapsulation is critical in food processing industry to protect bacterial viability as well as enhancing longevity in unfavorable environmental conditions. Several fermented dairy products, such as yogurt, cheese, cultured cream, and frozen dairy desserts, as well as biomass processing, will benefit from encapsulated probiotic

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bacteria (Vivek, 2013). Capsules, pills, suspensions, creams, and powders in the natural food sector will gradually use encapsulation technologies for direct consumption and external application of probiotics (Noruzi, 2016). On an industrial scale, encapsulating probiotic bacteria in foods poses scientific, microbiological, and financial problems, as well as customer behavior concerns. Seeking the right encapsulation process, healthy and efficient encapsulating products, and potent bacterial strains is the key obstacle in applying bacteria encapsulation to new foods in order to fulfill customer safeties (Pe´rez-Chabela et al., 2013). Encapsulation is anticipated to accelerate probiotics’ heat resistance, compression and shear stress resistance, and acid tolerance at 25 C in a variety of food matrices (Kavitake, Kandasamy, Devi, & Shetty, 2018). Fig. 25.9 represents some of the applications of bacterial cell encapsulation. LAB are used in dairy industry for fermenting milk to form dairy products such as yoghurt, butter, cream, and cheese. The fermentation process should be stopped at pH 5.5 to prevent over fermentation and spoilage of products (Rokka & Rantama¨ki, 2010). Thus by removing these bacteria from the mixture via gelatin microcapsules, the shelf life of the product increases. As gelatin provides high cell viability, the microbial cells are not harmed and may even be reused for next cell culture in fermentation (Kavitake et al., 2018).

25.3.8 Drug delivery The drug delivery via encapsulated cells is perhaps the most important feature of cell encapsulation (Yanagihara, 2005). It’s indeed probable to deliver/release the

FIGURE 25.9 Applications of cell encapsulation.

25.5 Future considerations

same volume of drugs in a monitored way with minimal adverse effects without having to use the machine again, thus improving the quality of life. Chitosan microencapsulation was being used in conjunction with many other natural polymers for the gradual release of pharmaceutical agents, proteins, growth factors, and medications (Mohammadi et al., 2011).

25.4 Conclusion We concluded that, after two decades of rigorous study, determining which polymer is best suited for therapeutic use remains a challenge. This is due to a lack of data on important details such as the polymer’s structure, the existence or absence of conflicting factors which trigger immune responses, enveloped cells toxicity, as well as polymer network’s permeability. Microencapsulation of microbial cells has recently been used in a variety of biotechnological processes. Microencapsulation is being used to immobilize cells for a number of uses due to its considerable benefits as well as flexibility with respect to a broad range of materials and approaches accessible. However, in the food industry, microencapsulation of probiotics has had the most market success. Encapsulated cells have also been used for fermentation of microbes as well as environmental protection/ decontamination, in addition to probiotics. Only alginate has been thoroughly researched and is currently suitable for use. In recent decades, a slew of new polymers for cell encapsulation have been introduced. All of these polymers were developed to solve problems that other polymers had, for instance, a lack of biotolerance in the host, unwanted cell death, failure to maintain adequate mechanical stability in vivo, as well as too poor permeability, interfered with cellular life. The fact that novel polymers have not been well described, with respect to application of cellular transplantation, and that investigations of applicability have often culminated in explanations of novel problems that must be solved in order to use the polymeric material for cell encapsulation has been a significant stumbling block. Alginate is now the single polymer with which a significant number of researchers have selected to investigate the factors that influence whether encapsulated cellular grafts succeed or fail.

25.5 Future considerations Encapsulation of microbial cells was being conducted by using a variety of compounds. Microbial cells have been encapsulated using a variety of novel microencapsulation techniques. The applicability of such approaches on a large-scale processing, indeed, needs to be determined. Microsphere stability is, however, a critical factor for their use in a variety of applications, and that it should be considered carefully. Following confinement inside the microspheres, cell physiology

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was shown to be dramatically altered. Different techniques for detecting and measuring altered physiology must be established and standardized. Another major challenge throughout this area is the development of nondestructive, precise, and fast methods for counting viable encapsulated cells. Whether other polymers suggested for encapsulation include comparable pollutants and whether this leads to the observed biotolerability is currently unclear. Being experienced with alginate, researchers believe that the existence as well as identification of pollutants in the polymeric material must be recorded in any encapsulation research. It will be challenging, but not impossible, to choose appropriate polymeric material for cellular immunoprotection as far as these parameters are still not stated.

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Carbon nanotubes based composites for biomedical applications

26

Sarika Verma1,2, Ramesh Rawat3, Vaishnavi Hada1, Ram Krishna Shrivastava3, Kunal Pal4, Sai S. Sagiri5, Medha Mili1,2, S. A. R. Hashmi1,2 and A.K. Srivastava1,2 1

Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India 2 Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India 3 Department of Chemistry, Institute for Excellence in Higher Education, Bhopal, Madhya Pradesh, India 4 Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 5 Department of Food Science, Agricultural Research Organization, Agro-Nanotechnology and Advanced Materials Research Center, the Volcani Institute, Rishon Lezion, Israel

26.1 Introduction Biomaterials act as a foundation for medical devices. There are various specific properties related to the nanoscale (Auffan et al., 2009) nanoparticles that have progressively been subjected to biomaterials. The chemical reactivity increases as a wide proportion of surface atoms, in comparison with those in bulk, which modifies their physicochemical properties. Among nanoparticles, carbon nanomaterials [carbon nanotubes (CNTs)] give fascinating combining properties like a very high chemical resistance, excellent mechanical properties, and lightweight (Wick et al., 2014). The notable attributes like chemical reliability and biocompatibility of carbon nanoparticles have fascinated scientists. The carbon nanoparticles play significant roles in drug delivery fields, desired therapeutics, enzymatic/ metabolic breakdown, identification of pathogens, antigen diagnosis, and tissue repair hyperthermia. From the past few years, CNTs have gained significant attention in scientific society owing to their absolute structures and invincible features like dimensional pie electron coupling, high conductivity, and smaller radius with mechanical strength (Kausar & Siddiq, 2014; Neves et al., 2012). These properties of CNT make it convenient in many fields such as electronics, automotive, and aeroplanes. The CNTs, an allotrope of carbon with a thin cylinder long tube

Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00018-8 © 2023 Elsevier Inc. All rights reserved.

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structure, are of two types—(single-wall carbon nanotubes) SWCNTs and (multiwall carbon nanotubes) MWCNTs (Kim, Chabot, & Aping, 2013). In the last decades the new direction of modern research, broadly defined as nanoscale-in-scale technology, has evolved as a recent trend among the researcher. Various techniques are used to synthesize CNTs nanocomposites, including the sol
gel process, the salvo-thermal process, the hydrothermal process, thermal treatment, microwave, irradiation, ultrasanitation, etc. Using various allied chemicals wherein, synergistically and simultaneously, the chemical reaction occurs among the multiple raw material components to obtain the appropriate suitable valuable product for the broad application spectrum (Che, Peng, Duan, Chen, & Liang, 2004; Sivaraj & Vijayalakshmi, 2017). Because of the above, we proceed in the novel process to develop and characterize CNTs based on biomedical applications. Further, the nanocomposites of CNT were described using various parameters, sophisticated complementary techniques for their physicochemical, morphological, thermal, electrical, etc., characterization. The main motive behind presenting this review is to address and bring forward the remarkable contribution of scientific advancements of CNT for biomedical applications. This will provide the direction to the latest trends in the scientific community for biomedical applications to get new approaches in carbon-based polymeric materials. The reported review also highlights CNT’s antibacterial properties, which proves the existence of carbon-based materials in the biomedical sector. Fig. 26.1 depicts the medical implementations of carbonbased nanomaterials (CBNs).

FIGURE 26.1 Biomedical applications of carbon-based nanomaterials (CBNs).

26.2 Carbon nanotube
based composites for biomedical applications

26.2 Carbon nanotube
based composites for biomedical applications Biomedical Science is a concept that focuses on the biological and chemical aspects of health. Because of innovative material discoveries, the biomedical sector became one of the most extraordinary alluring disciplines for scientists and researchers (Lamberti et al., 2015). CNTs have been used in drug stores and pharmaceutics since the start of the 20th century for drug delivery systems (DDS) in therapeutic applications. CNTs have been envisioned for utilization in many medical sectors, such as drug vectors, biomolecules, targeted therapies to cells or organs, tissue rejuvenation, and biosensor diagnostics and assessment (Hirlekar, Yamagar, Garse, Vij, & Kadam, 2009). CNTs are well-known for their unique, latest generation, and one-of-a-kind characteristics. They have a high surface area, good biocompatibility and versatility, and thermal, mechanical, and conductive that can be customized and functionalized based on the materials. Such substances are popular in the biomedical field. CNTs show antibacterial ability by which they can suppress the germination of bacteria. When the bacteria interact with CNTs, they pervade within the cell membrane, resist its activity, and destroy the cell feasibility of bacteria (Chen et al., 2013). Kang et al. noted the antibacterial efficacy of CNTs and they described that SWCNTs possess great potential for antimicrobial activity (Kang, Pinault, Pfefferle, & Elimelech, 2007; Kang, Herzberg, Rodrigues, & Elimelech, 2008). CNT has been used in biology and medicine to save people’s lives. When CNTs were combined with organic materials, they produced a good choice in the biomedical fields. CNT diagnostic applications include nerve tissue repairing, neural scaffolds, myocardial conduction, and cancer detection (Jain & Tiwari, 2021). In the biomedical field, the application of CNT brings few challenges to be met. The challenges start with the issue of safety, which implies using a very high purity of CNT to resist the probable deliverance of toxic ions while operating in any biological environment (Ignat et al., 2019; Saliev, 2019). The substantial hydrophobicity property of CNT makes it challenging to stabilize. As CNT has dispersion difficulty, another plan of action emerges from a dry powder, which limits or avoids dehydrating process. One more obstacle is viscosity; it increases when coupled to the appropriate disintegration of CNT in a fluid, even though at low concentration (Grady, 2006), making it more challenging to produce nanocomposite materials having a higher proportion of diffused CNT (Simon, Flahaut, & Golzio, 2019). Bajpai et al. (2020) developed advanced hybrid titanium compounds/FMWCNTs nanocomposites for biomedical applications. The distinctiveness of this study comprises implementing the basic materials like titanium nanoparticles with distinct shapes such as circular and nano-flowers nanoparticles for progressive nanocomposites A & B consisting of organic
inorganic titanium elements specifically titanium oxide and sodium titanium oxide, that are compliantly

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infused on F-MWCNTs which can be utilized in biomedical applications. The developed nanocomposite A showed antibacterial potential against gram 1 ve and gram 2 ve bacteria such as Staphylococcus aureus, Escherichia coli, Lactobacillus plantarum, Lactobacillus acidophilus, and Enterococcus faecalis by conducting a disk diffusion test. The advanced material is used in different implementations such as antibacterial performer, preventing from emissions like UV and X-rays during accomplishment of dental radiographic tests of sufferers.

26.2.1 Carbon nanotube nanocomposites for biosensors Because of their widespread use in health-care delivery, pharmaceutics, and food manufacturing, the advancement of biosensors is currently gaining interest in biomedical science and health-care applications. Biosensors were used to diagnose diseases, treat them, supervise patients’ conditions, and maintain the health of individuals. Biosensors have flourished as the cardiac health-care industry’s future. Biosensors could also be used to identify microorganisms such as bacteria, fungi, and viruses (Mohankumar, Ajayan, Mohanraj, & Yasodharan, 2021). CNTs have been termed “next-generation nanoprobes” because of their extraordinary structural, mechanical, electrical, and optical features (Tıˆlmaciu & Morris, 2015). They are well suited for biosensing applications due to their higher conductivity, excellent chemical steadiness (Zhao, Gan, & Zhuang, 2002), and fast electrontransfer rate (Lin, Lu, Tu, & Ren, 2004). The immobilization of biomolecules on the surface of CNT used biosensors is the fundamental component, which improves identification and signal transduction (Holzinger, Le Goff, & Cosnier, 2014; Hou et al., 2017; Jacobs, Peairs, & Venton, 2010; Kumar, Ahlawat, Kumar, & Dilbaghi, 2015; Wang et al., 2015; Yang, Chen, Ren, Zhang, & Yang, 2015; Zribi et al., 2016). Various CNT-glucose biosensors employed on glucose oxidase fusion were developed. CNT nonwoven fabrics were utilized by Zhu et al. (2014) to sense the presence of glucose-infused polyvinyl alcohol solution. Gonza´lez-Gaita´n, RuizRosas, Morallon, and Cazorla-Amoro´s (2017) focused their research on the changes that occurred in the structure of glucose which were having MWCNT coating on the composite material. The electrochemical biosensors based on CNT were developed to detect nitric oxide and sense epinephrine (Mphuthi, Adekunle, & Ebenso, 2016; Ulissi et al., 2014). Bisker et al. (2016) identified 20 different SWCNT COVID stages for identifying proteins in human blood. The results showed that the particular COVID stages could recognize fibrinogen with remarkable selectivity, resulting in a drop in SWCNT fluorescence intensity of .80% at saturation (Fig. 26.2A). Nevertheless, with a slight redshift, the transmittance remained unchanged (Fig. 26.2A). The oscillation frequency of the SWCNT sample before (Fig. 26.2B) and after (Fig. 26.2C) the fibrinogen addition showed that the fluorescent activation of SWCNT having a small radius was highly significant than the larger diameter nanotube. The identification of fibrinogen was evaluated in a human blood serum culture (Xuan, Thuy, Luyen, Huyen, & Tuan, 2017). The DNA

26.2 Carbon nanotube
based composites for biomedical applications

FIGURE 26.2 Schematic representation of SWCNT in tumour of mice. Reproduced by Maitiet al. 2019 under Creative Commons Attribution License.

conciliated surface-enhanced Raman scattering (SERS) characteristic of SWNTs was investigated by the Zhou Group, allowing for the ultrasensitive identification of a wide spectrum of ctDNA in human blood. SERS of SWNTs mediated by T-rich deoxyribonucleic acid (DNA) could detect a KRAS G12DM concentration as low as 0.3fM, with a recognition volume of 5 L from the sample volume (Zhou et al., 2016). SWCNTs are useful optical probes in biomedicine because of their photophysical features, such as fluorescence radiation in the near-infrared range and high photostability. Jena et al. (2017) created single-stranded DNA functionalized SWCNTs that reacted to the amount of lipid in the endosomal lumen of living cells. The lipid content of the SWCNTs was determined using a solvatochromic shift from their NIR photoluminescence (Jena et al., 2017).

26.2.2 Carbon nanotube nanocomposites for drug delivery DDS are strategies for bringing drugs to preferred tissues, organs, cells, and subcellular parts of the body to release the drug and uptake using a broad extent of drug delivery carriers. Its typical focus is to strengthen the pharmacological characteristics of targeted therapies and resolve issues like low solubility, drug accumulation, poor bioavailability, bad cellular uptake, absence of specificity, or to

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minimize medicinal toxicities. During 2015
18, substantial progress in drug delivery investigation was made, and breakthroughs in relevant areas such as medicinal chemistry, material sciences, and biological sciences were seen (Li et al., 2019). CNTs have risen to prominence among the numerous carbon allotropes as a supporting vehicle for carrying many therapeutic molecules into living cells due to their inherent shape, which allows for noninvasive perforation beyond biological layers (Chen et al., 2008; Das, Singh, Datir, & Jain, 2016; Liu, Zhao, Sun, & Chen, 2013; Panczyk, Wolski, & Lajtar, 2016). The drug particles are mostly connected to CNT sidewalls through coordinate/noncoordinate interaction of drug particles and functionalized CNT. However, every procedure has its own set of benefits and drawbacks. Because of the covalence, the drug-filled CNT remains firm in both extracellular and intracellular sections, indicating the marvelous results in absence of drug dissemination in the interior of the cellular micro environment of cancer cells flaw in the DDS. Noncovalent contact allows for administering the drug in the acidic environment of tumor locations, but it lacks stability in extracellular pH levels. As a result, using the internal chamber of CNT for drug release ensures that the drug is completely isolated from the physiological environment. External stimuli such as temperature, electric field, light, and a union have been explored to overcome the disparity in medication disintegrating in the tumor cell microenvironment. Shin and coworkers produced chitosan-functionalized CNT with thermosensitive polymer, (NIPAAm) poly-N-Isopropyl acrylamide, and 1-butyl-3-vinylimidazolium (NIPAAm-co-BVIm), then encapsulated the bovine serum albumin (BSA) at 37 C (body temperature) to test temperature-responsive biomolecule release. The BSA was released immediately above poly-lower VBIm’s critical solution temperature (LCST) of 38 C
40 C (Kang, Kim, & Shin, 2017). An electric field was employed by Shi et al. (2015) to emancipate ibuprofen from a hybrid hydrogel made up of sodium alginate (SA), bacterial cellulose (BC), and MWCNTs. Many medications have been loaded onto the CNT to date, as well as doxorubicin (Huang, Yuan, Shah, & Misra, 2011), paclitaxel (Singh, Mehra, & Jain, 2016), docetaxel (Chakraborty et al., 2014), oxaliplatin (Lee, Lin, Peng, & Shieh, 2016), and others, to establish their efficacy for in vitro and in vivo cancer treatments. Dai and coworkers have conducted substantial research on functionalized CNTs for medication delivery in vitro and in vivo (Dhar, Liu, Thomale, Dai, & Lippard, 2008; Liu et al., 2008). The team devised a new method for making CNT extremely dissolvable to trap therapeutic particles (Liu et al., 2008; Zeglis et al., 2015). Khandare and coworkers stated that drug-loaded MWCNT with calcium phosphate (CaP) crown might be used as a nano-capsule for the intracellular conveyance of an anticancer medication (Banerjee et al., 2015). Their presentation for the drug molecule administration from the nano-capsule is depicted in Fig. 26.2D. Fig. 26.2E reports the pH-activated CaP disintegration and drug administration in subcellular parts like lysosomes (pH-5). Risi et al. (2014) reported the well-organized discharge of a novel anticancerous medication on CNT over a long period. Xu et al. (2016) created an amine-terminated PEG

26.2 Carbon nanotube
based composites for biomedical applications

functionalized polydopamine (shell)-CNT (core) nano-system for drug administration to increase the biocompatibility of CNT (Mejri, Vardanega, Tangour, Gharbi, & Picaud, 2015).

26.2.3 Carbon nanotube nanocomposites for cancer treatment During the last decades, the picture of cancer treatment has changed greatly. The epoch in which surgical procedures and radiation therapy were the only beneficial ways to combat cancer growth has ended. A complicated scene is now arising in which cancerous molecular attributes appear to be the keystone of any medication. Moreover, the detailed description will assist in recognizing the critical importance of some iconic cancer therapies, such as surgical procedures, radioactivity, chemo and radiation, and hormonal therapy, which are now effectively interpreted in terms of the mechanisms underlying their effectiveness (Urruticoechea et al., 2010). Because of the various features, CNT is most popularly implemented in the biomedical sector. These are the most appealing possibilities for transporting chemotherapeutic medicines, DNA, and proteins (Adeli, Soleyman, Beiranvand, & Madani, 2013; Amenta & Aschberger, 2015; Eskandari, Hosseini, Adeli, & Pourjavadi, 2014; Hwang, Park, & Lee, 2017). CNTs are also effective photothermal agents due to their excellent NIR light absorption capabilities. Su et al. (2017) created MWCNT with iRGD-polyethyleneimine functionalization and candesartan conjugation (CD) (Zhang et al., 2017). The GdN@CQDs-MWCNTs’ 2 ve surface charge made it easier for them to bind to 1 ve charged DOX molecules. The nano drug’s cytosol release caused lung cancer cells to apoptosis by abrupting the mitochondrial membrane. Finally, the substance demonstrated therapeutic activity in vivo. Furthermore, therapeutic performance was induced by the localized heating effect caused by NIR irradiation (Song et al., 2016).

26.2.4 Carbon nanotube nanocomposites for tissue engineering Tissue engineering has tantalizingly provided the option of rejuvenating new tissue to cure a wide range of diseases and restrictions inside the human body. Nonetheless, despite improvements with in vitro and small mammal studies, advancement into biomedical has been sluggish. Many would suggest the ultimate goals, particularly in relieving symptoms for which there are currently no appropriate mainstream remedies, that may not be achieved due to imperfect system identification. In other words, existing practices may not even be capable of attaining self-sustaining tissue engineering (Williams, 2019). Supporting the regeneration of cells that need to renew dead or damaged tissues necessitates scaffolding nanocomposites characteristics. They must be cross-linked, mechanically resistant, have excellent biocompatibility, and allow for suitable cell attachment. Furthermore, nano-fillers such as CNT in those matrices increase the tensile features and adding over it also boosts the electric conduction (Andrews & Weisenberger, 2004). For instance, regulating the electro-active nature of cardiac

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or nervous cells throughout the cellular repairing process is very important. Yildirim, Yin, Nair, and Sun (2008) indicate that the incorporation of 1 wt.% of SWCNT in an alginate scaffold was sufficient to enhance the mechanical firmness given to natural tissues, and also increases cell adhesion and proliferation (Shin et al., 2011; Yildirim et al., 2008). Ahadian et al. investigated a coordinated Gelatin methacrylate (GelMA)/aligned MWCNT scaffold that improved the distinction of embryonic debris in cardiovascular cells, which means it could exhibit practical assistance for tissue therapy on stem cells (Ahadian et al., 2014, 2016). The improvement in the physical properties delivers a significant feature for bone tissue engineering and also in cardiac application in which electric conductance is important. Likewise, various researches proved that the CNT addition in biopolymer-based hydrogels can improve the maturation of cardiomyocytes for cardiac applications (Yu, Zhao, Huang, & Du, 2017) or cardiac patches (Pok et al., 2014). Additionally, Zhang et al. revealed the encouraging outcomes of SWCNT on osteoblast accretion in chitosan scaffold having crystalline hydroxyapatite, compared to pristine hydrogels and improves mechanical strength (Zhang et al., 2017). The scaffolds represented by Cancian et al. were developed by CNT in thermosensitive chitosan hydrogels and manifested suitable fluidity for conventional syringes (Cancian, Tozzi, Hussain, De Mori, & Roldo, 2016). Currently, Liu, Kim, Miller, Waletzki, & Lu (2018) reported a possible approach to fill the gap which ultimately resulted in roof spinal cord damage (Fig. 26.3). Nerve channel consisting of PEG functionalized CNT/oligo (poly (ethylene glycol) fumarate)

FIGURE 26.3 (a) Spinal cord of human body. (b) Conductive nerve conduits for injury treatment (spinal). (c) Structure of the conductive OPF-CNTpega hydrogel. Adapted from Simon et al. 2019 under Creative Coomons Attribution.

26.4 Future prospective

(OPF) nanocomposite had been developed by operating the injection molding approach. The reported hydrogel represents the improvement of tissue adhesion and augmentation, which proves that the nerve channels can help axon recovery (Liu et al., 2018).

26.3 Toxicity of carbon nanotubes CNTs have a broad spectrum of applications in biomedicine, including drug delivering systems, biomedical imaging, biosensors, tissue repairment, and therapies. CNTs are a new class of materials. Biological systems, however, continue to be harmed by their toxicity. Various studies on CNT toxicity have been set off to date (Allegri et al., 2016; Kobayashi, Izumi, & Morimoto, 2017; Liu et al., 2013; Madani, Mandel, & Seifalian, 2013). Several factors have been discovered to confer to the lethality of CNT in numerous investigations (Aldieri et al., 2013; Kostarelos, 2008; Koyama et al., 2009; Vittorio, Raffa, & Cuschieri, 2009). Some researchers created CNTs of various sizes and investigated their toxicity on cells and DNA (Raffa et al., 2008; Smart, Cassady, Lu, & Martin, 2006). Chronic recognition of long CNT causes critical swelling and growing fibrosis (Murphy et al., 2011). Furthermore, CNTs with a larger diameter and an equal average length have higher toxicity (Kolosnjaj-Tabi et al., 2010). SWCNT and MWCNT transported varied cytotoxic effects on cells due to their physiological and morphological differences (Di Giorgio et al., 2011; Fraczek, Menaszek, Paluszkiewicz, & Blazewicz, 2008). Furthermore, the stabilizing factors significantly influenced CNT toxicity (Kim et al., 2012; Nam, Kang, Kang, & Kwak, 2011). Independent CNTs tends to club in the existence of natural dispersants, resulting in lethality. In HUVEC cell lines, Jos and coworkers discovered that
COOH-functionalized SWCNT caused more toxicity than nonfunctionalized SWCNT (Praena et al., 2011). Li et al. (2013) found that strongly cationic-functionalized MWCNT has a more significant ability for lysosomal damage to their high cellular activation (Fig. 26.4; Ge et al., 2012). The lethality of CNT in cells has been studied extensively (Shareena, McShan, Dasmahapatra, & Tchounwou, 2018). Several characteristics like concentration, lateral dimension, surface ability, and functional groups, significantly impact its toxic level biologically (Alshehri et al., 2016; Chong et al., 2015; Li et al., 2014; Liang et al., 2016; Nurunnabi et al., 2013; Seabra, Paula, de Lima, Alves, & Dura´n, 2014; Tian et al., 2016; Wang et al., 2016).

26.4 Future prospective CNTs, as one of the most commonly employed kinds of nanomaterials, have attracted enormous research over the last two decades. CNTs are broadly used in several fields because of their mechanical, optical, electrochemical, and electrical

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FIGURE 26.4 Cellular and Molecular Mechanism of Toxicity of Carbon Nanotubes (CNT). Reproduced by Ge et al. 2012 under Creative Commons Attribution License.

capabilities. CNTs became attractive in biomedical engineering during the past few years because of their variable surface characteristics, size, and form. Attributed to the prevalence of both inorganic semiconducting capabilities and organic stacking features, CNTs are becoming a promising material. As a result, it can successfully interrelate with biomolecules while also responding to light. Various modifications in chemical procedures have been created and products were fully used in biological implementation as in drug delivering systems, tissue repairment, biomolecule identification, and cancer therapies due to their hazardous effect in the biological system. This review chapter underlines some of the improvements made in the usage of CNTs in biomedical applications. Furthermore, we emphasize the recently discovered essential properties of CNTs and their applications in better bioapplications in this work. Conversely, CNTs still have toxicity; more comprehensive research is needed to evaluate their toxicity and pharmacokinetics (Maiti, Tong, Mou, & Yang, 2019).

26.5 Conclusion CNTs usher in a new epoch of real-world applications while also addressing basic science at the nonmetric spectrum. CNTs are a type of nanomaterials that has crucial implementations in therapeutic applications and biomedical. Existing

References

tumor-desired deliveries employing CNT is a solution to cancer therapy’s unsettled obstacles. More research is needed to create a functionalized CNT transmission system for effective targeted brain medication. CNTs have proven to be unique in the development of stem cell transplantation and several cellular transport systems. Although functionalization of CNTs is successful in terms of good biocompatibility and reducing toxic effects, a thorough toxicological assessment of these nanomaterials is required. Furthermore, it necessitates a profound knowledge of the basic physiology governing CNT delivery. Some of these therapeutic strategies based on CNT will become a fact only if the adverse effects of CNT lethality are addressed thoroughly. In the upcoming years, there is indeed a possibility that superspecific and outstanding CNT-based systems will replace all commonly used diagnostic and biomolecular recognition systems. This review article explores CNTs and associated nanocomposites with biomedical applications. The above analysis and the review suggest that CBNs can be sufficiently utilized to make nanocomposites for biomedical applications. However, there are particular advantages, such as biocompatibility, tensile strength, nontoxic, etc. The reported present review work will open up the new direction and receive increasing consciousness shortly to develop advanced materials useful for antimicrobial application. This will lead to the modification era of making advanced materials from conventional microsphere to multifunctional nano-sphere.

Acknowledgment The recommended DST Grant-in-Aid for Scientific Research motivated this novel research work of Sarika Verma. The authors are also thankful to Director CSIR-AMPRI Bhopal for providing necessary instrumental, institutional facilities and encouragement.

Conflict of interest There are no conflicts to declare.

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CHAPTER

Cryogels as smart polymers in biomedical applications

27

O¨zlem Bic¸en U¨nlu¨er1, Ru¨stem Kec¸ili2, Rıdvan Say3,4 and Arzu Erso¨z1 1

Chemistry Department, Faculty of Sciences, Eski¸sehir Technical University, Eski¸sehir, Turkey 2 Department of Medical Services and Techniques, Yunus Emre Vocational School of Health Services, Anadolu University, Eski¸sehir, Turkey 3 Department of Chemistry, Faculty of Sciences, Anadolu University, Eski¸sehir, Turkey 4 Bionkit Co Ltd., Eski¸sehir, Turkey

27.1 Introduction Polymeric gels are semisolid systems with a three-dimensional (3D) network structure, usually formed using polymers via covalent or noncovalent bonds. Due to this structure, they show elastic properties. Polymeric gels have swelling property due to their hydrophilic structure. While the polymer matrix holds the liquid in the swelling process, the liquid in the gel provides free diffusion of other molecules. Besides swelling, elasticity, non-Newtonian rheological behavior, and mechanoelectrical effect are the important features of polymeric gels (Nayak & Das, 2018; Okay, Polymeric). The common method is to synthesize polymeric gels is the free-radical crosslinking polymerization method in aqueous media. Polymeric gels can be divided into the groups according to the cross-linking character: hydrogels, emulgels, organogels, xerogels, aerogels, and cryogels. Hydrogels are hydrophilic gels that are formed using hydrophilic monomer(s) or polymer(s) precursors containing hydrophilic groups either embedded or grafted. They are capable of absorbing a large volume of liquids or biological fluids without any loss of their physical structures (Hoffmanc¸, 2002; Mishra & Mishra, 2016; Peng, Chin, Tay, & Tchong, 2011), so hydrophilicity, higher swelling capacity, softness, and elasticity are their main features. Synthetic, semisynthetic, and natural polymers are employed in hydrophilic polymer synthesis (Ahmed, 2015; Maitra & Shukla, 2014). Organogels have a nonpolar phase immobilized within the spaces of the 3D network structure, due to gelators that enable physical interactions between certain types of compounds (Sahoo et al., 2011; Suzuki & Hanabusa, 2010). Emulsionbased gels are called emulgels. Emulsion is prepared by oil/water or water/oil types and gelation is incorporated with a gelling agent. Nonionic surfactants are used for the development of the emulgels’ characteristics. Emulgels are used in the development of drug carrier systems and investigating the drug release from Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00022-X © 2023 Elsevier Inc. All rights reserved.

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an external phase of the emulsion (Asija, 2013). Aerogels are generally sol
gel materials in which liquid consists of gel systems reacted with gas so, they have a high specific surface area, refractive index, ultralow thermal conductivity, etc. (Du, Zhou, Zhang, & Shen, 2013; Garcı´a-Gonza´lez, Alnaief, & Smirnova, 2011). Xerogels are the gels prepared by the sol
gel method to gain higher porosity and larger surface area with very small pore sizes. They are prepared at room temperature using different metal alkoxide precursors, water, and ethyl alcohol. Condensation procedure of the liquid alkoxide manages the preparation of the solid oxide network in xerogel formation (Czarnobaj, 2008). Since there are considerable research in polymeric gel preparation, the design, stability in the gel structure, and formation and network properties have still some problems. Low cross-linking degree causes to increase the pore size and accelerates the degradation of the polymeric gels. In this case, they become inactive polymers. In most polymeric gels, the kinetics of the gel volume change which is called the diffusive process is not as fast as required in many application areas. From this point, different types of techniques for the toughness of polymeric gels were used such as double network gels, hydrophobic interactions usage in the gel formation, and microsphere composite gel formation (Gong, Katsuyama, Kurokawa, & Osada, 2003; Huang et al., 2007; Okumura & Ito, 2001; Tuncaboylu, Sarı, Oppermann, & Okay, 2011). Among these polymeric gel synthesizing techniques, the free-radical cross-linking polymerization technique supplies the interconnected-macropore-structured gel formation. The polymeric gel prepared by free-radical cross-linking polymerization technique (Matsumoto, 2007) has a fast response rate. When a large amount of cross-linker is used, the obtained gels will not be flexible gels. Cryogelation is a specific and simple technique that allows the synthesis of interconnected porous gels with high rigidity, elasticity, and stability in various reaction conditions (Rogers & Bencherif, 2019). Polymeric gels that are prepared via cryogelation process are cryogels.

27.2 What is cryogel? Cryogels are interconnected supermacroporous gels that are synthesized according to cryogelation of monomeric or polymeric precursors at subzero temperature values. In fact, the term “cryogel” is the combination of two words: “cryo” and “gel.” The word “cryo” comes from Greek “cryos” that means cold or ice and “gel” means that semisoft matter. Studies on cryogels have been started in the 1970s and still have an important area in biotechnological studies. Due to the capability of the cryogels of migration of cells, blood, allowing the effective mass transport of consistency, and good mechanical properties under hydrophilic conditions, they become smart materials in the various research areas. The fact that the working limitations of other

27.3 Cryogel preparation method

polymeric gels are eliminated or minimized with cryogels provides a great advantage in terms of studies. Cryogels are sponge-like structured so that elastic and have the high absorption capacity naturally. Cryogels can easily be synthesized with various pore architecture, adjustable swelling properties, high hydrophilicity in terms of high compressibility, and elasticity related to the usage area. This variable architecture of the cryogels depends on the type or concentration of polymer (s) or monomer(s), cross-linker concentration, freezing and thawing conditions, and the way of freezing the cryogel solution. All these parameters affect the structure, pore distribution, or connection of the pores in the cryogel design (Erso¨z, ¨ nlu¨er, Do¨nmez, Hu¨r, & Say, 2014; Lozinsky et al., 2003; Shiekh, Andrabi, U ¨ nlu¨er, Erso¨z, & Say, 2019; Yavuz, Singh, Majumder, & Kumar, 2020; Su¨mbelli, U ¨ Unlu¨er, Erso¨z, & Say, 2020). Cryogels can be synthesized in different morphology such as monolithic columns, membranes, disks, sheets, etc. Their porosity distribution can be controlled and immobilization of active molecules to their interconnected pores can be easily performed. Fig. 27.1 shows the cryogel structure, shapes, porosity, and porosity changes. The interconnected macropores or supermacropores provide the cryogel spongy and elastic morphology with a pore compressed without any cracks in their microstructure size range from 10 to 200 μm. Via the macroporous elastic structure resulted in high rate of cross-linking and mesh structure with thick pore walls, they can be compressed without any deformation in their microstructure, giving them stability compared to other polymeric gels. Fig. 27.2 shows the squeezing test of cryogel. As seen in Fig. 27.2, cryogel can easily gain its shape after the squeezing process and lose the water in its macropores with a compressing process due to its high hydrophilic character.

