Nanomaterials in Healthcare 103234475X, 9781032344751

Table of contents :
Cover
Half Title
Title Page
Copyright Page
Dedication
Contents
Preface
Acknowledgments
List of Reviewers
Editors
Contributors
1. Introduction to Nanomaterials and Their Scope in Drug Delivery
1.1. Introduction
1.2. Types of Nanoparticles
1.2.1. Metallic Nanoparticles
1.2.2. Lipid-Based Nanoparticles
1.2.3. Carbon-Based Nanoparticles
1.2.4. Polymer-Based Nanoparticles
1.2.5. Silica-Based Nanoparticles
1.3. Application of Nanoparticles
1.3.1. Antibacterial
1.3.2. Cancer
1.3.3. Neurodegenerative
1.3.4. Infectious Disease
1.3.5. Immunotherapy
1.4. Nanoparticles Toxicity
1.4.1. Metallic-Based Nanoparticles
1.4.2. Lipid-Based Nanoparticles
1.4.3. Silica-Based Nanoparticles
1.4.4. Carbon-Based Nanoparticles
1.4.5. Polymer-Based Nanoparticles
1.5. Prospects and Challenges
References
2. Application of Nanomaterials in Medicine: A Clinical Perspective
2.1. Introduction
2.1.1. History of Nanomedicine
2.1.2. Advantages of Nanomaterials in Clinical Medicine
2.1.3. Disadvantages of Nanomaterials in Clinical Medicine
2.1.4. Latest Trends in Nanomedicine
2.2. Nanomaterials Used in Communicable Diseases
2.2.1. Tuberculosis
2.2.2. HIV/AIDS
2.2.3. Influenza
2.3. Nanomaterials Used in Non-Communicable Diseases
2.3.1. Cardiovascular Diseases
2.3.2. Diabetes
2.3.3. Neurodegenerative Diseases
2.3.4. Autoimmune Disorders
2.4. Nanomaterials Used in Cancer
2.5. Nanomaterials for Imaging, Screening, and Diagnosis
2.6. Tackling COVID-19 Using Nanotechnology
2.7. Nanomedicine and Associative Technologies
2.7.1. Additive Manufacturing
2.7.2. Artificial Intelligence, Machine Learning, and Bioinformatics
2.7.3. Robotics, Automation, and IoT
2.8. Ethical Concerns about Nanomedicines
2.9. Clinical Trials and Approvals of Nanomedicine
2.10. Conclusion
References
3. Advancement of Polymer-Based Nanocarrier System in Drug Delivery
3.1. Introduction
3.2. Types of Polymer-Based Nanocarriers
3.2.1. Classification of Polymer Nanocarriers Based on Affinity to Water
3.2.1.1. Hydrophilic
3.2.1.2. Hydrophobic
3.2.1.3. Amphiphilic
3.2.2. Classification of Polymer Nanocarriers Based on Source
3.2.2.1. Natural
3.2.2.2. Synthetic
3.2.3. Classification of Polymer Nanocarriers Based on Charge
3.2.3.1. Cationic
3.2.3.2. Anionic
3.2.3.3. Charge reversible polymers
3.3. Application of Polymeric Nanoparticles in Drug Delivery
3.3.1. Oral Drug Delivery
3.3.2. Vaginal Drug Delivery
3.3.3. For Cancer Therapy
3.3.4. Ocular Delivery
3.4. Conclusion and Future Prospect
References
4. Liposomes and Lipid Structures: Classification, Characterization, and Nanotechnology-Based Clinical Applications
4.1. Introduction
4.2. Need for Liposomes
4.3. Evolution through Advancements
4.3.1. Solid Lipid Nanoparticles (SLNs)
4.3.2. Nano Lipid Carriers (NLCs)
4.4. Conventional Methods for Liposome Preparation
4.4.1. Film-Hydration Method
4.4.2. Double-Emulsification Method
4.4.3. Reverse Phase Evaporation Method
4.4.4. Solvent Injection Method
4.4.5. Detergent Dialysis Method
4.5. Novel Methods for Liposome Preparation
4.5.1. Supercritical Fluid (SCF) Technology
4.5.2. Dual Asymmetric Centrifugation Techniques (DAC)
4.5.3. Membrane Contactor Technology
4.5.4. Microfluidic Technique
4.6. Characterization of Liposomes
4.6.1. Morphological Characterization
4.6.2. Stability Study
4.6.3. Encapsulation Efficiency
4.6.4. In Vitro Drug Release
4.6.5. Freeze-Drying
4.6.6. Miscellaneous Methods
4.7. Liposomes for Treatment of Various Diseases
4.7.1. Cancer
4.7.2. Reproductive Organs
4.7.3. Developmental Disorders
4.7.4. Arthritis and Bone Abnormalities
4.7.5. Wound Healing
4.7.6. Antimicrobial Diseases
4.7.7. Nervous System Disorders
4.8. Clinical Applications of Liposomes Concerning Nanotechnology
4.9. Future Prospective
4.10. Conclusion
References
5. Functionalized Carbon-Based Nanoparticles for Biomedical Application
5.1. Introduction
5.2. Types and Properties of Carbon-Based Nanomaterials
5.2.1. Fullerenes
5.2.1.1. Structural dimension
5.2.1.2. Physical property
5.2.1.3. Mechanical property
5.2.1.4. Electrical property
5.2.1.5. Thermal property
5.2.1.6. Optical property
5.2.1.7. Chemical property
5.2.2. Carbon Nanotubes
5.2.2.1. Structural dimension
5.2.2.2. Physical property
5.2.2.3. Mechanical property
5.2.2.4. Electrical property
5.2.2.5. Thermal property
5.2.2.6. Optical property
5.2.2.7. Chemical property
5.2.3. Graphene and Its Derivatives
5.2.3.1. Structural dimension
5.2.3.2. Physical property
5.2.3.3. Mechanical property
5.2.3.4. Electrical property
5.2.3.5. Thermal property
5.2.3.6. Optical property
5.2.3.7. Chemical property
5.2.4. Nanodiamond
5.2.4.1. Structural dimension
5.2.4.2. Physical property
5.2.4.3. Mechanical property
5.2.4.4. Electrical property
5.2.4.5. Thermal property
5.2.4.6. Optical property
5.2.4.7. Chemical property
5.2.5. Carbon Dots
5.2.5.1. Structural dimension
5.2.5.2. Physical property
5.2.5.3. Mechanical property
5.2.5.4. Electrical property
5.2.5.5. Thermal property
5.2.5.6. Optical property
5.2.5.7. Chemical property
5.3. Synthesis of Functionalized Carbon-Based Nanoparticles
5.3.1. Synthesis
5.3.2. Exohedral Functionalization