27.3 Cryogel preparation method Cryogels are prepared via cryogelation technique. In cryogelation process, polymerization between the monomer(s) or polymer(s) precursor and the cross-linker formed in subzero temperatures in the presence of the reaction initiators and this process is called as the freezing process. After freezing, the formed polymeric ice crystals are left for the thawing process at room temperature. After thawing process, the formed polymeric gel is called cryogel. In brief, cryogelation technique is a freezing-thawing process to obtain interconnected macroporous structured cryogels. Cryogels are totally different in morphologic aspect compared with polymeric gels obtained in nonfrozen reactions due to cryogelation technique. An aqueous solvent is used in cryogel formation. Initially, monomer(s) and polymer (s) or their mixtures as cryogel precursors are mixed in an aqueous solvent and the mixture cooled. After the cooling process, an appropriate cross-linker is added to the mixture. After the addition of the reaction initiator chemicals such as ammonium persulphate (APS)/N,N,N0 N0 -Tetramethylethylenediamine (TEMED),

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FIGURE 27.1 Cryogels can be synthesized in different morphology in terms of size, shape, and porosity. (A) Different cryogel formations such as beads, monoliths, disk, injectable, and sheet as a schematic representation. (B) Cryogels in different forms. (C) Cryogel porosity diversity as schematic representation. (D) SEM micrographs of the cryogels showing usage of different concentrations formed different porosity in the structure (Parvaiz, Syed, Anamika, Majumder, & Kumar, 2021). SEM, Scanning electron microscopy. Reproduced from Parvaiz, A. S., Syed, M. A., Anamika, S., Majumder, S., & Kumar, A. (2021). Designing cryogels through cryostructuring of polymeric matrices for biomedical applications. European Polymer Journal, 144, 110234, with the permission.

27.3 Cryogel preparation method

FIGURE 27.2 Compression of a cryogel. (A) Dried cryogels. (B) Swelling profile of the cryogel in water. (C) Recovery of water from cryogel pores. (D) Deswallon cryogels (Loo et al., 2013). Reproduced from Loo, S-L., Fane, A., Lim, T., Krantz, W., Liang, Y., Liu, X., & Hu, X. (2013). Superabsorbent cryogels decorated with silver nanoparticles as a novel water technology for point-of-use disinfection. Environmental Science & Technology 47(16):9363
9371, with the permission.

the whole cryogel solution is incubated at subzero degrees for cryopolymerization process and ice crystal formation. It should be emphasized that ice crystals are the precursors of the macropores in the structure. After cryopolymerization process at subzero temperatures (216 C or 218 C), the formed frozen polymer system is thawed at room temperature. In the polymeric lattice, the ice crystals, which will form the porogen structure, melt and interconnected macroporosities in the cryogel structure are formed during the thawing process. After this, the formed cryogel is washed with deionized water several times. The obtained cryogels can be stored in 0.2% NaN3 solution at 4 C to prevent bacterial growth until use. Fig. 27.3 shows the steps of cryogelation process as the initial system when the cryogel precursors are dissolved in aqueous media, frozen system when the polymerization process has occurred at subzero temperatures and cryogel forma¨ nlu¨er, tion at room temperature following the thawing process (Say et al., 2012; U ¨ zcan, & Uzun, 2014). Also, the change in 3D structure versus volume, porosity, O and average pore size is shown. In the synthesis, cryogelation temperature, cooling rate, cross-linking type, etc. affected the formed cryogel. These parameters and effects to the cryogel characteristics are given in Table 27.1.

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FIGURE 27.3 Cryogel formation process (A) Representation of cryogelation process. (B) a. Different shaped cryogels and b. SEM image of cryogel that shows macropores. (C) Cryogel formation based on the change in 3D structure affected various parameters. SEM, Scanning electron microscopy. Reproduced from Parvaiz, A. S., Syed, M. A., Anamika, S., Majumder, S., Kumar, & A. (2021). Designing cryogels through cryostructuring of polymeric matrices for biomedical applications. European Polymer Journal, 144, 110234, with the permission.

27.4 The precursors in cryogel preparation

Table 27.1 Effects of variable parameters on cryogel morphology and characteristic. Parameters in cryogel preparation Cross-linking method

Cryogelation temperature

The cooling rate of the cryogel The molecular weight of the polymeric precursor

Effects of the parameters on cryogel morphology 2 The physical cross-linking strategy provides the gel formation with insufficiently large pores 2 The chemical cross-linking strategy provides sufficiently large pores but may cause potential cytotoxicity 2 Increasing cryogelation temperature provides the large pore size and high pore wall width and pore density 2 Increasing in cooling rate provides the decreasing pore size 2 Increasing the molecular weight of the polymer precursor provides increasing gel stiffness and decreasing in molecular weight of the polymer precursor provides the pore size increases

27.4 The precursors in cryogel preparation The precursors used in cryogel synthesis could be divided into three categories as natural, semisynthetic, and synthetic precursors. The precursors must be dissolved in aqueous reaction media. During the cryogelation process, the chosen solvent should provide the prepared cryogel solution to crystallize rather than vitrify it. As the result, macropores do not occur in the cryogel structure. For the formation of the cryogenic structure, the choice of solvent as water and preparing precursor solutions in aqueous media is the important point. So, the water-soluble natural, semisynthetic, and synthetic precursors are widely used in cryogel synthesis. Gelatin, silk, collagen, and chitosan are some of the examples of natural polymers used in cryogel synthesis. For gelatin and collagen, hydroxyl, carboxyl, amide groups, and amino are the functional groups where the main functional groups are primary amino, ether, amide, and hydroxyl groups for chitosan. Carboxylic acid and the amino groups are the functional groups for silk. In accordance with the functional groups in those precursors, different cross-linkers are used in cryogelation process. When gelatin or chitosan is used as a precursor, methylene bis acrylamide (MBAAm) or glutaraldehyde (GA) is used in cross-linking step. 2-Hydroxyethyl methacrylate (HEMA), methacrylic acid (MAAc), ethylene glycol dimethacrylate (EGDMA), polyvinyl alcohol (PVA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), acrylamide (AAm), N,N-dimethyl acrylamide, N,N-dimethyl acrylamide (DMA), acrylic acid (AAc), and N-isopropyl acrylamide are some of the synthetic precursors that are used in cryogel synthesis as precursors in combination with MBAAm, GA, or biodegradable cross-linkers following the functional groups

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on the monomers in precursor
cross-linker interaction process (Baimenov, Berillo, Poulopoulos, & Inglezakis, 2020; Tripathi & Melo, 2019). A single precursor using such as a natural and/or synthetic polymer made the cryogel structure the simplest types of cryogel. Two or more polymers mixing homogenously provide the synthesizing composite cryogels and macro, nano, or microparticles can be added to this composite cryogel solution to make embedded those particles into cryogel macropores. Via a proper cross-linker, cryogelation process takes place and a composite cryogel structure is formed that has a unique characteristic which is suitable for the different research areas. These composite cryogel platforms are capable of combining the advantageous parts of each precursor used in the preparation of composite cryogels (Memic et al., 2019).

27.5 The cross-linking strategy in cryogel preparation A typical cryogelation process is composed of the steps of preparation of precursor solution, the interaction of the cross-linker with precursor, and freezingthawing process. To obtain a polymer network in cryogel, the cross-linking process has an important role. Two main mechanisms, physical and chemical, are used in cross-linking step in cryogel formation. Covalent bonding and cross-linking of monomers or polymers or the mixture of polymer(s)/monomer(s) occur in chemical cross-linking. The reactive types in cryogel solutions undergo a polymerization process between ice crystals at subzero temperature values in the chemical cross-linking process (Bencherif et al., 2012). Contrarily, the physical cross-linking strategy consists of hydrophobic or ionic interactions between the polymer precursor chains. Polymers are either cross-linked after they are condensed around ice crystals or before cryotreatment in physical cross-linking depending on the polymerization kinetics of the polymers (Shih et al., 2018). However, the physical cross-linking procedure is a simple and rapid process and cryogels synthesized without any chemical reactions, the formed cryogel macropores are usually small (,10 μm) and they have unpredictable features such as gel stability based on the formation, degradability, and gel integrity. This feature restricts the usage area of the physical cryogels. Besides this, chemical cryogels, namely, chemically cross-linked cryogels, are stable and have defined properties such as stability, degradability, rigidity, etc. So, the chemical cross-linking strategy is the most preferred technique for cryogel preparation. The free-radical polymerization technique is the widely used technique among chemical cross-linking methods (Henderson, Ladewig, Haylock, McLean, & O’Connor, 2013). In this process, a continuous polymeric network is formed around the ice crystals by the reaction of free radicals and precursors (Kelly & Zweben, 2000). In addition to the cross-linkers causing the freeradical formation, an initiator system should be introduced to polymerization solution for delaying or slowing the polymerization kinetics prior to the ice crystal formation. APS/TEMED pair is used as radical initiator pair for cryogel fabrication. APS/

27.6 Characterization of cryogels

FIGURE 27.4 Formation of AAm-based cryogel network via free-radical cryopolymerization. Reproduced from Sabbagh, F., & Muhamad, I. I. (2017). Acrylamide-based hydrogel drug delivery systems: Release of Acyclovir from MgO nanocomposite hydrogel. Journal of the Taiwan Institute of Chemical Engineers, 72:182
193, with the permission.

TEMED initiator system in addition to the cryogel solution provides the cross-linking reactive polymer block formation starting by generating the free radicals. Fig. 27.4 (Sabbagh & Muhamad, 2017) shows the schematic representation of AAm-based cryogel synthesis mechanism. Another way of cross-linking strategy is photo-cross-linking. Photoinitiator decomposes when it is exposed to the light and provides the preparation of cryogels via radical polymerization (Nair, 2016). For example, polydimethylsiloxane (PDMS)-based cryogels are synthesized by polymerization 218 C through freeradical polymerization using UV light. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linking method is another way in cross-linking step of cryogel synthesis. This cross-linking type can only be used in the presence of protein such as gelatin or the matters consist of amino and carboxyl groups as the functional groups in their structure (Newland et al., 2015; Tao et al., 2017).

27.6 Characterization of cryogels After the synthesis of cryogel via cryogelation technique, the characterization process is important to prove the unique structure and investigate the difference from

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the other polymeric gels. Surface area and porosity determination are the important characterization processes. Nitrogen porosimetry is used to determine the pore volume, and distribution and surface area of the synthesized cryogels using various techniques based on nitrogen adsorption
desorption isotherms. Based on the nitrogen gas adsorption to the macropores in cryogel, Brunauer
Emmett
Teller (BET) equation is convenient for the determination of the pore features in the structure (Tekin, Uzun, Sahin, ¸ Bekta¸s, & Denizli, 2011). The surface area of the cryogels is variable due to the precursors, cross-linking agent and degree, time of cryogelation, etc. Table 27.2 shows the specific surface area of various p(HEMA) cryogels determined according to the BET equation. Another characterization method for the determination of surface morphology and porosity of the cryogels is microscopy imaging. The scanning electron microscopic (SEM) image of cryogels indicates the interconnected macropores, pore distribution, and pore size. Also, the thickness of the pore walls is investigated using SEM images. The cryogel samples prepared for SEM analysis are as follows: The synthesized cryogels are dried before the analysis and the fragment of dried cryogel sample was placed on an SEM holder and gold was sputtered onto prepared samples for gold film coating during 2 min. The coated cryogel sample mounted in the SEM and scanned to investigate the surface morphology (Erso¨z ¨ nlu¨er et al., 2014; Yavuz et al., 2020). In Fig. 27.5, SEM image of et al., 2014; U an p(HEMA) cryogel and binano enzyme immobilized cryogel is shown as an example. To determine the functional groups such as -NH2, -SO3, -OH, -COOH, and -CONH2 in cryogel structure, Fourier transform infrared spectroscopy (FTIR) is the most widely used technique. The sample was prepared as a drying cryogel sample before the analysis. The dry cryogel sample is ground in a mortar and Table 27.2 Specific surface area of p(HEMA)-based cryogels.

Cryogel composition

BET surface area (m2/g polymer)

p(HEMA)

27.2

p(HEMA-co-MAH) p(HEMA-co-DEAE) p(HEMA-co-PEI) p(HEMA)/p(GMA-coEDMA) p(HEMA-co-MAGA)/p (HEMA) p(AA-co-MA-co-AAc)

18.6 9.3 8.9 18.4

Kavoshchian, Üzek, Uyanık, S Ëenel, and Denizli (2015) Jalilzadeh and S Ëenel (2016) Wang and Sun (2013) Wang and Sun (2013) Wang and Sun (2013)

29.2

YeË silova, Osman, Kara, and Özer (2018)

21.9

Kim and Lee (2019)

BET, Brunauer
Emmett
Teller.

References

27.6 Characterization of cryogels

FIGURE 27.5 SEM image of (A) HEMA cryogel and (B) binano enzyme embedded HEMA cryogel. SEM, Scanning electron microscopy. Reproduced from Su¨mbelli, Y., U¨nlu¨er, O¨. B., Erso¨z, A., & Say, R. (2019). Synergistic effect of binanoenzyme and cryogel column on the production of formic acid from carbondioxide. Journal of Industrial and Engineering Chemistry, 76:251
257, with permission.

mixed with KBr. The prepared cryogel-KBr tablet is prepared and placed on the device. The dry cryogel sample is placed directly into the sample part and functional group analysis is performed for analysis using the ATR part of the device. In the study of Baimenov et al. (2020), the peak at 3524 cm21 corresponds the vibration band of a hydroxyl group, the peak at 2955 and 1262 cm21 indicated the an aliphatic C-H group and ester band, respectively, for FTIR analysis of the p(HEMA) cryogel. In addition, a frequency at 1728 cm21 shows the carbonyl group. Swelling analysis of the cryogels is a characterization method to determine the pore volume and porosity percentage due to their hydrophilic features. For the determination of pore volume which indicates the ratio of porous sample air volume to porous sample total volume, the cyclohexane uptake method is used. According to this method, cryogel sample is dried first and immersed in cyclohexane at room temperature. Mass of dried cryogel (Md) and mass of wet cryogel (Mw) are determined. Using Eq. 27.1, pore volume is detected. Pore volume 5

Mw gel 2 Md gel Md gel

(27.1)

The porosity percentage of the cryogel samples is determined according to the swelling performance of cryogel in deionized water. For this purpose, the mass of the dried cryogel sample was recorded and immersed in deionized water. Then the mass of wet sample is recorded. Using Eq. 27.2, the porosity percentage is

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FIGURE 27.6 p(AMPS) hydrogel (A) 0.1% cross-linking and (B) 10% cross-linking swelling feature by water uptake during the time. Reproduced from Baimenov, A., Berillo, D. A., Poulopoulos, S. G., & Inglezakis, V. J. (2020). A review of cryogels synthesis, characterization and applications on the removal of heavy metals from aqueous solutions. Advances in Colloid and Interface Science, 276:102088, with permission.

determined. Msub indicates the submerged mass of cryogel sample (Sharma, Bhat, Vishnoi, Nayak, & Kumar, 2013). %Porosity 5

Mw gel 2 Md gel x100 Mw gel 2 Msub

(27.2)

An example of the swelling capacity of the cryogels was shown in Fig. 27.6, accompanied by 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) cryogel. To investigate the balance between porosity and mechanical strength mechanical test is applied to the cryogels as the other characterization process. High porosity adversely affects mechanical properties, while low porosity leads to mechanical instability and fluid leakage from the cryogel. Compression analysis is used to investigate the mechanical properties of the cryogels. In compression analysis uniaxial stress is applied to the cryogel and Young’s modulus is determined. The Young’s modulus of the cryogel is directly related to its bulk stiffness and it is known as uniaxial stress 2 strain compression value (Shirbin, Karimi, Jun-An Chan, Heath, & Qiao, 2016). Fig. 27.7 shows the relation the swelling degree and Young’s modulus via a stress
strain graph for two types of cryogel.

27.7 The biomedical applications of the cryogels 3D matrices that have unique structural features are mostly used in various biomedical applications. Polymeric gel scaffolds, traditionally hydrogels, are extensively employed in this area; however, their applications are limited due to weak mechanical features and limited molecular diffusion of the samples through the gels. So,

27.7 The biomedical applications of the cryogels

FIGURE 27.7 (A) Swelling process; (B) swelling test; (C) stress
strain curve; (D) Young’s modulus of two different cryogels: A—the more porous cryogel and B—the more stiffed cryogel structure. Reproduced from Shirbin, S. J., Karimi, F., Jun-An Chan, N., Heath, D. E., & Qiao, G. G. (2016). Macroporous hydrogels composed entirely of synthetic polypeptides: Biocompatible and enzyme biodegradable 3D cellular scaffolds. Biomacromolecules, 17(9), 2981
2991, with the permission.

cryogels are emerging as smart alternative matrices for biomedical applications. The advantages of the cryogels are their capability of synthesizing in different pore morphology with various pore sizes and modulating the swelling capacity and capillary action, high compressible features, and elastic structure, which make them smart materials in various biomedical applications. The cell separation process, chromatography column material, environmental studies, biomedical and tissue engineering, regenerative medicine, etc. are the application areas of cryogels. In this part of the chapter, the application of cryogels in biomedical applications due to their structure that is formed by cryostructuring and designing will be discussed.

27.7.1 Cryogels in bioseparation process In bioseparation process, cryogels can be used as column materials due to their porous and mechanic strength, eliminating the column backpressure. The yield of

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the separation process is high due to effective separation. Besides these, the easy modification of the cryogel macropores allows the affinity interactions with the target molecule, thus the bioseparation process is applied in one step instead of several comprehensive stages. The whole plasma matrix or complex samples can be through the cryogel column and the separation is high yielded. Chromatographic separation is more effective by eliminating some steps that would cause substance loss during matrix preparation. Thus the separated biomolecule in high purity obtained and it can be used as precursors for different biomedical applications. The structure of cryogels can be combined with molecularly imprinting molecules to increase the functionality and specific recognition sites of the biomolecules in the bioseparation process. The molecular imprinting method provides the synthesis of robust, easy to use, reusable, and has high specific polymers, namely, molecular imprinted polymers (MIP) for the usage in diagnosis, monitoring, and screening of the target biomolecules for biomedical applications (Hou et al., ¨ nlu¨er, Erso¨z, Denizli, Demirel, & Say, 2013; Zhang, Jıang, Jiu-Tong, & 2021; U Qiong, 2021). In molecular imprinting process, the monomers mixture and template molecule are cross-linked. After the polymerization process, template molecule is removed from the polymeric network. A cavity specific to the target molecule is formed by removing the target molecule process and molecular beads that are specific to the target molecule are formed. These synthesized MIPs can be embedded into cryogel network in cryogel synthesis procedure in the step of mixing precursor with a cross-linker and can be used in the recognition and purification of biomolecules from the matrix. The selective immunoglobulin G (IgG) removal is one example of cryogel and MIP combination. IgG is found in richlevel immunoglobulin type and has an important role in the treatment and diagnosis of various diseases. For IgG removal, IgG imprinted polymer has been synthesized and embedded into the poly(N-isopropyl acrylamide-N-methacryloyl-(l)histidine-based cryogels. These monolithic cryogels used in IgG purification with high recognition of IgG molecules with the controlled binding and elution steps. According to the data, the binding capacity is found as 106.1 mg/g at pH 7.4 and 40 C (Perc¸in, Idil, & Denizli, 2019). In another study of consisting a combination of MIP particles and cryogel for selective removal of the target molecule is hyaluronic acid (HA) separation from the matrix using D-glucuronic acid particle imprinted polymers embedded into ¨ nlu¨er et al., 2013). First, D-glucuronic acid imprinted polymers have cryogel (U been synthesized to obtain specific cavities for HA as glucuronic acid is part of it. Then, MIP particles have been embedded into polyacrylamide cryogel structure (Fig. 27.8). HA was purified from fish eye samples and Streptococcus equi RSKK 679 cell culture using the MIP-embedded polyacrylamide cryogels as the column material in fast protein liquid chromatography. The maximum HA adsorption capacity was found to be 318 mg/g.

27.7 The biomedical applications of the cryogels

FIGURE 27.8 SEM image of (A) and (B) p(AAm) cryogel, (C) and (D) embedded MIP particles into p (AAm) cryogel. MIP, Molecular imprinted polymers; SEM, scanning electron microscopy. Reproduced from U¨nlu¨er, O¨. B., Erso¨z, A., Denizli, A., Demirel, R., & Say, R. (2013). Separation and purification of hyaluronic acid by embedded glucuronic acid imprinted polymers into cryogel. Journal of Chromatography B, 934, 46
52, with permission.

Molecular imprinting process can be formed directly in the macropores of the cryogels. There are some examples in literature based on directly molecular imprinting on the surface of the macropores of the cryogels as biomedical applications. The selective recognition of myoglobin in blood serum is achieved using myoglobin imprinted cryogels. A new approach based on lanthanide-chelate cross-linked MIP cryogel polymer plays a major role in myoglobin separation in the presence of other biomolecules in the serum sample. N-methacryloyl amidoantipyrine-Ce(III) used as the complex functional monomer and p(HEMA) as the precursor of the cryogel. The binding capacity of the synthesized cryogels was found as 68 mg/g and stability has remained after 12 binding-eluting cycles. It was observed that the prepared cryogels have high selectivity toward myoglobin through hemoglobin, lysozyme, and cytochrome c (Dolak et al., 2018). In another study, protein-imprinted cryogel columns have prepared for the lysozyme separation process (Rabieizadeh, Kashefimofrad, & Naeimpoor, 2014). Lysozymeimprinted polyacrylamide cryogel columns have been synthesized containing 8% w/v of total monomers and 0.3% w/v of lysozyme. The adsorption capacity of the

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synthesized cryogel was found as 36.3 mg lysozyme per gram of cryogel at pH 5 7.0. Antibodies from plasma can be separated via modified cryogel columns. The cyanogen bromide (CNBr) activation method is used to embed the antibodies or proteins onto cryogel macropores. The modification of the macropores for increasing the functionality of the cryogels in separation studies is the most used technique. In the study of Alkan, Bereli, Baysal, and Denizli (2010), protein A carrying p(HEMA) cryogels has been prepared for selective isolation of autoantibodies from rheumatoid arthritis patient plasma. Protein A was covalently bonded onto p(HEMA) cryogels using the CNBr activation method. The prepared protein A immobilized cryogel was used to remove IgM antibody from rheumatoid arthritis plasma via passing the whole plasma through the cryogel column. The maximum IgM adsorption was found as 42,7 mg/g and the target antibody was repeatedly adsorbed and eluted many times without any decrease in the efficiency of the cryogel column. Thus it is clear that the cryogel embedding systems prepared by the combination of cryogels with materials having different functional groups or directly with large polymeric structures such as MIP are highly effective materials for the isolation of proteins or antibodies. Another advantageous part of the usage cryogels in this type of analysis is that they can be used repeatedly remaining their effectiveness because of their tailored structure according to each separation analysis.

27.7.2 Cryogels in wound dressing applications In designing wound dressing materials and fast hemostasis for uncontrolled nonpressing surface hemorrhage, cryogels are attractive materials due to their tissue adhesion feature. When cryogels are synthesized with biocompatible monomer(s) or polymer(s) precursors and combine the suitable materials they can be used in wound dressing applications due to their flexible, stretchable, and porous structure. In the study of Li, Zhang, Liang, He, and Guo (2020) antibacterial and antioxidant tissue-adhesive cryogels has been developed for wound healing and nonpressing surface hemostasis. The prepared cryogels are quaternized chitosan (QCS) and polydopamine (PDA) cryogels that are capable of blood cell and platelet adhesion in the treatment of wound healing. The synthesized cryogels were used in the rat liver injury model, standardized circular rabbit liver section model, and pig skin laceration model in this study. It was found that the synthesized QCS/PDA cryogel that contains 20 mg/mL QCS and 2.0 mg/mL PDA has good hemostatic performance. Bilayered cryogels are introduced in the literature in wound healing treatments (Priya et al., 2016). The synthesized bilayer cryogels was used in not only wound healing but also the skin regeneration process as graft materials. In bilayer concept, top layer and bottom layer consist of polyvinylpyrrolidone-iodine (PVP-I) and gelatin cryogel, respectively. The PVP-I layer of the bilayer cryogel is to supply the antiseptic part and the gelatine layer

27.7 The biomedical applications of the cryogels

is to regenerative part. The gelatin cryogel part showed high mechanical strength compatible with the PVP-I cryogel layer which supplies the iodine release for suppressing the microbial growth. The gelatin part of the cryogel serves as the supporting media for cell attachment and proliferation of fibroblast cells and keratinocytes and it is the macroporous media for loading the microparticles or bioactive materials in skin regeneration process. In the literature, the development of tannic acid (TA) particle-embedded (HEMA) cryogels is another study in the field of wound dressing cryogel research (Sahiner, Sagbas, Sahiner, & Silan, 2017). TA is a polyphenol and has -OH groups which supplied the TA structure antioxidant and antimicrobial feature. The structure of TA consists of a glucose molecule that is connected to 10 gallic acids via ester bonds. When p(HEMA) cryogels combine with TA particles, cryogel structure gains antibacterial feature and this makes the cryogels usable in a wound healing process to get rid of bacterial contamination effect because open wounds are sensitive to bacterial colonization, the development of biocompatible, elastic, porous, and stable antibacterial materials is important. In the mentioned study, p(TA) particles in different particle sizes were synthesized via microemulsion methods and embedded into super porous p (HEMA) cryogels. Then, the usage of these synthesized antibacterial materials in wound healing has been investigated. Fig. 27.9 shows the p(HEMA) cryogels and the potential usage of TA embedded p(HEMA)cryogels. Bioadhesive property of the cryogels has also been demonstrated in this figure. Using the advantages of the cryogels such as tissue-like water content, flexibility that provides their high tolerance in the wound area, Go´rska, Krupa, & Majda (2021) developed cryogel dressing for the treatment of wounds. PVA cryogel membranes were loaded with resveratrol which is an antiinflammatory compound to use in active wound dressing. Controlled release of resveratrol from the macropores of PVA cryogel membranes could accelerate the healing of chronic wounds. In this study, a different method is used for the solubilization of resveratrol due to its low water solubilization process. Resveratrol was dissolved in labrasol which consisted of the combination of PVA and propylene glycol. This solution was synthesized as the cryogel freezing at (280 C) followed by thawing during six cycles. After this procedure, the cryogenic membranes with interconnected macropores, including resveratrol and elastic structure, were produced. The synthesized PVA cryogels are the new active wound dressing materials due to resveratrol ingredient as the antiinflammatory agent. Cryogels could be combined with nanoparticles to increase their functionality in the related research area. For instance, zinc-oxide (ZnO) nanoparticles were embedded onto PVA cryogel macropores to gain the cryogels antibacterial property (Chaturvedi, Bajpai, Bajpai, & Singh, 2016). These synthesized antibacterial cryogels were used as wound dressing materials and have the antibacterial potential that is much important feature for injury treatments. It was indicated that PVA-ZnO combined cryogel has antibacterial activity against Gram-positive and Gram-negative bacteria besides the whole cryogel system is biocompatible. This type of cryogel that is combined with nanoparticles has the advantage of both

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FIGURE 27.9 Digital image of swollen p(HEMA) cryogels, and p(TA) particles-embedded p(HEMA) cryogel composites that have the potential as tissue cover material. Reproduced from Sahiner, N., Sagbas, S., Sahiner, M., & Silan, C. (2017). P(TA) macro-, micro-, nanoparticle-embedded super porous p(HEMA) cryogels as wound dressing material, Materials Science and Engineering: C, 70(1):317
326, with the permission.

biocompatibility of the precursor and the property of the nanoparticle brings out the usage of them for medical applications.

27.7.3 Cryogels in tissue engineering applications Cryogels have the advantage for mostly tissue engineering applications, including cell separation and usage in bioreactor and designing scaffolds. Natural polymers in cryogel synthesis have low toxicity and promote cellular interaction but have some disadvantageous parts such as low mechanical features and high cost. Synthetic polymers used in tissue engineering applications have good stability and mechanical strength; however, they have the toxicity potential and probability

27.7 The biomedical applications of the cryogels

of producing hazardous products during degradation (O’Brien, 2011). The precursor materials have to be chosen based on the suitability for designing tissue engineering studies—the combination of the advantageous parts of the natural and synthetic materials.

27.7.3.1 Cryogels as bioreactors Cryogels can be used as bioreactors due to their surface area-to-volume ratio pro¸ imen & Denizli, 2012; Kumar, Tripathi, & Jain, viding the culturing the cells (C 2011). The usage of the cryogels as a bioreactor was investigated in different cell lines. HEMA-lactate-dextran cryogels were synthesized based on the cryogelation method and used as the bioreactor for bone applications (Bolgen et al., 2008). Osteoblast like cells (MG-63) were seeded for investigating the parameters specific to bone generation such as alkaline phosphatase (ALP) activity, cell viability, and morphology. The improved ALP activity was obtained that shows that cryogel cell seeding media is suitable for a bone generation. Chitosan and poly (N-isopropyl acrylamide), poly (NiPAAm)-chitosan, and poly acrylonitrile-co-N0 vinyl-2-pyrrolidone (poly(AN-co-NVP) cryogels have been investigated for the suitability of the bioreactor for fibroblasts (COS-7) and human liver hepatocarcinoma cells (HepG2) cells (Jain et al., 2015). It was found that the synthesized cryogels were suitable and allow the growth of HepG2 cells for providing the artificial liver tissue. Different polymeric materials can be combined to obtain a more suitable composition for bioreactor usage. For instance, a smart cryogel design with a combination of hydrophilicity and hydrophobicity allows static culture of hybridoma cell lines with the presence of charged surface (Jain, Karande, & Kumar, 2011). Polyacrylamide-chitosan cryogels were tested for the growing medium of hybridoma cells and antibody production was obtained in the culture media. The efficient monoclonal antibody production has been achieved in different shapes (monolith, disks, and beads) of the polyacrylamide-chitosan cryogels. Long-term cultivation of hybridoma cells lines on polyacrylamide-chitosan cryogels in all three formations was achieved under static culture conditions. The monolithic cryogel bioreactor performed most efficiently and produced a total of 57.5 mg of monoclonal antibody. Thus the efficiency of the cryogels as bioreactors on the production of monoclonal antibody has been proved.

27.7.3.2 Cryogels in cell separations Cryogels have an advantage over other polymeric monolithic gel columns based on their low pressure and lack of diffusion resistance. Viscous sample such as whole blood or plasma can be applied to the monolithic cryogels without any pretreatment organization. Fig. 27.10 shows the flow-through of the blood sample from the monolithic cryogel without any pretreatment. The interconnected supermacropores of monolithic cryogels allow convective migration of large particles such as cells, making them candidates for cell affinity chromatography. In this type of chromatographic separation, cryogel macropores enhanced with the immobilization ligands for the interaction of cell surface

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FIGURE 27.10 (A) Blood sample flow throughout the cryogel column. Column image 1 shows before application, Column images between 2 and 5 show during the application, and Column image 6 shows after application. During the analysis, blood sample was passed through the synthesized cryogel. (B) Fractions after flow through were collected (Ertu¨rk & Mattiasson, 2014). Reproduced from Ertu¨rk, G., & Mattiasson, B. (2014). Cryogels-versatile tools in bioseparation. Journal of Chromatography. A, 1357:24
35, with permission.

receptors with ligands in the separation process. The functionalized cryogels is used a chromatographic column material in cell separation. As the specific example of the cell affinity chromatography, protein A ligand was immobilized of epoxy-containing cryogel monoliths. After the target cells were labeled with specific antibodies, the cells were separated via the interaction of antibody-protein A interaction, while cell sample was passing through the cryogel column. The captured cells were recovered in high yields and remained their viability due to the sponge-like elastic structure of the cryogels. This method is a fast and reproducible method for cell separation without any deformation in both

27.7 The biomedical applications of the cryogels

cryogel and cells (Kumar & Srivastava, 2010). CD34 human acute myeloid leukemia KG-1 cells can be separated using the cell affinity chromatography using immobilized protein A cryogels as column material. For separation of CD 34 cells, the interaction of anti-CD34 antibody which is found on the cell surface with protein A is the main interactions (Hixon, Lu, & Sell, 2017). Protein A immobilization step obtained with mixing the protein A and precursor polymer solutions cryopolymerization. This cryogel preparation and usage in chromatographic separation as the column material for whole-cell separation indicates the importance of the cryogels in cell affinity chromatography. Fig. 27.11 shows the chromatographic separation of CD34 human acute myeloid leukemia KG-1 cells. Another cell separation process consisted of the separation of the cells from the mixture of Escherichia coli and Saccharomyces cerevisiae using concanavalin A immobilized cryogel monolith columns (Dainiak, Galaev, & Mattiasson, 2006). Both types of the cell sample were prepared in equal amounts and applied to the cryogel column. In chromatographic analysis, S. cerevisiae cells were bound to the cryogel matrix via the interaction of cell surface and concanavalin A and E. coli cells passed through the column without any interaction with the column. The bound S. cerevisiae cells are collected from the cryogel macropores by introducing 0.3 M methyl α-d-manno-pyranoside. The collected S. cerevisiae cells and flow-through E. coli cells were analyzed by counting and determination of the activity. The results indicated that using tailored cryogels is an effective method for the cell separation process.

27.7.3.3 Cryogels as tissue scaffolds In tissue engineering research, it is very important to develop new and effective materials, to develop materials that can be easily prepared in practice and mimic real nature, based on completely biocompatible materials. Cryogels are frequently used in the field of biomedicine and the design of biodegradable materials due to their soft and flexible structures, as well as their adjustable pore size. Based on the application area in tissue engineering such as scaffold designing or mimicking the natural tissue, the capability of the easy modification of the macropores to increase the surface area or gain affinity to the cryogels make them stand out as materials to be used in tissue engineering (Andac, Plieva, Denizli, Galaev, & Mattiasson, 2008; Henderson et al., 2013). Tissue engineering focuses on the usage of 3D scaffolds to repair the tissue and encourage regrowth at the wound site. A scaffold provides a polymeric network mimicking the native tissue, like an extracellular matrix (ECM) analog. Ideal synthetic scaffolds should have good biocompatibility, suitable mechanical properties, and biodegradability and promote cell adhesion and proliferation to allow displacement with native tissue (Chan & Leong, 2008; Dhandayuthapani, Yoshida, Maekawa, & Kumar, 2011). Cryogel tissue scaffolds are one of the scaffold types among the various fabricated tissue scaffolds such as hydrogels, decellularized structures, electrospun fibers, etc. The uniqueness of the cryogels are being popular as the scaffolds for

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FIGURE 27.11 SEM images of (A) bound CD34 human acute myeloid leukemia KG-1 cells to the protein A
PVA cryogel and (B) throughout macropores is shown. SEM, Scanning electron microscopy. Reproduced from K. R. Hixon, T.Lu, S. A. Sell, A comprehensive review of cryogels and their roles in tissue engineering applications, Acta Biomaterialia, 2017, 62, 29
41, with permission.

regeneration of skin, bone, nerve, cartilage regeneration, and tendon tissue types (Dhandayuthapani et al., 2011). For bone regeneration study, mineralized cryogels using the poly (ethylene glycol)-co-2-hydroxyethyl methacrylate precursor have been synthesized via cryocopolymrization process (Liu et al., 2019). The synthesized cryogels was used as the scaffold material by inducing osteogenic differentiation of mesenchymal stem cells (MSCs). The osteogenic differentiation was monitored ALP activity and the

27.7 The biomedical applications of the cryogels

expression of related osteogenic gene markers such as ALP and osteocalcin during 14 days. The mineralized poly (ethylene glycol)-co-2-hydroxyethyl methacrylate cryogels served as the bone tissue scaffold and also the implantation of this scaffold repaired the bone defect in 4 weeks. In tissue engineering studies using cryogels a new perspective which includes the combination of cryogelation and 3D bioprinting technique was developed. 3D bioprinting technology in tissue engineering studies provides a more suitable scaffold design for controllable layer thickness and number in the scaf¨ nlu¨er et al., 2022). Cryogelation profold for cell attachment and proliferation (U cess gained more porous and controlled thickness to the desired tissue scaffold type. Gelatin-HA-based 3D bioprinted cryogenic scaffolds are the suitable cell attachment and proliferation media for fibroblasts cells (L-929 Mus musculus, mouse purchased from American Type Culture Collection). For designing 3D bioprinted cryogel scaffolds, the first step is to find the optimum combination of gelatin and HA mixture for bioprintability. Second, cross-linking of gelatin-HA cryogel solution takes place. After 3D bioprinted scaffolds were fabricated, immediately cross-linking at subzero temperatures obtained. Thawing at room temperature followed the freezing process provided the cryogel formation. Fibroblasts cells seeded the fabricated gelatin-HA scaffolds. The cell attachment and proliferation study proved that 3D bioprinted cryogenic Gel
HA scaffolds suitable candidates for cell growth without any cytotoxic effects. In bone tissue repair investigations, cryogel with poly-ε-caprolactone (PCL) combination is used (Van Rie, Declercq, & Van Hoorick, 2015). Gelatin as cryogel precursor is used and PCL which is used for enhancing the stability and mechanical strength was added to the cryogel structure. Besides the combination of different precursors, a combination of the cryogelation and 3D modeling technology is used to make a more definite shape and layered cryogels in order to have suitable osteoblast carriers. The biocompatibility and proliferation of the cells were monitored using live/dead staining assay and histology. The attachment of viable cells onto the PCL-gelatin cryogel network and good colonization on the PCL-gelatin 3D printed cryogels are suitable carriers for the target study. In scaffold designing, cryogels structures can be combined with nanoparticles to form scaffold as culture media for different cell lines. For instance, HA-based cryogels combine with halloysite nanotubes (HNTs) in different compositions due to HA biological functions such as interaction with different cells via cell surface glycoprotein performing the proliferation, cellular stimulation of migration, etc. Besides, HA has an important role in cell differentiation and cell development through receptors, tissue hydration, cellular homeostasis, and so on (Bourguignon, Zhu, & Zhu, 1998; Iwata, 1993). HNTs are two-layered natural clay with a hollow nanotubular shape. The combination of clay and HA in scaffold structure provides a new aspect in cell differentiation or proliferation in tissue engineering studies (Suner et al., 2019). In the study of Suner et al. (2019), the scaffolds are synthesized, including the different ratio of HA and HNTs, by cryogelation method and they were investigated as the cell culture media for rat MSCs, human

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FIGURE 27.12 The growth of rat MSC, HeLa, and HCT116 cells on HA-HNTs scaffolds with different ratio. Stained with DAPI shows the nuclei of cells while fluorescein diacetate (FDA) shows the scaffold. MSC, Mesenchymal stem cells. Reproduced from Suner, S. S., Demirci, S., Yetiskin, B., Fakhrullin, R., Naumenko, E., Okay, O., . . . Sahiner, N. (2019). Cryogel composites based on hyaluronic acid and halloysite nanotubes as scaffold for tissue engineering. International Journal of Biological Macromolecules, 130, 627
635, with permission.

cervical carcinoma cells (HeLa) and human colon cancer cells (HCT116). The scaffolds were characterized as blood compatibility, mechanical strength, biodegradability, and porosity, they were used in cell proliferation studies. The proliferation profiles of the cells on the scaffolds were investigated by DAPI and fluorescein diacetate staining and confocal laser scanning images. Fig. 27.12 indicates the usage of the cryogel scaffolds for different cell lines as proliferation media. Cryogel HA scaffolds combined with HNTs showed excellent adhesion, viability, proliferation, and the growth of various types of cell lines. The most important point of this study is that increasing the concentration of HNTs embedded into HA cryogels increases the MCSs cells growth. So, cryogels are attractive materials due to their interconnected macropores, high thermal and mechanical

27.7 The biomedical applications of the cryogels

stability, and biocompatibility in tissue engineering and embedding other biomaterials onto macropores enhanced their usage in cell viability, cell proliferation, and adhesion effects. Cryogels can also be used as scaffolds in neural tissue regeneration studies. Gelatin or dextran precursors linked to the main component of brain ECM, in cryogelation step and the formed scaffolds can be used as bioactive materials in neural tissue regeneration (Jurga et al., 2011). These scaffolds can promote the differentiation of human cord blood-derived stem cells (CBSCs) into artificial neural tissue in vitro. Neural tissue studies aim to develop the supporting materials in neurogenesis and reconstruction of lost brain tissue when large lesions and cavities occur. In the study of Jurga et al. (2011), it was found that 3D cryogel network was the medium for the encouragement of neural stem cell differentiation into electrically active neurons. The differentiation of the CBSCs into neural stem cells on cryogel scaffolds is obtained in three steps. The first step is the isolation of stem cells from cord blood samples, the second step is performing stem cells to Nestin-positive neuroblasts (green), and the third stage is differentiation into mature neural-like cells (positive for MAP2 and NeuN). Fig. 27.13 shows cryogel scaffolds for brain cell survival and stem cell differentiation to design an artificial brain tissue model in vitro. The dextran- and gelatin-based immobilized laminin cryogel scaffolds are 3D bioactive scaffolds in neural tissue engineering and brain repairing studies by using the advantage of containing tissue-specific porosity and mechanical properties. Gelatin-based cryogel scaffolds can be the 3D porous network in the differentiation of adipose-derived mesenchymal stem cells toward hepatocyte-like cells (Gandomani, Lotfi, Tamandani, Arjmand, & Alizadeh, 2017). In the first step of the study, human adipose-derived mesenchymal stem cells isolated and cultured. Second, gelatin cryogel scaffold was synthesized using glutaraldehyde as a crosslinker in cryogelation process. Last, cell attachment and proliferation and differentiation parameters were investigated onto gelatin cryogel scaffolds. Gelatin cryogel scaffolds promoted hepatogenic differentiation of human adipose-derived mesenchymal stem cells due to the 3D interconnected macroporous network of the cryogel architecture. Fig. 27.14 shows the differentiation of stem cell to hepatocytes on gelatin cryogel scaffolds. Hepatocyte differentiation was also followed by hepatic gene expression using real-time polymerase chain reaction on 7, 14, and 21 days. According to the data, hepatic differentiation of hADSCs was improved in cryogel scaffold that was biocompatible and merged the differentiation in microenvironment aspect. Overall, cryogels are smart materials in the tissue engineering research area with the capacity of fabrication of scaffolds for various cell types in combination with desired mechanical strength for different tissue types and interconnected porous which is enabled cell attachment and proliferation.

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FIGURE 27.13 Artificial brain tissue generated from CBSCs on the cryogel scaffolds. CBSCs images indicating neural cell differentiation in cryogel scaffold after 4 weeks. (A) Confocal scanning shows the distribution of glial cells in red and Nestin in green color in the 3D scaffold. (B) Confocal image of distribution of GFAP, glial fibrillary acidic protein-positive glial cells, (C) MAP2 cells, (D) S100beta positive glial cells. CBSCs, cord blood-derived stem cells. Reproduced from Jurga, M., Dainiak, M. B., Sarnowska, A., Jablonska, A., Tripathi, A., Plieva, F. M., . . .McGuckin, C. P. (2011). The performance of laminin-containing cryogel scaffolds in neural tissue regeneration. Biomaterials, 32(13), 3423
3434, with permission.

27.7.4 Cryogels in drug release applications Cryogels have a great interest in drug delivery systems because they have spongy scaffolds to promote the delivery of the biomolecules without mass loss. The goal of the cryogel studies in drug delivery systems to develop cytocompatible porous

27.7 The biomedical applications of the cryogels

FIGURE 27.14 Differentiation steps of hADSCs to hepatocytes. (A) SEM image of gelatin cryogel scaffold (a) adhesion and growth of hADSCs on the gelatin cryogel scaffold (b). (B) Proliferation of hADSCs using MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide on tissue culture polystyrene and gelatin cryogel scaffold during 12 days. During the culture time, cell numbers increased in both groups. (C) Morphological images of hADSCs after hepatic differentiation (a), 3D gelatin cryogel scaffold (b), and undifferentiated hADSCs (c). SEM, Scanning electron microscopy. Produced from Gandomani, M. G., Lotfi, A. S., Tamandani, D. K., Arjmand, S., & Alizadeh, S. (2017). The enhancement of differentiating adipose derived mesenchymal stem cells toward hepatocyte like cells using gelatin cryogel scaffold. Biochemical and Biophysical Research Communications, 491(4):1000
1006, with permission.

polymers obtaining the controlled release of the relevant drugs. Besides this, designing effective and low-cost drug delivery systems have importance in the biomedical research area. Researchers are conducting studies to investigate the optimum combination of porous, biocompatible cryogels that can provide a controlled release for the drug of interest to find the optimum parameters. Bioactive peptides, proteins, and hydrophilic drugs easily can be loaded onto cryogel macropores because of the cryogels’ high swelling capacity, controllable drug diffusion rate, and physical properties. Adjustable interconnection by incorporating the second polymer or monomer to form interlocking cryogel networks that can alter the degree of cross-linking of cryogel pores is the advantageous part of designing drug delivery cryogel systems. Proteins and drugs can be encapsulated as cryogel formation or entrapped in densely onto cryogel macropores to be released for degradation from the cryogel polymeric network. For releasing of small drug molecules, the releasing parameters controlled over the optimization of cryogel formation, such as swelling features, pore sizes and distributions, stiffness, or the softness, depending on the precursor and cross-linker concentration. Additional drug releasing control

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mechanism over cryogels can be enhanced by introducing second polymer or monomers considering their functional benefit to the system. Among the natural polymers that are used in cryogel, gelatin has an importance due to easy modification of gelatin in drug release. The modification and processing of gelatin change its electrostatic properties. This feature leads the releasing of different charged molecules via gelatin cryogel modification so there is increase in the number of the investigation of drug release studies of various drugs. The cross-linking strategies and types, even the amount of cross-linkers during the cryogelation phase, are important parameters in the use of cryogels in controlled drug release systems, as they will affect the porosity and therefore swelling degree of the cryogels. Drug release profiles of the cryogels can be enhanced using molecularly imprinted cryogels. In this strategy, specific drug binding sites are imprinted onto cryogel macropores and make the cryogel structures more specific to controlled drug releasing. Metal chelators, specific protein interactions, and ionic interactions are the main effects in drug binding onto cryogels in this method. Here, some studies related with drug release profiles of cryogels have been described. For diclofenac sodium and indomethacin drugs, 3D biocomposite cryogels based on chitosan and clinoptilolite were synthesized via cryogelation technique and the potentials as drug carrier has been investigated (Dinu, Cocarta, & Dragan, 2016). The cumulative release of diclofenac sodium from the prepared cryogel monoliths was found as lower than 5% at pH 1.2 and higher than 70% at pH 7.4. Indomethacin cumulative release was found 6% within the first hour in phosphate buffer saline (PBS) from composite cryogels. Molecularly imprinted cryogels were also used as drug release systems (Bakhshpour, Yavuz, & Denizli, 2018). Mitomycin C (MMC) imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)- histidine methyl ester)
Cu(II) (PHEMAH-Cu(II)) cryogel membranes were prepared for drug delivery of MMC using the cryogelation technique under partially frozen conditions. In the synthesis of cryogel the precursor and cross-linker (HEMA/MBAAm) ratio changed at the constant amount of MMC as template molecule to determine the effect of cross-linker and pore size depending on the amount of cross-linker in drug release. Increasing the amount of crosslinker caused the more rigid and cross-linked structure of cryogel. Different freezing temperatures such as 214 C, 218 C, 220 C, and 222 C in cryogelation were experienced and drug release performance of each cryogels was investigated. The pore size of the cryogel membranes increased with increasing polymerization temperature and the drug release rate of MMC increased with the increasing amount of drug. Vitamin B12 releasing property was investigated from the synthesized cryogel combination of precursors dextran methacrylate (DEX-MA) and polyethylene glycol dimethacrylate (PEG-DMA) via cryogelation technique. Swelling and mechanical property and drug release profile of Vitamin B12 were investigated (Pacelli et al., 2021). The natural polysaccharide dextran (DEX) and the synthetic

27.7 The biomedical applications of the cryogels

polymer polyethylene glycol (PEG) was combined and used as precursors in order to form a novel drug delivery system for Vitamin B12. 5.0 mg Vitamin B12 was added to the cryogel precursor solution of DEX-MA/PEG-DMA and APS/ TEMED initiator pairs were added to form Vitamin B12-loaded cryogel. The prepared cryogel was placed into PBS solution at 37 C, the releasing performance was monitored using high-performance liquid chromatography. Fig. 27.15 shows the Vitamin B12 releasing performance from cryogel and hydrogel formulations. According to the graphs, cryogels exhibited better-releasing media for Vitamin B12 with a slower rate diffusion compared to the related hydrogels. Besides, the synthesized cryogels were cytocompatible and this binary polymeric system with the combination of PEG-DMA and DEX-MA was the effective drug delivery platform. They could be used as the drug-releasing scaffolds for localized therapeutic applications.

FIGURE 27.15 Vitamin B12 releasing profiles from cryogel and hydrogel in PBS (pH 7.4) at 37.0 C 6 1.0 C. In the graphics indicated the comparison between (A) hydrogels of DEXMA/PEG-DMA 2.5% and DEX/PEG-DMA 5%, (B) cryogels of DEX-MA/PEG-DMA 2.5% and DEX/PEG-DMA 5%, (C) hydrogels and cryogels made of DEX-MA/PEG-DMA 2.5%, and (D) hydrogels and cryogels made of DEX-MA/PEG-DMA 5% were observed. PBS, Phosphate buffer saline. Reproduced from Pacelli, S., Muzio, L. D., Paolicelli, P., Fortunati, V., Petralito, S., Trilli, J. . . . Casadei, M. A. (2021). Dextran-polyethylene glycol cryogels as spongy scaffolds for drug delivery. International Journal of Biological Macromolecules, 166:1292
1300, with permission.

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Cryogels can be used as the drug carrier and tissue scaffold at the same time. In this field of use, the viability and proliferation of the cells in the scaffold can be checked while monitoring the controlled drug release. Thu, the effect of drug release on cells or the parameters affecting them are also examined simultaneously. For instance, kefiran cryogels have been synthesized for investigating the usage in tissue engineering studies and controlled drug release media by attachment of human adipose-derived stem cells and loading diclofenac sodium, respectively (Radhouani et al., 2019). In the first step, the water-soluble glucogalactan kefiran polymer was extracted from kefir grains, then kefiran polymerbased cryogel was synthesized via cryopolymerization method. Kefiran polymer is a natural polymer with high biocompatibility feature that can be used in tissue engineering studies. For diclofenac sodium-loaded cryogel synthesis, kefiran polymer was mixed with the diclofenac sodium solution and followed by freezing-drying process in the presence of cross-linker and initiators. Human adipose-derived stem cells were attached to the obtained kefiran cryogels and controlled drug releasing profile and cell proliferation have been investigated. The results of the study indicated that the developed cryogel system with drugloaded in combination with scaffolding was an important tool in both tissue engineering and controlled drug release investigations area in the biomedical aspect.

27.8 Conclusion As detailed in this chapter, it is obviously seen that cryogels have a great potential for various area in biomedical applications. One of the most important advantageous part of using cryogels is that cryogel-based materials have unique and adjustable features. Selection of polymers as precursors, solute concentrations, cooling rate and temperatures, and the amount of cross-linker are the controllable parameters in cryogel synthesis and provide them to be synthesized according to the research with highly adjustable physicochemical properties and large interconnected pores. On the basis of these features, cryogels can be synthesized in different shapes such as monolithic columns, membranes, and used in bioseparation of whole blood cells, proteins, controlled drug releasing media, scaffolds in tissue engineering, etc. in biomedical applications. The fact that cryogels are cost effective, manufacturability, reproducibility, scalability, and stable materials makes them standout materials.

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290. Available from https://doi.org/10.1080/00914037.2020.1825083. ¨ . B., Erso¨z, A., Denizli, A., Demirel, R., & Say, R. (2013). Separation and purifi¨ nlu¨er, O U cation of hyaluronic acid by embedded glucuronic acid imprinted polymers into cryogel. Journal of Chromatography B, 934, 46
52. ¨ ., O ¨ zcan, A., & Uzun, L. (2014). Preparation of a novel hydrophobic affinity ¨ nlu¨er, O U cryogel for adsorption of lipase and its utilization as a chromatographic adsorbent for fast protein liquid chromatography. Biotechnology Progress, 30(2), 376
382. Wang, C., & Sun, Y. (2013). Double sequential modifications of composite cryogel beds for enhanced ion-exchange capacity of protein. Journal of Chromatography. A, 1307, 73
79. ¨ ., U ¨ . B., Erso¨z, A., & Say, R. (2020). Anti-LDL antibody-nanoparticles ¨ nlu¨er, O Yavuz, S. O embedded cryogel for low density lipoprotein-depletion from hypercholesterolemic human serum. Separation Science and Technology, 55(10), 1786
1794. ¨ zer, E. T. (2018). Molecularly imprinted particle Ye¸silova, E., Osman, B., Kara, A., & O embedded composite cryogel for selective tetracycline adsorption. Seperation and Purification Technology, 200, 155
163. Zhang, P., Jıang, L.-Y., Jiu-Tong, M. A., & Qiong, J. I. A. (2021). Application of molecular imprinting technology in post-translational modified protein enrichment. Chinese Journal of Analytical Chemistry, 49(1), 24
33.

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Naturally derived ceramics polymer composite for biomedical applications

28

E. Shinyjoy1, S. Ramya1, P. Saravanakumar2, P. Manoravi3, L. Kavitha1 and D. Gopi2 1

Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India 2 Department of Chemistry, Periyar University, Salem, Tamil Nadu, India 3 Materials Chemistry and Metal Fuel Cycle Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamil Nadu, India

28.1 Introduction Biomaterials are those which are gaining much interest in the arena of biomedicine due to their physiochemical and biocompatible properties. They play a major part in reinstating, replacing, or repair of the damaged tissues or body parts to increase the life expectancy (Agarwal, Singh, Mohan, Mandal, & Bhatia, 2020; Song et al., 2018). Generally, biomaterials are classified as metals, polymers, ceramics, and composites based on their nature and applicability (Fig. 28.1). Metallic biomaterials are used when the implants are subjected to high mechanical load-bearing applications. The polymeric materials are utilized when the material need to be highly flexibile (Kumar et al., 2020; Love, 2017; Teo et al., 2016). Whereas, the ceramic materials are very significant because of their biocompatibility and anticorrosion property. These characteristics make them to be used for bone substitutes. Composite materials are generally used to improve the interaction between tissue and implant. Among the biomaterials, ceramic materials are most widely used for the reconstruction of damaged of skeletal and bone disorders (Bose, Banerjee, & Bandyopadhyay, 2017; Eliaz, 2019; Eliaz & Metoki, 2017). In particular, calcium phosphate based bioceramic material, notably hydroxyapatite (HAP), is suitable for orthopedic implants and dental materials. This is due to their high chemical similarity with human bone. They possess excellent bioactivity, high biocompatible properties, etc. (Al-Sanabani, Madfa, & Al-Sanabani, 2013; Shi et al., 2021). HAP (Ca10(PO4)6OH2), an important calcium phosphate group, has very similar chemical combination to bone mineral and also demonstrated as promising bone replacement material due to its exceptional bioactivity, high Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00012-7 © 2023 Elsevier Inc. All rights reserved.

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classification of biomaterials FIGURE 28.1 Classification of biomaterials.

FIGURE 28.2 Applications of hydroxyapatite.

osseointegration, good biocompatibility, and nontoxicity (Bal, Kaito, Korkusuz, & Yoshikawa, 2020). HAP, when placed in the body, can integrate with the surrounding tissues deprived of causing any adverse effects like poisonousness, swelling, or any foreign body response. For this purpose, HAP is used in variety of biomedical applications like bone and dental fillers, sutures, lenses, coating material for implants, etc. (Fig. 28.2). There are many synthetic methods for the

28.1 Introduction

preparation of HAP but there is a wide exploration for the synthesis of simplistic, inexpensive, and ecological method (Chen et al., 2015; Ronan & Kannan, 2017). Sometimes chemical synthesis of HAP leads to little complication, and, moreover, the HAP preparation by chemical sources is biologically unsafe. Compared to synthetic-derived HAP, the biowaste-derived HAP is nonstoichiometric due to the occurrence of trace elements such as Na21, Mg21, Zn21, K1, Si21, Ba21, F2, and CO322. This makes the naturally derived HAP analogous to the arrangement of human bones. But the use of HAP in implant application possesses some limitations like lack of antibacterial property that leads to implant failure. The bone implant related infections are very difficult to be treated and often end with the revision surgery for the removal of the implant. So, the substitution of minerals like Ag, Zn, and Ce into HAP is adopted for the successive application of implant. Among the mineral ions, Zn is believed to have superior antibacterial and nontoxic property (Gopi, Sathishkumar, Karthika, & Kavitha, 2014; Granito et al., 2018; Ramya, Shinyjoy, Kavitha, Kannan, & Gopi, 2016; Saravanakumar, Sutha, Kavitha Louis, Manoravi, & Gopi, 2020). It is well evident from the report that natural HAP is more bioactive than chemically synthesized HAP which is owing to the occurrence of mineral ions in HAP. The trace mineral ions are more essential for the bone regeneration and bone formation process. The added advantage is that the naturally derived HAP is considered ecofriendly due to their more sustainability, low cost, and large availability which in turn leads to positive contribution to economy, environment, and good health (Khiri et al., 2016; Mignardi, Archilletti, Medeghini, & Devito, 2020). Therefore researchers nowadays focus on the synthesis of HAP from biowaste materials (Fig. 28.3) like fish bones, bovine bones, crab shells, egg shells, sea corals, etc. (Abdulrahman et al., 2014). To overcome the limitations and to utilize HAP for load-bearing applications, HAP can be used in composite with polymeric materials. This composite is believed to have enhanced mechanical and bioactive properties compared with single components (Simionescu & Ivanov, 2015; Singhvi1, Zinjarde, & Gokhale, 2019). Polymeric materials like polylactic acid, poly(vinylpolypyrrolidone) (PVP), polyanionic cellulose, and biopolymers like polysaccharides, protein, chitosan, and gelatin are reported to be suitable polymers for the development of composite with HAP mainly due to their calcium binding property. Among the polymers, PVP has significant properties like low toxicity, biocompatibility, and its structural similarity to proteins, and finds its application in orthopedics and dentistry (Gopi, Kanimozhi, & Kavitha, 2015; Gritsch et al., 2019; Kim, Hwangbo, Koo, & Kim, 2020; Qi, Cheng, Ye, Zhu, & Aparicio, 2019; Salama, 2019). Since the use of natural resources and biowaste has rapidly expanded, the utility of biopolymers in the composite is also used (Fig. 28.4). Biopolymers are those which are nontoxic, biocompatible, bioactive material that can be recycled. Due to the enhanced bioactivity, eco-friendly nature, carboxymethyl cellulose (CMC) is one of the biopolymers that can be compatible with HAP for

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FIGURE 28.3 Natural sources of calcium.

biomedical applications. CMC has excellent mechanical and biological properties which enhance the application of composite in various biomedical applications (Andreeben & Steinbu¨chel, 2019; Baryła, Kulikowska, & Bernat, 2020; Gopi, Bhuvaneshwari, Indira, & Kavitha, 2013; Pagliano, Ventorino, Panico, & Pepe, 2017). Thus the use of naturally available biowaste material in the formation of composite is adopted in this chapter. Though the chapter has provided wide scope in terms of biomaterials, bioceramic, and biopolymers (Adeogun et al., 2018; Hammood, Hassan, & Alkhafagy, 2019; Moretto et al., 2020), we deal with the fabrication of zinc-substituted HAP (Zn-HAP)/cellulose nanocrystals (CNC) biocomposite, synthesis of crab shell derived HAP/PVP/aloe vera (AV) biocomposite, and electrophoretic deposition (EPD) of HAP/CMC/sodium alginate (SA) composite on Ti alloy (Gopi et al., 2013; Sridevi et al., 2019). Firstly, in the fabrication of Zn-HAP/CNC biocomposite, HAP is derived from country eggshell, whereas CNC were obtained from regular waste sources like bamboo fiber as per the established chemical routes. The Zn-HAP/CNC composite is believed to provide a combined mechanical and biocompatible property. The presence of Zn-HAP in the composite will show a main role in the enhancement of the antibacterial property, while the CNC improves the mechanical property of the composite. The synthesis of crab shell derived HAP/PVP/ AV biocomposite is achieved, for enhanced mechanical, antibacterial, and biocompatible properties. Fig. 28.5 shows the graphical representation of the synthesis of HAP/PVP/AV composite (Sridevi, Sutha, Kavitha, &

28.1 Introduction

FIGURE 28.4 Naturally derived biopolymers.

Gopi, 2020). The presence of polymer plays a major role in providing the mechanical property, good biocompatibility, and very low toxicity. To make the composite more advantageous with excellent antimicrobial property, AV is used in combination with the HAP and polymer. The presence of amino acids in AV will have a key part in the regeneration of damaged bone tissues. Also, the lectin present in AV helps in improving cell proliferation. Also, the chapter deals with the development of HAP/CMC/SA biocomposite over the Ti alloy by EPD method (Fig. 28.6). The HAP in the composite is biogenically derived from the thermal decomposition of Rohu fish bones. For the enhanced biocompatibility of HAP, CMC—a biopolymer which is derived from rice husk—is used in the composite with HAP. In addition to this, SA in the composite enhances the biocompatibility and biodegradability of composite. Thus the HAP/CMC/SA biocomposite coated Ti alloy will be an eminent candidate for various biomedical applications. Overall, the naturally derived ceramics polymer composite will be most eminent candidate for various biomedical applications (Mathina, Shinyjoy, Kavitha, & Gopi, 2020).

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FIGURE 28.5 Graphical representation of synthesis of hydroxyapatite/poly(vinylpolypyrrolidone)/aloe vera composite.

28.2 Preparation of biogenic-derived biocomposites 28.2.1 Materials Analytical grade reagents were used to synthesis of biogenic-derived biocomposite by using various biowaste materials. For this purpose, Zn(NO3)2 and (NH4)2HPO4 were used as the source of zinc and phosphate ions, respectively. In addition to this, NaOH, ClCH2COOH, PVP, H2SO4, ethanol, HNO3, H3PO4, NaNO2, and SA were purchased and used without any further purification. For deposition, Ti alloy was used as the metallic implants.

28.2.2 Various biocomposites from biowaste materials Many diverse biocomposites, which involve various types of biogenic materials, are used to prepare biogenic-derived substituted or/and HAP/nanofiber/biopolymer biocomposite. This book chapter presents various aspects about the biogenicderived biocomposites prepared for biomedical applications (Mathina et al., 2020; Sridevi et al., 2019, 2020).

28.2 Preparation of biogenic-derived biocomposites

FIGURE 28.6 Graphical representation of the fabrication of hydroxyapatite/carboxymethyl cellulose/ sodium alginate composite on Ti alloy.

28.2.3 Zinc-substituted hydroxyapatite/cellulose nanocrystals biocomposite A successful deposition of Zn-HAP/CNC biocomposite on Ti alloy was achieved by Sridevi et al. (2019) through EPD method. Zn-HAP and CNC were synthesized according to the standard procedure from Gopi et al. (Shi et al., 2021). Prior to EPD procedure, the fixed amount of Zn-HAP and CNC mixture is added into ethanol/water (40 mL) solution for the preparation of Zn-HAP/CNC biocomposite. The above biocomposite mixture was then stirred and ultrasonicated to obtain clear dispersion. After this procedure, EPD method was carried out on Ti alloy at room temperature under DC voltage. A platinum electrode and Ti alloy was used as anode and cathode, respectively. The Zn-HAP/CNC biocomposite deposition was carried out by a fixed constant voltage of 20 V for deposition time of 5 min. After EPD, the Zn-HAP/CNC biocomposite coating was rinsed with deionized water and subsequently dried in desiccators.

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28.2.4 Hydroxyapatite reinforced with polyvinylpyrrolidone/aloe vera biocomposite Mathina et al. (2020) prepared HAP from crab shell and its composites HAP/PVP (1, 2, and 3 wt.% of PVP) and CS-HAP/PVP/AV (HAP/PVP/AV) at three different concentrations of 0.5, 1, and 1.5 wt.% AV. They have synthesized HAP/PVP/ AV using 1, 2, and 3 wt.% of PVP in 4 g of HAP and stirred for 4 h at 40 C and then ultrasonically dispersed to ensure clear HAP/PVP composite solution. After this procedure, 0.5, 1.0, and 1.5 wt.% of AV powder was blended to the above HAP/PVP composite. The HAP/PVP/AV composite was regularly maintained at pH 10 and was ultrasonicated for 2 h to achieve clear dispersion. The HAP/PVP/ AV composite suspension was filtered and subsequently dried in microwave oven at 80 C for 12 h and complete into a fine HAP/PVP/AV composite powder. Fig. 28.7 shows the formation mechanism of HAP/PVP/AV composite.

28.2.5 Hydroxyapatite/carboxymethyl cellulose/sodium alginate biocomposite Sridevi et al. (2020) have synthesized the pure HAP from the bones of Labeo Rohita fish, extraction of CMC from rice husk, and HAP/CMC/SA biocomposite at different concentrations like 1, 2, and 3 wt.% of SA. For the synthesis of HAP/CMC/SA biocomposite, the fixed amount of HAP and CMC derived from biogenic samples were added in the ethanol/water (1:2 ratio) mixture and the solution was continuously agitated for 12 h to obtain a clear suspension. To the HAP/CMC composite, three various concentrations of 1, 2, and 3 wt.% SA were added at pH 7 by the addition of NaOH/HCL. Further, the HAP/ CMC/SA mixture was continuously stirred for 24 h to attain uniform suspensions. Fig. 28.8 shows the reaction mechanism of the HAP/CMC/SA biocomposite. During the EPD procedure for HAP/CMC/SA biocomposite on Ti alloy, the fixed amount of HAP/CMC/SA biocomposite electrolyte is added into ethanol/water mixture. The deposition of HAP/CMC/SA biocomposite on Ti alloy was carried out for 5 min at the fixed voltage of 30 V under the DC power supply system. After deposition of HAP/CMC/SA biocomposite, the coated sample was rinsed with deionized water, dried, and after stored in desiccator. Fig. 28.8 shows the reaction mechanism for the fabrication of HAP/CMC/SA composite on Ti alloy.

28.2.6 Characterization The nature of the Zn-HAP/CNC, HAP/PVP/AV, and HAP/CMC/SA biocomposites were investigated using Fourier transform infrared spectrometer (FTIR, Nicolet 380) over the range from 4000 to 400 cm21. The phase composition and the crystallinity of the Zn-HAP/CNC, HAP/PVP/AV, and HAP/CMC/SA biocomposites were evaluated by the X-ray diffraction (XRD) spectrometer. The surface morphology and elemental composition of the Zn-HAP/CNC, HAP/PVP/AV, and

28.2 Preparation of biogenic-derived biocomposites

FIGURE 28.7 Schematic representation of the formation of hydroxyapatite/poly(vinylpolypyrrolidone)/aloe vera composite.

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FIGURE 28.8 Reaction mechanism for the fabrication of hydroxyapatite/carboxymethyl cellulose/sodium alginate composite on Ti alloy.

28.3 Results and discussion

HAP/CMC/SA biocomposite samples were observed by high-resolution scanning electron microscope (HRSEM) equipped with energy dispersive X-ray (EDX) spectrometry technique. •

Bioactivity assessment

The bioactivity assessment of the Zn-HAP/CNC, HAP/PVP/AV, and HAP/ CMC/SA biocomposites was tested by simulated body fluid (SBF) immersion method. The SBF was prepared through Kukubo’s standard protocol method at pH 7.4. The Zn-HAP/CNC, HAP/PVP/AV, and HAP/CMC/SA biocomposite samples were soaked in SBF medium at various days at room temperature. The soaked samples were further analyzed using HRSEM to confirm the formation of apatite layer on the surface (Mathina et al., 2020; Sridevi et al., 2019, 2020). •

Mechanical studies

The microhardness and adhesion tests of as-prepared Zn-HAP/CNC, HAP/ PVP/AV, and HAP/CMC/SA biocomposite samples have been investigated by using standard scratch tester according to ASTM F1044-05 standard and Vickers diamond indenter (HMV2T, Shimadzu), respectively (Mathina et al., 2020; Sridevi et al., 2019, 2020). •

Antibacterial activity

The antibacterial activities of all the Zn-HAP/CNC, HAP/PVP/AV, and HAP/ CMC/SA biocomposite samples have been investigated against two bacterial strains such as Staphylococcus aureus and Escherichia coli by disk diffusion method (Mathina et al., 2020; Sridevi et al., 2019, 2020). •

In vitro cell viability analysis

The in vitro cell viability test was used to examine the proliferation of MG63 osteoblast-like cells from human osteosarcoma cells (MG63) on the Zn-HAP/ CNC, HAP/PVP/AV, and HAP/CMC/SA biocomposites. After the incubation period, the tested plates were observed at 570 nm and the percentage cell viability related to the control well was calculated using the formula (Mathina et al., 2020; Sridevi et al., 2019, 2020).

28.3 Results and discussion Several biowaste materials have been used to formulate HAP nanoparticles through various fabrication techniques such as sonochemical, thermal decomposition and mechanochemcial synthesis, etc. The conversion of biowaste material into its valuable products requires high temperature procedure. However, the conventional chemical preparation techniques have certain serious environmental issues, due to the usage of large quantity of chemicals and by-products. The development of

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nanophase HAP particles and its biopolymer composite from biowaste materials was attempted through simple procedures. When compared with the conventional chemical synthesis, the biowaste-derived biocomposites exhibit potential properties because of their enhanced reactivity, biocompatibility, rapid cell growth, bioactivity, and easy interaction with the surrounding tissues. In this chapter, various biowaste-derived HAP/biopolymer composites were discussed for their physicochemical, morphological, and biological properties.

28.3.1 Egg shell derived hydroxyapatite/cellulose nanocrystals biocomposite •

FTIR analysis

The FTIR spectra shown in Fig. 28.9 indicate the specific peaks of HAP, ZnHAP, and Zn-HAP/CNC biocomposite derived from biowaste materials. The IR spectrum shows the specific characteristic peaks of the phosphate group vibrations for ν1 ν4. The spectrum reveals the presence of hydroxyl group in HAP. The obtained peak reveals the formation of biocomposites of Zn-HAP and CNC polymer composite.