5.3.2.1. Covalent functionalization
5.3.2.2. Non-covalent functionalization
5.3.3. Endohedral Functionalization
5.4. Risk-Assessment of Functionalized Carbon-Based Nanoparticles
5.5. Application of Functionalized CBNs
5.5.1. Biosensors
5.5.2. Drug Delivery
5.5.3. Therapy
5.5.3.1. Role in tissue engineering and regenerative medicine
5.5.3.2. Role as free radical scavengers
5.5.3.3. Role as an antimicrobial
5.5.3.4. Role in cancer therapy
5.6. Future Prospects and Challenges
References
6. Engineered Magnetic Nanoparticles: Challenges and Prospects
6.1. Introduction
6.2. Synthesis of Magnetic Nanoparticles (MNPs)
6.2.1. Physical Method
6.2.2. Chemical Method
6.2.3. Biological Synthesis Method
6.3. Properties and Application
6.3.1. Characteristics of Magnetic Particles
6.3.1.1. Particle size
6.3.1.2. Particle density
6.3.1.3. Particle shape
6.3.1.4. Magnetic property
6.3.2. Application of Magnetic Nanoparticles
6.3.2.1. Hyperthermia
6.3.2.2. Photothermal therapy
6.3.2.3. Drug delivery
6.3.2.4. Infection treatments
6.3.2.5. Magnetic resonance imaging
6.4. Conclusion
References
7. Nano Metal-Organic Frameworks as a Promising Candidate for Biomedical Applications
7.1. Introduction
7.2. Synthesis of NMOFs
7.2.1. Solvothermal Synthesis
7.2.2. Microemulsion Synthesis
7.2.3. Microwave-Assisted Synthesis
7.2.4. Ultrasound/Sonochemical Synthesis
7.2.5. Electrochemical Synthesis
7.2.6. Mechanochemical Synthesis
7.3. Biofunctionalization of NMOFs
7.4. NMOFs for Drug Delivery and Targeted Tumor Therapy
7.4.1. pH-Responsive Drug Delivery
7.4.2. Temperature-Responsive Drug Delivery
7.4.3. Ion-Responsive Drug Delivery
7.4.4. ATP-Responsive Drug Delivery
7.4.5. Redox-Responsive Drug Delivery
7.5. NMOFs for Bio-Imaging
7.6. Conclusions and Outlook
Acknowledgments
References
8. Porous Silica Nanoparticles for Targeted Bio-Imaging and Drug Delivery Applications
8.1. Introduction
8.2. Strategies for Functionalization of Silica Hybrid Nanocarriers
8.3. Drug Delivery Applications of Silica Nanohybrid
8.3.1. Silica Polymer Nanohybrid for Drug Delivery
8.3.2. Silica Nucleic Acid Nanohybrid for Drug Delivery
8.4. Silica Protein Nanohybrid for Drug Delivery
8.5. Silica Peptide Nanohybrid for Drug Delivery
8.6. Silica Quantum Dot for Drug Delivery
8.7. Silica Magnetic Nanohybrids for Drug Delivery
8.8. Clinical Trials for Silica-Based Nanoformulations
8.9. Conclusion
References
9. Recent Advancement of Multifunctional ZnO Quantum Dots in the Biomedicine Field
9.1. Introduction
9.2. Structure, Properties, and Fabrication Methodologies
9.2.1. Importance of Structure-Property Synergism of ZnO QDs
9.2.1.1. Optical characteristics
9.2.1.2. Physiochemical properties and surface chemistry
9.2.1.3. Biological features
9.2.2. Synthesis Routes: Trade-Offs and Accomplishments
9.2.2.1. Wet-chemical approaches (hydrothermal, sol-gel, microwave-assisted synthesis, continuous flow synthesis)
9.2.2.2. Bio-synthesis: Green and sustainable synthetic scheme
9.3. Advancements of ZnO QDs in Biomedical Domains
9.3.1. Targeted Drug Delivery and Point-of-Care Diagnostics
9.3.2. Treatment of ROS-Mediated Disorders
9.3.3. Wound Healing and Engineered Tissue Regeneration
9.3.4. ZnO QDs with Anti-Microbial Potential
9.3.5. Sensing and Imaging Applications in Biology
9.3.6. Cancer Theranostics
9.4. Future Prospects and Challenges
References
10. Relevant Properties of Metallic and Non-Metallic Nanomaterials in Biomedical Applications
10.1. Introduction
10.2. Structural Engineering of Nanoparticles
10.2.1. Size of Nanoparticles
10.2.2. Shape of Nanoparticles
10.2.3. Surface of Nanoparticles
10.2.4. Structural Tuning for Biomedical Applications
10.2.4.1. Magnetic resonance imaging (MRI)
10.2.4.2. Surface-enhanced raman spectroscopy (SERS)
10.3. Ceramic Biomaterials
10.3.1. Hydroxyapatites as Biomaterials
10.3.2. The Surface Features of Hydroxyapatites
10.3.3. The Effect of the Surface Structure of Hydroxyapatites on the Adsorbed Proteins Structure
10.3.4. Luminescent Lanthanide Hydroxyapatite-Based Nanomaterials
10.3.5. Silica-Based Nanomaterials
10.3.6. Silica Surface Structure
10.3.7. Silica in the Drug Delivery Field
10.4. Overview
References
11. Exosomes and Their Theragnostic Applications in Healthcare
11.1. Introduction
11.2. Sources for Exosome Isolation
11.3. Mechanism of Exosome Biogenesis
11.4. Structure, Composition, and Function of Exosomes
11.5. Exosomes for Theragnostic Applications
11.5.1. Native Exosomes for Theragnostic Applications
11.5.2. Engineered Exosomes for Theragnostic Applications
11.6. Absorption and Distribution of Exosome-Based Theragnostic System
11.7. Challenges Related to Exosomes for Theragnostic Application
11.8. Conclusion and Future Prospective
References
12. Nanogels for Theranostic Applications in Healthcare
12.1. Introduction
12.2. Applications of Nanogels
12.2.1. Nanogels for Targeted Drug Delivery
12.2.1.1. Active targeting
12.2.1.2. Passive targeting
12.2.2. Nanogels for Stimuli-Responsive Drug Delivery
12.2.2.1. Temperature-responsive nanogels
12.2.2.2. pH-responsive nanogels
12.2.2.3. Light-responsive nanogels
12.2.2.4. Magnetic-responsive nanogels