FIGURE 28.9 Infrared spectroscopic analysis of the biocomposites.

28.3 Results and discussion

In addition to this, the polymer composite formation with the Zn-HAP also observed through the detailed characteristic peaks for the stretching vibrations C O, C O C, CH2, and C H in CNC. The CNC-incorporated biocomposite shows the resultant peaks for all the composites reveals the formation of composites. In addition, a peak shift was also observed for the Zn and CNC incorporation in the HAP structure. It is also observed that the received peaks support the formation of biocomposites by mixing Zn-HAP and CNC composite without any other interfacial bonding. •

XRD analysis

The phase compositional analysis of HAP, Zn-HAP, and Zn-HAP/CNC biocomposite is depicted in Fig. 28.10. The XRD pattern illustrates the characteristic peaks of specific hexagonal phase structure for HAP. The XRD peaks for Zn-HAP in the diffraction range of 15 30 indicate the phase pure formation of HAP and then for the CNC at the range of 30 45 . The XRD pattern for CNC observed at the 2θ values of 16.5 and 21.2 shows the presence of CNC phase without other secondary phase formation. Further, the biocomposite formation of Zn-HAP with CNC does not exist any interfacial bonding.

FIGURE 28.10 X-ray diffraction pattern of the biocomposites.

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•

SEM and EDX investigations

The morphological evaluation of the biocomposite-coated Ti alloys obtained through EPD and the elemental analysis is exhibited in Fig. 28.11. The morphological investigation reveals a nonuniformly distributed spherical particle on the Ti alloy surface with high aggregation was observed. Finally, the formation of Zn-HAP biocomposite with CNC shows a considerable morphological change in the coating surface. Zn-HAP/CNC composite exhibits a small granule structure on the surface on the dense surface coverage was observed. This is because of the formation of composite with the ceramics and polymer matrix on the surface of the metallic implant. Further, the compositional analysis of the Zn-HAP/CNC composite coated Ti alloy implants was performed using energy-dispersive X-ray spectroscopy (EDAX) spectrum and showed the presence of Ca, Mg, Zn, C, O, and P without any additional components.

FIGURE 28.11 FESEM micrographs of (A) hydroxyapatite (HAP), (B) Zn-HAP, and (C) Zn-HAP/cellulose nanocrystals (CNC) composite coated Ti alloy. (d) Energy-dispersive X-ray spectroscopic spectrum of the Zn-HAP/CNC.

28.3 Results and discussion

•

Mechanical characterization

The microhardness to investigate the adhesion strength of the Ti alloys coated with HAP, Zn-HAP, and Zn-HAP/CNC biocomposite was estimated through hardness indenter. The adhesive strength analysis showed that the Zn-HAP/CNC (12.5 MPa) biocomposite coated Ti alloy sample exhibits a better adhesive strength than the Zn-HAP (9.8 MPa) and HAP (8.7 MPa) coating. It was observed that the incorporation of polymer in the bioceramics structure induces higher adhesion strength between the implant surface and the bioceramic coating. •

Antibacterial activity

The antibacterial activity of the biocomposites were investigated against the bacterial strains like E. coli and S. aureus for Zn-HAP/CNC biocomposite. The bacterial resistance behavior of the composite were analyzed by the clear zone formation around the disk are evaluated. The formation of higher inhibition zone E. coli (8 mm) and S. aureus (7 mm) for Zn-HAP/CNC biocomposite indicates the enhanced antibacterial activity. The improved antibacterial activity of the biocomposite is due to the existence of Zn ion in the HAP structure. Interestingly, the Zn-HAP/CNC biocomposite shows potentially improved antibacterial activity against E. coli than the S. aureus. For this reason, Zn-HAP/CNC composite with outstanding antibacterial activity is a potential material for biomedical applications.

28.3.2 Crab shell extracted hydroxyapatite/poly (vinylpolypyrrolidone)/aloe vera biocomposite •

FTIR analysis

The various concentrations of PVP and AV in HAP/PVP/AV composites are investigated by FTIR spectroscopy to determine the functional nature of the composite, and the obtained spectra are shown in Fig. 28.12. The spectrum for biowaste-derived HAP shown in Fig. 28.12 exhibits the stretching and bending vibrations peaks that correspond to the P-O groups. The FTIR spectra of different concentrations of PVP containing HAP are shown in Fig. 28.12A. The corresponding PVP peaks along with the HAP peaks confirms the composite formation. The C 5 O peaks present in the polymer matrix influence a mild shift in the peak position to the lower frequency, which confirms the hydrogen bond formation between PVP and HAP composite. The FTIR spectra obtained for HAP/PVP containing different concentration of AV are depicted in Fig. 28.12B. In addition to the peaks that correspond to HAP/ PVP, an additional peak supporting the interaction of HAP/PVP with the AV was observed. This additional peak confirms the composite formation of HAP/PVP/ AV. Therefore the FTIR spectrum for HAP, PVP, and AV exhibits the characteristic peaks indicating the formation of HAP/PVP/AV biocomposite.

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FIGURE 28.12 Fourier transform infrared spectra of (A) hydroxyapatite (HAP) and HAP/poly (vinylpolypyrrolidone) (PVP), and (B) HAP/PVP/aloe vera at various concentrations.

•

XRD analysis

The acquired XRD patterns for the HAP/PVP/AV biocomposite containing various concentrations of PVP and AV are depicted in Fig. 28.13A and B. The corresponding peaks obtained for HAP reveals the formation of hexagonal phase structure without any additional phase detection. Additionally, the pattern also shows the peaks correspond to the PVP and AV for the HAP/PVP/AV biocomposite, indicating a strong interface between the ceramics and polymer matrix. Furthermore, it is also observed that a peak shift of the reflection plane such as 002 and 211 of the HAP for the PVP and AV is due to the bonding between the components. Hence, the HAP/PVP/AV biocomposite shows the characteristic peaks of HAP without any additional phase change with respect to the incorporation of PVP and AV. •

SEM analysis

The microstructural evaluation of the biowaste-derived HAP and its biocomposite formation with PVP and AV analyzed through SEM morphological analysis is shown in Fig. 28.14. The morphological characterization reveals welldispersed highly oriented spherical-shaped HAP particles. The composite formation of HAP with the PVP polymer matrix also exhibits the discrete particles along with the formation of agglomerated surface. The increase in

28.3 Results and discussion

FIGURE 28.13 X-ray diffraction pattern of hydroxyapatite (HAP) and different concentrations of (A) HAP/ poly(vinylpolypyrrolidone) (PVP) and (B) HAP/PVP/aloe vera.

the concentrations of PVP in the HAP/PVP biocomposite up to 2 wt.% shows many interconnected pores. The pores surface structure formation will indicates a potential improvement in biological properties. The increase in PVP concentration in the HAP/ PVP composite leads to the formation of large aggregates with irregular shape. Finally, the addition of AV in the HAP/PVP composite revealed a distinct change in the morphology. The lower concentration of the AV in HAP/PVP depicts the formation of uneven clumsy particles. Due to the high moisture content in AV, the higher concentration in the HAP/PVP matrix induces a gel-like structure formation. The elemental analysis of the HAP/PVP/AV composite measured through EDAX analysis is shown in Fig. 28.14. The EDAX analysis confirms the elements in the composite and the as-synthesized composite is pure. •

Mechanical characterizations

The most important consideration in the development of ceramic polymer biocomposite is to enhance the mechanical integrity of the material for various biomedical applications which may be associated with required mechanical properties in high load-bearing biomedical implant applications. Mechanical property of HAP, HAP/PVP, and HAP/PVP/AV composite is assessed and the values 1 wt.% PVP in HAP is found to be higher (i.e.,) 159 Hv The results evidenced an increase in the PVP concentration up to 2 wt.% in the HAP/PVP (163 Hv) biocomposite, which demonstrates an increase in the hardness value, and further

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FIGURE 28.14 Microstructural evaluation of (A) hydroxyapatite (HAP), (B D) different concentrations of poly(vinylpolypyrrolidone) (PVP) in HAP/PVP biocomposite, (E G) different concentrations of aloe vera (AV) in HAP/PVP/AV biocomposite, and (H) energy dispersive X-ray spectrum of HAP/PVP/AV.

28.3 Results and discussion

increase in the PVP concentration reduces the hardness values of the composite which may be due to the large aggregates of the composites. Moreover, the addition of AV in the HAP/PVP biocomposite shows a similar hardness value for all the compositions. •

Contact angle measurements

The results of contact angle measurement carried out to access the surface wettability of the biocomposites are shown in Fig. 28.15. The obtained water droplet image for the analysis reveals that the synthesized samples are hydrophilic in nature. From this investigation, it has been found that the pure HAP derived from biogenic waste materials exhibit higher contact angle values which are to be decreased for the biocomposites with PVP and AV inclusion. As a result of this, it is concluded that the inclusion of AV in the biocomposites tends to increase the hydrophilic nature of the composite. •

Antibacterial activity

Antibacterial activity of the HAP/PVP/AV biocomposites was investigated through the different bacterial strains and the biocomposites should have bacterial inhibitory activity without changing its biocompatibility. For this reason, the

FIGURE 28.15 Contact angle of (A) hydroxyapatite (HAP) and (B and C) HAP/poly(vinylpolypyrrolidone) (2 and 1 wt.%) spreading of water droplet images.

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antibacterial activity of the biocomposites was investigated for various concentrations against the bacterial strains. The antibacterial activity of the biocomposite was measured through the clear zone formation around the disk. For all the materials, the HAP/PVP/AV biocomposite demonstrates an enhanced potential antibacterial activity which is because of the presence of AV in the HAP/PVP biocomposite. Because the compounds like anthraquinone, acemannan, and salicyclic acid present in the AV extract demonstrate a direct bacterial inhibition activity. Likewise, the antibacterial activity of S. aureus (23 mm) is higher than that of E. coli (22.4 mm) which is because of the difference in cell wall between the bacterial strains. From these results, it is concluded that the HAP/PVP/AV biocomposite depicts an anticipating antibacterial composite for biomedical implant applications. •

In vitro cytocompatibility analysis

In general, a ceramic used for the biomedical applications should possess the biocompatibility and have to accelerate the cell proliferation. To appraise the biocompatibility, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for the HAP, HAP/PVP, and HAP/PVP/AV biocomposites via MG63 cells. The biocomposites cultured with cells were observed by fluorescence microscopy to investigate the cell proliferation after 1, 3, and 5 days of incubation (Fig. 28.16). The result reveals a high absorbance value for the HAP/PVP/AV incubated for 3 days than the other samples. The increase in cell proliferation of the biocomposite is due to the presence of HAP and AV in the biocomposite structure which leads to the better cell viability and enhanced cell proliferation. Basically, MG 63 cell lines can be easily attached and proliferate with the HAP-based composites. The maximum percentge of cell viabiity was observed for 1 wt.% of HAP/PVP/ AV composite as shown in Fig. 28.16. Even though HAP is a naturally available bone mineral, it helps in the increase of the biocompatibility of the composites. Along with the HAP, the AV addition in the HAP structure also plays a significant role in the improvement in the biocompatibility of the composite. Hence, the as-prepared HAP/PVP/AV is found to be a better composite in biomedical applications.

28.3.3 Fish bone derived hydroxyapatite/biopolymer composite •

FTIR spectroscopic analysis

The functional, structural, and compositional nature of the biowaste-derived HAP and HAP/polymer biocomposites were investigated through FTIR spectroscopy, and the obtained spectrum is shown in Fig. 28.17. The obtained FTIR spectrum reveals the specific functional peaks of the HAP and its polymer composites. FTIR analysis for HAP sample shows low intense peaks for the OH group which reveals the lesser hydration level of biowaste-derived HAP. While

28.3 Results and discussion

FIGURE 28.16 (A) Percentage cell viability of the hydroxyapatite (HAP)/poly(vinylpolypyrrolidone) (PVP)/ aloe vera (AV) against HOS MG63 cell line. (B) Fluorescence microscopic images of HAP/ PVP/AV composite for different days of incubation.

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FIGURE 28.17 Fourier transform infrared spectra of (A) HAP, (B) HAP/CMC, (C) HAP/CMC/SA (1%), (D) HAP/CMC/SA (2%) and (E) (C) HAP/CMC/SA (3%) biocomposites .

incorporation of polymer in HAP structure, the composite formation shows high intense absorption peaks for the OH region. Moreover, the IR spectrum of all the concentrations reveals specific stretching and bending vibrational peaks at the desired frequencies indicates the phosphate ions. Subsequently, the incorporation of CMC/SA polymer for the formation of composite with HAP accelerates a peak shift in the direction of lesser wave number which confirms the interconnected element bond formation among the organic and calcium in the inorganic phase structure. Along with the peak shift, the introduction of CMC and SA biopolymers in the HAP structure shows a reduction in intensities with peak broadening for the biocomposites which also indicates the interaction of polymeric compound with the HAP.

28.3 Results and discussion

Furthermore, the spectra of HAP/polymer biocomposites confirm the formation of clear peaks at 1600 cm21 for the absorption of COO group. Likewise, the increasing SA concentration influences the phosphate peaks broadening which reveals that the SA composition plays a potential result on growth rate of the HAP. This result confirms that the coating of HAP/CMC/SA composite on the Ti alloy can be an effective composition for improved biomedical applications. •

X-ray diffraction investigation

The phase characteristic peaks of the biocomposites investigated by XRD are indicated in Fig. 28.18. XRD pattern of HAP particles indicates the formation of the hexagonal phase structure with high crystalline orientation. The HAP/CMC composite with different SA concentrations exhibited similar hexagonal phase structure. The phases for the obtained HAP composites was matched well with the standard ICCD card (file No: 09-0423).

FIGURE 28.18 X-ray diffraction pattern of (A) HAP, (B) HAP/CMC, (C) HAP/CMC/SA (1%), (D) HAP/CMC/ SA (2%) and (E) (C) HAP/CMC/SA (3%) biocomposites.

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The observed stronger high intense peak in the diffraction pattern corresponds to the (211) plane at 31.77 because of the crystallographic structure and orientation of the biocomposites. The XRD pattern also affirms the formation of phase pure biocomposites. In addition, the composite formation of HAP with the polymeric materials leads to decrease in peak intensity with respect to the increasing the concentration of the SA in the HAP/CMC composites with reduction in crystallinity. More interestingly, the HAP/polymer composite does not exhibit any specific peaks and affirms the formation of crystalline HAP. •

Microstructural evaluation

The morphological and microstructural characterization of the Ti alloy coating with HAP and its biocomposites was carried out using Field Emission Scanning Electron Microscopy (FESEM). The FESEM micrograph of biocomposite coating obtained using EPD is depicted in Fig. 28.19. The HAP derived from biowaste materials formed coating on the Ti alloy implant is revealed in Fig. 28.19A. The HAP coating on the implant surface shows the presence of spherical morphology. The HAP containing CMC composite coating demonstrates a dense deposition. The SA introduction and increase in the concentration in the HAP/CMC composite increase the porosity percentage which is due to the influence of the coating parameters and the interaction between the organic and inorganic components. The coating also shows a homogeneously covered surface morphology without any coating defects and nonporous structure. Further, the lower SA concentration in HAP/CMC exhibits the deposition of a solid agglomerated surface with minimal porosity, while increasing the SA concentration the porosity percentage also increased and shows a meso and macroporous fiberlike coating with extensive porosity for the SA concentrations of 2% and 3%. The microstructural change in the structural property is because of the influence of the anodic deposition of the polymeric composite containing more number of carboxyl groups. Furthermore, the polymeric addition in HAP biocomposite improves the formation of porous surface structure which will be highly beneficial for the cellular interaction between the implant surface and the biological tissues. •

In vitro bioactivity assessment

The bioresorbability of the HAP/polymer composite coated Ti alloy implant was investigated by immersing the coated specimens in the SBF medium for 14 days under humidified environment. The bioresorbability of the biowaste-derived HAP and HAP/CMC/SA polymer biocomposite was obtained by the in vitro apatite layer deposition on the implant surface. The apatite layer deposition on the surface of the implant was investigated through the FESEM analysis revealed in Fig. 28.20 which indicates that the immersion of the implant in biological fluid stimulates the apatite deposition on the implant-coated surface. The agglomerated white layer growth on the surface of the implant reveals the bioactivity of the coated implant. The micrograph confirms that the implant coated with all concentrations of the biocomposite exhibits interaction with the SBF medium and induces the apatite layer deposition.

28.3 Results and discussion

FIGURE 28.19 Field Emission Scanning Electron Microscopic images of (A) HAP, (B) HAP/CMC, (C) HAP/CMC/SA (1%), (D) HAP/CMC/SA (2%), (E) (C) HAP/CMC/SA (3%) biocomposites and (F) EDAX image of HAP/CMC/SA (2%).

The bioresorbability analysis shows a formation of specific surface morphological apatite layer deposition which indicates the spherical-shaped apatite layer deposition and this indicates the biomineralization behavior of the implant surface in the SBF medium. The introduction of CMC/SA in the HAP bioceramics enhances the apatite growth. At the maximum of 2% SA in the HAP/CMC biocomposite, exhibits a fully covered apatite deposition on the implant surface and the further increase reduces the rate of apatite deposition which is due to the higher dissolution behavior of the organic polymer matrix.

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FIGURE 28.20 FESEM images of SBF growth after 14 days (A) Biogenic derived HAP, (B) HAP/CMC, and (C-E) HAP/CMC/SA biocomposite at different (1, 2 & 3%) concentrations of SA coating on Ti alloy.

•

Microhardness analysis

The mechanical integration of the biogenically derived biocomposite coated Ti alloy is investigated. The microhardness analysis indicates a lower hardness value for the HAP (163 Hv)-coated implant and depicted an increase in the hardness value for the Ti alloy coated with HAP/CMC (169 Hv), HAP/CMC/1% SA

28.3 Results and discussion

(182 Hv), and HAP/CMC/2% SA (191 Hv) biocomposite. Further, the increase in polymer concentration in the HAP/CMC/SA biocomposite exhibits a reduction in hardness value for the 3% SA which may be because of the lack of strong interfacial bonding. •

Antibacterial analysis

For the biological applications, the antibacterial vulnerability of the biowastederived HAP is essential. Therefore the surface modifications are required to enhance its surface and compositional properties for enhanced biological properties. The bacterial activity of the HAP and HAP/CMC/SA polymer composites is depicted in Fig. 28.21.

FIGURE 28.21 Antibacterial activity of HAP/CMC, HAP/CMC/SA (1%), HAP/CMC/SA (2%), HAP/CMC/SA (3%) biocomposite against (A) S. Aureus and (B) E. Coli.

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The antibacterial activity analysis indicates an improved bacterial resistance against S. aureus, a Gram-positive strain and a lower bacterial resistance was observed against E. coli, a Gram-negative bacterial strain. The SA with the concentration of 2% shows a maximum of bacterial zone inhibition for both pathogens. Furthermore, the higher inhibition zone formation for the HAP/CMC/SA biocomposites against S. aureus is because of the surface negative charge. The electrostatic interaction of the composite because of the negative charge of the SA for the Gram-positive bacterial strain leads to the enhanced bacterial resistance behavior against S. aureus. Therefore the as-proposed HAP/CMC/SA composite would be a better alternative for the biomedical applications with enhanced potential properties.

FIGURE 28.22 Optical microscopic images of the HAP/CMC/SA biocomposite containig 2% SA at different concentrations (A) Control, (B) 12.5, (C) 25, (D) 50, (E) 100 and (F) 200 μg.

Acknowledgments

•

In vitro cytotoxicity assay

The cytocompatibility of the HAP/CMC/SA composite containing 2 wt.% of SA was investigated through in vitro cytotoxicity assay on MG63 cell line is shown in Fig. 28.22. The obtained analysis result indicates that the cell viability analysis leads to the elongation of the cell culture with the biocomposite. Due to the high cytocompatibility, there is no considerable phonotypical change with the sample concentration. Furthermore, the sample containing higher concentration of HAP/CMC/SA exhibits an enhanced cell viability which indicates that the obtained composition is highly biocompatible to the cell culture and improves the osteoblast induction with the surrounding tissues. Of all the composites, the HAP/polymer biocomposite derived from egg shell biowaste materials can be an effective alternative biomaterial for potential orthopedic implant applications.

28.4 Conclusion With the advancement in the application of biomaterials in the field of orthopedics and dentistry, different bone-like materials are fabricated which can be utilized as fillers and coating materials for metallic implants. For decades, researchers pay more attention toward the usage of composites (inorganic-polymer) in various applications than towards utilization of single components for biomedical applications. In general, these composites play a key role in combining the advantage of both the inorganic and polymer components in which inorganic components like HAP possess excellent bioactivity, osteoconductivity, biocompatibility, etc., whereas polymers exhibit good mechanical properties. This combination of properties from HAP and polymer in composites will makes the composite exhibit enhanced mechanical, biocompatible, and more bioactive properties. In this chapter, we summarized the fabrication of simple, eco-friendly, and valuable biocomposite for various biomedical applications. The naturally derived HAP and the polymer are more economic than the synthetically derived HAP/polymer composite. Also, the additional advantage is that the process of fabrication is not complicated. The results thus obtained concluded that the fabrication of HAP/polymer composite is nontoxic, mechanically stable, and have good biocompatible property. It is also noteworthy to highlight that in this chapter, we have utilized the biowaste materials to produce the potential biomaterial and composite. Thus it can be concluded that the as-fabricated Zn-HAP/CNC, HAP/PVP/AV, and HAP/CMC/SA biocomposite can act as a promising candidate for biomedical applications.

Acknowledgments The author D. Gopi acknowledges the major financial support from the Department of Science and Technology (DST-SERB, Ref. No.: EMR/2017/003803), University Grants

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Commission-Department of Atomic energy (UGC-DAE CSR, Ref. No. CSR-KN/CRS-118/ 2018 19/1056, Dated: 26.12.2018). E. Shinyjoy acknowledges the University Grants Commission for Dr. D.S. Kothari Fellowship, New Delhi, India (No. F.4-2/2006 (BSR)/ CH/18-19/0078; Dated: 06-02-2019); and S. Ramya (F.4-2/2006 (BSR)/CH/17-18/0170, Dated: 25.09.2018) acknowledges the University Grants Commission for Dr. D.S. Kothari Fellowship, New Delhi, India. Also, the authors acknoweldges S. Sutha for her contribution.

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Molecularly imprinted polymers (MIPs) for biomedical applications

29

Ru¨stem Kec¸ili1, O¨zlem Bic¸en U¨nlu¨er2, Arzu Erso¨z2,3 and Rıdvan Say3 1

Department of Medical Services and Techniques, Yunus Emre Vocational School of Health Services, Anadolu University, Eski¸sehir, Turkey 2 Chemistry Department, Faculty of Science, Eski¸sehir Technical University, Eski¸sehir, Turkey 3 Bionkit Co Ltd., Eski¸sehir, Turkey

29.1 Introduction The rapid advancements of biomedical sciences enabled the elongation of life expectancy and led to the urgent need of diagnosis and identification of the health problems in early stage. For the efficient management of these problems, the advanced drug delivery system-based therapies and the efficient screening of the medical process as well as bio-imaging became a vital issue (Mehta, Desai, Basu, Kumar Singhal, & Kumar Kailasa, 2021; Xiao et al., 2021). Smart materials such as molecularly imprinted polymers (MIPs) are highly cross-linked engineered powerful materials that feature 2D and 3D cavities that exhibit great affinity and selectivity towards a specific compound created by molding the multidimensional cavities around this target compound (template) during the polymerization process and then removing the template from the cross-linked polymeric network afterwards (Dolak, Kec¸ili, Hu¨r, Erso¨z, & Say, 2015; Kec¸ili & Hussain, 2018; Kec¸ili, Yılmaz, Erso¨z, & Say, 2020; Kupai, Razali, Bu¨yu¨ktiryaki, Kec¸ili, & Szekely, 2017; Sellergren, 2001). MIPs can be successfully employed as a highly selective and sensitive component in many sensor systems (Emir Diltemiz, Kec¸ili, Erso¨z, & Say, 2017; Kec¸ili & Denizli, 2021; Moein, 2021; Wang, Liang, & Qin, 2020) and also in many separation methods such as chromatography and solid-phase extractions (Haginaka, 2008; Madikizela, Tavengwa, & Chimuka, 2018; Say, Kec¸ili, & Erso¨z, 2016; Tamayo, Turiel, & Martı´n-Esteban, 2007) due to its specificity towards a target analyte. MIPs also offer a degree of customizability since it can be tailored to be selective towards different types of compounds depending on the nature of the imprinted compound. MIPs are also called as “plastic antibodies” or “synthetic receptors.” The mechanism is similar to that involved in antibodies or enzymes where the sensitive recognition is based on the 3D shape and the chemical interactions between the target compound and the functional Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00008-5 © 2023 Elsevier Inc. All rights reserved.

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monomer of the MIP, resembling the lock and key concept. Thus, MIPs were regarded as “antibody mimics” and displayed great advantages over natural antibodies for sensor technologies due to their highly cross-linked structure, high stability, sensitivity, and robustness. In addition, MIPs can take advantage of the wide range of polymers with different properties to design and fabricate the controlled drug delivery and bio-imaging systems (Abdollahi, Khalafi-Nezhad, Mohammadi, Abdouss, & Salami-Kalajahi, 2018; Inutsuka, Okamoto, & Yoshimi, 2019; Marcelo, Ferreira, Viveiros, & Casimiro, 2018; Mokhtari & Ghaedi, 2019; Vaneckova, Bezdekova, Han, Adam, & Vaculovicova, 2020). This chapter presents an overview of the design and preparation of novel materials based on molecular imprinting technology and their use in biomedical applications. It starts with the brief description and demonstration of the molecular imprinting technology. Then, the latest advancements in MIP-based biomedical applications (i.e., drug delivery, bioimaging, cancer therapy and sensor applications) are presented. In the final section, conclusions and future trends are discussed.

29.2 Molecular imprinting technology Molecular imprinting is a technique that is applied for the design and development of highly selective polymeric materials towards the target compounds (Bai & Pan, ¨ zgu¨r, 2021). The obtained polymers 2021; Kec¸ili et al., 2011, 2014; Kec¸ili, 2018; O using this technology also called “molecularly imprinted polymers (MIPs)” possess ligand-recognition characteristics reminiscent of those of biological counterparts such as antibody-antigen recognition (Alexander et al., 2006). This has led to MIPs often being referred to as artificial antibodies and even as enzyme mimics in cases when they exhibit catalytic features. MIPs selectively recognize and bind the target compound, usually with high affinity and specificity comparable to those observed in biology. In the molecular imprinting technology, a well-defined composition of polymerizable functional monomer/s and cross-linker are copolymerized in the existence of the target compound (template) in a porogenic solvent chosen to solubilize and stabilize the prepolymerization mixture. After polymerization, the template is extracted from the polymeric structure by washing with a suitable solvent, revealing reflective recognition regions towards the target compound which are complementary to the template in terms of 3D shape and functionality. The molecular imprinting technology is schematically shown in Fig. 29.1.

29.2.1 Key parameters for the preparation of molecularly imprinted polymers There are many parameters that influence the molecular imprinting process and impact on MIP performance. Thus, the nature of the template, and the choice of the chemical reagents, that is, functional monomer, cross-linker. and solvent is crucial

29.2 Molecular imprinting technology

FIGURE 29.1 The schematic depiction of the molecular imprinting technology. (He et al., 2021) Reproduced with permission from He, S., Zhang, L., Bai, S., Yang, H., Cui, Z., Zhang, X., & Li, Y. (2021). Advances of molecularly imprinted polymers (MIP) and the application in drug delivery, European Polymer Journal, 143, 110179.

in order to get efficient functional MIPs, as are temperature and pressure (Piletsky et al., 2002, 2004). The template is at the center of MIP preparation and the choice of functional monomer is critical as it must have chemical groups that can interact with complementary functional groups of the template. The extensively preferred functional monomers in MIP synthesis participate in hydrogen bonding interactions. Fig. 29.2 shows the most widely preferred functional monomers that exhibit acidic, basic, and neutral features in the preparation of MIPs. On the other hand, initiators also have crucial role in the MIP preparation process. Fig. 29.3 shows the common initiators preferred in the preparation of MIPs. When heat or UV-light is applied, these initiators rapidly decompose into free radicals as represented in Fig. 29.4. The unpaired electrons exist on these generated free radicals and has great ability to attack the functional monomers as well as cross-linkers and initiate the polymerization reaction. Another key factor is the cross-linker that is also very crucial in the polymerization process and responsible firstly for fixing and controlling the morphology of the polymeric structure, secondly for stabilizing the imprinted binding sites and lastly for providing mechanical stability and cavity rigidity of the obtained polymer. The functional monomer to cross-linker ratio should be high enough to maintain stability and the porousness of the recognition sites, thus, greater the

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CHAPTER 29 Molecularly imprinted polymers (MIPs)

Basic

Methacrylic acid (MAA)

4-Vinylpyridine

Trifluoro methacrylic acid

1-Vinylimidazole

4-Vinylbenzoic acid

4-Vinylbenzamidine

(4-VP)

Neutral

748

2-Hydroxyethylmethacrylate (HEMA)

Acrylamide (AAm)

Methyl methacrylate (MMA)

FIGURE 29.2 Common functional monomers preferred in the preparation of molecularly imprinted polymers.

2,2’-azodiisobutyronitrile (AIBN)

2,2’-azobis-(2,4-dimethylvaleronitrile) (ABDV)

FIGURE 29.3 Common initiators preferred in the preparation of molecularly imprinted polymers.

degree of cross-linker the more rigid the polymeric structure. The most commonly employed technique for the MIPs is the free radical polymerization technique which is triggered by an initiator either by heat also called “thermolysis” and UV radiation also called “photolysis.” On the other hand, the porogen is responsible for bringing together all prepolymerization components including template, functional monomer/s, cross-linker, and initiator. It also enables the effective formation of pores in the macroporous polymeric network. Common cross-linkers preferred in the preparation of MIPs are given in Fig. 29.5.

29.2 Molecular imprinting technology

FIGURE 29.4 Decomposition reaction of azobisisobutyronitrile (AIBN) by applying UV light/heat.

Ethylene glycol dimethacrylate (EGDMA)

Divinylbenzene (DVB)

Trimethylolpropane trimethacrylate (TRIM)

Pentaerythritol triacrylate (PETRA)

FIGURE 29.5 Common cross-linkers preferred in the preparation of molecularly imprinted polymers.

29.2.2 Approaches for the preparation of molecularly imprinted polymers There are three different approaches for the synthesis of MIPs which differ depending on the interactions between the functional monomer/s and the target compound (template) involved in the imprinting process. These approaches are covalent, noncovalent, and semicovalent imprinting techniques. The covalent approach involves the formation of covalent bonds between the functional monomer/s and template prior to polymerization. Then, the template is removed from the polymeric structure. During the rebinding process, the covalent bonds are reformed to bind the template to the polymer network. The first example of covalent imprinting was

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reported by Wulff, Sarhan, and Sarhan (1972). In their study, 4-nitrophenyl-α-Dmannopyranoside was conjugated to 4-vinylbenzeneboronic acid as the template and copolymerized with ethylene dimethacrylate and methyl methacrylate. After polymerization, the boronic acid ester was cleaved, and the 4-nitrophenyl- α -Dmannopyranoside was extracted from the polymeric structure. The results showed that the prepared imprinted polymer strongly and selectively binds the template during the rebinding process. This strategy was further developed by Shea and Thompson (1978). The main superiority of this approach is the homogeneity of the binding sites due to the orientational control and interaction stability provided by covalent bonds. This has been further studied by Shimizu and coworkers (Umpleby, Bode, & Shimizu, 2000). Moreover, this approach accomplishes higher binding constants due to covalent bond types such as boronate ester (Wulff, Vesper, Grobe-Einsler, & Sarhan, 1977), ketal/acetal (Shea & Dougherty, 1986), and Schiff’s base (Wulff, Best, & Akelah, 1984). However, this technique has some drawbacks including the requirement for significant synthetic effort and usually slow kinetics of bond formation and cleavage making the removal of the template quite difficult. Moreover, there is a limited number of reversible covalent bond-types available. In addition, matching the template and functional monomers functionalities for covalent imprinting can be difficult. These drawbacks limit the application of this approach. In the early 1980s, Mosbach and coworkers introduced a second strategy that became known as noncovalent imprinting (Arshady & Mosbach, 1981). In this approach, the template interacts with a functional monomer through noncovalent interactions (i.e., hydrogen bonding, van der Waals forces, ionic or π-π interactions) during the imprinting and rebinding processes. Prior to polymerization, the template and functional monomers are self-assembled by dissolving them in an appropriate solvent. The template is removed by washing the prepared MIPs with a suitable solvent or a mixture of solvents. In this approach, the rebinding process of the template to the imprinted sites of the MIP is based on noncovalent interactions. Non-covalent imprinting is the predominant method used today due to its simplicity, faster template rebinding, and ease of extraction of the template from the cross-linked polymeric structure. In addition, a large number of functional monomers and cross-linkers are commercially available which is one of the main advantages of this approach. In 1995, Whitcombe et al. developed a hybrid approach, combining the superiorities of the covalent and the noncovalent techniques called “semi-covalent imprinting” (Whitcombe, Rodriguez, Villar, & Vulfson, 1995). In this technique, the template is covalently coupled to functional monomer/s during polymerization process whereas rebinding takes place with noncovalent interactions. During the semicovalent imprinting process, a linker group known as “sacrificial spacer” is used between the template and the functional monomer to prevent steric hindrance. Sacrificial spacer is then removed along with the template. Depending on the intended application, MIPs can be efficiently synthesized in a variety of formats. MIPs have been conventionally prepared as monolithic form by

29.3 Applications of molecularly imprinted polymers

applying bulk polymerization technique where the resultant solid monoliths are crushed, ground, sieved to an appropriate particle size and subsequently packed in a chromatographic column. Although this approach is facile, the preparation steps are time consuming, tedious, unsuitable for the production in large-scale, and the ground particles are irregular in size and shape. To overcome these drawbacks of bulk polymerization technique, more sophisticated MIP formats such as nanofibers (Kim & Chang, 2011; Rajhans, Gore, Siddique, & Kandasubramanian, 2019), nanoparticles (Pardeshi, Dhodapkar, & Kumar, 2014; Wang et al., 2020), microbeads ¨ zer, Osman, & Yazıcı, 2017), membranes (Dong et al., (Say et al., 2003; Tu¨may O 2020; Zhang et al., 2019) and cryogels (Andac¸, Galaev, & Denizli, 2016; Dolak et al., 2020; Su¨mbelli, Kec¸ili, Hu¨r, Erso¨z, & Say, 2021) were designed and fabricated applying different strategies including electrospinning, precipitation polymerization, suspension polymerization, and cryopolymerization.

29.3 Applications of molecularly imprinted polymers in biomedical science 29.3.1 Drug delivery The delivery of the drugs is commonly investigated in the area in which the delivery of the drug compound to its target region is performed. In addition, the delivery efficiency is carefully controlled. Furthermore, various drug compounds need to be administered over an extended time also called “controlled release” to obtain the highest therapeutic impact of the target drug which are fastly metabolized and efficiently eliminated from the body after administration. One of the interesting applications of MIPs as efficient platforms is the delivery of the target drugs (Alvarez-Lorenzo & Concheiro, 2006; Luli´nski, 2017). MIPs exhibit excellent features including high affinity, selectivity and loading capacity for the target drug compound, great stability under extreme conditions such as highly acidic and basic pH environment. The great affinity of the MIPs towards the target drug compound lead to increase in the residence time of the drug in vivo and in vitro. These unique features make MIPs as powerful platforms for drug delivery applications. Various reported applications are briefly demonstrated in the following. In a research performed by Anirudhan, Divya, and Nima (2013), release of thiamine hydrochloride was conducted by employing the synthesized selective MIPs towards the target drug. In this research, firstly, the preparation of 3methacryloxypropyltrimethoxysilylated montmorillonite was performed and then MIP in the layers of the silylated montmorillonite was synthesized. Itaconic acid and ethylene glycol dimethacrylate (EGDMA) were employed as the functional monomer and cross-linker, respectively. The experimental data exhibited that the prepared silylated montmorillonite-based MIP towards thiamine hydrochloride can be effectively used for the controlled release of thiamine hydrochloride. The

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controlled release of thiamine hydrochloride was investigated at two different physiological environment. The highest release value (88.2%) was achieved at pH 7.4 within 8 h because of the highest MIP swelling behavior. Korde and colleagues reported to design and development of nanoporous MIP nanoparticles (nanoMIPs) for the controlled release of amygdalin (AD) which is an anticancer drug (Korde, Mankar, Phule, & Krupadam, 2019). In this research, the functional monomer 2-vinylpyridine (2-VP) and cross-linker EGDMA were polymerized in the presence of the target drug AD. The synthesized nanoMIPs were successfully used for the efficient release of AD (Fig. 29.6). The release values of the target drug AD from the prepared nanoMIPs in deionized H2O were 0.095, 0.120, 0.180, and 0.205 μg within 5, 30, 180 and 300 min, respectively. The similar results were achieved using nanoMIPs in pH 2.0 and pH 7.0 buffer solutions. In an interesting study conducted by Cegłowski and coworkers (Cegłowski, Kurczewska, Ruszkowski, Libersk, & Schroeder, 2019), the effect of cross-linker on binding features, and release behavior towards doxorubicin and cytotoxicity of MIPs was investigated. In this study, EGDMA and TRIM were used as the crosslinker while MAA and 2-hydroxyethyl methacrylate (HEMA) were chosen as comonomers. The obtained highly hydrophilic and cross-linked MIPs could effectively release the target drug doxorubicin. MIP prepared by using the cross-linker EGDMA displayed three times higher binding behavior for doxorubicin than the one achieved by using the cross-linker TRIM. The release kinetics of the target drug doxorubicin from the prepared MIPs was mainly dependent on the medium pH and the cross-linker was chosen. Same researchers also prepared MIPs and tested their drug release behavior towards paclitaxel (Cegłowski, Kurczewsk, Ruszkowski, & Schroeder, 2019). For this purpose, paclitaxel imprinted MIPs were prepared by using the functional

FIGURE 29.6 The synthesis of nanoporous molecularly imprinted polymer nanoparticles for the controlled release of amygdalin. Reproduced with permission from Korde, B. A., Mankar, J. S., Phule, S., & Krupadam, R. J. (2019). Nanoporous imprinted polymers (nanoMIPs) for controlled release of cancer drug. Materials Science & Engineering C 99, 222 230.

29.3 Applications of molecularly imprinted polymers

monomers MAA and HEMA and the cross-linkers EGDMA and TRIM. The prepared MIP were efficiently applied for the controlled release of the target drug paclitaxel from the polymeric network. Under optimum conditions (pH 7.4), the maximum release of the paclitaxel was achieved by employing the MIPs synthesized by using TRIM as the cross-linker. An et al. prepared a konjac glucomannan-based MIP for the effective recognition and controlled release of anticancer drug 5-fluorouracil (An, Kang, Zhang, Guan, & Tian, 2020). In the reported work, thermosensitive and biocompatible MIP was synthesized by using N-isopropyl acrylamide as the functional monomer and polysaccharide konjac glucomannan. The obtained experimental results confirmed that the developed polysaccharide-based thermosensitive MIP can sensitively and selectively recognize the target drug 5-fluorouracil. The highest binding capacity was achieved within 12 h. In vitro studies on the controlled release of 5-fluorouracil were carried out in pH 7.4 phosphate buffer environment and the obtained cumulative release of the target drug was higher at 25 C than that at 38 C. In a work conducted by Shadabfar, Abdouss, and Khonakdar (2020), 5fluorouracil selective-magnetic moleculary imprinted Fe3O4@SiO2 nanoparticles were synthesized for the controlled drug release and cancer treatment applications. For this aim, MAA and TRIM were used as the functional monomer and cross-linker, respectively. In vitro experiments on the controlled release of 5fluorouracil were conducted in phosphate buffer solutions (pH 3.0 and pH 7.4) at 37 C. The results confirmed that 5-fluorouracil was efficiently released from the prepared magnetic moleculary imprinted Fe3O4@SiO2 nanoparticles. In addition, the prepared magnetic nanoparticles displayed 61% cytotoxicity towards cancer cells while they didn’t exhibit considerable impact on normal cells. Ji et al. developed hollow magnetic moleculary imprinted Fe3O4@SiO2 nanoparticles for the efficient binding and controlled release of silybin (Ji et al., 2020). In their work, researchers used functional monomer MAA and cross-linker EGDMA for the synthesis of MIP layer on the surface of magnetic Fe3O4 nanoparticles wrapped by mesoporous silica particles. The prepared magnetic moleculary imprinted Fe3O4@SiO2 nanoparticles exhibited great recognition and release behavior towards the target drug silybin. The highest drug binding capacity was achieved as 15.40 mg/g. The experiments on the controlled release of silybin showed that the rate of the molecularly imprinted magnetic nanoparticles-based release process reached the equilibrium value within 14 h at pH 8.2. In a crucial study carried out by Kim, Yun, Shim, and Yoon (2020), a polysaccharide-based molecularly imprinted biomaterial was prepared for the controlled release of atenolol. For this objective, imprinted biomaterial towards atenolol was prepared by using polyvinyl alcohol (PVA), mungbean starch and various plasticizers including glycerol and citric acid. The release of the target drug atenolol from the prepared polysaccharide-based molecularly imprinted biomaterial was conducted using artificial skin. The results confirmed that the cumulative release of atenolol was higher at pH 4.0 than that at pH 10.0. The drug

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release process was rised with a relatively steady rate over time. The controlled release of the target drug was achieved around 96.2% to 99.1% within 2 weeks. In another crucial research Sedghi, Ashrafzadeh, and Heidari (2021) synthesized pH-sensitive graphene oxide (GO)/-based MIPs towards curcumin. In their research, vinyl groups modified-GO was synthesized applying different modification stages and the researchers used the functional monomers acrylated β-cyclodextrin (β-CD) and acrylic acid (AA) for the synthesis of the selective MIPs on the surface of vinyl groups modified-GO (Fig. 29.7). The achieved results showed that GO-based MIPs are great nanomaterials for the efficient release of curcumin from the polymeric structure which was dependent on the medium pH. The obtained release value was around 30% at pH 5.5 while 10% of the curcumin was released at pH 7.4. Silva et al. prepared silicone-based molecularly imprinted hydrogels towards moxifloxacin that is an antibiotic used for the treatment of bacterial eye infections (Silva et al., 2021). In this study, the functional monomer AA was employed for the preparation of a set of moxifloxacin imprinted hydrogels. The prepared hydrogels exhibited a great recognition and release behavior towards the target antibiotic moxifloxacin. In vitro drug release studies were carried out using a microfluidic cell to mimic the ocular surface and the outputs from these studies confirmed that the prepared moxifloxacin imprinted hydrogels are quite effective against S. epidermidis and S. aureus for approximately 14 days.

FIGURE 29.7 The schematic representation of the preparation of pH-sensitive GO-based MIPs towards curcumin. Reproduced with permission from Sedghi, R., Ashrafzadeh, S., & Heidari, B. (2021). pH-sensitive molecularly imprinted polymer based on graphene oxide for stimuli actuated controlled release of curcumin, Journal of Alloys and Compounds, 857, 157603.

29.3 Applications of molecularly imprinted polymers

In another work on the drug release, application of MIPs was reported by Wang et al. (2021). In their work, the researchers designed and developed MIPs for controlled release of S-sulpiride which is an antipsychotic drug used for the treatment of schizophrenia. MIPs towards S-sulpiride were synthesized by using a novel functional monomer N-acryloyl-tryptophan and cross-linker EGDMA. The highest binding of the drug was obtained as 226.2389 μmol/g. The experiments on the controlled release of S-sulpiride exhibited that the rate of the drug release process fastly rised over the time within 30 min. Then, it slowly reduced until 2 h. In addition, the rate of the cumulative drug release at pH 1.0 was higher than that at pH 6.8 and 7.0 which confirms that the acidic conditions enhanced the release of the target drug S-sulpiride.

29.3.2 Bio-imaging and cancer therapy Bio-imaging and cancer therapy are another successful applications of MIP-based materials. Bio-imaging enables the careful observation and evaluation of the biological processes in the wide range which vary between the subcellular scale and experimental animal scale (Janib, Moses, & MacKay, 2010). By incorporating a suitable fluorescent probe to the bio-imaging platform, this process provides effective and sensitive diagnosis of a number of diseases in the early stages as well as monitoring the response of the treatment process. MIPs can be successfully employed as powerful materials for the bio-imaging and cancer therapy applications (Haupt, Rangel, & Bui, 2020; Vaneckova et al., 2020). As one of the first and crucial attempts on the design and fabrication of novel imaging systems based on MIPs, Asadi and colleagues reported the preparation of brain-targeted magnetic MIPs towards olanzapine (Asadi et al., 2016). In this work, a fructose-based biodegradable cross-linker was preferred for the synthesis of magnetic MIPs composed of Fe3O4, SiO2 nanoparticles and fluorescein isothiocyanate (FITC) (Fig. 29.8). The obtained data from in vivo experiments on the release of the target drug olanzapine confirmed that the developed magnetic MIPs with biodegradable feature exhibit high affinity and controlled release behavior towards olanzapine. In another work carried out by Yin, Li, Ma, and Liu (2017), a MIP-based photothermal therapy approach was proposed for the first time. In this approach, Au nanorods were chosen as the core nanomaterial and sialic acid was preferred for the synthesis of surface imprinted Au nanorods. The prepared sialic acid imprinted Au nanorods were effectively accumulated at the tumor. When applying near-infrared laser to the tumor for a while, the targeted sialic acid imprinted Au nanorods which were accumulated at the tumor region efficiently absorb the applied near-infrared laser energy and convert it to heat which causes to tumor ablation during a number of photothermal therapy treatments. Zhang and coworkers designed and developed a luminescence nanoparticle La3Ga5GeO14 (LGGO) modified with selective MIP layer for trichlorfon (Zhang et al., 2019). MIP layer was prepared by using the functional monomer MAA and

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FIGURE 29.8 The synthesis of magnetic MIPs composed of Fe3O4, SiO2 nanoparticles, and fluorescein isothiocyanate (FITC) for the controlled delivery of olanzapine applying external magnetic field. Reproduced with permission from Asadi, E., Abdouss, M., Leblanc, R. M., Ezzati, N., Wilson, J. N., & Kordestani, D., (2016). Synthesis, characterization and in vivo drug delivery study of a biodegradable nanostructured molecularly imprinted polymer based on cross-linker of fructose, Polymer, 97, 226 237.

cross-linker EGDMA. After removal of the template trichlorfon from the polymeric network, the obtained LGGO/MIP nanocomposite was effectively used as a powerful nanocarrier for the drug binding and successfully applied for the fluorescence-based in vivo bio-imaging applications as schematically depicted in Fig. 29.8. The researchers concluded that the developed LGGO/MIP nanocomposite exhibited a long persistent luminescence feature and great penetration behavior in mice which ensures its high potential for bio-imaging applications (Fig. 29.9). In another research conducted by Qin et al. (2020), an effective approach was proposed for the synthesis of MIP-based fluorescent zeolitic imidazolate framework-8 (FZIF-8) for the controlled release of anticancer drug doxorubucin

29.3 Applications of molecularly imprinted polymers

FIGURE 29.9 The schematic depiction of the La3Ga5GeO14 (LGGO) synthesis, modification process of its surface with selective molecularly imprinted polymer (MIP) layer towards the target pesticide trichlorfon and in vivo bio-imaging application of the developed LGGO/MIP nanocomposite. Reproduced with permission from Zhang, D.-D., Liu, J.-M., Sun, S.-M., Liu, C., Fang, G.-Z., & Wang, Z. S. (2019). Construction of persistent luminescence-plastic antibody hybrid nanoprobe for ın vivo recognition and clearance of pesticide using background-free nanobioimaging. Journal of Agricultural and Food Chemistry 67, 6874 2 6883.

and targeted bioimaging application. For this objective, firstly, encapsulation of the target drug doxorubicin and carbon quantum dots into the FZIF-8 was performed. In the next stage, the synthesis of MIP layer was performed on the previously synthesized core nanomaterial. CD59 cell membrane glycoprotein was chosen as the template for the preparation of selective MIP layer that allows the prepared doxorubicin loaded- MIP/ FZIF-8 to be enriched to tumor regions through efficient detection of MCF-7 cancer cells. The drug release and imaging behavior of the developed doxorubicin loaded-MIP/ FZIF-8 having fluorescent feature was investigated in the in vivo and in vitro tests. The achieved results confirmed that doxorubicin loaded-MIP/FZIF-8 exhibited great biocompatibility, biodegradability, and high performance on the controlled release of the target drug doxorubicin.

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The researchers also noted that the developed MIP-based fluorescent nanomaterial can be successfully employed for the imaging and treatment of tumors. In a crucial research carried out by the group of Haupt (Demir et al., 2018), carbon dots coated with MIP layer were prepared for the targeting and imaging of cancer cells. In their research, firstly, the synthesis of N-doped carbon dots was carried out using starch as the carbon source via hydrothermal approach. In the next step, the synthesis of MIP layer was performed on the surface of the synthesized N-doped carbon dots using glucuronic acid as the template since it is an epitope of hyaluronan which is one of the potential biomarker for various cancers. In this research, the developed carbon dots/MIP nanocomposites were successfully used for the efficient targeting and bioimaging of the hyaluronan on fixated human cervical cancer cells. Jia et al. demonstrated the synthesis of silanized silica nanoparticles/MIP nanocomposite for the fluorescence-based targeted bioimaging and controlled drug release into the pancreatic cancer BxPC-3 cells (Jia et al., 2019). In this work, bleomycin and 71 2 80 peptide of human fibroblast growth factor-inducible 14 modified with glucose were chosen as dual templates for the effective binding of bleomycin to the pancreatic cancer BxPC-3 cells that causes overexpression of human fibroblast growth-factorinducible 14. In vitro targeted bio-imaging experiments confirmed that silanized silica nanoparticles/MIP nanocomposite exhibited excellent specificity and targeting behavior towards the BxPC-3 cells. Liu and coworkers design and developed fluorescent conjugated-MIP nanoparticles towards sialic acid for the targeted bio-imaging of cancer cells (Liu et al., 2017). In this research, firstly, poly(fluorene-alt-benzothiadiazole) (PFBT) was conjugated with phenylboronic acid (PBA) that exhibit a great binding affinity towards sialic acid. Then, the target compound sialic acid was imprinted into the PFBT/ PBA nanopolymeric structure. After removal of the sialic acid from the polymeric network, sialic acid imprinted fluorescent nanoparticles were successfully applied for the bioimaging of DU 145 cancer cells via binding of sialic acid groups overexpressed in cancer cells to the imprinted fluorescent nanoparticles (Fig. 29.10).

29.3.3 Sensing and separation processes Another interesting application field of MIPs is the detection and separation of the target compounds such as proteins, biomarkers and pharmaceuticals, etc. in biological samples. For example, Bagheri and his colleagues developed a selective magnetic composite nanosensor for ephedrine (EP) detection in serum, urine and pharmaceutical samples (Bagheri, Pajooheshpour, Afkhamic, & Khoshsafar, 2016). In this study, firstly, the preparation of core shell Fe3O4/SiO2/TiO2 (FST) nanostructure was carried out and then magnetic FST-MIP composite sensor was prepared by polymerization of the functional monomer methyl methacrylate (MMA) on the template EP on the surface of carbon paste electrode. The synthesized magnetic composite nanosensor showed an excellent binding affinity towards EP and the detection limit was achieved as 0.0036 μM.

29.3 Applications of molecularly imprinted polymers

FIGURE 29.10 The synthesis of sialic acid imprinted fluorescent nanoparticles and recognition of the target cancer cell. Reproduced with permission from Liu, R., Cui, Q., Wang, C., Wang, X., Yang, Y., & Li, L., (2017). Preparation of sialic acid-imprinted fluorescent conjugated nanoparticles and their application for targeted cancer cell imaging, ACS Applied Materials & Interfaces 9 3006 2 3015.

Piloto et al. demonstrated the synthesis of MIP/quantum dots (QDs)-based fluorescent membranes for the detection of myoglobin (Piloto et al., 2021). MIPs were conjugated with QDs and then assembly of the cellulose membranes with MIP/QDs was done (Fig. 29.11). The synthesized MIP/QDs-based fluorescent membranes were successfully applied for the sensitive detection of myoglobin in serum samples. The calculated detection limit was 3.08 pg/mL. In another crucial study on the recognition and separation of myoglobin in serum samples by using MIP-based cryogels was conducted by the research group of Say (Dolak et al., 2018). In their study, N-methacryloyl amidoantipyrine-Ce (III) was used as the lanthanide chelate-based complex functional monomer for the preparation of myoglobin imprinted cryogels. The binding capacity of the synthesized cryogels was found as 68 mg/g and stability has remained even after 12 binding-eluting cycles. In another recently published research (Canpolat et al., 2021), the same research group demonstrated the synthesis of molecularly imprinted cryogels for the effective recognition and separation of cytochrome c in serum samples. For this aim, the researchers chose N-methacryloyl amidoantipyrine-Ce (III) as the lanthanide chelate-based complex functional monomer for the preparation of imprinted cryogels towards the target protein cytochrome c. The synthesized cryogels exhibited great selectivity and recognition behavior towards cytochrome c in the existence of other competing proteins including myoglobin and hemoglobin. The obtained binding capacity was 98.33 mg/g.

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FIGURE 29.11 The schematic demonstration of the assembly of the cellulose membranes with molecularly imprinted polymer/quantum dots (MIP/QDs). (A) Raw QDs-modified membranes, (B) MIP/QDs-modified membranes before binding of myoglobin, (C) MIP/ QDs-modified membranes after binding of myoglobin. Reproduced with permission from Piloto, M. L., Ribeiro, D. S. M., Rodrigues, S. S. M., Santos, J. L. M., Sampaio, P., & Sales, G. (2021). Imprinted fluorescent cellulose membranes for the on-site detection of myoglobin in biological media, ACS Appl. Bio Mater. (In press).

Chen et al. demonstrated the preparation of a MIP-based magnetic graphene oxide (GO) for the selective extraction of glycoprotein from physiological fluids (Chen et al., 2021). For this purpose, magnetic GO modified with 2,4-difluoro-3formyl phenylboronic acid was chosen as the supporting matrice for the immobilization of the glycoprotein horseradish peroxidase. For the synthesis of magnetic GO, FeCl2U4H2O was reacted with FeCl3U6H2O in the presence of GO. The reaction was conducted at 80 C for 1 h amd the medium pH was 9.0. On the other hand, acrylamide was used as the functional monomer for the preparation of selective MIP towards the target glycoprotein. The achieved results confirmed that the prepared MIP-based magnetic GO displayed excellent extraction behavior with a binding capacity of 200.88 mg/g. Wang and coworkers published a research on the synthesis of MIP nanoparticles-based fluorescent test strips for the sensitive detection of dopamine (DA) (Wang et al., 2019). In this work, firstly carbon QDs and CdTe QDs were synthesized and successfully embedded into the silica nanoparticles. In the next step, MIP layer exhibiting dual fluorescent emission were prepared on the surface of silica nanoparticles by using the backbone monomer acrylamide (AAm), the functional monomer 4-vinylphenylboronic acid (VPBA) and the cross-linker

29.3 Applications of molecularly imprinted polymers

methylene bisacrylamide (MBAAm). Finally, MIP nanoparticles-based fluorescent test strips were prepared via coating of MIP nanoparticles on the surface of filter papers (Fig. 29.12). The results showed that the developed MIP nanoparticles-based fluorescent test strips can be successfully used for the recognition of DA in serum samples. The detection process was carried out withing a very short time (3 min) using a quite small volume of sample (10 μL). The achieved detection limit was 100 150 3 1029 M.

FIGURE 29.12 (A) The synthesis of molecularly imprinted polymer (MIP) nanoparticles-based test strips towards DA, (B) visual sensing process of DA using the fabricated test strips. Reproduced with permission from Wang, J., Dai, J., Xu, Y., Dai, X., Zhang, Y., Shi, W., Sellergren, B., & Pan, G. (2019). Molecularly imprinted fluorescent test strip for direct, rapid, and visual dopamine detection in tiny amount of biofluid, Small 15, 1803913.

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In another important research (Qi et al., 2019), Qi and and coworkers prepared a paper-based electrochemical sensor having selective MIPs towards carcinoembryonic antigen. The researchers effectively applied the developed paper-based electrochemical sensor bearing MIP as the recognition component for the detection of carcino-embryonic antigen in serum samples. The sensor response was linear in the concentration range from 1.0 to 500.0 ng/mL and the detection limit was found to be 0.32 ng/mL. In a study reported by Mohamed, Khashaba, El-Wekil, and Shahin (2019), a new green magnetic MIP based on the cross-linking of chitosan was designed and prepared for the selective separation of memantine from human serum before its determination by fluorimetry. The linearity range was obtained in the range between 1.84 and 95.0 ng/mL with a low detection limit (0.6 ng/mL). The prepared green magnetic MIP was applied for the efficient recognition of memantine in human serum samples and pharmaceutical tablets with recoveries of 97.6 6 2.9 and 100.8 6 3.0, respectively. Bilici et al. demonstrated the preparation of magnetic MIP for the sensitive detection of metoclopramide in urine samples (Bilici, Badak, Zengin, Suludere, & Aktas, 2020), The prepared magnetic MIP was successfully used for recognition of metoclopramide through the formation of a charge-transfer complex between the eluted metoclopramide and picric acid. The magnetic MIP showed linear response in the range from 5.0 to 150.0 ng/mL. The detection and quantitation limit values were calculated to be 1.5 and 4.95 ng/mL, respectively. Toudeshki and colleagues designed and developed a new MIP on the surface of magnetic MWCNTs for the selective recognition and preconcentration of metformin, one of the most common drugs prescribed for type-2 diabetes, in biological fluids (Toudeshki, Dadfarnia, & Haji Shabani, 2019). The developed MIPbased magnetic MWCNTs showed linear response in the range from 0.5 to 50.0 μg/L. The achieved detection limit was 0.13 μg/L. ¨ zku¨tu¨k et al., 2016), the research group of In another crucial research (Birlik O Say demonstrated the development of MIP-based potentiometric sensor for the recognition of sarcosine that is a potential prostate cancer biomarker. In their research, methacryloylamido histidine (MAH) was chosen as the functional monomer. The developed MIP-based potentiometric sensor exhibited excellent affinity, sensitivity, and selectivity towards sarcosine. The sensor response was linear in the concentration range from 1022 to 1026 mM. The response time and detection limit was achieved as ,2 min and 1.35 3 1027 mM, respectively.

29.4 Conclusions and future perspectives It is no doubt that MIPs harbor interesting applications in biomedical science and therefore there’s a significant attention in the field of interdisciplinary sciences with rapidly emerging applications ranging from sensing to drug delivery,

References

therapy, and bioimaging. The rapid advancements in the design and preparation of novel MIPs having excellent features can substantially affect the evaluation and analyzing ability of scientists and researchers, and can deliver exciting developments to biomedical sciences. Major challenges of the drug delivery, bioimaging and sensing systems are their efficient, biocompatible, sensitive, selective, cost-effective and timeeffective applications in biomedical field. This chapter aims to answer these challenges to the scientists and researchers for proper implementation of MIPs as powerful platforms for biomedical applications. MIP-based sensor, drug delivery, and therapy as well as bioimaging applications continuously being progressed by rapid scientific and technological advancements such as development of new functional monomers, functionalization & fabrication technologies, novel nanomaterials for the preparation of MIP-based nanocomposites, etc. These rapid advancements will certainly enable for the design and development of more biocompatible, powerful, low-cost, stable, sensitive and rapid MIP-based sensor, bioimaging, drug delivery, and therapy systems.

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CHAPTER

30

Natural biopolymer scaffolds for bacteriophage delivery in the medical field

Ana Mafalda Pinto1,2, Marisol Dias2, Lorenzo M. Pastrana2, Miguel A. Cerqueira2 and Sanna Sillankorva2 1

Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, Portugal 2 INL—International Iberian Nanotechnology Laboratory, Braga, Portugal

30.1 Introduction Since the early 20th century, there has been an intensive pursuit for biomaterials with unique physicochemical properties and a high degree of biocompatibility for applications in the biomedical and pharmaceutical industries (Puscaselu, Lobiuc, Dimian, & Covasa, 2020). Plants, animals, bacteria, and fungi all synthesize biopolymers as part of their natural life cycle. These natural sources are relatively low-cost, readily available due to their renewable nature, making these types of materials extremely appealing to high-value industries (Puscaselu et al., 2020). Materials from natural resources have, for instance, been produced to support, improve, or replace damaged tissues (Lee & Mooney, 2012; Raus, Nawawi, & Nasaruddin, 2021), and other biomedical applications include use in wound healing or prosthetics. On the other hand, synthetic polymers and ceramics or even metal alloys were far more used during the previous century (Pavlatou, 2020). Because of the interesting properties of biopolymers, a variety of systems based on proteins (e.g., albumin, whey protein, casein, collagen, gelatin, and soy) and polysaccharides (e.g., alginate, chitosan, and cellulose) have been studied for biomedical delivery applications. In addition, biopolymer carriers are very used to deliver biological/chemical compounds such as vitamins, bioactive agents, and probiotics. Encapsulation of phages, viruses specific to bacteria, has gained vast interest in the last two decades. As a result, phages have been encapsulated in synthetic polymers and natural biopolymers. The latter is focused on this chapter along with factors in the encapsulation, storage, and delivery process that may challenge phage viability.

30.2 Phage therapy Phages are viruses that only kill bacteria. Phages are the most diverse bacterial viruses found ubiquitously in nature, with an estimated 1031 total particles Advances in Biomedical Polymers and Composites. DOI: https://doi.org/10.1016/B978-0-323-88524-9.00019-X © 2023 Elsevier Inc. All rights reserved.

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(Ho, Fabijan, Lin, Maddocks, & Iredell, 2020; Pirnay, 2020). In 1915, Frederick Twort published the first paper describing the antibacterial activity of these virallike agents, and in 1917, Felix d’Herelle coined the term “bacteriophage” (phage) [see Kutter et al. (2010) and Maciejewska, Olszak, and Drulis-Kawa (2018) for a more detailed review in phage therapy]. In 1919, d’Herelle, in an unprecedented trial, explored the administration of virulent phages directly to a patient suffering from a major infection (Altamirano & Barr, 2021; Petrovic Fabijan et al., 2020). Felix d’Herelle developed a study that used an oral concentrate of phages therapeutically to treat Shigella enteritis (bacillary dysentery) (Petrovic Fabijan et al., 2020). Afterward, phage therapy was instantly applied and commercialized as a valuable therapeutical approach to face bacterial infections (Kutter et al., 2010; McCallin, Sarker, Sultana, Oechslin, & Bru¨ssow, 2018; Qadir, Mobeen, & Masood, 2018). However, the recent notoriety of phage therapy was quickly hindered by the discovery and intense research of antibiotics with their broad spectrum of action and ease to produce on a large scale (Altamirano & Barr, 2021; Pirnay, 2020). Currently, 96% of the phages identified fall into the order of Caudovirales, which are characterized as being double-stranded DNA viruses with a tail appendage that can be differentiated into lytic and temperate phages (Maciejewska et al., 2018; Roach & Debarbieux, 2017). Caudovirales were for many years only classified in three families (Siphoviridae, Myoviridae, and Podoviridae) (Kakasis & Panitsa, 2018), but the International Committee on Taxonomy of Viruses (ICTV) has revised the currently available data, dividing Caudovirales into 14 different families. These 14 families further include 73 subfamilies, 927 genera, and over 2800 species (ICTV, 2021). Phages directly impact gene transference for the evolution of bacterial species; more precisely, they are moved by a horizontal gene transfer with host and other phages (Canchaya, Fournous, Chibani-Chennoufi, Dillmann, & Bru¨ssow, 2003). A phage’s life cycle starts with its adsorption to the specific bacterial host receptor(s) on the cell surface and injection of the nucleic acid into the bacterial cytoplasm (Fig. 30.1) (Pinto, Silva, Pastrana, Ban˜obre-Lo´pez, & Sillankorva, 2021). After, phages will undergo lytic or lysogenic infections in the bacteria (Altamirano & Barr, 2021). During a lytic infection cycle, the phage genome is replicated, proteins are synthesized, and progeny particles are assembled inside the bacterium. The host cells eventually lyse facilitated by several viral proteins that destabilize the bacterial envelope (e.g., holins and endolysins), disrupting the cell wall and releasing the phage progeny, which will infect other bacteria. The lysogenic cycle consists of integrating the genetic material of the phage into the bacterial genome, where it is replicated together with the DNA of the host cell. The genetic content of the phage is transmitted to all daughter cells without causing the lysis of the bacterium. The phage remains dormant in the bacterium until the lytic cycle is induced, usually caused by external stimuli (e.g., stress or cellular damage processes of the bacterial host; ultraviolet (UV) light or mitomycin exposure), ending the cycle through lysis of the bacteria (Harada et al., 2018).

30.2 Phage therapy

FIGURE 30.1 The lytic and lysogenic life cycles of phages. Reprinted from Pinto, A. M., Silva, M. D., Pastrana, L. M., Ban˜obre-Lo´pez, M., & Sillankorva, S. (2021). The clinical path to deliver encapsulated phages and lysins. FEMS Microbiology Reviews. https://doi.org/10.1093/ femsre/fuab019.

The extensive use of antibiotics in the last decades has resulted in a significant increase in antibiotic resistance, which is recognized as a threat to public health worldwide (Martins, Toleman, & Gales, 2020). Therefore the research and development of novel therapeutic strategies against bacterial infections are critical (Altamirano & Barr, 2021). Phage therapy is again in the spotlight as an alternative treatment of bacterial infections and can replace or complement antibiotics to treat multidrug resistant bacterial infections. The benefits of using phages compared to antibiotics are vast (Table 30.1). Nevertheless, there are inherent disadvantages in using phages related to their limited host range and the high frequency of phage-resistant mutants that can arise during treatment, both of which can be circumvented using cocktails of multiple phages (Jault et al., 2019).

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Table 30.1 Characteristics of phages and antibiotics as antibacterials. Phages

Antibiotics

Specificity and selectivity—only toward target bacterial species, not disturbing the commensal microflora. Minimization of the possible secondary infections Self-replication—auto-dosing through upon contact with the host bacteria at the site of infection, avoiding the need for repeated administration Minor side effects—insignificant side effects due to the release of endotoxins upon bacterial lysis

No specificity—targets pathogenic microorganisms and normal microflora and may lead to severe secondary infections

Specific phage resistance—the targeted bacterial species may become resistant to the phage, but other phage species will be still able to kill Easy search—new phages can be found, produced, and used against bacterial resistant species

Multiple doses—easy metabolization and removal from the organism. No accumulation at the site of bacterial infection Multiple side effects—allergies, intestinal disorders, secondary infections, adverse effects on the kidney and the liver, among others Resistance to antibiotics—provoke resistance and multidrug resistance in several species and genera of bacteria Length and expensive search—difficult to manufacture new antibiotics, particularly against antibiotic-resistant bacteria

Adapted from Sarhan, W. A., & Azzazy, H. M. E. (2015). Phage approved in food, why not as a therapeutic? Expert Review of Anti-Infective Therapy, 13(1), 91 101. https://doi.org/10.1586/ 14787210.2015.990383.

How can phage therapy advance as a mainstream antibacterial for standard medical strategy? The answer is not easy since each administration pathway and medical condition has its specific constraints, and every patient might have different bacterial susceptibility to phages (Da˛browska, 2019; Ryan, Gorman, Donnelly, & Gilmore, 2011). Phages need to be effective, stable, and safe for use in therapy (Altamirano & Barr, 2021). To this date, only a few clinical trials have investigated the use of phages to treat different bacterial (e.g., urinary tract infections, diarrhea, wound infections, and chronic otitis media) (Jault et al., 2019; ¨ ngga˚rd, & Harper, Leitner et al., 2020; Sarker et al., 2016; Wright, Hawkins, A 2009). These trials revealed that phages are safe, causing no evident side effects (Chan, Stanley, Modak, Koff, & Turner, 2021), but their efficacy in trials has not been assured. This was mostly because phage susceptibility was not studied before the trials, leading to a phage activity not more effective than that obtained in placebo treatments. Nonetheless, phage efficacy has been demonstrated in various compassionate and case reports, particularly when administered topically, for instance, to chronic wounds [see Pinto, Cerqueira, Ban˜obre-Lo´pez, Pastrana, and Sillankorva (2020) for more information]. The patients administered with phages, as a last resource, usually have bacterial infections not responsive to antibiotics (McCallin et al., 2019).

30.2 Phage therapy

30.2.1 Regulatory approval of phage therapy Phage therapy is still not seen with therapeutic potential, mostly due to safety and regulatory approval concerns (Sarhan & Azzazy, 2015). The nonexistence of precise guidelines for their regulatory approval is a major obstacle for their adoption as part of mainstream medicine (Sarhan & Azzazy, 2015), requiring conventional rules and codes to be revised (Fauconnier, 2019). In addition, a challenge for using phages as antimicrobials is their specificity and host range, which is limited to the bacteria that carry their complementary receptor (Lin, Koskella, & Lin, 2017; Moelling, 2020). Sometimes, the type of phages needs to be changed during a clinical trial; however, the approved phage cocktail composition may not be changed without new approval (Moelling, 2020; Sarhan & Azzazy, 2015). These regulation challenges lead to a difficult process of accepting phage therapy by pharmaceutical companies (Ferna´ndez, Gutie´rrez, Garcı´a, & Rodrı´guez, 2019). For example, if phages are developed under a personalized medicine model, additional intellectual property challenges will need to be addressed, and consequently, it will discourage investment from companies (Anomaly, 2020). In 2009, the FDA approved the first phase I clinical trial, where a phage cocktail preparation combining eight different phages was applied to treat venous leg ulcers (Rhoads et al., 2009). The results of this trial evidenced the safety of the phage cocktail used. In addition, the PhagoBurn trial, where a cocktail of lytic anti-Pseudomonas aeruginosa phages was applied in burn wound infections, also showed no significant adverse effects on volunteers (Jault et al., 2019). In 2020, the FDA cleared a phase 1/2 phage trial, submitted as an Investigational New Drug (IND) application (Voelker, 2019). This trial will investigate intravenous administration of phage therapy on multidrug-resistant and complicated bacterial infections and will be conducted by the University of California San Diego in collaboration with AmpliPhi Biosciences Corporation. Investigators and clinicians have explored different approaches to accelerate patient access to phage therapy when needing an urgent new treatment (Fauconnier, 2019). In Europe, some of these sporadic cases were conducted with the phage therapy treatments under Article Section 37 (Unproven Clinical Practice Interventions) of the Helsinki Declaration developed by the World Medical Association (Pirnay et al., 2018). This article allows the compassionate phage use if life-threatening or long-term (chronic) or in complicated disabling diseases that cannot be treatable with the current therapies (Pirnay et al., 2018).