12.2.3. Nanogels for Poorly Water-Soluble Drugs
12.2.4. Nanogel for Gene Delivery
12.2.5. Nanogels for Brain Drug Delivery
12.2.6. Nanogels in Diagnosis and Imaging
12.3. Challenges and Future Perspective
12.4. Summary and Conclusion
References
13. Theranostic Application of Nanofibers in Tissue Engineering
13.1. Introduction
13.2. Nanofiber-Based Scaffold for Drug Delivery
13.3. Nanofiber-Based Stem Cell Therapy and Labeling
13.4. Nanofiber-Based Scaffold Construction and Modification
13.5. Multifunctional and Smart Nanofiber-Based Scaffolds
13.6. Conclusion and Future Prospects
References
14. Role of Nanomaterials in Biosensing Applications
14.1. Introduction
14.2. Biosensors: An Overview
14.3. Nanomaterials - Characteristic Features for Biosensing Applications
14.4. Nanomaterials-Based Biosensing for In-Vitro Diagnostics
14.4.1. Metal Nanoparticles
14.4.2. Metal Oxide-Based Nanomaterials
14.4.3. Carbon-Based Nanomaterials
14.4.4. Nanocomposites
14.5. Challenges and Future Prospects
14.6. Conclusion
References
15. Application of Two-Dimensional Materials for Cancer Theranostic
15.1. Introduction
15.2. Properties of 2D Nanomaterials
15.3. Synthesis of 2D Nanomaterials
15.4. Application of 2D Nanomaterials in Cancer Therapy
15.4.1. Graphene and Its Derivative
15.4.2. Two-Dimensional Transition Metal Dichalcogenides (TMDCs)
15.4.2.1. Molybdenum disulfide (MoS2)
15.4.2.2. Tungsten disulfide (WS2)
15.4.2.3. MXenes
15.4.2.4. Xenes
15.4.3. Black Phosphorus (BP)
15.4.4. Boron Nitride (BN)
15.4.5. Metal Oxide Nanosheets
15.4.5.1. Manganese dioxide (MnO2)
15.4.5.2. Molybdenum oxide (MoOx)
15.4.5.3. Zinc oxide (ZnO)
15.4.5.4. Iron oxide (IO)
15.4.6. Layered Hydroxides (LDH)
15.4.7. Metal Organic Framework (MOF)
15.5. Conclusion
References
16. Solid Lipid Nanoparticles: Towards Emerging Cancer Nanomedicine
16.1. Introduction
16.2. Characteristics
16.3. Methods of Preparation of Solid Lipid Nanoparticles for Cancer Nanomedicine
16.3.1. High Shear Homogenization
16.3.1.1. Hot homogenization
16.3.1.2. Cold homogenization
16.3.2. Solvent Emulsification Technique
16.3.3. Ultrasonication or High-Speed Homogenization
16.3.4. Double Emulsion Method
16.3.5. Spray Drying Method
16.3.6. Supercritical Fluid
16.3.7. Microemulsion-Based SLNs' Preparation
16.4. Routes of SLNs' Delivery
16.4.1. Transdermal/Topical
16.4.2. Oral
16.4.3. Parenteral
16.4.4. Pulmonary
16.4.5. Brain
16.5. Toxicology and Clearance
16.6. Applications
16.6.1. Breast Cancer
16.6.2. Lung Cancer
16.2.3. Colon Cancer
16.7. Conclusion
References
17. Gold Nanoparticles for Cancer Therapy and Diagnosis
17.1. Introduction
17.2. Synthesis of Gold Nanoparticles
17.3. Gold Nanoparticles for Cancer Therapeutic Application
17.3.1. Gold Nanoparticles for Drug Delivery and Nucleic Acid Delivery
17.3.2. Photodynamic Therapy
17.3.3. Photothermal Therapy
17.3.4. Gold Nanoparticle-Based Combined Cancer Therapy
17.4. Application of Gold Nanoparticles in Cancer Diagnosis
17.4.1. Bio-Imaging
17.4.1.1. Computed tomography (CT)
17.4.1.2. Magnetic resonance imaging (MRI)
17.4.1.3. Nuclear imaging
17.4.1.4. Fluorescence imaging (FI)
17.4.1.5. Photoacoustic imaging (PA)
17.4.2. Biosensing
17.5. Gold Nanoparticles as Theragnostic Agents
17.6. Clinical Status of Gold Nanoparticle Formulations
17.7. Safety Concerns and Challenges for Application of Gold Nanoparticle in Healthcare
17.8. Conclusion
References
18. Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment
18.1. Introduction
18.1.1. Self-assembly of Peptide
18.1.2. Targeting Peptides
18.2. Peptide-Based NPs in Cancer Therapeutics
18.2.1. Peptide-Based NPs for Gene Delivery/Cytotoxic Drug
18.2.2. Peptidomimetics with Chemotherapy
18.2.2.1. Peptide hormones-based drug conjugates
18.2.2.2. Peptide-based NPs vaccines for immunotherapy
18.3. Peptide-Based NPs in Cancer Theragnostics
18.3.1. Targeting Peptides
18.3.2. Environment Responsive Peptides
18.3.3. Cell-Penetrating Peptides (CPPs)
18.3.4. Peptide Receptor Radionuclide Therapy (PPRT)
18.4. Peptide-Based Nanoparticles
18.5. Cell-penetrating Particles
18.6. CPPs: Protein Delivery in Cancer
18.7. Conclusion and Future Prospects
References
19. Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor: Safe Nanomedicines
19.1. Cell-Inspired Systems
19.1.1. Exosomes
19.1.2. Cell-Derived Nanovesicles
19.2. Lipid-Based Systems
19.2.1. Solid Lipid Nanoparticles (SLNs)
19.2.2. Coordination Micelles
19.2.3. Filomicelles
19.3. Bacteria-Inspired Systems
19.3.1. Cellular Ghost
19.3.2. Microbots
19.3.3. Recombinant Bacteria
19.4. Hydrogel-Based Systems
19.4.1. Alginate-Based Hydrogel
19.4.2. Interpenetrating and Semi-Interpenetrating Polymer Network (IPN) Hydrogels
19.4.3. Imprinted Hydroxyethyl Methacrylate (HEMA) Hydrogels
19.5. Virus-Inspired Systems
19.5.1. Viral Gene Vectors
19.5.2. Virus-Like Particles
19.5.3. Virosomes
19.6. Mammalian Cell-Based Systems
19.6.1. RBC
19.6.2. Stem Cells
19.6.3. Platelets
19.6.4. Macrophages
19.6.5. Lymphocytes
19.7. Application in Cancer Therapy
19.8. Biological Effects and Toxicity of Biomimetic Nanovesicles
19.9. Conclusions
19.10. Limitations and Future Research
References
Index