30.2.2 Phage application in medicine There is a need to perform randomized controlled trials to reduce the bias and confirm the medical value of phage therapy products. Such analysis can identify the most relevant factors that lead to the success or failure of a clinical trial (Go´rski, Borysowski, & Mie˛dzybrodzki, 2020; Go´rski, Mie˛dzybrodzki, et al., 2020;

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Pirnay & Kutter, 2021). In the last years, some case reports in patients have been published (Fig. 30.2). Overall, phage therapy was used as a last resort according to Article Section 37 of the Declaration of Helsinki or under emergency IND applications and informed consent by the patients who underwent treatment. There has been an increase in case reports in the last years and the main bacterial species targeted have been Staphylococcus aureus, P. aeruginosa, and Klebsiella pneumoniae. The infections tackled included infections in the integumentary system (e.g., infected wounds and ulcers), respiratory system (e.g., cystic fibrosis, recurrent pneumonia), urinary system (e.g., urinary tract infections), skeletal system (e.g., bone, periprosthetic joint, and prosthetic knee infections), among others. Also, a few clinical trials have been completed or are currently active or recruiting participants (Fig. 30.3).

FIGURE 30.2 Case reports published in the last years targeting different bacterial species and infections. The data presented correspond to the year of the publication.

30.2 Phage therapy

FIGURE 30.3 List of completed, ongoing, and upcoming clinical trials (according to data provided in NIH clinicaltrials.gov).  Recruiting; †not yet recruiting.

In general, the clinical trials using phage therapy against different bacterial species are randomized, placebo-controlled, and double-blind clinical trials. There are, however, two observational trials that are either case-controlled or cohort. Some of the trials from 2020 are still recruiting patients. Some of them are due to being submitted only late in the year 2020. This is, for instance, the case of the 2020 study directed to the prosthetic joint, joint implant, and bone infections that was first submitted on November 25 and estimated to start on December 20. Also, some studies have been delayed due to the SARS-CoV-2 caused pandemic that made patient recruiting and keeping up with the trial starting deadlines more difficult. Similar to the case reports published, there has been an increase in clinical trials in the last few years, and their results are expected with anticipation. In addition, there are already programed clinical trials that will start later in the current year (2021) or next year. Therefore some of the upcoming clinical trials are already recruiting patients while others are in an earlier stage and not yet recruiting. Interestingly, there is an ongoing trial with COVID-19 patients with expanded access testing an intermediate-size population using phages under the IND treatment protocol. Therefore the increase in published clinical trial data and case reports will escalate clinical guidelines for phage therapy.

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CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery

30.3 Bacteriophage encapsulation Phages consist of a nucleic acid molecule surrounded by a protein envelope and are unstable particles that can be degraded and inactivated by environmental conditions (e.g., temperature, pH, and ionic strength or in the presence of UV light and proteases) (Loh et al., 2020). Encapsulation, a process in which active agents are enclosed in a carrier material, has been chosen to improve phage stability by shielding them from environmental conditions. A well-designed process of formulation and encapsulation is essential to prevent damage and ensure stabilization of the viral capsids and DNA/RNA components to avoid phage viability losses during manufacturing procedures. In addition, the carrier must endure adverse environmental conditions upon their administration and carry the phage to the specific infection site (Malik et al., 2017). For this, phages must be screened for various conditions, which may vary depending on the purpose of use, and may include assessing storage temperature, period of storage, varied pH conditions, among others. Most phages are stable at 4 C, while others remain stable at temperatures as low as 220 C or 280 C if a preservative agent (e.g., glycerol) is used to avoid the formation of crystal structures, or even stable at room temperature or 37 C (Alvi, Asif, Tabassum, Abbas, & Rehman, 2018). When preserved in liquid, certain phages can resist long periods (Ferna´ndez et al., 2019), but another way of storing is in dry powder formulations, which can be done by lyophilization or spray drying (Manohar & Ramesh, 2019). However, it is important to remember that not all phages can endure the drying process, resulting in a substantial loss of their titer (Gonzalez-Menendez et al., 2018). The choice of polymers can be a challenge due to phage inactivation issues. For instance, chitosan can inactivate phage particles, making them avirulent, but it also inhibits Escherichia coli and may disturb phage reproduction phases (Kochkina & Chirkov, 2000). The effect of different degrees of polymerization of chitosan on Bacillus thuringiensis phage 1 97A caused inhibition of infection and phage inactivation due to the chemical properties of chitosan (Kochkina, Surgucheva, & Chirkov, 2000). Infection inhibition depended inversely on the polymerization degree, while the extent of inactivation was proportional to the polymerization degree. In addition, this polymer inactivated more phage T2 than T7 (Kochkina et al., 2000). Although the direct contact of phages with chitosan causes their inactivation, chitosan has been frequently used, for instance, in the outer layer of core-shell particles to provide stability. The oral administration of phages presents many challenges to free phages, including the exposure to the acidic stomach environment, the action of bile, enzymes in the digestive tract, and their short perpetuity inside the digestive tract (Colom et al., 2017). Encapsulation offers more advantages than disadvantages. Encapsulation not only acts as a storage stabilizer limiting phage proteins and nucleic acid degradation but can also serve as a way of prolonging phage circulation time inside the patient for the treatment of systemic infections, prophylactic treatments, or to

30.3 Bacteriophage encapsulation

combat intracellular infections (Harada et al., 2018; Loh et al., 2020; Malik et al., 2017). Also, phage encapsulation of orally administered phages has been extensively studied as an alternative to free phage administration, protecting phages from adverse acid gastric conditions, including enzymatic attack, hydrolysis by the low pH, and inactivation by immune system components (Dini, Islan, & Castro, 2014). Furthermore, several delivery systems can assist the encapsulated cargo in penetrating the tissue, which can be difficult with free compounds. Several factors influence the therapeutic effectiveness and are mostly related to release and delivery behaviors (Tibbitt, Dahlman, & Langer, 2016). For instance, to successfully decrease the number of pathogenic bacteria at an infection site, phages, at high doses, must be administered in a regulated manner. This presents a significant difficulty in terms of formulation and delivery (Malik et al., 2017). Also, due to the harsh environment encountered in the gut, phages can be quickly inactivated if not properly formulated.

30.3.1 Encapsulation of phages in natural polymers Several natural protein and polysaccharide biopolymers have been used for phage encapsulation targeting topical infections (e.g., wounds and burn wounds) and gastrointestinal (GI) infections and disorders (Table 30.2). Proteins, such as collagen, gelatin, and keratin, can act as functional materials, for instance, in wound healing, and have been continuously investigated due to their exceptional features (Kumar, Rajendran, Houreld, & Abrahamse, 2018). They can be used as carriers of different molecules, such as antimicrobial agents, and achieve good wound healing results (Kumar et al., 2018). Collagen, also widely applied as a wound dressing, presents high benefits in increasing the production of fibroblasts, improving the bioavailability of fibronectin, supporting and preserving leukocytes, macrophages, fibroblasts, and epithelial cells (Pallaske, Pallaske, Herklotz, & Boese-Landgraf, 2018). In addition, collagen has a great capacity to maintain the microenvironment status and speed wound healing (Pallaske et al., 2018). Polysaccharides are complex carbohydrates highly applied in several biotechnological fields and are considered unique raw materials due to their low cost and great availability (Tayeb, Amini, Ghasemi, & Tajvidi, 2018). Aside from that, these biopolymers present other interesting properties such as nontoxicity, biocompatibility, biodegradability, chelation, and absorption capacity that support their application as healing agents (Hamedi, Moradi, Hudson, & Tonelli, 2018). Alginate is a natural anionic polysaccharide derived from brown seaweed that is extensively applied to produce a variety of encapsulation systems due to infection and physicochemical properties such as extensive availability, resistance to contamination, and simplicity, combined with a desirable cost-effective production (Messaoud et al., 2016; Traffano-Schiffo, Aguirre Calvo, Castro-Giraldez, Fito, & Santagapita, 2017). This biopolymer has been widely used for healing and regenerating human tissue, particularly for wound dressing, due to its excellent

777

Table 30.2 In vitro clinical phage therapy approaches using proteins and polysaccharides as phage carriers. Type

Reference

Application

Materials

Pathogen

Phage

System

Method

Main outcome

Proteins

Cheng et al. (2018)

Wound healing

Collagen/ polycaprolactone

E. coli

T4

Fibers

Electrospinning

Dini et al. (2014)

Gastrointestinal (GI) delivery

Enterohemorrhagic and Shiga toxinproducing E. coli

CA933P

Microbeads

Homogenization

Shen et al. (2021)

Wound healing

Emulsified pectin (EP) and emulsified alginate gel microbeads Ca-alginate

E. coli

HZJ

Fibers

3D-plotting system

Barros et al. (2020)

Tissue regeneration and infection prevention and control

Ca-alginatenanohydroxyapatite

E. faecalis

vB_EfaS_LM99 (LM99)

Hydrogel

Casting

Zhang et al. (2020)

Wound healing

E. coli

T7

Hydrogel

Casting

Soykut et al. (2019)

GI delivery

Oxidized sodium alginate(OSA), gelatin, and hyaluronic acid (HA) Na-alginate

Polycaprolactone/collagen I B decomposed completely in vivo in 8 weeks with no deleterious effects on muscle or subcutaneous tissues. EP had a higher swelling capacity and slower matrix degradation. EP had better phage protective capability. Encapsulated HZJ phages showed no cytotoxicity and promoted cell proliferation and adhesion compared to free phage. Phage-loaded hydrogels had no effect on osteoblastic cells’ proliferation and morphology, leading to a higher osteogenic and mineralization response. High inhibition of adhesion and colonization to femoral tissues by multidrug-resistant E. faecalis. ABgel had good self-healing properties and improved healing ( . 40% of the wound tissue regenerated).

Bacillus subtilis (BS), S. Enteritidis (SE), S. Typhimurium (ST)

Microcapsules

Spray drying

Phage stability increased after encapsulation.

´ Sliwka et al. (2019)

GI delivery

Mannitol-alginate

E. coli

BS (φ19 5; F11 1; 6 1; 6 2); SE (F5 3; F5 4; AK-1; AK-2; AK-3 1; Lake2 2); ST (Lake2 1; IC B1 1) T4

Macrospheres

Extrusion

Moghtader, Æ Egri, and Piskin (2017)

GI delivery

Ca-alginate (coatings with chitosan and polyethyleneimine)

E. coli

T4

Beads

Extrusion

Encapsulated T4 phage titers decreased only a little in an acidic environment. Polyethyleneimine coating increased the stability under GI tract conditions.

Polysaccharides

Sarhan and Azzazy (2017) Leung et al. (2016)

Wound healing

Honey, polyvinyl alcohol, chitosan

P. aeruginosa

PS1

Nanofibers

Electrospinning

Pulmonary infections

P. aeruginosa

PEV2, PEV40

Powders

Spray freeze drying (SFD) and spray drying (SD)

Bean et al. (2014)

Wound healing

Trehalose, mannitol, and Lleucine (SprayDried Respirable Powders) Agarose/ Hyaluronan

S. aureus

K

Hydrogel

Casting technique

Ma et al. (2012)

GI delivery

Ca-alginate

S. aureus

K

Microspheres

Extrusion

Matinkhoo, Lynch, Dennis, Finlay, and Vehring, (2011) Kumari, Harjai, and Chhibber (2010) Yongsheng et al. (2008)

Pulmonary infections

Trehalose spraydried particles (Spray-Dried Respirable Powders)

Burkholderia cepacia (KS4-M KS14) and P. aeruginosa (KZ, D3)

KS4-M, KS14, and cocktails of phages KZ/D3 and KZ/D3/ KS4-M

Powders

Spray-drying

Wound healing

Hydroxy propyl methyl cellulose

K. pneumoniae

Kpn5

Hydrogel

Casting technique

GI delivery

Chitosanalginate—pig gut

S.Typhimurium DT104

Felix O1

Microspheres

Extrusion

HPCS-BV/PS1 combined with bee venom had the highest antibacterial activity. SD method caused less phage reduction, and the resulting powders achieved better aerosol performance for PEV2 Hyaluronidase aided phage release by degrading the hyaluronic acid methacrylate in the top layer of the hydrogel, allowing bacteria to be killed. Protective encapsulating agents increased phage viability during drying. Encapsulation increased phage survivability upon exposure to simulated gastric conditions. Spray-drying with sugars at low temperatures (40 C 45 C) avoided phage inactivation and produced an aerodynamic particle diameter of ,3 μm, appropriate for delivery to the lungs. Single-dose of phage significantly reduced mice mortality.

Encapsulated phage titers only decreased 0.67 logs at pH 2.4 and were fully maintained after 3 h exposed to bile solutions.

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CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery

properties, including bacteriostatic, antiviral, fungistatic, nontoxic, high absorbent, nonallergic, breathable, hemostatic, and biocompatible (Raus et al., 2021; Tavakoli & Klar, 2020). Some previous reports already demonstrated that using the combination of alginate and gelatin had numerous attractive features (e.g., biocompatibility and biodegradability) for various biomedical applications, especially for wound dressings (Sarker et al., 2014). It has also been shown that alginates can interact with other natural polyelectrolytes such as pectin and form strong complexes and create hydrogels in the presence of divalent cations (Sandoval-Castilla, Lobato-Calleros, Garcı´a-Galindo, Alvarez-Ramı´rez, & Vernon-Carter, 2010). This interaction can improve alginate beads’ mechanical and chemical stability and raise the encapsulation efficiency, for instance, of orally administered compounds (Sandoval-Castilla et al., 2010). Alginate presents some problems, including poor cell material interactions and poor biodegradability (Leal-Egan˜a, Dietrich-Braumann, Dı´az-Cuenca, Nowicki, & Bader, 2011). Thus alginate blends with other polymers (e.g., gelatin) are used to create covalent cross-linking to overcome this problem (Sarker et al., 2014). The alginate chitosan interaction has been systematically investigated to create a more effective bead for drug delivery (Bhattarai, Shrestha, & Dhandapani, 2011). For instance, alginate is known to shrink in low pH and dissolve in higher pH. On the other hand, chitosan dissolves at low pH and is insoluble in higher pH ranges (Takka & Gu¨rel, 2010). The alginate-chitosan complexation minimizes leakage of the encapsulated compound from the produced beads (Bhattarai et al., 2011). Agarose is extensively used as scaffolds in tissue engineering owing to its exceptional biocompatibility combined with a great tissue-mimicking performance (Go´rski, Borysowski, et al., 2020; Go´rski, Mie˛dzybrodzki, et al., 2020). Moreover, this polymer’s functional groups and stiffness can preserve cellular adhesion and propagation. Also, it has a modifiable water absorption aptitude, giving cells the capacity for a suitable activity (Salati et al., 2020). However, agarose used as hydrogels has some drawbacks, such as lack of cell adhesiveness and poor biodegradability (Roach, Nover, Ateshian, & Hung, 2016), and thus often combined with other polymers to circumvent these disadvantages (Salati et al., 2020).

30.3.2 Phage encapsulation for wound healing applications The regeneration and healing of damaged skin can be supported by biomaterials (Murray, West, Cowin, & Farrugia, 2019). However, the implanted biomaterials are seen as foreign materials being recognized by myeloid cells that attempt to eliminate them (Sheikh, Brooks, Barzilay, Fine, & Glogauer, 2015). These signals are received by the adaptive immune system, leading to rejection or chronic inflammation. Therefore although the idyllic biomaterial for wound healing should reduce foreign body and rejection responses, it should also not impede the inflammatory responses having a key role in the healing process (Liu et al., 2020). Thus biomaterials can be manipulated to increase the host’s healing

30.3 Bacteriophage encapsulation

capacity (e.g., tissue regeneration by the polarization of macrophages toward a regenerative, antiinflammatory, constructive M2 macrophage phenotype) (Lee, Byun, Madhurakkat Perikamana, Lee, & Shin, 2019; Wu et al., 2019). The protein-based polycaprolactone/collagen I nanofibers (PCL-ColI), varying in PCL and ColI concentrations, have been used to incorporate phage T4 to eradicate E. coli infections and undertake wound hemostasis (Cheng et al., 2018). Homeostatic studies demonstrated that PCL-ColI membranes with more than 70% collagen were more homeostatic, having significantly shorter homeostatic times combined with a smaller amount of bleeding. The PCL-ColI membranes with 30% of PCL and 70% of collagen had the best antibacterial effect in vitro, and the in vivo biocompatibility assays revealed that they presented the fastest degradation time. Inflammatory and necrotic cells were found in the surrounding wound area after 1 week of implantation in mice, and myofibroblast and hair follicle tissues were recovered 8 weeks later. In a recent study, a locally isolated phage named HZJ was incorporated into alginate hydrogel fibers and used to create three-dimensional (3D) wound dressings with potential antibacterial properties to target H5α E. coli (Shen et al., 2021). Phages were encapsulated in a hydrogel matrix to ensure a proper tail orientation and availability for targeting the bacteria host effectively. Results demonstrated that 85% and 90% of the lytic activity of phages was preserved after encapsulation. Indeed, the phage-loaded alginate samples released 10% of their encapsulated phage particles within 24 h. Also, compared to phage-free samples, phage-loaded hydrogel fiber samples presented fewer E. coli cells after 2 h of exposure to the samples. Moreover, the presence of HZJ phages inside the alginate hydrogel fibers showed no cytotoxicity levels, promoting cell proliferation and adhesion significantly juxtaposed to phage-frees (P , .01). Mammalian cells have no receptors that interact with alginate molecules, and so alginate dressings are less likely to stick to wounds, causing discomfort during dressings changes (Lee & Mooney, 2012). The fibrous structure of the alginate hydrogels makes them excellent for removing exudate from wounds and enables a controlled release of therapeutic substances (Yang et al., 2018). Also, a phage-based 3D hydrogel dressing released phages slowly and suppressed the growth of bacteria for 24 h (Shen et al., 2021). An alginate-nanohydroxyapatite (Alg-nanoHA) hydrogel was used to encapsulate LM99 phage for local phage delivery with tissue regeneration properties and infection control of multidrug-resistant Enterococcus faecalis (Barros et al., 2020). The data obtained in this work showed that LM99 phage was efficiently encapsulated inside the Alg-nanoHA hydrogels by ionic cross-linking without compromising phage structure and viability. Also, phage viability in the hydrogels remained steady for up to 7 days, and they were successfully released from the composites. Furthermore, in vivo studies in a rabbit model showed good biocompatibility and safety for subcutaneously implanted hydrogels. In addition, phages and hydrogels did not hinder osteoblastic cells’ viability, proliferation, and morphology in vitro (Barros et al., 2020).

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CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery

Another work developed an injectable hydrogel (ABgel) composed of oxidized sodium alginate (OSA), hyaluronic acid, and gelatin to encase acidic fibroblast growth factor (aFGF) and phage T7. This hydrogel had antibiosis and skin regenerating features, ideal for fighting antibiotic-resistant bacteria and improving wound regeneration (Zhang et al., 2020). The rapid hydrogel formation caused by the gelatin and OSA reaction formed stable hydrogels with exudate-absorption capacity while providing a biocompatible microenvironment for aFGF and phage. In vitro tests demonstrated that ABgel improved wound healing, with wounds gradually improving after 3 days, and more than 40% of the wound tissue regenerated. Moreover, the antimicrobial activity of ABgel significantly inhibited microbial growth with no undetectable bacterial levels in wounds after 7 days (Liu et al., 2020). Chitosan is another biopolymer used in wound healing due to its biocompatibility, biodegradability, hemostatic activity, and a great capacity to accelerate tissue regeneration (Dai, Tanaka, Huang, & Hamblin, 2011). A study developed nanofibers composed of honey, polyvinyl alcohol, chitosan (HPCS) to be applied as wound dressings (Sarhan & Azzazy, 2017). The nanofibers produced by the electrospinning technique were loaded with bee venom (BV), propolis, or P. aeruginosa specific phage (PS1). BV and phage combined nanofibers (HPCS-BV/PS1) had enhanced antibacterial activity and the highest biocompatibility and wound healing activity (Sarhan & Azzazy, 2017). Furthermore, this formulation showed the best antibacterial activity against all strains tested and reached a nearly total killing of multidrug-resistant P. aeruginosa. An agarose-hyaluronic acid methacrylate (HAMA) hydrogel system was produced to release S. aureus phage K in the event of hyaluronidase’s (HAase) presence (Bean et al., 2014). HAMA was used as a barrier to prevent the early release of phages from the agarose layer without HAase. Although a few phages passed through the hydrogel pores, the vast majority were preferentially released in the presence of HAase. One interesting result of this work was that, in the presence of a non-HAase generating bacterial strain, the HAMA layer remained intact, indicating that this hydrogel system uses a semiautomated trigger-release treatment (Bean et al., 2014).

30.3.3 Phage encapsulation to prevent and manage gastrointestinal diseases Poor bioavailability is still a serious concern for many drugs and vaccines delivered through the oral route due to their low solubility, degradation by the stomach’s harsh acidic environment, or proteolytic enzymes of the GI tract (George, Shah, & Shrivastav, 2019). Therefore the application of biodegradable polymers such as alginate and chitosan, among others, in the development of nontoxic and efficient oral carrier systems has been regarded as one of the most popular options for oral drug delivery, circumventing the bioavailability drawbacks (Yan, Zhou, Yu, Jin, & Zhao, 2020).

30.3 Bacteriophage encapsulation

Low-methoxylated pectin emulsified microbeads with oleic acid have high phage encapsulation efficiency protecting phages against acidic conditions (Dini, Islan, de Urraza, & Castro, 2012). A comparison between emulsified lowmethoxylated pectin and alginate microbeads to deliver phages orally demonstrated that the emulsified pectin resulted in harder and more cohesive gel microbeads than emulsified alginate (Dini et al., 2014). Emulsified alginate was the least resistant to acidity but had a higher encapsulation efficiency. In aqueous conditions, emulsified pectin demonstrated significant swelling capacity and slower matrix degradation, indicating that the oleic acid is mostly found on the surface of emulsified pectin microbeads (Dini et al., 2014). Furthermore, the emulsified pectin matrix had a higher phage protective capacity than the emulsified alginate in acidic environments. In addition, the formation of the oleic acid coating in pectin matrices enhances matrix stiffness. These authors conducted modeling studies, concluding that this encapsulation system is plausible to be generalized for the controlled release of high molecular weight chemicals, drugs, and therapeutic agents (Dini et al., 2014). T4 phage was encapsulated inside modified alginate beads coated with two polycations polymers (chitosan or polyethyleneimine) to increase phage stability for a controlled release in the GI compartment (Moghtader et al., 2017). Phages encapsulated in these beads achieved over 90% loading capacities. The coating with those the polycationic polymers increased phage stability under simulated GI tract conditions [e.g., simulated gastric fluid (SGF) and bile salts], especially in the polyethyleneimine coating. Tests demonstrated that phages were released from the beads, which were active at basic pH. The results further concluded that polyethyleneimine was considered a better coating than chitosan, having increased phage stability at low pH values and achieving higher phage release rates (Moghtader et al., 2017). The conjugation between alginate and polyethyleneimine also leads to an ionic interaction forming a polyelectrolyte complex membrane, which can act as a physical obstacle to drug release from the alginatepolyethyleneimine beads (Setty, Sahoo, & Sa, 2005). The blocking of the surface pores possibly explains the increased membrane thickness and consequent reduction of the swelling of the beads resulting in a prolonged release of the drug (Setty et al., 2005). In a different approach, mannitol-alginate-dried macrospheres were used to ´ et al., 2019). The main objecencapsulate phage T4 for GI applications (Sliwka tive was to produce microspheres to ensure the great viability of phages during the harsh conditions to which they are exposed in the digestive system. The viability of T4 phages internalized in mannitol-alginate wet and dried formulations were studied for up to 4 weeks of storage at room temperature. Then, this preparation was tested in simulated GI conditions with an acid environment (pH 2.5), where they revealed a slight decrease in their number (5.02 3 1010 PFU/mL). The results showed that adding 0.3 M mannitol might improve phage protection against the severe GI environment and enable many T4 phages to ´ remain active (Sliwka et al., 2019). This study showed that mannitol-alginate-dried

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CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery

microspheres could be considered a carrier for oral phage therapy applications to reduce intestinal colonization of pathogens and be a good future designed technology for the future. Different Bacillus subtilis, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis), and S. enterica subsp. enterica serovar Typhimurium (S. Typhimurium) phages microencapsulated using sodium alginate were evaluated (Soykut et al., 2019). Microencapsulation prevented phages in a simulated GI, stomach, and bile salt environment. Nonetheless, phage titer decreases were observed with a maximum decrease of 2.29 logs for B. subtilis phages, 1.71 logs for S. Enteritidis phages, and 0.60 logs for S. Typhimurium phages. In contrast, free phages were inactivated already after 15 min of incubation. Besides, the stability of phages was increased after being microencapsulated inside the alginate microcapsules, with high stability levels for more than 3 h of incubation in a bile salt medium. Phage titer discrepancies between microencapsulated and free phages reached 3 log units (Soykut et al., 2019). An oral delivery system composed of calcium carbonate (CaCO3) microparticles co-encapsulated with phage K into alginate microspheres was developed to control intestinal colonization of S. aureus (Ma et al., 2012). Encapsulated phage K titers decreased 2.4 log units compared to the free phage, totally degraded upon exposure to SGF (1 h, pH 2.5). In addition, coencapsulation of phage K with CaCO3 protected phage K in acidic environments compared to encapsulation in alginate alone. Applying CaCO3 as an antacid excipient to the alginate microspheres enhanced the persistence of loaded phage K in the acid conditions with a reduction of only 0.17 log units after 2 h of exposure to SGF at pH 2.5. A possible explanation for this improvement is that the CaCO3 increases the strength of the alginate gel networks causing a more neutral microclimate inside the microspheres. In the last decades, CaCO3-based microparticles have been employed as a novel delivery carrier for drugs and bioactive compounds due to their good sustained release and high stability (Qiu et al., 2012). Protective compounds such as trehalose, sucrose, skim milk, and maltodextrin were evaluated and shown to increment the viability of encapsulated phage K when dried, varying with the type and concentration of each incorporated additive (Ma et al., 2012).

30.4 Conclusions and future perspectives Phage therapy is a promising technique because phages can kill infectious superdrug-resistant pathogens. Nevertheless, phage therapy is only successful if the phage formulation has activity toward the infectious bacteria due to the limited host range. This demands a careful identification of the pathogens before phage administration. Furthermore, the loss in viability throughout storage and the conditions they need to withstand once administered in humans can restrict the medical use of phages on a large scale. Besides, the target-site-specific

30.4 Conclusions and future perspectives

delivery and pharmacokinetics may make recurrent administration necessary. Antibody-mediated phage inactivation and clearance by the recipient’s reticuloendothelial system may also occur. For this reason, expanding our understanding of phage biology offers the most promising chances for the effective incorporation of phage treatment as an alternative or supplementary therapeutic method. Studies have shown that pure phages are unstable in suspension and lose their activity when processed and stored. Thus their integration into various biomaterial constructions is important to allow their use in treating various infections. Phage delivery needing to pass through the gut necessitates using an acid-resistant material, such as alginate, for an effective phage deposition. Varied phage-specific responses can occur when phages are embedded, encapsulated, or adsorbed in or onto carrier materials and exposed to harmful conditions. Before any phage can be placed on or onto a biomaterial, it must undergo extensive compatibility and characterization tests. Nonetheless, given the bacterial resistance to antibiotics, new and innovative methods of releasing phages are likely to emerge in clinical trials and, ideally, in clinics to help reduce the burden of infection on global health. Furthermore, as a synergistic approach, phages can be employed in conjunction with antibiotics. The two combined agents may act additively, meaning that their combined efficacy is equivalent to the total of their separate effects. Also, synergistically, they can work together, resulting in greater efficacy than when used separately. Adopting a combination strategy may improve bacterial eradication, allow a more efficient diffusion of both into bacterial biofilms, and a reduction in the risks of phage and bacterial resistance development can be achieved, thus extending the therapeutic utility of antibiotics. However, few conclusions have been reached so far about this synergy between antibiotics and phages since antibiotics with identical mechanisms of action, such as suppression of cell wall synthesis, may have different results when coupled with certain phages, and should be further studied. Natural products derived from microbial, animals, or plants with antimicrobial properties, such as honey and BV, have also been used against antibiotic-resistant bacteria. BV, for instance, that has been encapsulated together with phages, has antioxidant, antibacterial, antiviral, and analgesic properties, being used to treat a variety of diseases with great ability to improve wound healing. The use of phages and honey in combination is advantageous because it gathers the antibiofilm activity of honey and the ability of phages to lyse specific bacteria. Therefore the combined delivery of phage and natural antimicrobials is a promising antimicrobial alternative (Alves, Cerqueira, Pastrana, & Sillankorva, 2020; Oliveira et al., 2017; Oliveira, Sousa, Silva, Melo, & Sillankorva, 2018; Pimchan, Cooper, Eumkeb, & Nilsson, 2018) that can provide additional synergic benefits. However, as with most antimicrobials, several factors can impact their efficacy, including tissue penetration, maximum plasma concentration, and bioavailability, that require investigation. In Table 30.2, numerous studies are included where encapsulated phages were used. Unfortunately, the majority are in vitro studies, and only a few test the phage formulations in vivo. This poses an issue because, even though numerous

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research has demonstrated the therapeutic potential of phages in vitro, more evidence is needed to provide a strong regulatory case for clinical usage. Further in vivo studies are necessary to validate the promising in vitro results. Another issue with phage treatment is the regulatory restrictions that exist in the pharmaceutical industry. The laws for conventional medical products are problematic for phage-based products, so new guidelines for producing and creating effective phage products and their administration should be created.

Funding Ana Mafalda Pinto acknowledges the Portuguese Foundation for Science and Technology (FCT) grant SFRH/BD/138138/2018. SS acknowledges national funding by FCT, through the individual scientific employment program (2020.03171.CEECIND). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit.

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Sarhan, W. A., & Azzazy, H. M. E. (2017). Apitherapeutics and phage-loaded nanofibers as wound dressings with enhanced wound healing and antibacterial activity. Nanomedicine: Nanotechnology, Biology, and Medicine, 12(17), 2055 2067. Available from https://doi.org/10.2217/nnm-2017-0151. Sarker, B., Papageorgiou, D. G., Silva, R., Zehnder, T., Gul-E-Noor, F., Bertmer, M., . . . Boccaccini, A. R. (2014). Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties. Journal of Materials Chemistry B, 2(11), 1470 1482. Available from https://doi.org/10.1039/ c3tb21509a. Sarker, S., Sultana, S., Reuteler, G., Moine, D., Descombes, P., Charton, F., . . . Bru¨ssow, H. (2016). Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: A randomized trial in children from Bangladesh. EBioMedicine, 4, 124 137. Available from https://doi.org/10.1016/j.ebiom.2015.12.023. Setty, C. M., Sahoo, S. S., & Sa, B. (2005). Alginate-coated alginate-polyethyleneimine beads for prolonged release of furosemide in simulated intestinal fluid. Drug Development and Industrial Pharmacy, 31(4 5), 435 446. Available from https://doi. org/10.1080/03639040500214647. Sheikh, Z., Brooks, P. J., Barzilay, O., Fine, N., & Glogauer, M. (2015). Macrophages, foreign body giant cells and their response to implantable biomaterials. Materials, 8(9), 5671 5701. Available from https://doi.org/10.3390/ma8095269. Shen, H. Y., Liu, Z. H., Hong, J. S., Wu, M. S., Shiue, S. J., & Lin, H. Y. (2021). Controlled-release of free bacteriophage nanoparticles from 3D-plotted hydrogel fibrous structure as potential antibacterial wound dressing. Journal of Controlled Release, 331. Available from https://doi.org/10.1016/j.jconrel.2021.01.024. ´ Sliwka, P., Mituła, P., Mituła, A., Skaradzi´nski, G., Choi´nska-Pulit, A., Niezgoda, N., . . . Skaradzi´nska, A. (2019). Encapsulation of bacteriophage T4 in mannitol-alginate dry macrospheres and survival in simulated gastrointestinal conditions. LWT, 99, 238 243. Available from https://doi.org/10.1016/j.lwt.2018.09.043. Soykut, E. A., Tayyarcan, E. K., Evran, S., ¸ Boyacı, ˙I. H., C ¸ akır, ˙I., Khaaladi, M., & Fattouch, S. (2019). Microencapsulation of phages to analyze their demeanor in physiological conditions. Folia Microbiologica, 64(6), 751 763. Available from https://doi. org/10.1007/s12223-019-00688-1. Takka, S., & Gu¨rel, A. (2010). Evaluation of chitosan/alginate beads using experimental design: Formulation and in vitro characterization. AAPS PharmSciTech, 11(1), 460 466. Available from https://doi.org/10.1208/s12249-010-9406-z. Tavakoli, S., & Klar, A. S. (2020). Advanced hydrogels as wound dressings. Biomolecules, 10(8), 1 20. Available from https://doi.org/10.3390/biom10081169. Tayeb, A., Amini, E., Ghasemi, S., & Tajvidi, M. (2018). Cellulose nanomaterials—binding properties and applications: A review. Molecules (Basel, Switzerland), 23(10), 2684. Available from https://doi.org/10.3390/molecules23102684. Tibbitt, M. W., Dahlman, J. E., & Langer, R. (2016). Emerging frontiers in drug delivery. Journal of the American Chemical Society, 138(3), 704 717. Available from https:// doi.org/10.1021/jacs.5b09974. Traffano-Schiffo, M. V., Aguirre Calvo, T. R., Castro-Giraldez, M., Fito, P. J., & Santagapita, P. R. (2017). Alginate beads containing lactase: Stability and microstructure. Biomacromolecules, 18(6), 1785 1792. Available from https://doi.org/10.1021/ acs.biomac.7b00202.

References

Voelker, R. (2019). FDA approves bacteriophage trial. The Journal of the American Medical Association, 321(7), 638. Available from https://doi.org/10.1001/ jama.2019.0510. ¨ ngga˚rd, E. E., & Harper, D. R. (2009). A controlled clinical Wright, A., Hawkins, C. H., A trial of a therapeutic bacteriophage preparation in chronic otitis due to antibioticresistant Pseudomonas aeruginosa: A preliminary report of efficacy. Clinical Otolaryngology, 34(4), 349 357. Available from https://doi.org/10.1111/j.17494486.2009.01973.x. Wu, R. X., He, X. T., Zhu, J. H., Yin, Y., Li, X., Liu, X., & Chen, F. M. (2019). Modulating macrophage responses to promote tissue regeneration by changing the formulation of bone extracellular matrix from filler particles to gel bioscaffolds. Materials Science and Engineering C, 101. Available from https://doi.org/10.1016/j. msec.2019.03.107. Yan, X., Zhou, M., Yu, S., Jin, Z., & Zhao, K. (2020). An overview of biodegradable nanomaterials and applications in vaccines. Vaccine, 38(5), 1096 1104. Available from https://doi.org/10.1016/j.vaccine.2019.11.031. Yang, K., Han, Q., Chen, B., Zheng, Y., Zhang, K., Li, Q., & Wang, J. (2018). Antimicrobial hydrogels: Promising materials for medical application. International Journal of Nanomedicine, 13, 2217 2263. Available from https://doi.org/10.2147/IJN. S154748. Yongsheng, M., Pacan, J. C., Wang, Q., Xu, Y., Huang, X., Korenevsky, A., & Sabour, P. M. (2008). Microencapsulation of bacteriophage Felix O1 into chitosan-alginate microspheres for oral delivery. Applied and Environmental Microbiology, 74(15), 4799 4805. Available from https://doi.org/10.1128/AEM.00246-08. Zhang, J., Ge, J., Xu, Y., Chen, J., Zhou, A., Sun, L., . . . Ning, X. (2020). Bioactive multiengineered hydrogel offers simultaneous promise against antibiotic resistance and wound damage. International Journal of Biological Macromolecules, 164. Available from https://doi.org/10.1016/j.ijbiomac.2020.08.247.

793

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acetobacter, 83
84 Acetobacter xylinum, 83
84 Acidic fibroblast growth factor (aFGF), 782 Acinetobacter baumannii, 376 Acrylamides (AAm), 578, 681
682, 760
761 Acrylic acid (AA), 19, 161, 681
682, 754 Acrylonitrile butadiene styrene (ABS), 133
134, 160, 315 Activated carbon, 273
274 Actuators used on conducting polymers, 267
272 Additive manufacturing (AM), 19
20, 159, 319
321, 336
337, 469 Adeno-associated virus (AAV), 513
514 Adenocarcinoma cells, 9
10 Adenovirus serotype 5 (Ad5), 524 Adsorption method, 626 Aerogels, 675
676 Agarose, 780 Agarose-hyaluronic acid methacrylate hydrogel system (Agarose-HAMA hydrogel system), 782 Aggrecanase-degradable hydrogel, 239 Agriculture and food processing, 647
648 Agrobacterium, 72
74, 83
84 Alginate, 69
72, 111
113, 134
135, 353
355, 479
481, 635, 777
780, 782 alginate-based self-assembled nanoparticles, 353
355 alginate/BC hydrogel, 94 nanogel, 231 Alginate-nanohydroxyapatite hydrogel (AlgnanoHA hydrogel), 781 Aliphatic urethane diacrylate (AUD), 170 Alkaline phosphatase (ALP), 693 Aluminum (Al), 36 Alzheimer’s diseases, 238
239 Ambient parameters, 440 American society of testing and manufacturing (ASTM), 321
322 Ammonia sensor, 627 Ammonium persulphate (APS), 677
679 Amoxicillin, 483 Amperometric biosensor, 620 Amygdalin (AD), 752 Amylopectin, 401
403 Analytes, 618 Angiogenesis, 542
544

Anionic clays, 624
625 Antibacterial hemostatic activities (AHAs), 235
236 Antibody-based biosensors, 628 Anticancer drugs, 78 Antifouling, 576 mechanism, 567
569 natural, 568
569 strategies, 567
568 Antimicrobial peptides (AMPs), 595
597 Antiplatelet therapy, 382 Antiviral peptide polymer, 595
597 Antiviral polymers (AVPs), 592
601, 603
604 application of, 601
608 drug delivery system, 601
603 food packaging, 607
608 polymers in protective application, 603
606 classification of virus, 592t Aptamers, 618 aptamer-based biosensors, 625 Aqueous electrolytes, 276 Arachidonic acid, 549
550 Arginine-glycine-aspartic acid sequence (RGD sequence), 443 Articles, analysis of citations of, 75
80, 77t Aspergillus niger, 250
251 Atherosclerosis diseases, 238
239 Atomic force microscopy (AFM), 400
401, 419
424 flexural testing, 421
423 FTIR analysis, 419
420 Izod impact test, 423
424 tensile testing, 420
421 thermogravimetric analysis, 424 Autoclave forming of thermoplastic composites, 406
407 Automated fiber/tape placement process, 57
58 Automated tape laying (ATL), 57
58 Azobenzene, 234 Azotobacter, 69
72

B Bacillus, 508 B. subtilis, 784 B. thuringiensis, 776 Bacterial cellulose (BC), 72
74, 83
84, 96, 662
663 bacterial cellulose-AuNPs, 98
99

795

796

Index

Bacterial cellulose (BC) (Continued) BC-Ag nanocomposites, 101
102 BC-MMT composites, 86 BC-TA-Mg composites, 104 BC-ZnO nanocomposite films, 90 BC/BS biocomposite, 92 BC/ZnO nanocomposite films, 91
92 biocomposite scaffolds, 95 biomedical applications of, 84
105, 86t films, 103
104 Bacterial cellulose and chitosan (BC-Chi), 92 Bacterial cellulose whiskers (BCW), 93 Bacterial cellulose-silver sulfadiazine composites (BC-AgSD), 102
103 Bacterial cellulose/alginate (BCA), 94
95 Bacterial cellulose/silk fibroin (BC/SF), 97 Bacterial membrane (BM), 92 Bacterial nano-cellulose (BNC), 144
145 Bacteriophage, 769
770 encapsulation, 776
784 phage encapsulation for wound healing applications, 780
782 phage encapsulation to prevent and manage gastrointestinal diseases, 782
784 of phages in natural polymers, 777
780 Baker
Lonsdale model, 476 Banana/sisal fiber, 415
416 Bee venom (BV), 782 Benzophenone (BP), 170 β-cyclodextrin (β-CD), 754 β-thiopropionate cross-linker, 571 Bifidobacterium B. bifidum, 644
645 B. longum, 636 B. pseudolongum, 636 Bilayered cryogels, 690
691 Binders, 277 jetting 3D printing, 142
143 Bio-imaging, 755
758 Bio-sensing, 348, 356 Bioabsorbable polymer, 41 Bioactives, 473 Bioaffinity sensors, 620
621 Bioavailability synthesis, 645
646 Bioceramics for gene delivery, 526
531 Biocompatibility, 1, 7, 40, 191 on skin, 51 Biocompatible materials, functionalization of shape-memory polymers by, 307
308 Biocomposites, 42
43, 92. See also Composites applications of, 43
53 biomedical composites, 40 biomedical polymer composites, 42
43 from biowaste materials, 716

and classification, 40
43 requirements and parameters for biomedical applications, 40
42 Biodegradability, 7 polymer, 41 Biodegradable matrices, 401
404 modification or recycling polymer matrices, 404 Biodegradable polymers, 41, 45
46, 401
403, 782 Biodegradation, 528
529 of hydrogel, 191 Biofilm, 563
565 reactors, 635 Biofouling, 563
565 Biogenic-derived biocomposites, 716
721 characterization, 718
721 materials, 716 preparation of, 716
721 Bioinks, 186
188 Biological oxygen demand sensor (BOD sensor), 627
628 Biomaterials, 1
2, 109
110, 143
144, 205
206, 657
658, 711 Biomedical antifouling polymer nanocomposites, 569
576 Biomedical application(s), 131
133, 165
166, 746 biogenic-derived biocomposites for, 716 CNTs-based composites for, 659
665 cryogels, 686
704 Biomedical composites, 40, 43
45 fabrication techniques of, 54
58 Biomedical polymer composites, 42
43 classification of polymers and composites, 2
3 fabrication techniques polymer composites, 3
7 natural biomedical composites, 42
43 polymers and composites for biomedical applications, 7
22 synthetic biomedical composites, 43 Biomolecular component of biosensor, 625
626 Biopolymers, 61
62, 76
77, 554, 618, 713, 769 biopolymer-based composites for drug delivery, 61
62 biopolymer-based hydrogels, 663
664 for development of biosensors, 622
625 matrices, 552 Bioprinted hydrogels in tissue engineering and drug screening, 209
210 Bioprinting, 183
184 parameters, 186 speed, 185
186 technology, 183
186, 193
196 Bioreceptors, 620
621 Bioremediation, 635

Index

Bioresorbability analysis, 735
736 Biosensors, 617 biomolecular component, 625
626 biopolymers for development, 622
625 characteristics, 621
622 classification, 618
621 CNTs nanocomposites for, 660
661 fundamentals, 618 principle, 618 merits and limitation of, 630 recent applications of, 627
630 recent trends in, 626
627 Bioseparation process, cryogels in, 687
690 Biowaste, 713 Bladder inflation molding (BIM), 407 Blank-holders, 409 Bombyx mori. See Silkworms (Bombyx mori) Bone, 446, 534
535 cement, 46
47 formation process, 713 grafting, 49 healing, 100, 483 of human body, 45
46 regeneration, 483 tissue engineering, 198, 401
403 tissue regeneration, 483 Bone marrow mesenchymal stem cells (BMSCs), 13 Bone morphogenetic proteins (BMPs), 534 Bovine serum albumin (BSA), 662
663 Bragg’s equation, 470
471 Brain-derived neurotropic factor (BDNF), 441 Brunauer
Emmett
Teller theory (BET theory), 683
684 Buccal drug delivery, 479
482 polymer composites for buccal drug delivery, 482t 1,4-butanediol diglycidyl ether (BDDGE), 445
446

C Caffeic acid, 548
549 Calcium carbonate (CaCO3), 517
518, 784 Calcium fluoride, 47 Calcium phosphate (CaP), 517
518, 662
663 calcium phosphate
based bioceramic material, 711 cement, 51
53 glass, 134
135 Calcium silicate, 47 Calcium spirulan, 595 Calixarene, 238
239 Cancer, 478, 531
532 CNTs nanocomposites for cancer treatment, 663

prevention, 646
647 therapy, 755
758 Candesartan conjugation (CD), 663 Candida albicans, 101
102 Capillary encapsulation method, 639
640 Carbon, 405
406 carbon-based biopolymers, 624 functionalization of shape-memory polymers by carbon fillers, 306
307 grease, 269 materials, 273
274 nanomaterials, 657
658 nanoparticles, 657
658 nanospheres, 624 power, 269 sphere, 273
274 Carbon black (CB), 364
365 Carbon fiber-reinforced composite (CFR composite), 50 Carbon fibers (CFs), 46, 277
279, 306 polysulfone composites, 46 Carbon nanofibers (CNFs), 306 Carbon nanotubes (CNTs), 269, 625, 657
658 based composites for biomedical applications, 659
665 CNTs nanocomposites for biosensors, 660
661 CNTs nanocomposites for cancer treatment, 663 CNTs nanocomposites for drug delivery, 661
663 CNTs nanocomposites for tissue engineering, 663
665 CNT-glucose biosensors, 660
661 CNT/PANI hybrid film, 272 fibers, 270
272 toxicity of, 665 Carbon spacer lengths (CSLs), 578 Carbon-based nanomaterials (CBNs), 618, 624, 658 Carboxybetaine (CBs), 579
581 Carboxylic acid, 681
682 Carboxymethyl cellulose (CMC), 11
12, 479
481, 713
714 Carboxymethyl chitosan (CMCh), 228 Cardiac tissue engineering, 203 use of electro-spun fibers in, 453
455 Cardiomyocytes (CMs), 441 Cardiovascular diseases (CVD), 381, 532
533 Cardiovascular gene therapy, 532
533 Carnosol, 548
549 Carrageen, 595 Carrageenan, 594
595 Cartilage tissue, 239

797

798

Index

Cartilage tissue (Continued) engineering, 198
203 Carvacrol, 546 Catheters, 565 Cationic poly (2-(dimethylamino) ethyl acrylate) (PDMAEA), 527
528 Cationic polymers, 479
481 Caudovirales, 770 Cell affinity chromatography, 693
694 cell
biomaterial interactions, 366 cryogels in cell separations, 693
695 culture substrates, 446 encapsulation, 635
636 transplantation, 635 viability, 204
205 Cellulose, 10
12, 67
69, 72
74, 83
84, 353
355 fibers, 399
400 hydrogel membranes, 10
11 Ceramic particle
dispersed polymer composites, 401. See also Naturally derived ceramics
polymer composite characterization, 416
424 atomic force microscopy, 419
424 Charpy impact strength test, 418
419 structural properties, 416
418 curing of composites, 411
412 different types of, 412
416 fabrication of ceramic particulate
dispersed composites, 406
410 matrices used in ceramic particle
dispersed polymer composites, 401
405 reinforcements used in, 405
406 Ceramics, 19, 47, 132 Cerebrovascular disease, 381 Chain mobility, 301
302 Charge storing method of conducting polymers, 272
273 Charging
discharging mechanism, 272 Charpy impact strength test, 418
419 Chemical exfoliation mechanism, 270
272 Chemical oxidation polymerization method, 273
274 Chitin, 446 Chitosan, 9
10, 67
69, 111
113, 134
138, 228, 231
232, 247
248, 250
251, 253
254, 353
355, 446
447, 452, 479
481, 593, 598, 647, 681
682, 713, 782 chitosan-based nanoparticles, 247
248 chitosan/XG-based mechanically enhanced scaffolds, 506 microencapsulation, 648
649 polyelectrolyte nanocomposite system, 353
355

polysaccharide, 250
251 properties, 250
254 Chitosan, 1,3-diethyl-2-thiobarbituric acid (CSDETBA), 9
10 Chitosan nanoparticles (CSNPs), 80, 252
253, 441, 639 Chitosan-1,3-diethyl-2-thiobarbituric acid-PCL (CS-DETBA-PCL), 13 Chondrocytes, 647 cells, 96
97 Chromatographic separation, 687
688, 693
694 Chymotrypsin inhibitor 2 (CI2), 580
581 1,8-Cineole, 549
550 Citalopram-loaded gelatin nanocarriers (CGNs), 452
453 Citrobacter freundii, 90 Clarithromycin, 254
255 Clay, 347, 405 clay-based nanocomposites, 412
415 Closed molding method, 410 Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR-associated (Cas), 532 Coacervation process, 639 Coauthorship analysis, 64
66, 65t, 66t, 67t Coaxial electrospinning method, 553
554 Cobalt, 304
305 Coefficient of thermal expansion (CTE), 270
272 Col1a1 gene, 198 Collagen (Col), 7, 19, 111
113, 135
138, 227, 401
403, 544
546, 550
551, 555
556, 681
682, 777 Collagen/hydroxyapatite (CHA), 19
20 Colorectal cancer (CRC), 646
647 Combinational therapies, 351
352 Composites, 31
32, 35, 66
67, 318
319, 399, 465
466. See also Biocomposites advantages of, 35
38 consolidation of many parts, 37 corrosion resistance, 36 design flexibility, 35
36 dimensional stability, 37 durable, 38 high strength, 36 high-impact strength, 36
37 light weight, 36 low thermal conductivity, 38 nonconductive, 37 nonmagnetic, 37 radar transparent, 37 strength related to weight, 36 applications of, 38
39 aerospace/aircrafts, 38 appliances, 38

Index

automobile and transportation, 38 biocomposites, 43
53 electricity, 39 environmental, 39 infrastructure, 38
39 biocomposites, 40
43 for biomedical applications, 7
22 classification of, 2
3, 3f, 40
43 curing of, 411
412 fabrication methods, 54
58, 406
410 functionalization of shape-memory polymers by history of, 33
35 composite material in daily life, 35 fiberglass in 20th century, 34 limitation of, 39 polymers, 449
450, 450t using shape-memory polymers, 303
308 Compression molding, 56
57, 409
410 Computational studies of biomedical antifouling polymer nanocomposites, 577
581 antifouling properties of polymers using, 577
578 polyzwitterions, 579
581 surface hydration effect on antifouling properties, 578
579 Computer numeric controlled machining (CNC machining), 316 Computer-aided design software (CAD software), 159 Conductimetric biosensor, 620 Conducting polymers, 279
280, 618. See also Thermoresponsive polymers; Shapememory polymers (SMP) electrochromic materials and devices, 285
289 energy harvesting based on polymer, 279
283 energy storage from, 272
279 organic light-emitting diodes, 283
285 sensors and actuators used on, 267
272 Conjugated polyelectrolytes (CPEs), 285 Conjugated polymers, 265
266, 287 Contemporary rapid prototyping systems, 321
329 available rapid prototyping systems, 323
329 FDM, 327 LOM, 327 SLA, 327
329 SLM, 325
326 SLS, 324
325 classification based on type of material used, 324t examples of processes, 323t rapid prototyping process based on form of raw material, 324t Continuous compression molding process, 409

Conventional fabrication process, 337
338 Conventional machining process, 316 Cooccurrence analysis, 66
75 Copolymers, 284
285 Copper (Cu), 273 Coprecipitation-hydrothermal method, 466
467 Cord blood-derived stem cells (CBSCs), 698
699 Cornea, 248
249 Corneal tissue engineering procedures, 447 Coronary artery bypass graft surgery (CABG surgery), 382 Coronary heart disease, 381 Corrosion, 40 Covalent bonding, 682
683 Covalent imprinting technique, 749
750 Coxsackie virus and adenovirus receptor (CAR), 524 Crab shell
extracted hydroxyapatite/poly (vinylpolypyrrolidone)/aloe vera biocomposite, 725
730 antibacterial activity, 729 contact angle measurements, 729 FTIR analysis, 725 mechanical characterizations, 727 SEM analysis, 726 in vitro cytocompatibility analysis, 730 XRD analysis, 726 Critical solution temperature (CST), 225
226, 372 Cross-linking cryogels, 682
683 density, 189
190 hydrogel, 379 method, 626 polymerization method, 675
676 strategy in cryogel preparation, 682
683 Crosslinkers, 191
192 Cryogel(s), 379
380, 675
677 biomedical applications of, 686
704 in bioseparation process, 687
690 in drug release applications, 700
704 in tissue engineering applications, 692
699 in wound dressing applications, 690
692 as bioreactors, 693 in cell separations, 693
695 characterization of, 683
686, 684t cross-linking strategy in cryogel preparation, 682
683 precursors in cryogel preparation, 681
682 preparation method, 677
680 morphology and characteristic, 681t tissue scaffolds, 695
699 Cryopolymerization process, 677
679 Curing, 410 of composites, 411
412

799

800

Index

Cyamopsis tetragonolobus, 506
507 Cyanogen bromide activation method (CNBr activation method), 689
690 Cyanovirin-N, 597 Cystic fibrosis, 565 Cytochrome, 689
690 Cytokines, 549
550 Cytotoxicity, 103
104

D D-glucuronic

acid, 72
74, 688 506
507 Deacetylation, 501
502 Decellularized ECM (dECM), 135
138 Delivery process, 769 Dendrimers, 599
601 Dental field, 47
49 Deoxyribonucleic acid (DNA), 228, 660
661 DNA-based cryogel network, 379
380 DNA-based vaccines, 531
532 Deposition process, 279
280, 344 Dextran (DEX), 353
355, 598, 702
703 dextran-based immobilized laminin cryogel scaffolds, 699 Dextran methacrylate (DEX-MA), 702
703 Dextrin, 598 Diabetes mellitus, 544
546 Dialysis bag approach, 473 Diaphragm forming, 407 Diarrhea, 644
645 Dibutyltin chloride, 599 Diethyl fumarate (DEF), 140
141 Differential scanning calorimetry (DSC), 301
302, 472 Differential thermal analysis, 404 Digital light processing (DLP), 138, 166
167 Dimethyl amino methyl styrene (DMAMS), 605 Direct-ink-write (DIW), 170 Discharge process, 277 Dispersion
deposition mechanism, 277
279 Dopamine (DA), 378
379, 760
761 Doping method, 265
266 Dorzolamide, 255 Droplet bioprinting, 185 Drug delivery systems (DDSs), 61
62, 352
353, 363, 365, 375, 444
445, 592
593, 601
603, 659 applications in drug delivery, 477
483 characterization and drug release properties, 470
477 determination of drug loading into composites, 473 estimation of drug release from composites, 473
474 D-mannopyranose,

Fourier transform infrared spectroscopy, 471 mathematical treatment of drug release kinetics, 474
476 mechanisms for controlling drug release from composites, 476
477 scanning electron microscopy, 472 thermal analysis, 472 X-ray diffraction, 470
471 synthesis of polymer composites, 466
470 Drug loading capacity (DLC), 473 Drug(s), 473, 638, 701
702 delivery, 99
100, 170
177, 224, 226
227, 249, 350
356, 354t, 648
649, 751
755, 777
780 for bone tissue regeneration, 483 CNTs nanocomposites for, 661
663 vehicles, 61
62 discovery studies, 183 drug-composite system, 469 drug-loaded scaffold, 452
453 particles, 365 release cryogels in drug release applications, 700
704 mechanism, 476 system, 171 screening, 206
208, 207t bioprinted hydrogels in, 209
210 studies, 183 Dry dipping method, 274
275 Drying process, 776 Dual responsive smart nanocarriers, 380 Dynamic light scattering, 470 Dynamic mechanical analysis (DMA), 301
302

E Egg shell
derived hydroxyapatite/cellulose nanocrystals biocomposite, 722
725 antibacterial activity, 725 FTIR analysis, 722 mechanical characterization, 725 SEM and EDX investigations, 724 XRD analysis, 723 Elastic forces, 189
190 Elastomer, 32 actuators, 164
165 Electro-spun polymers in neural tissue engineering, use of, 450
455 Electrochemical actuators, 267
270 Electrochemical biosensors, 620, 660
661 Electrochemical oxidation methods, 265
266 Electrochemical polymerization method, 265
266 Electrochromic devices, 285
289 Electrochromic materials, 285
289

Index

Electrochromism, 285
286 Electroluminescence, 283 Electrolyte force strategy, 270 system, 269
270 Electron beam (E-beam), 54 Electron beam melting (EBM), 325
326 Electron transporting layers (ETL), 283, 285 Electronic nose (E-Nose), 629 Electronic tongue (E-tongue), 629 Electrophoretic deposition (EPD), 79
80, 713
714 Electrospinning, 4, 468
469, 553
554, 640
641 applications of electro-spun fibers in tissue engineering applications, 450
455 coaxial electrospinning system, 5f parameters influencing fiber production, 436
440 polymers for fabrication of electrospun fibers, 440
450 composite and hybrid, 449
450 natural polymers, 444
449 synthetic polymers, 440
444 principle of, 434
435 theory of, 433
434, 434t Electrospun fibers, 436 applications of electro-spun fibers in tissue engineering applications, 450
455 polymers for fabrication of, 440
450 use of electro-spun fibers in cardiac tissue engineering, 453
455 Electrostatic spraying process, 434 Electrothermal fiber actuators, 270
272 ELISA process, 448 Emulgels, 675
676 Emulsification process, 638 Emulsion, 675
676 electrospinning method, 553
554 emulsion-based gels, 675
676 process, 638 Encapsulation, 637
644, 647
648 of phages in natural polymers, 777
780 in vitro clinical phage therapy approaches, 778t Energy dispersive X-ray spectrometry technique (EDX spectrometry technique), 718
721 Energy harvesting based on polymer, 279
283 Enfuvirtide (Enf), 597 Engineered tissue, 166
168 hard tissue regenerative implants, 168 soft tissue regenerative implants, 166
167 Enterococcus faecalis, 659
660, 780
781 Entrapment efficiency (EE), 473 Entrapment method, 626

Enzyme-responsive polymers (ERPs), 237
239, 240t applications, 238
239 Enzymes, 618 biosensors, 620 enzyme-responsive polyion complex nanoparticle, 238
239 Ephedrine (EP), 758 Epithelization process, 550
551 Epoxy, 400
401 Escherichia coli, 9
10, 90, 508, 659
660, 695, 721, 776 Essential oils (EOs), 542, 546
557 essential oil-loaded biopolymeric films for wound healing applications, 554
557 essential oil-loaded films, 552
554 mechanisms of promoting wound healing by, 550
551 preparation of essential oil-loaded films, 552
554 wound healing physiology, 542
546 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), 683 Ethylene glycol dimethacrylate (EGDMA), 681
682, 751
752 Ethylene-vinyl acetate, 300 Eugenol, 546 Eukaryotic cells, 639
640 Ex vivo delivery, 516
517 genetic therapy, 516 External stimuli, 662
663 Extracellular matrix (ECM), 135
138, 166, 186
187, 365
366, 542
544, 550
551, 636, 695
696 Extrusion-based 3D printing, 139
140 FDM, 139 3D plotting, 140 Eye anatomy and protection mechanism of, 248
249, 248f glasses, 41

F Fabrication, 318, 327, 410 of ceramic particulate
dispersed composites, 406
410 methods of composite fabrication, 406
410 methodology, 316, 319, 410 polymers for fabrication of electrospun fibers, 440
450 techniques of biomedical composites, 54
58 techniques polymer composites, 3
7 Fatigue, 400
401

801

802

Index

Feline calicivirus (FCV), 605 Feline herpes virus (FHV-1), 605 Fermentation process, 501
502 Ferric ion, 504 Ferrite, 405
406 Fetal bovine serum (FBS), 572 Fiber-reinforced composites (FRCs), 2, 318
319 Fiber-reinforced polymer composites (FRPCs), 337
338, 399
400 Fiberglass, 34 in 20th century, 34 composites, 35
36 Fibers, 399
403 Fibrin, 380 fibrin/HA interpenetrating network hydrogel, 380 Fibroblasts, 542
544, 647 Fibroin, 447 Fibronectin, 227 Fibrous composites, 31
32 Filament binding, 56
57 Fillers, 4 Film-forming emulsion, 552
554 Finite element analysis (FEA), 166
167 First-order release kinetic model, 475 Fish bone
derived hydroxyapatite/biopolymer composite, 730
739 antibacterial analysis, 737 FTIR spectroscopic analysis, 730 microhardness analysis, 736 microstructural evaluation, 734 in vitro bioactivity assessment, 734 in vitro cytotoxicity assay, 739 X-ray diffraction investigation, 733 Flexural testing, 421
423 Fluorescein isothiocyanate (FITC), 755 Fluorescent zeolitic imidazolate framework-8 (FZIF-8), 756
758 Fluoride polymers, 16 5-fluorouracil, 78, 376
377, 753 Food and Drug Administration (FDA), 13, 353
355, 501
502 Food packaging, 607
608 biodegradable polymer composite, 608t Foodborne pathogens, 607 Fouling, 563 fouling-degradation, 567
568 fouling-release, 567
568 fouling-resistance, 567
568 4D printing, 160
161 application of, 166
177, 172t drug delivery implants, 170
177 engineered tissue constructs, 166
168 medical devices, 169
170

polymers for, 161
166 Fourier transform infrared spectroscopy (FTIR), 92, 470
471, 684
685, 718
721 analysis, 412
415, 419
420 Free-radical polymerization technique, 682
683 Fucoidan, 595 Functionalization of shape-memory polymers by biocompatible materials, 307
308 by carbon fillers, 306
307 by magnetic particles, 304
305 by silicate, 304 Functionally graded material (FGM), 318 Fused deposition modeling (FDM), 134
135, 138
139, 327 Fused filament fabrication (FFF), 138

G Galactan, 595 Gallium, 204 Gas-permeable polymeric membranes, 18
19 Gastrointestinal diseases, phage encapsulation to prevent and manage, 782
784 Gelatin (Gel), 90
91, 111
113, 135
138, 401
403, 444
446, 453
454, 681
682, 697, 713, 777 gelatin-based cryogel scaffolds, 699 gelatin-based immobilized laminin cryogel scaffolds, 699 gelatin-HA-based 3D bioprinted cryogenic scaffolds, 697 Gelatin methacrylate (GelMA), 663
664 Gelation, 375, 528 Gellan gum, 111
113, 501
504 Gene delivery, 514
517 applications of, 531
535 methods and techniques used in gene delivery, 517
526 polymers and bioceramics for, 526
531 systems, 513
514, 519 Gene therapy, 383, 513
516, 531 Genipin, 135
138 Genome editing, 515
516 Gentamicin, 483 Germline gene delivery systems for, 515 gene therapy, 515
516 Glass fibers, 43
45 glass fiber
based polymeric composites, 399
400 and resins, 56
57 Glass transition temperature (Tg), 412
415 Gluconacetobacter, 72
74 Glucose oxidase (GOx), 617 Glutaraldehyde (GA), 453
454, 681
682

Index

Glycerol, 401
403 Glycolic acid, 529
530 Glycosaminoglycans (GAGs), 189 Gold (Au), 273, 347, 517
518 Gold nanoparticles (AuNPs), 98
99, 365
366 Gold nanorod-coated MSN-bearing photosensitizer (AuNR-coated MSN-bearing photosensitizer), 352 Granulation tissue, 542
544 Granules, 401
403 Graphene, 269, 273
274, 347 Graphene nanoplatelets (GNPs), 16
17 Graphene oxide (GO), 171, 284
285, 347, 356, 760 Graphite, 269 Green composite materials, 400
401 Guar gum, 506
508

H Hair follicle stem cells, 550
551 Halloysite nanotubes (HNTs), 697 Hand layup methods, 410 molding, 54 Healing process, 780
781 Heart attack, 382 Hematopoietic stem cells (HSC), 516
517 Hemoglobin, 689
690 Hemostasis, wound healing phases, 386 Heparin (Hep), 19 heparin-based system, 353
355 Hepatocarcinoma cells (HepG2), 693 Hereditary diseases, 516
517 Herpes virus, 597 High-density lipoprotein cholesterol (HDL-C), 381
382 High-density polyethylene (HDPE), 45
46, 400
401 High-pressure processing, 408 High-resolution scanning electron microscope (HRSEM), 718
721 High-temperature curing, 411
412 Higuchi mathematical model, 448 Hixon Crowell model, 475
476 Hole transporting layer (HTL), 283 Human bone marrow mesenchymal stem cells (hMSCs), 167 Human cervical carcinoma cells (HeLa), 697 Human colon cancer cells (HCT116), 697 Human embryonic kidney 293 (HEK-293), 205
206 Human endometrial stem cells (hEnSCs), 443 Human immunodeficiency virus (HIV), 595

Human iPSCs (hIPSCs), 448 Human mesenchymal stem cells (hMSCs), 198 Human simplex virus (HSV), 593 Human umbilical artery smooth muscle cells (HUASMC), 204
205 Human umbilical vein ECs (HUVECs), 167, 204
205 Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), 167 Hyaluronan, 9 Hyaluronic acid (HA), 9, 74
77, 96, 104
105, 111
113, 192
193, 235
236, 380, 443, 570
571, 598, 688, 782 Hyaluronidase (HAase), 782 Hybrid composites, 415
416 Hybrid polymers, 363
364, 449
450 Hydrochloric acid, 593 Hydrocolloids, 401
403 Hydrogel(s), 99
100, 162
163, 186
189, 375
376, 528, 675
676, 686
687 applications of, 193
208 bioinks, 188
193 for 3D bioprinting, 194t bioprinted hydrogels in tissue engineering and drug screening, 209
210 composites, 479, 482 fabrication, 378
379 nanocomposites, 356 natural hydrogel bioinks, 192f scaffolds, 191 Hydrophilic drugs, 700
701 Hydrophilic properties, thermoresponsive polymers, 371
372 Hydrophobic properties, thermoresponsive polymers, 371
372 Hydrothermal method, 466
467 Hydroxyapatite (HAp), 17, 20
21, 45
46, 111
113, 134
135, 143
144, 198, 307
308, 711 hydroxyapatite/carboxymethyl cellulose/sodium alginate biocomposite, 718 hydroxyapatite/ultra-high molecular
weight PE composites, 22 reinforced with polyvinylpyrrolidone/aloe vera biocomposite, 718 Hydroxyethyl cellulose (HEC), 11
12 2-hydroxyethyl methacrylate (HEMA), 681
682, 752 Hydroxylapatite, 307
308 Hydroxypropylmethyl cellulose (HPMC), 602
603 Hypericum perforatum oil (HPO), 556
557 HPO-loaded chitosan films, 556

803

804

Index

I

L

Ibuprofen, 11
12 Imidazoles, 411
412 Immunofluorescence assay, 448 Immunoglobulin G (IgG), 688 Immunosensors, 620
621, 628 In situ delivery, 517 fabrication of interpenetrating network, 380 polymerization method, 277
279, 467
468 scaffolds, 371 In vitro cell viability analysis, 721 In vivo delivery, 517 Inflammation tissues, 376
377 wound healing phases, 386
387 Infrared spectroscopy, 80 Injection molding, 55 Injection-compression technique, 408 Insulin-producing cells (IPCs), 448 Integrins, 366 Intelligent polymers, 223
224, 384 Interglobular domain (IGD), 239 International Committee on Taxonomy of Viruses (ICTV), 770 Interpenetrating networks, 379
380 Interpenetrating polymeric networks (IPN), 165
166, 190
191 Intestinal tract health, 644
645 Intrinsically conductive polymers (ICPs), 265
266 Investigational New Drug (IND), 773 Ionic liquids (ILs), 276 ionic liquid
based enzyme biosensor, 623
624 Ionic polymer metal composites (IPMC), 269
270 ι-carrageenan, 594
595 Irgacure 651, 164
165 Irgacure 819 (IRG 819), 161
162 Itaconic acid (IA), 347, 751
752 Izod impact test, 423
424

Lactic acid bacteria, 529
530, 645
646, 648 Lactobacillus GG, 644
645 L. acidophilus, 659
660 L. casei, 644
645 L. plantarum, 638, 659
660 L. reuteri, 645
646 L. rhamnosus, 645
646 λ-carrageenan, 594
595 Laminated object manufacturing (LOM), 327 Laminates, 3 Laminin, 227 Laponite, 624
625 Laser engineered net shaping (LENS), 321, 325
326 Laser-assisted bioprinting (LaBP), 138, 143, 184
185 Latex technology, 6
7 Layer-by-layer assembly (LbL), 344, 347
350, 349t, 353
355 Layer-by-layer self-assembly method, 642
643 Layered double hydroxide (LDH), 518 Lentivirus (LV), 513
514 Levofloxacin, 255 Light energy, 283 Light-emitting diodes (LEDs), 283 Linalool, 546 Lipase immobilization, 94 Lipids, 401
403, 563
565 lipid-lowering therapy, 381
382 molecules, 520 vesicles, 520 Lipofection systems, 520 Lipoplexes, 520 Lipopolysaccharides, 546 Liposome gene delivery systems, 520
521 Liquid crystal elastomer (LCE), 164
165 Liquid molding, 55 Lithium-ion batteries (LIBs), 272, 277 Low-density lipoprotein cholesterol (LDL-C), 381
382 Low-density polyethylene (LDPE), 4 Low-pressure processing techniques, 406 Lower critical solution temperature (LCST), 225
226, 363
364 polymers, 373, 384 principle for thermoresponsive polymers showing, 371
372 Lysozyme, 689
690 lysozyme-imprinted polyacrylamide cryogel columns, 689
690