Citation preview

Nanomaterials in Healthcare This cutting-edge reference book discusses the biomedical applications of nanomaterials. It covers different types of nanoparticles, such as polymeric nanoparticles, lipoidal nanoparticles, and metallic nanoparticles. It discusses the current trends and challenges in the development of safe biomedicines. The book reviews FDA-approved medicines, nanohybrid systems for early-stage diagnosis and treatment of diseases, advanced approaches of cost-effective bio-imaging, and theragnostics. It also covers the basic design and fundamental understanding of surface-engineered biomedicine. The book is meant for experts in the healthcare industry as well as post-graduates in biomedical engineering and nanotechnology.

Nanomaterials in Healthcare

Edited by

Rohit Srivastava Department of Biosciences and Bioengineering Indian Institute of Technology Bombay, Mumbai, India

Sujit Kumar Debnath Department of Biosciences and Bioengineering Indian Institute of Technology Bombay, Mumbai, India

Rajendra Prasad School of Biochemical Engineering Indian Institute of Technology - BHU Varanasi, Uttar Pradesh, India

First edition published 2024 by CRC Press 2385 NW Executive Center Drive, Suite 320, Boca Raton, FL 33431 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2024 selection and editorial matter, Rohit Srivastava, Sujit Kumar Debnath, and Rajendra Prasad; individual chapters, the contributors Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. ISBN: 978-1-032-34475-1 (hbk) ISBN: 978-1-032-34481-2 (pbk) ISBN: 978-1-003-32236-8 (ebk) DOI: 10.1201/9781003322368 Typeset in Times by MPS Limited, Dehradun

We dedicate this book to our parents and mentors.

Contents Preface Acknowledgments List of Reviewers Editors List of Contributors

ix xi xiii xv xvii

1 Introduction to Nanomaterials and Their Scope in Drug Delivery Pankaj Kumar, Uzma Afreen, and Sujit Kumar Debnath

1

2 Application of Nanomaterials in Medicine: A Clinical Perspective Arnab Ghosh and Alyssa Gomes

17

3 Advancement of Polymer-Based Nanocarrier System in Drug Delivery Pallavi Kiran, Abhinanda Kar, Arpita Banerjee, and Arzoo Puri

35

4 Liposomes and Lipid Structures: Classification, Characterization, and Nanotechnology-Based Clinical Applications Suditi Neekhra, Priyanka Maske, and Roshan Keshari 5 Functionalized Carbon-Based Nanoparticles for Biomedical Application Monalisha Debnath, Swati Patil, and Sujit Kumar Debnath 6 Engineered Magnetic Nanoparticles: Challenges and Prospects Roshan Keshari and Bhingaradiya Nutan 7 Nano Metal-Organic Frameworks as a Promising Candidate for Biomedical Applications Dhruv Menon, Swaroop Chakraborty, Prateek Goyal, Eugenia Valsami-Jones, and Superb K. Misra 8 Porous Silica Nanoparticles for Targeted Bio-Imaging and Drug Delivery Applications Dhwani Rana, Raghav Gupta, Bharathi K., Rupali Pardhe, Nishant Kumar Jain, Sagar Salave, Rajendra Prasad, Derajram Benival, and Nagavendra Kommineni 9 Recent Advancement of Multifunctional ZnO Quantum Dots in the Biomedicine Field Sayoni Sarkar, Jaisen Lokhande, and Sujit Kumar Debnath

55

75

101

115

133

155

vii

viii Contents 10 Relevant Properties of Metallic and Non-Metallic Nanomaterials in Biomedical Applications Parisa Fatehbasharzad, Pavlo Ivanchenko, Ola El Samrout, and Jaime Gómez Morales

175

11 Exosomes and Their Theragnostic Applications in Healthcare Abhishekh Tiwari, Zainab Godhrawala, and Atul Chaskar

195

12 Nanogels for Theranostic Applications in Healthcare Vaishali Pawar, Amreen Khan, Shruti Pendse, Rupali Bagale, Akshara Adapa, and Padmini Chandra

211

13 Theranostic Application of Nanofibers in Tissue Engineering Atul Chaskar, Namrah Azmi, and Dhriti Shenoy

225

14 Role of Nanomaterials in Biosensing Applications Jasmeen Kaur and Menam Pokhrel

239

15 Application of Two-Dimensional Materials for Cancer Theranostic Barkha Singh and Ritika Uday Gaitonde

259

16 Solid Lipid Nanoparticles: Towards Emerging Cancer Nanomedicine Amreen Khan, Vaishali Pawar, Rupali Bagale, Shruti Pendse, and Akshara Adapa

281

17 Gold Nanoparticles for Cancer Therapy and Diagnosis Janhavi Devrukhkar and Jasmeen Kaur

297

18 Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment Pallavi Kiran, Vibha Kumari, Baishali A. Jana, and Prachi Kulkarni

317

19 Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor: Safe Nanomedicines Fatemeh Salemizadehparizi, Rajendra Prasad, and Berivan Cecen

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Index

353

Preface Nano(bio)materials have been recognized as safe imaging and therapeutics platforms for both preclinical and clinical trials. In early 1990s, nanoimaging agents were developed for non-invasive imaging of targeted cells in living subjects. Imaging-integrated therapeutic agents known as “theranostics” have also been proposed as smart platforms for site-selective diagnostics and treatment with minimum dose requirements. However, precise diagnostics and therapeutics of targeted site is a major challenge. Various targeting agents such as folic acid, RGD peptide, antibodies, HA, proteins, etc., have been applied to improve the stability and selective targeting ability of hybrid nanomaterials. A few nanoparticles have been approved for clinical trials and also approved by FDA. On the other hand, nanomaterial-based photothermal therapy, photodynamic therapy, chemotherapy, tissue repair or regeneration, etc., have gained tremendous attention in healthcare which are discussed in this book. Several other parameters such as scalability, reproducibility, stability, toxicity, etc., which are essential for translational research, have been covered in this book. This is the first comprehensive book on various engineering and characteristic approaches of nanomaterials in healthcare. This book deals with known and unknown facts of translational medicines and their future goals. Overall, this book will be beneficial for biomedical students, scientists, and researchers of academia and biotechnology industries. For preparing this book, we have gathered international experts’ opinion in the field of functional nanomaterials for medical applications. Chapters are collected from national and international authors. Introduction to nanomaterials and their translational scope have been addressed in Chapters 1 and 2. Chapters 3–5 describe the importance of soft and non-metallic nanosystems for targeted bioimaging and therapeutic applications. Leading scientists describe the importance of surface engineered inorganic nanoparticles in targeted imaging, drug delivery, and cancer therapeutic in Chapters 6–10. Now, inherent biocompatibility and selective tumor targeting are key challenges for clinically relevant biomedicine/ nanomedicine which are covered in Chapters 11 and 12. Chapters 13–15 discuss the imaging-integrated therapeutic systems known as theranostics platforms. One of clinically acceptable system, viz., lipid nanoparticles for cancer nanomedicine is discussed in detail in Chapter 16. Plasmonic nanomaterials for cancer nanomedicine are discussed in Chapter 17. Chapters 18 and 19 are written by molecular theranostics experts who have worked extensively to develop clinically acceptable systems. This book may help various researchers and investigators in the area of translational research on nanoparticles to learn about imaging and therapeutics importance, which are essential for the study of early stage diagnosis and treatment. This book will be beneficial for graduate and medical students who are learning about the cutting edge technologies. Nanomedicine investigators, biomedical engineers, radiologist, and oncologists will be able to utilize the concept of technologies described in this book in developing cost-effective novel imaging and therapeutics.

ix

Acknowledgments We acknowledge our reviewers for reviewing chapters and contributing authors from national and international top-tier institutes. We acknowledge the support of the Department of Biotechnology, Government of India. We are grateful to the Shanti Swarup Bhatnagar Prize 2021 in Medical Sciences and Abdul Kalam National Fellowship along with The Royal Society of Chemistry and Biology, and Indian National Science Academy. We also acknowledge the support of the top-tier institutes such as Indian Institute of Technology and Bombay and Indian Institute of Technology (BHU), Varanasi, India.