J Jute fiber-reinforced unidirectional thermoplastic composite, 318
319

K

κ-carrageenan, 594
595, 598 Kapton (polyimide), 280 Kefiran polymer, 704 Keratin, 777 Kidney, 533
534 Klebsiella pneumoniae, 89
90, 774 Korsmeyer
Peppas equation, 476

Index

M Macula, 248
249 Magnesium hydroxide, 347 Magnetic biosensors, 620 Magnetic particles, functionalization of shapememory polymers by, 304
305 Magnetization, 304
305 Maltodextrin, 784 Manual lay-up molding, 409 Maraviroc (MRV), 600
601 Mass of dried cryogel (Md cryogel), 685 Mass of wet cryogel (Mw cryogel), 685 Matrix metalloproteinases (MMPs), 239 Melt extrusion technique, 4 Membrane forces, 409 fouling, 563 Mesenchymal stem cells (MSCs), 168, 198, 696
697 Mesoporous silica nanoparticles (MSNs), 347, 483, 519 Mesopotamian settlers, 33
34 Metal, 132, 273, 400
401 nanoparticles, 483, 517
518, 618 Metal containing polymers, 599 Metaloorganic frameworks (MOFs), 276 Methacrylate hyaluronan
polyacrylamide hydrogels (MHA
PAAm hydrogels), 235
236 Methacrylic acid (MAA), 573, 681
682 Methacryloyl-l-lysine (MLL), 575
576 Methacryloylamido histidine (MAH), 762 2-Methacryloyloxyethyl phosphorylcholine (MPC), 18 Methicillin-resistant Staphylococcus aureus (MRSA), 377 Methyl methacrylate (MMA), 758 Methylene bisacrylamide (MBAAm), 681
682, 760
761 Methylene blue dye (MB dye), 647 remediation capability, 647 remediation from water, 647 Micelles, 377
378 Microbial cell encapsulation applications, 644
649 encapsulation method, 637
644 microcapsule properties for cell encapsulation, 637f Microbial sensors, 621 Microbubble gene delivery systems, 521
523 Microencapsulation, 784 Mitomycin C (MMC), 702 Molecular dynamics (MD), 577
578 Molecular imprinting technology, 746
751

Molecular mechanics (MM), 577
578 Molecularly imprinted polymers (MIPs), 688, 745
746 applications in biomedical science, 751
762 approaches for preparation of, 749
751 key parameters for preparation of, 746
748 MIP-based photothermal therapy approach, 755 Monomers, 265
266, 327
328 Monte Carlo (MC), 577
578 Montmorillonite, 504 Moxifloxacin, 754 Mucin, 252 Multidrug resistance gene 1 (MDR1), 531
532 Multifunctional fabrication process, 336 Multiwalled carbon nanotubes (MWCNTs), 347, 657
658

N

N,N,N0 N0 -Tetramethylethylenediamine (TEMED), 677
679 N,N-dimethyl acrylamide, N,N-dimethyl acrylamide (DMA), 681
682 N-(1-(2,3-dioleyloxy) propyl)-N,N,Ntrimethylammonium chloride (DOTMA), 520 N-acetyl-D-glucosamine, 67
69, 72
74, 250
251 N-alkyl-N-methyl-p-nitroaniline, 268 n-butyl acrylate (BA), 170 n-butylamine, 164
165 N-hydroxyethyl acrylamide (HEAA), 578 N-hydroxymethyl acrylamide (HMAA), 578 N-hydroxypentyl acrylamide (HPenAA), 578 N-hydroxypropyl acrylamide (HPAA), 578 N-isopropyl acrylamide, 681
682 N-trimethyl chitosan nanoparticles, 254
255 Nano drugs, 351 Nano-biosensor, 628 Nano-drug delivery system (NDDS), 532
533 Nano-fillers, 80, 663
664 Nanobiotechnology, 350 Nanocarriers, 376
377 Nanoceramics, 483 Nanocomposites, 78, 343, 465
466 of CN, 658 drug delivery systems, 350
351 films, 90, 503 materials, 343 scaffolds of sodium alginate/XG, 506 Nanofibers, 4, 267, 273
274, 553
554, 782 Nanogel engineering, 569
573 Nanohydroxyapatite, 483 Nanoparticles (NPs), 267, 376
377, 530
531, 574, 601
602, 638, 657
658 drug, 351

805

806

Index

Nanoparticles (NPs) (Continued) gene delivery systems, 517
519 mesoporous silica nanoparticles, 519 nanoparticle-based biopolymer composites, 623
624 nanoparticle-mediated ocular drug delivery system, 247
248 polymer grafting on/from modified surface of, 346
347 Nanoprecipitation process, 638 Nanosystem, 17
18 Natural antifouling, 568
569 Natural biomedical composites, 42
43 Natural biopolymers, 42, 135
138, 483 scaffolds bacteriophage encapsulation, 776
784 phage therapy, 769
775 Natural fibers, 42
43 Natural gums (NG), 497
498, 500
508 gellan gum, 501
504 applications, 502
504 guar gum, 506
508 applications, 507
508 scientometric analysis, 499
500 XG, 504
506 applications, 504
506 Natural polymers, 111
113, 133, 189, 247
248, 370, 444
449, 682, 692
693. See also Synthetic polymers blends and composites of, 133
138 natural polymers
based composites, 135
138 cellulose, 10
12 chitosan, 9
10, 446
447, 527 collagen, 7 and composites, 7
12 encapsulation of phages in, 777
780 gelatin, 444
446 HA, 9 silk, 7
8, 447
449 Natural resources, 713 reinforcement from, 405 Natural rubber (NR), 93 Naturally derived ceramics
polymer composite. See also Ceramic particle
dispersed polymer composites preparation of biogenic-derived biocomposites, 716
721 results, 721
739 crab shell
extracted hydroxyapatite/poly (vinylpolypyrrolidone) /aloe vera biocomposite, 725
730 egg shell
derived hydroxyapatite/cellulose nanocrystals biocomposite, 722
725

fish bone
derived hydroxyapatite/biopolymer composite, 730
739 Naviculan, 595 Near-infrared light (NIR), 167 Necrosis, 84 Neoagarohexaose (NA6), 605 Neural tissue engineering, 205
206 use of electro-spun polymers in, 450
455 Neuronal cells, 451
452 N-hydroxysuccinimide cross-linking method (NHS cross-linking method), 683 Nickel foam (Ni foam), 273, 304
305 Nigella sativa oil (NSO), 444 Nitric oxide synthases, 549
550 Nitrobacter sp., 627 Nitrogen porosimetry, 683
684 Nitrosomonas sp., 627 Non-VV (nVV), 517, 525
526 Nonbiodegradable matrices, 404
405 thermoplastics, 404 thermosetting, 405 Nonviral gene delivery systems (nVGD systems), 513, 523
526 Nucleic acid polymers (NAPs), 597 Nucleic acids, 344
345, 465, 618 biosensor, 621, 628 Nutrient synthesis, 645
646 Nylon, 315

O Ocular delivery, chitosan nanoparticles in, 254
257, 256t Oligo ethylene glycol (OEG), 572 Oligo(poly (ethylene glycol) fumarate) (OPF), 664
665 Oligodeoxy nucleotides, 525
526 Open contact molding method, 54
55 Open molding method, 410 Ophthalmic drug delivery, 479 polymer composites for ocular drug delivery, 481t Optical biosensors, 619 Oral delivery system, 784 Organic light-emitting diodes, 283
285 Organogels, 675
676 Organotin polymers, 599 Orthopedic tissues, 46
47, 109
110 Orthoses, 144
145 Osteogenic protein-1 (OP-1), 534 Osteoprogenitor cell morphology, 100 Ovarian cancer, diagnosis of, 93 Oxide-based biopolymer composites, 624
625 Oxidized sodium alginate (OSA), 782

Index

Oxygen electrode, 617 Oxygenases, 549
550

P Paddle method, 474 Particulate-reinforced composites, 412
415 Patient-specific scaffolds, 117
120 Pectin, 777
780 Penicillium chrysogenum, 250
251 Perfluorocyclobutane-based copolymers (PFCBbased copolymers), 284
285 Peripheral arterial disease, 381 Peroxidases, 549
550 Personal protective equipment (PPE), 603
604 pH-sensitive smart polymers, 228
232 applications, 230
232 pH-responsive polymers, 233t Phage encapsulation to prevent and manage gastrointestinal diseases, 782
784 for wound healing applications, 780
782 Phage therapy, 635, 769
775 characteristics of phages and antibiotics as antibacterials, 772t phage application in medicine, 773
775 regulatory approval of, 773 Phenylalanine ethyl esterification, 353
355 Phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO), 163
164 Phenylboronic acid (PBA), 758 10-phenylphenothiazine, 347 Phosphate buffer saline (PBS), 702 Phosphobetaine (PB), 579
580 Phosphorothioate oligonucleotides (PS-ONs), 597 Photo-initiators, 191
192 Photochemistry, 232
234 Photoisomerization process, 234 Photolysis, 747
748 Photonic crystals, 288 Photopolymerization technique, 329 Photosensitive polymers, 232
236 applications, 234
236 in biomedical fields, 237t Photovoltaic devices, 279 π-conjugated polymers, 287
288 Piezoelectric biosensor, 619 Plasmid DNA, 525
526 Plastic antibodies. See Molecularly imprinted polymers (MIPs) Platelet-rich plasma (PRP), 441 Poisson’s ratio, 170 Poly (glycerolsebacate) (PGS), 453 Poly (lactic acid) (PLA), 453, 529
530 Poly (N-iso propyl acrylamide) (PNIPAM), 528

poly N-isopropyl acrylamide (NIPAM), 165
166 Poly-(L-lysine) (PLL), 529, 570
571 Poly-2-hydroxyethyl methacrylate hydrogel system, 238
239 Poly-amido amine dendrimers (PAMAM), 529 Poly-styrene-co-ethylene-co-butylene-co-styrene (SEBS), 268 Poly(1,8-octanediol-co-citrate) (POC), 453 Poly(2-alkyl-2-oxazoline), 372, 374 Poly(2-oxazolines), 363
364 Poly(2-phenylethylmethacrylate)-grafted cellulose nanocrystals (MxG-CNC-g-PPMA), 378 Poly(2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl methacrylate) (PTMA), 277
279 Poly(2,5-dimethoxyaniline) (PDMA), 287
288 Poly(3-hexylthiphene) (P3HT), 279
280 Poly(3-hydroxubutyrate-co-4-hydroxubutyrate), 95 Poly(3-methylthiophene) (P3MT), 287
288 Poly(3,4-ethylene dioxythiophene), 452 Poly(3,4-ethylenedioxythiophene) (PEDOT), 98
99, 267, 287
288 Poly(acrylamide-co-acrylonitrile), 377
378, 504 Poly(acrylic acid-co-acrylate), 378 Poly(acrylic acid) (PAA), 228
230, 348 Poly(allylamine hydrochloride), 348
350 Poly(anthraquinonylsulfide)/grapheme, 277
279 Poly(aspartamides), 598 Poly(butylcyanoacrylate), 376
377 Poly(dimethylaminoethyl methacrylate), 228
230 Poly(ether), 372, 374 Poly(ethylene glycol) (PEG), 11
12, 17
18, 363
364, 572, 578, 580
581, 599, 702
703 Poly(ethylene oxide)-b-PSPA, 234
235 Poly(ethyleneglycol) methacrylate (PEGMA), 573 Poly(fluorene-alt-benzothiadiazole) (PFBT), 758 Poly(glycolic acid) (PGA), 15 Poly(L-lactic acid) (PLLA), 13
15, 441
442 Poly(lactic acid-co-trimethylene carbonate) (P (DLLA-TMC)), 168 Poly(lactic acid) (PLA), 21, 42
43, 111
113, 133
134, 315, 400
401, 453
455, 529
530, 713 Poly(lactic-co-glycolic acid) (PLGA), 15
16, 171, 230
231, 401
403, 440
441, 529
531 Poly(methacrylic acid) (PMA), 228
230 Poly(methyl methacrylate) (PMMA), 15, 43 Poly(MPC-co-dodecyl methacrylate) (PMD), 18 Poly(N,N-(dimethylamino)ethyl methacrylate) (PDMAEMA), 372, 374 Poly(N,N-diethyl acrylamide), 226 Poly(N-(2-hydroxyethyl)acrylamide) (pHEAA), 579

807

808

Index

Poly(N-(3-hydroxypropyl)acrylamide) (pHPAA), 579 Poly(N-(5-hydroxypentyl) acrylamide) (pHPenAA), 579 Poly(N-alkyl-substituted acrylamide), 372
373 Poly(N-hydroxyethylacrylamide-based nanogels) (polyHEAA-based nanogels), 572 Poly(N-hydroxymethyl acrylamide) (pHMAA), 579 Poly(N-isopropyl acrylamide) (pNIPAAm), 225
226 Poly(N-isopropylacrylamide) (PNIPAM), 363
364, 376, 693 Poly(N-vinyl alkylamine), 226 Poly(N-vinylcaprolactam) (PNVCL), 226, 363
364 Poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), 374 Poly(oligo(ethylene glycone)(methylether) (meth) acrylate), 372 Poly(p-phenylene vinylene) (PPV), 266
267 Poly(polyethylene glycol citrate-co-Nisopropylacrylamide)(PPCN), 377 Poly(propylene fumarate) (PPF), 139 Poly(sodium 4-styrenesulfonate) (PSSA), 266
267, 348
350, 605 Poly(sulfobetaine acrylamide), 356 Poly(vinylpyrrolidone) (PVP), 453
454, 598, 713 Polyacetylene (Pac), 265
266, 277
279 Polyacrylamide (PAMs), 579 polyacrylamide-chitosan cryogels, 693 Polyacrylonitrile-co-N0 -vinyl-2-pyrrolidone cryogels (poly(AN-co-NVP cryogels), 693 Polyamides, 400
401, 607 Polyampholytes, 573 Polyaniline (PANI), 266
267, 269
270, 272, 277
280 Polyanionic carbosilane dendrimers (PCDs), 600
601 Polyanionic cellulose, 713 Polybetaines, 573 Polycaprolactone (PCL), 13, 111
113, 133
134, 168, 376
377, 442
443, 452
453, 697 Polycaprolactone diacrylate (PCLDA), 161
162 Polycaprolactone/collagen I nanofibers (PCLColI), 781 Polycarbonate (PC), 160, 315 Polydimethylsiloxane (PDMS), 268, 683 Polydopamine (PDA), 576, 690
691 Polyesters, 400
401 Polyether ether ketone (PEEK), 400
401 Polyether/polyurethane (PE-PU), 165
166 Polyethylene (PE), 18, 364
365, 607 Polyethylene glycol diacrylate (PEGDA), 167

Polyethylene glycol dimethacrylate (PEG-DMA), 17
18, 702
703 Polyethylene glycol methylacrylate (PEGMA), 347 Poly(ethylene glycol) methyl ether methacrylate (PEGMA), 681
682 Polyethylene oxide (PEO), 364
365, 647 Polyethylene terephthalate (PET), 276
277, 607 Polyethyleneimine (PEI), 285, 384, 525, 529
531 Polyethyleneimine ethoxylated (PEIE), 285 Polyglycerol sebacate (PGS), 14
15 Polyhexamethylene guanidine hydrochloride (PHMG-Cl), 89
90 Polyhydroxy alkanoates (PHA), 401
403 Polylactide, 300 Polymer dispersed liquid crystals (PDLCs), 288 Polymer poly(2-(diethylamino)ethylmethacrylate) (PDEAEMA), 231 Polymer solar cell (PSC), 279
280 Polymer(s), 4, 19, 32, 67
69, 132, 138, 247
248, 265
267, 346, 353
355, 371, 400
401, 465, 597, 599, 638, 662
663 for 4D printing, 161
166 aggregation, 363 for biomedical applications, 7
22 blend scaffolds of GuG/gelatin, 507
508 chains, 300
301, 346, 371
372 classification of, 2
3, 3f composites, 3, 303, 399, 466, 478 materials, 2 matrices, 32 synthesis of, 466
470 energy harvesting based on, 279
283 for fabrication of electrospun fibers, 440
450 for gene delivery, 526
531 grafting on/from modified surface of nanoparticles, 346
347 nanocomposites, 343
346, 565
566 polymer-based composites, 31
32, 466 polymer-based electronic devices, 267 polymer-coated hollow sulfur particles, 266
267 polymer-drug conjugates, 597
598 polymer-magnetic nanocomposite, 469
470 in protective application, 603
606 using computational methods, 577
578 Polymercaptan, 411
412 Polymeric composites and blends application of value-added polymers, 143
145 blends and composites of natural and synthetic polymers, 133
138 current challenges and possible solutions, 145
146 synergistic approach in polymeric materials, 132
133

Index

3D printing techniques employed to print polymeric materials, 138
143 Polymeric gels, 675, 677
679 scaffolds, 686
687 Polymeric materials, 2, 132
133, 265, 713 synergistic approach in, 132
133 Polymeric micelles, 377 Polymeric nanoparticles, 376
377 Polymerization, 346
347 of PNVCL, 373 process, 265
266 Polymersomes, 380 Polymethylmethacrylate (PMMA), 400
401 Polynucleotides, 622 Polyparaphenylene (PPP), 277
279 Polypeptides, 622 Polyphenylsulfone, 160 Polyphosphate (PolyP), 605 Polypropylene, 403
404, 607 Polypropylene oxide (PPO), 374 Polypyrrole (PPy), 269
270, 272, 279
280, 452, 454 Polysaccharides, 72
75, 78
79, 465, 501
502, 506
507, 563
565, 592
595, 622, 713, 769, 777
780 Polystyrene, 607 Polysulfone (PSF), 18
19, 400
401 Polytetrafluoroethylene, 300 Polythiophene (PTh), 269
270, 272, 277
280 Polyurethanes (PUs), 133
134, 268, 401
403, 443
444 Polyuria, 280 Polyvinyl alcohol (PVA), 4, 111
113, 198, 382, 452, 454
455, 469
470, 598, 647, 681
682, 753
754 GG-based nanofibrous scaffolds, 502
503 poly(vinyl alcohol)-based polymer electrolytes, 270 Polyvinyl chloride (PVC), 404, 607 Polyvinylidene fluoride (PVDF), 16
17 Polyvinylpyrrolidone-iodine (PVP-I), 690
691 Polyvinylpyrrolidone/aloe vera biocomposite, hydroxyapatite reinforced with, 718 Polyzwitterions (PZs), 579
581 Pores, 41, 90
91 Porous bacterial cellulose using agarose microparticles (pBC-M), 96
97 Porous carbon materials, 273
274 Porphyromonas gingivalis, 21 Postprocessing, stages of RP, 320
321 Potentiometric biosensors, 620 Powder bed fusion, 141
143 binder jetting or powder liquid 3D printing, 142
143

SLS, 142 Powder liquid 3D printing, 142
143 Preheating technology for stamp-forming processes, 408
409 Preprocessing, stages of RP, 319
321 Printability, 191
193 Probiotics, 645
646, 769 antimicrobial potential, 646 bacteria, 644
645 encapsulation, 647
648 Programmable laser scanning method, 276
277 Proliferation, wound healing phases, 387
388 Protein(s), 465, 563
565, 568, 700
702, 713, 769, 777 biopolymer, 447 fouling, 568 protein A ligand, 693
694 protein
protein interactions, 595
597 synthesis, 253
254 Proteolytic enzymes, 237
238 Prototyping technique, 316 Pseudocapacitors, 272
273 Pseudomonas, 69
72 P. aeruginosa, 9
10, 90, 238
239, 255, 508, 774 P. syringae, 89
90 Pullulan, 353
355, 598 Pultrusion techniques, 409
410 Pupil, 248
249

Q Quantum dots (QDs), 759 Quaternary benzophenone-based amide (QBAm), 605 Quaternary benzophenone-based ester (QBEst), 605 Quaternized chitosan (QCS), 690
691 Quercetin, 548
549

R Radio frequency (RF), 34 Rapid prototyping (RP), 315 advancements in, 334
339 comparison with traditional manufacturing, 338 fiber-reinforced polymer composites, 337
338 functionally graded materials using rapid prototyping, 338 improvement of product quality, 335 improvement on versatility of rapid prototyping, 335 multifunctional fabrication process, 336 applications, 329
334

809

810

Index

Rapid prototyping (RP) (Continued) contemporary rapid prototyping systems, 321
329 preprocessing, process, and postprocessing in, 319
321 printable and embeddable functions, 336
337 actuations, 336 antennas and electromagnetic structures, 337 energy storage, 337 propulsion, 337 sensors, 336 thermal management, 336 Rat bone marrow mesenchymal stem cells (rMSCs), 444 Reaction injection molding (RIM), 55, 408 Reactive oxygen species (ROS), 352, 544
546, 548
549 Reactive thermoplastic (RTM), 407 Receptor, 618 Recombinant adeno connected virus (rAAV), 534 Recombinant DNA technology, 383 Recovery process, 300 Recycling polymer matrices, 404 Reduced graphene oxide (RGO), 17 Regenerative medicine, 183
184, 226
227, 375, 635 Resin transfer molding (RTM), 54
55, 409
410 Resonant biosensors, 619 Retrovirus (RV), 513
514 Reversible addition-fragmentation chain transfer (RAFT), 572 Reversible electrochemical redox reactions, 277 Rheumatic heart diseases, 381 Rhizobium, 83
84 Ribavirin-loaded polyanions, 602 Ribozymes, 525
526 Room-temperature curing, 411 Rosmarinic acid, 548
549 Rubber, 32

S S-glycoprotein, 595 S-sulpiride, 755 Saccharomyces cerevisiae, 695 Salmon muscle and slowing down lipid oxidation, 94 Salmonella enterica, 607 Sarcina, 72
74, 83
84 Scaffolds, 183, 388, 780 fabrication methods, 114
117, 115t 3D-(bio)printing techniques, 116t fabrication of, 114
120 patient-specific scaffolds, 117
120 scaffold fabrication methods, 114
117

properties and characterization, 110
114 treatment of cartilage lesions, 112t Scanning electron microscopy (SEM), 448, 470, 472, 684 analysis, 416
418 imaging, 441 Scientometric analysis, 62
80, 499
500 Seebeck effect, 280
282 Selective laser melting (SLM), 325
326 Selective laser sintering (SLS), 138, 142, 324
325 Self-assembled polymer nanocomposites applications in biomedical science, 350
356 drug delivery, 350
356, 354t methods of preparation of, 345
350 layer-by layer assembly technique, 347
350 polymer grafting on/from modified surface of nanoparticles, 346
347 Semicovalent imprinting technique, 749
750 Sensing, 758
762 Sensors on conducting polymers, 267
272 Separation processes, 758
762 Shape memory hydrogels (SMHs), 171 Shape memory material, 93 Shape-memory effect (SME), 300 Shape-memory polymers (SMP), 163
164, 299
302. See also Conducting polymers; Thermoresponsive polymers categorization of, 302 composites using, 303
308 cross-linking, 300
301 limitations of, 308
312 recovery force and work capacity, 310
312, 311t recovery time and activation process, 308
310 mechanism of shape-memory polymers, 302
303 thermal transitions, 301
302 Sheet molding compound (SCM), 56
57 Shigella enteritis, 769
770 Short interfering ribonucleic acids (siRNA), 384, 525
526 Sialic acid, 247
248, 252 Sialon, 405
406 Silica, 347, 405 nanofiber, 450
451 Silica nanoparticles (SNPs), 231, 519 Silicate, functionalization of shape-memory polymers by, 304 Silicon carbide (SiC), 405
406 Silicon nitride (Si3N4), 405
406 Silk, 135
138, 447
449, 681
682 silk-based polymers, 111
113 Silk fibroin (SF), 7
8, 447, 454
455

Index

Silk sericin (SS), 104
105 Silkworms (Bombyx mori), 7
8 Silymarin-zein (SMN-Zein), 94 Simulated body fluid immersion method (SBF immersion method), 721 Simulated gastric fluid (SGF), 783 Single sugar α-linked glucuronic acid-based oligosaccharide combined with bacterial cellulose (SSGO/BC), 100
101 Single-wall carbon nanotubes (SWCNTs), 657
658 Skin, 376, 385
386, 446 biocompatibility on, 51 tissue engineering, 203
204, 388 Smart materials, 363, 745
746 Smart mechanical actuators, 378 Smart polymers (SP), 223
224 applications of TSSPs, 226
228 enzyme-responsive polymers, 237
239 photosensitive polymers, 232
236 TSSPs, 225
226 Sodium (Na), 94
95 Sodium alginate (SA), 662
663 Sodium titanium oxide, 659
660 Sol
gel process, 412
415 Solution mixing technique, 4
5 Solution parameters, 438
440 concentration, 438
439 conductivity/surface charge density, 439
440 molecular weight, 439 surface tension, 439 Somatic cells, 516 Somatic gene therapy, 516
517 Sonochemical techniques, 721
722 Sphingomonas elodea, 501
502 Spin coating, 344 Spinal cord injury (SCI), 77
78 Spirooxazine, 234 Spiropyran, 234 Spray drying method, 643
644 Spray-up method, 410 Stamp-forming processes, preheating technology for, 408
409 Standard Tessellation Language format (STL format), 319
320 Staphylococcus S. aureus, 9
10, 79
80, 90, 255, 365
366, 508, 659
660, 721, 774 S. epidermidis, 92 Starch, 401
403 Static contact angle (SCA), 103
104 Stereolithography (SL), 138, 159
160, 315, 327
329

contemporary rapid prototyping techniques in summarized form, 331t layer thickness and surface roughness, 332t Stereolithography apparatus-1 (SLA-1), 159
160 Storage process, 299
300 Streptococcus thermophilus, 644
645 Stromal cell
derived factor-1 (SDF-1), 377 Subtractive manufacturing method, 320
321 Sucrose, 784 Sulfated polysaccharides, 595 Sulfobetaine (SB), 579
580 sulfobetaine-based polymers, 356 Supercapacitors, 272
274, 276 Superhydrophobic surfaces, 569, 576 Surface hydration effect on antifouling properties, 578
579 Surface modification of grafted polymer, 344 Surface plasmon resonance (SPR), 617 Surface-enhanced Raman scattering (SERS), 660
661 Surface-initiated ATRP (SIATRP), 346
347 Swelling forces, 189
190 of hydrogel, 189
190 Synthetic biomedical composites, 43 Synthetic polymers, 13
18, 111
113, 133, 135
138, 370, 440
444, 527
531, 682, 692
693. See also Natural polymers blends and composites of, 133
138 PCL, 13 PEG, 17
18 PLGA, 15
16 PLLA, 13
15, 441
442 PMMA, 15 poly L-lactic-co-glycolic acid, 440
441 polycaprolactone, 442
443 polyurethane, 443
444 PVDF, 16
17 synthetic polymers
based composites, 134
135 thermoresponsive polymers, 528
531 polyethylenimine, 529
531 Synthetic receptors. See Molecularly imprinted polymers (MIPs) Synthetic resources, reinforcements from, 405
406 System parameters, 436
438 applied voltage, 437 collector types, 438 flow rate, 437 tip to collector distance, 437
438

T Tannic acid (TA), 690
691 Taylor’s cone, 433

811

812

Index

Teflon, 280 Temperature-sensitive smart polymers (TSSPs), 225
226 applications of, 226
228 thermoresponsive polymers, 229t pH-sensitive smart polymers, 228
232 Tenofovir (TFV), 600
601 Tensile testing, 420
421 Tetracycline, 483 Tetracycline hydrochloride loaded bacterial cellulose composite membranes (BC-TCH), 103 2,2,6,6-tetramethyl-4-piperidinol grafted PAA (gPAA), 605 Therapeutic delivery systems, 350
351 Thermal analysis, 472 Thermal biosensors, 619
620 Thermally induced phase separation (TIPS), 206
208 Thermo-sensitive nanoparticles, 78 Thermoelectric generator, 280
282 Thermogravimetric analysis (TGA), 404, 424, 447, 472 Thermolysis, 747
748 Thermoplastic polyurethane nanofibers (TPU nanofibers), 161, 444 Thermoplastic resin transfer moulding (TP-RTM), 407 Thermoplastics, 315, 327, 404 autoclave forming of thermoplastic composites, 406
407 BIM, 407 blank-holders and membrane forces, 409 composites, 406 autoclave forming of, 406
407 continuous compression molding, 409 diaphragm forming, 407 high-pressure processing, 408 injection-compression technique, 408 low-pressure processing techniques, 406 methods for, 406
409 molecules, 32 preheating technology for stamp-forming processes, 408
409 resins, 32 RTM, 407 thermoplastic composites considering vacuum forming, 406 Thermoresponsive aluminum composite films (Thermoresponsive Al composite films), 378 Thermoresponsive behaviors, 371
374 principle for thermoresponsive polymers showing UCST and LCST, 371
372

type of thermoresponsive polymers, 372
374 Thermoresponsive hydrogels, 375
376, 479 Thermoresponsive polymeric composites, 364
366 form of thermoresponsive polymers and, 374
380 films, 378
379 hydrogels, 375
376 interpenetrating networks, 379
380 micelles, 377
378 nanoparticles, 376
377 polymersomes, 380 mechanisms, 366
374 Thermoresponsive polymers, 165
166, 363
364, 372
375, 528
531. See also Conducting polymers; Shape-memory polymers (SMP) applications of, 381
388 form of polymeric composites and, 374
380 films, 378
379 mechanisms, 366
374 poly(2-alkyl-2-oxazoline), 374 poly(ether), 374 poly(N,N-(dimethylamino)ethyl methacrylate), 374 poly(N-alkyl-substituted acrylamide), 373 poly(N-vinylcaprolactam), 373 poly(oligo(ethylene glycol) methyl ether methacrylate), 374 principle for thermoresponsive polymers showing UCST and LCST, 371
372 Thermosets, 32 composites, 406 Thermosetting closed molding, 410 methods for, 409
410 open molding, 410 Three-dimension (3D), 188
189 bioprinting, 188, 198 applications, 188 system, 375
376 cell culture model, 183 network network structure, 675 polymer nanoparticles, 569 plotting, 140 porous polymer matrix, 498 printed biomimetic CHA, 19
20 printed scaffolds, 315 printers, 318 printing, 131, 160
161, 320
321, 469
470 extrusion-based 3D printing, 139
140 laser-assisted bioprinting, 143 methods, 333 powder bed fusion, 141
143

Index

systems, 315, 320 techniques employed to print polymeric materials, 138
143 technology, 132
133, 318, 336 vat polymerization, 140
141 scaffolds, 90
91 structures, 109
110 3D-(bio)printing method, 114
117, 120 wound dressings, 781 3-dimensional microporous regenerated bacterial cellulose/gelatin (3DMP rBC/G), 90
91 Threshold energy, 186 Thymol, 546 Tissue engineering, 2, 45
46, 94
98, 160
161, 183
184, 192
193, 204
205, 226
227, 327, 363, 365
366, 375, 446, 498, 647 applications in, 7 approaches, 183 bioprinted hydrogels in, 209
210 CNTs nanocomposites for, 663
665 cryogels in tissue engineering applications, 692
699 electro-spun fibers in tissue engineering applications, 450
455 fabrication of, 114
120 scaffolds properties and characterization, 110
114 treatment of cartilage lesions, 112t Tissue grafts, 109
110 Tissue regeneration, 109
110, 162
163 Tissue remodeling, wound healing phases, 388 Titanium carbide, 405
406 Titanium oxide, 659
660 Transducer, components of biosensor, 618 Transfemoral prostheses (TP), 49 Transforming growth factor-gene delivery (TGFgene delivery), 535 Transmission electron microscopy, 470 Transplanted cells, 635
636 Trehalose, 784 Triboelectric generator, 280 Tricalcium phosphate, 111
113 β-tricalcium phosphate/poly(lactic acid-cotrimethylene carbonate) (TCP/P(DLLATMC) ), 168 Trichloromethane (TCM), 444 2,2,2-trifluoroethanol (TFE), 444 Trimethylamine, 605 Tryptophan, 574
575 Tumor growth inhibition (TGI), 598 Tumor-targeted drug therapy, 478
479 polymer composites for tumor-targeted drug therapy, 480t Twin-screw extruder, 4

Tylor’s Cone, 468 Tyrosine, 574
575 2-dimensional cell culture (2D cell culture), 183

U Ultrasound, 521
522 ultrasound-based gene delivery systems, 522
523 Ultraviolet (UV), 320 light, 770 radiation, 54, 234
235 Unsaturated polyester resin (UPR), 404
405 Upper critical solution temperature (UCST), 225
226, 363
364 polymers, 384 principle for thermoresponsive polymers, 371
372

V Vaccinia virus, 597 Vacuum bag molding, 409
410 Vacuum forming technique, thermoplastic composites considering, 406 Vacuum molding process, 406
407 Vacuum resin assisted transfer molding (VARTM), 55
56 Value-added polymers, application of, 143
145 Vancomycin, 483 Varicella zoster virus (VZV), 599 Vascular endothelial growth factor (VEGF), 513
514, 542
544 Vascular stenosis, 169
170 Vascular tissue engineering, 204
205 VAT polymerization, 138, 140
141, 336 Venous thromboembolism, 381 4-vinylphenylboronic acid (VPBA), 760
761 2-vinylpyridine (2-VP), 752 Viral gene delivery systems, 523
526 Viral vectors (VVs), 514 Virion, 523
524 VoSviewer software, 64 Vroman effect, 368
369

W Water methylene blue dye remediation from, 647 in oil emulsion, 638 vapor transmission, 608 water-solubilizing agent, 597
598 water-soluble thermoresponsive polymers, 372 Web of Science (WoS), 62, 498
499 Wettability, 576 Wolff’s law, 45
46 Wound dressings, 100
105, 554

813

814

Index

Wound dressings (Continued) cryogels in wound dressing applications, 690
692 Wound healing, 385
388 applications, 86
92 functional groups present in different types, 87t of thermoresponsive polymers in wound healing, 388 by EOs, 550
551 essential oil-loaded biopolymeric films for, 554
557 phases, 385
388 hemostasis, 386 inflammation, 386
387 proliferation, 387
388 tissue remodeling, 388 physiology, 542
546

X X-ray diffraction (XRD), 470
471, 718
721

X-ray fluorescence spectroscopy (XRF spectroscopy), 94
95 Xanthan gum (XG), 499
500, 504
506 Xanthomonas campestris, 504 Xerogels, 675
676

Y Young’s modulus, 86
89, 686

Z Zero-order release kinetic model, 475 Zidovudine, 598 Ziegler
Natta method, 265
266 Zinc oxide (ZnO), 405
406, 691
692 Zinc oxide nanorods (ZnO-NRs), 16
17 Zirconium dioxide, 405
406 ZnO-NR-decorated GNPs (ZNGs), 17 Zwitterionic nanomaterials, 573
576