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List of Reviewers

Dr. Dr. Dr. Dr. Dr. Dr. Dr.

Puneet Khandelwal Prem Prakash Arpan Pradhan Atul Sharma Qing He Mahadeo Gorain Ipsita Chinya

Dr. Chetan Borkhataria Dr. Malaykumar Chotaliya Dr. Karan Varshney Dr. Abhay Dharamsin Dr. Chintan Tank Dr. Punit Bhatt Dr. Vivek Borse

Dr. Jayrajsinh Sarvaiya Dr. Saumendu Deb Roy Dr Prithviraj Chakraborty Dr. Sunny Shah Dr. Vivek Patel

Senior Postdoctoral Fellow, The Johns Hopkins University School of Medicine Senior Postdoctoral Fellow, Meharry Medical College Postdoctoral Fellow, Emory University, USA Senior Postdoctoral Fellow, Tufts University, USA Staff Scientist, Tufts University, USA Senior Scientific Fellow, NCCS, Pune, India Assistant Professor and HOD Assistant Professor and HOD, DR. V. R. Godhania College of Engineering & Technology, Porbandar Assistant Professor, B. K. Mody Government Degree Pharmacy College, Rajkot, Gujarat, INDIA Assistant Professor, R K School of Pharmacy, Rajkot, Gujarat, India Doctor of Medicine (MD) Candidate at Deakin University & Research Officer at Monash University Principal, Parul Institute of Pharmacy, Dean, Faculty of Pharmacy, Parul University, Vadodara, Gujarat, India Head of Department, Faculty of Pharmacy, Dr. Subhash Technical Campus, Junagadh, Gujarat, India Assistant Professor, Department of Pharmacology, Faculty of Pharmacy, Dharmsinh Desai University, Nadiad, Gujarat, India DST INSPIRE Faculty, NanoBioSens Lab, Department of Medical Devices, National Institute of Pharmaceutical Education And Research (NIPER) Hyderabad, Hyderabad, Telangana, India Associate Professor, National Forensic Sciences University, Gandhinagar, Gujarat, India Principal, Mata Gujri College of Pharmacy, Kishanganj, Bihar, India Professor, Dept. of Pharmaceutics, Mata Gujri College of Pharmacy, Mata Gujri University, Kishanganj, India Assistant Professor Assistant Professor, B K Mody Government Pharmacy College, Rajkot, Gujarat, India Manager R&D, Formulation Research & Development Non-Orals, Sun Pharmaceutical Industries Ltd, Gujarat, India

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Editors Dr Rohit Srivastava is a Dr Shanti Swarup Bhatnagar awardee currently working as a professor and head at the Indian Institute of Technology Bombay. He completed his master of science and PhD degree in biomedical engineering from Louisiana Tech University, Ruston, LA, the USA, for which he received the best student of the year award in 2005. Dr Srivastava holds various awards such as Healthcare Innovation Worldcup Award 2013, DBT IYBA Award 2013, DBT Tata Innovation Fellowship, Stanford MedTech Award 2016, DBT National Bioscience Award 2016, Shri Om Prakash Bhasin Award 2018, Abdul Kalam Technology Innovation National Fellowship, etc. Additionally, Dr Rohit Srivastava is an elected fellow of the Royal Society of Chemistry (FRSC), fellow of the Royal Society of Biology (FRSB), fellow of NASI, and fellow of INAE, etc. His lab has graduated 35 PhD students and 75 master’s students at IIT Bombay. His NanoBios lab at IIT Bombay comprises 22 PhD students, 7 master’s students, 5 post-docs, 3 interns, and 5 staff. Dr Srivastava has published more than 200+ articles in international journals and conference proceedings with an h-index of 37. Further, he has participated in several book chapters and books. Nationally and internationally, his lab receives grants for most of all advanced science and technology domains. More than 130+ of his current ideas have been submitted as patents, and design applications to the U.S. and Indian Patent Office, with several of them approved. Dr Sujit Kumar Debnath is working as an industrial post-doctoral fellow at Applied Materials India Pvt Ltd. He has completed his PhD in pharmacy from Gujarat Technological University, Ahmedabad, Gujarat, in 2018. Afterward, he joined as an institute post-doctoral fellow at the Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India. He has more than 14 years of teaching and research experience. Dr Sujit works on smart and novel drug delivery systems using nanoparticulate approaches. He is well-versed in drug delivery, pre-clinical studies, toxicity, chromatographic estimation, and many more. He holds the GUJOST award from Gujarat Council on Science and Technology, Govt. of Gujarat under the Department of Science and Technology (DST). Nationally and internationally, Dr Sujit has been actively engaged in different collaborative projects. His contribution to science has gotten a brilliant reception from the worldwide scientific community. He has published 24 articles in various highimpact renowned journals and 3 book chapters in reputed publishers. He is frequently appointed as a guest lecturer. He also serves as an active reviewer in many reputable journals. Dr Rajendra Prasad is currently working as an assistant professor at the Indian Institute of TechnologyBHU, Varanasi, India. Before this position, he had postdoctoral experience from (i) Tufts University, Medford, MA, USA; (ii) Technion-Israel Institute of Technology, Haifa & NOVA Medical School, Faculdade de Ciências Médicas Campo Mártires da Pátria, Lisboa, Portugal; and (iii) Indian Institute of Technology, Bombay, Powai, India. He works in cancer theranostics for solid tumor imaging and ablation. In 2017, Dr. Prasad earned his PhD degree from CSIR-National Chemical Laboratory, Pune, India, on multifunctional hybrid nanomaterials design for cancer theranostics. Dr Prasad mainly focuses on clinically relevant nanomedicines for cancer diagnosis and therapeutics. He has designed chemically conjugated multimode contrast and therapeutic agents such as liposomes, NanoBiosomes, Gold Nanorods, porous silica, drugconjugated hybrids, etc., and has investigated them in pre-clinical models. He is well-versed in pre-clinical trials and light-mediated combination cancer therapeutics. To date, he has published about 23 research articles in top-tier journals, about 11 patents, 7 trademarks, 5 journal cover arts, and 2 book chapters with an h-index of 13. Dr Prasad holds awards like the best paper award, PBC Fellow, SAS Fellow, etc.

xv

Contributors Akshara Adapa NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Uzma Afreen Birla Institute of Technology Mesra, Jharkhand, India Namrah Azmi Dept. of Physics Faculty of Natural Sciences Norwegian University of Science and Technology Trondheim, Norway Rupali Bagale NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Arpita Banerjee Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Derajram Benival Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India Berivan Cecen Department of Biomedical Engineering and Department of Mechanical Engineering Rowan University Glassboro, New Jersey, USA

Swaroop Chakraborty Geography, Earth and Environmental Sciences University of Birmingham Birmingham, UK Padmini Chandra NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Atul Chaskar National Centre for Nanoscience and Nanotechnology University of Mumbai Mumbai, Maharashtra, India Monalisha Debnath Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Janhavi Devrukhkar NanoBios Laboratory Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Parisa Fatehbasharzad Molecular and Preclinical Imaging Centers Department of Molecular Biotechnology and Health Sciences University of Torino Torino, Italy and Department of Neuro- and Sensory Physiology University Medical Center Göttingen Göttingen, Germany

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xviii Contributors Ritika Uday Gaitonde Department of Life Sciences Ramnarain Ruia Autonomous College Mumbai, Maharashtra, India Arnab Ghosh Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Zainab Godhrawala National Centre for Nanoscience and Nanotechnology University of Mumbai Mumbai, Maharashtra, India

Baishali A. Jana Indian Institute of Technology Bombay Mumbai, Maharashtra, India Bharathi K. Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India Abhinanda Kar Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Alyssa Gomes Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Jasmeen Kaur NanoBios Laboratory Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Prateek Goyal Materials Engineering Indian Institute of Technology Gandhinagar, Gujarat, India

Roshan Keshari Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Raghav Gupta Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India

Amreen Khan NanoBios Lab Department of Biosciences and Bioengineering and Center for Research in Nanotechnology and Science Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Pavlo Ivanchenko Department of Chemistry and Interdepartmental Nanostructured Interfaces and Surfaces (NIS) Centre University of Torino Torino, Italy and Vrije Universiteit Brussel (VUB) ETEC Department MOBI Research Group Brussels, Belgium Nishant Kumar Jain Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Pallavi Kiran Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Nagavendra Kommineni Center for Biomedical Research Population Council New York, New York, USA Prachi Kulkarni Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India

Contributors xix Pankaj Kumar Indian Institute of Technology Bombay Mumbai, Maharashtra, India Jaisen Lokhande Department of Pharmacology Tilak Municipal Medical College and Lokmanya Tilak Municipal General Hospital Mumbai, Maharashtra, India Priyanka Maske Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Dhruv Menon Materials Engineering Indian Institute of Technology Gandhinagar, Gujarat, India Superb K. Misra Materials Engineering Indian Institute of Technology Gandhinagar, Gujarat, India Jaime Gómez Morales Laboratorio de Estudios Cristalográficos Instituto Andaluz de Ciencias de la Tierra (CSIC-UGR) Granada, Spain Suditi Neekhra Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Bhingaradiya Nutan Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Rupali Pardhe Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India

Swati Patil Department of Pharmacology Lokmanya Tilak Municipal General Hospital and Lokmanya Tilak Municipal Medical College Mumbai, Maharashtra, India Vaishali Pawar NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Shruti Pendse NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Menam Pokhrel NanoBios Laboratory Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Arzoo Puri Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Dhwani Rana Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India Sagar Salave Department of Pharmaceutics National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gujarat, India

xx Contributors Fatemeh Salemizadehparizi Department of Biomedical Engineering Binghamton University Binghamton, New York, USA Ola El Samrout Department of Chemistry and Interdepartmental Nanostructured Interfaces and Surfaces (NIS) Centre University of Torino Torino, Italy and Laboratoire de Reactivité de Surface LRS Sorbonne Université Paris, France Sayoni Sarkar Centre for Research in Nanotechnology and Science and NanoBios Lab Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Dhriti Shenoy National Centre for Nanoscience and Nanotechnology University of Mumbai Mumbai, Maharashtra, India

Barkha Singh Department of Biosciences and Bioengineering and Centre for Research in Nano Technology & Science (CRNTS) Indian Institute of Technology Bombay Mumbai, Maharashtra, India Vibha Kumari Department of Biosciences and Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India Abhishekh Tiwari National Centre for Nanoscience and Nanotechnology University of Mumbai Mumbai, Maharashtra, India Eugenia Valsami-Jones Geography, Earth and Environmental Sciences University of Birmingham Birmingham, UK

Introduction to Nanomaterials and Their Scope in Drug Delivery

1

Pankaj Kumar1, Uzma Afreen2, and Sujit Kumar Debnath1 1 2

Indian Institute of Technology Bombay, Mumbai, Maharashtra, India Birla Institute of Technology, Mesra, Jharkhand, India

Contents 1.1 1.2

Introduction Types of Nanoparticles 1.2.1 Metallic Nanoparticles 1.2.2 Lipid-Based Nanoparticles 1.2.3 Carbon-Based Nanoparticles 1.2.4 Polymer-Based Nanoparticles 1.2.5 Silica-Based Nanoparticles 1.3 Application of Nanoparticles 1.3.1 Antibacterial 1.3.2 Cancer 1.3.3 Neurodegenerative 1.3.4 Infectious Disease 1.3.5 Immunotherapy 1.4 Nanoparticles Toxicity 1.4.1 Metallic-Based Nanoparticles 1.4.2 Lipid-Based Nanoparticles 1.4.3 Silica-Based Nanoparticles 1.4.4 Carbon-Based Nanoparticles 1.4.5 Polymer-Based Nanoparticles 1.5 Prospects and Challenges References

DOI: 10.1201/9781003322368-1

2 2 3 3 4 4 4 4 4 5 6 6 7 7 8 8 9 9 10 10 11

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2 Nanomaterials in Healthcare

FIGURE 1.1 Nanoparticles with functionalized therapeutic molecules or act as a carrier for drug molecule.

1.1 INTRODUCTION Recently, the augmentation of nanoparticles (NPs) in the scientific community circumvents the insolubility, instability, side effects, drug loss, and systemic distribution. Other than this, nanoparticles can be deployed for diagnosis as well. Scientifically, NPs are defined by European Commission as A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm–100 nm [1].

As shown in Figure 1.1, NP design is subject to therapeutic purposes, for example, antibody engineered with NP target-specific ligand of disease, drug delivery, nucleic acid delivery, vaccine delivery, and others that have been discussed in detail in this chapter. The therapeutic capability of NPs depends on their size and shape by altering physicochemical properties and surface area. Therefore, NP adaptation to interact with biological molecules is complemented. The scope of this chapter is the introduction of na­ noparticles such as metallic-based, lipid-based, carbon-based, polymer-based nanomaterials, and silicabased nanoparticles used in the different spectrum of biology and healthcare, maneuvering it to overcome constraints from drug or entirely noble designed NP for treatment of disease or diagnosis. Here, the discussion is limited to the structure of NPs and their application (antibacterial, cancer, food and agri­ culture, neurodegenerative disease, and immunotherapy) along with the toxicity that arises from them. Nonetheless, there is a multitude of factor that determines the therapeutic wherewithal, such as materials, size, methods of nanoparticle formulation, route of administration, physicochemical properties, and other factors [2]. The flexibility provided by NPs for modification in size, shape, and surface makes wide use in different domains, including treatment of disease to vaccine delivery. Most recently, the SARS-CoV-2 vaccine was manufactured within 3 months of its sequencing as an mRNA vaccine encapsulated to lipid nanoparticles, showing 90% efficacy and acting as a boon for the pandemic [3].

1.2 TYPES OF NANOPARTICLES Nanoparticles can be calssified into different types according to the size, morphology, chemical, and physical properties (Figure 1.2). Broadly, these nanoparticles are two types: organic and inorganic.

1 • Introduction to Nanomaterials and Their Scope in Drug Delivery 3

FIGURE 1.2 Different types of nanoparticles.

1.2.1 Metallic Nanoparticles Metallic nanoparticles (MNPs) contain a metal core of inorganic metal or metal oxide shelled with inorganic or organic material or metal oxide. The unique physicochemical property of MNPs is the perfect candidate for imaging as well as magnetic separation depending on the metal. It can be different types like gold, silver, iron, copper, and zinc. The synthesis of metallic NP is processed through a topdown approach (bulk molecules reach the nanosize molecule) or the bottom-up approach (a simpler form of nanoparticles is considered for NP formulation). The chemical methods have been employed for the synthesis of metallic nanoparticles that are the Turkevich method, Brust-Schiffrin method, and Seed growth method. Turkevich and Brust-Schiffirin method results in the spherical form of NPs, where as Seed growth results in rods or oval-shaped. The green synthesis involved, whereas microorganism, plant, or their product have been employed for the formation of the nanoparticles [4]. Contrary to chemical methods of synthesis, green synthesis is environmentally affable and utilizes bacteria, actinomycetes, fungi, and plants from the biological point of view. Green synthesis approaches include the poly­ saccharide method, tollens method, polyoxometalate method, and biological method [5,6].

1.2.2 Lipid-Based Nanoparticles The lipid carrier system includes nanoemulsion, liposomes, solid lipid nanoparticles (SLN), and nanostructured lipid (NLC). The difference in their structures is due to micelles’ arrangement. The liposomes are the first lipid carrier systems during the evolutionary trajectory of discovery to the introduction of solid lipid nanoparticles and nanostructured lipid carriers. Liposomes have an aqueous core surrounded by a double phospholipid layer. Nanoemulsions have a liquid hydrophobic core surrounded by a lipid monolayer, and NLC/SLN constitutes a solid hydrophobic core surrounded by a monolayer [7]. Lipid nanoparticles can be prepared by different methods: solvent evaporation tech­ nique, cold high-pressure homogenization, hot-high-pressure homogenization, and ultrasonic solvent emulsification technique. Temperature, pressure, and solvent are the key changeable parameters for these techniques [8].

4 Nanomaterials in Healthcare

1.2.3 Carbon-Based Nanoparticles Carbon has several technological applications, including developing synthetic materials and drug delivery. Carbon nanotubes, graphene oxide, and graphene quantum dots are the commonly used carbonbased nanomaterials (CBNs) studied in the biomedical field due to their structural variability and physical and optical properties [9,10]. CBNs can be synthesized using different techniques: mechanical exfoliation, chemical exfoliation, chemical vapor deposition, electric arc discharge, and laser ablation. The mechanical exfoliation needed adhesive tape to exfoliate from graphene and transfer it to other surfaces. In contrast, chemical exfoliation requires adding a chemical group to convert graphite to graphene. In the electric arc method, the potential difference is applied in presence of high voltage and low pressure in between graphite rods. Laser ablation graphite is utilized to make carbon nanotube [11].

1.2.4 Polymer-Based Nanoparticles Polymeric nanoparticles have a size range from 1 to 1000 nm containing entrapped drug or biological active compound within or surface-adsorbed polymeric core. Consideration of polymeric nanoparticles is due to their lower cytotoxicity and their degradable nature. The categorization can be intended into nature-based polymers and chemical-based polymers. Natural polymeric material includes starch, chit­ osan, cellulose, and alginate. In contrast, chemosynthesis material includes polylactic acid (PLA), polyurethane, and poly(lactic-glycolic acid) (PLGA), polymethyl methacrylate resin (PMMA), etc. The biosynthesised polymeric nanomaterials incorporate microbial enzyme for the synthesis of nanoparticles. Some frequently used polymeric nanoparticles fabrication methods are solvent evaporation, emulsifi­ cation, nanoprecipitation, reverse salting out, and solvent diffusion [12].

1.2.5 Silica-Based Nanoparticles Silica is one of the abundant elements. The synthesis of these nanoparticles is divided into two parts chemical and biogenic. Biogenic synthesis utilizes biological entities to synthesize these nanoparticles [13]. Whereas chemical synthesis has different categorizations based on the process involved and the chemical used. There are a few chemical synthesizing processes: stober method, mesoporous silica nanoparticle, hollow or core-shell, shaped particles, etched particles, and surface modification technique [14].

1.3 APPLICATION OF NANOPARTICLES Scientific study of nanoparticles in last decade in wide array of the field. It includes alternative for treatment of multi drug resistance (MDR) to disease such as cancer, Alzheimer’s disease, Parkinson’s disease, and infectious disease as well. The recent application of nanoparticles in various fields are described in Figure 1.3.

1.3.1 Antibacterial With the advancement of knowledge, the antibacterial properties of nanoparticles and their impact have been widely studied. Silver NPs show antibacterial attributes in contrast to gold NPs. Although gold NPs acquired bactericidal properties against multidrug resistance (MDR) gram-positive and negative bacteria

1 • Introduction to Nanomaterials and Their Scope in Drug Delivery 5

FIGURE 1.3 Representation of application of nanoparticles [ 15].

when functionalized with the cationic agent [16,17]. Furthermore, copper nanoparticles containing PVA have shown a noncytotoxic impact in human corneal cells with antibacterial properties that may provide scope in healthcare after a comprehensive study [18]. The bactericidal properties were influenced due to cell wall disruption, intracellular proteins muddle, oxidative stress imbalance, and others. The facile synthesis procedure for metallic nanoparticles might be a promising tool to conquer the MDR in healthcare that is limited to drugs only. Double-fold growth inhibition demonstrated by SLN engulfing anti-tubular drugs such as rifampicin, pyrazinamide, and isoniazid, tested on M. marinum opposing individual drug counterparts [19]. Doxycycline encapsulated SLN reduced B. meitensis from the liver and spleen [20]. Tetracycline hydro­ chloride adsorbed on porous hollow silica spheres showed antibacterial activity against E. coli and S. aureus [21]. Using biomimetic adhesive property, silica NP loaded with antibacterial agent (benzalk­ onium chloride) displayed improved antibacterial properties enabling the scope of mask fabrication [22]. The antibacterial activity of carbon dots tested, synthesized from vitamin C, has shown broad antibacterial activity [23]. Likewise, copper oxide decorated carbon nanoparticles have antibacterial activity against gram-positive bacteria as well as gram-negative bacteria [24]. Sometime the composite nanoparticles containing biodegradable materials like PLA and polybutylene adipate terephthalate (PBAT) demonstrate improved antibacterial activities. Carboxymethylcellulose (CMC) attach to silver due to its cationic property show the antibacterial property [25]. Not only metalic NP, polymer (chitosan)-based nanoparticles have shown antibacterial against Staphylococcus [26].

1.3.2 Cancer Uncontrolled growth of cells at a particular body site is known as cancer. The presence of a lump in the organs fixed at a specific area is considered a benign tumor; however, the capability to move away from the site is considered a malignant tumor. The encouraging engagement of NPs towards cancer therapeutics in recent times has progressed. Iron nanoparticles and dextran-coated iron NP with the aid of ultrasound, have shown depletion in tumors in vivo and in vitro due to hyperthermia [27,28]. Additionally, hyperthermia exercises to ameliorate controlled drug release from magnetoliposomes consisting of doxorubicin experimented in glioma cells both in-vitro and in-vivo [29]. Also, iron-based nanoparticles act as a vehicle for the endogenous tumor antigen to lymph node producing a robust immune response against cancer [30]. Irinotecan carried by lipid nanoparticles engineered with chitosan has shown a cytotoxic effect on HCT-116 cells and conformity of sustained drug release at colon pH, suggesting therapeutic use in colon

6 Nanomaterials in Healthcare cancer [31]. In one of the studies, lipid nanoparticles with targeting antibody-like EGFR, containing antiE6/E7 siRNA molecule reduced the 50% tumor in xenografted human papilloma virus (HPV) compared to those without targeting an antibody [32]. Mesoporous NPs containing doxorubicin were synthesized for prostate cancer. The framework of pH sensitized CaCO3 was imbedded to control the drug release at tumer site for better anti-tumor effect [33]. Chitosan was also used to cap mesoporous silica consisting of curcumin, demonstrating anticancer effectiveness against glioblastoma cells [34]. Multifunctional mesoporous silica nanoparticles containing both siRNA and miRNA photosensitized with indocyanine green to facilitate endosomal escaping and surface conjugation to iRGD peptide for better cellular penetration and improving anti-tumor activity against breast tumor [35]. Folic acid decorated via graphene oxide, a carrier of doxorubicin, has shown the synergistic impact of drug and photothermal therapy in tumor declination in Balb/c mice [36]. Multiwalled carbon nanotubes engineered to carry iron as well as doxorubicin for the treatment of cancer have demonstrated specific drug delivery [37]. Furthermore, the carbon dots with modification of hyaluronic acid, as targeted delivery also carried doxorubicin acted in an acid-sensitive manner [38].

1.3.3 Neurodegenerative Neurodegenerative disease is a defect in the brain’s neuronal cells or peripheral nervous system. Alzheimer’s and Parkinson’s disease are the most common ones. Until now, there is no cure for the neurodegenerative disease, but the NPs bolstered the treatment by overcoming intimidating blood-brain barrier (BBB) and as drug carriers to diagnostic agents. Oleic acid molecules overlay to iron NPs tied up with N-isopropyl acrylamide derivative (NIPAm-AA) tagging with small hairpin (shRNA) and nerve growth factor (NGF) binds (NP-NIPAm-AA-NGF (pDNA)) display its effect on Parkinson diseasepreventing α-syn synthesis by shRNA, guided by NGF to surpass BBB [39]. Furthermore, iron nano­ particles surrounded by bovine serum albumin (BSA) ornate with sialic acid adhere to a β-amyloid biomarker of Alzheimer’s, accomplished as a diagnostic agent [40]. To increase the stability of the drug, astaxanthin-loaded lipid nanoparticles were prepared for Alzheimer’s disease [41]. The availability of resveratrol was increased 4.5 times in the brain in the form of SLN compared to free resevatrol [42]. Similarly, to overcome the erythropoietin (EPO) insolubility and clearance time, EPO encapsulated with lipid nanoparticles is not only able to cross the blood-brain barrier but also restore the cognitive ability of mice in Alzheimer’s disease [43]. Curcumin encapsulated SLNs have demonstrated oxidative stress through activation of the BCL2 family and P38MAPK path­ ways by surpassing the blood-brain barrier in the epileptic mice model [44]. An approach has been adopted to surpass acid labile substances in the gastric condition. Mesoporous NP was prepared with levodrop drug and fatty acid. These nanoparticles were not affected by the gastric acidic pH condition while released this drug at pH 7.4 [45]. Peptide low-density lipoprotein receptor (LDLR), as an outer covering helped in transcytosis to cross the blood-brain barrier (BBB) in vitro, reflecting a good candidate for neurodegenerative study [46]. Moreover, mesoporous silica nanoparticles loaded berberine coated with lipids prevent acetylcholine esterase activity and amyloid fibrillation [47]. Other types of nanoparticles like CBNs have shown synapse gene expression in short-term exposure but at long-term exposure causing cell death in the neuron [48]. Polymer-based NP decorated with antiAβ1–42 targeting Alzheimer’s disease have shown a decrease in the level of amyloid-β peptide (Aβ) and complete correction of memory disease [49].

1.3.4 Infectious Disease Infectious disease is prevalently caused due to viruses, bacteria, parasites, or their products. This infection can be contagious. Some NPs have been exploited to surpass the MDR microbes. These nanoparticles act as a transporter for drugs and biomolecules for better therapeutic purposes than

1 • Introduction to Nanomaterials and Their Scope in Drug Delivery 7 individual drugs. NPs are used as immune enhancers. Silver NPs designed with tannic acid represent oneself as adjuvants, increasing the protection against the herpes simplex virus. The protection is due to the response NPs engender HSV-specific humoral and cell-mediated immunity [50]. Co-delivery of mannose and HIV peptide is designed with gold NP to uplift the immune response through peptide presentation to dendritic cells [51]. Doxycycline encapsulated lipid nanoparticles upsurge vitamin D serum in chronic brucellosis through mannose [52]. The lipid nanoparticles encapsulating rifampicin not only increased the ther­ apeutic index but also increased internalization due to lectin, a mannose receptor, present in the macrophage acting as capable of tuberculosis treatment [53]. Carrying biomolecules, the SLNs con­ sists of RNA unmodified versions of c7D11, c8A and c6C that indicated their existence as antibodies in rabbits after a day of intramuscular injection protecting poxviruses [54]. Moreover, si-RNA of SARS-CoV-2 encapsulated with SLNs inhibits almost 90% of the infection [55]. Mesoporous silica nanoparticles was used as a cargo carrier of rifampicin for S. aureus infections [56]. Functionalization of an amino group on mesoporous silica nanoparticles loaded with shikimic and quercetin have shown strong anti-influenza activity [57]. Composite nanoparticles engineered involving carbon nanotube and graphene functionalized with amphotericin B have shown better anti-leishmanial activity [58]. Various infectious diseases have been positively tested against the Klebsiella apnominia, Paeroginosa, and H. pylori [59].

1.3.5 Immunotherapy The enhancement of immune response to eliminate the disease employing NPs are aided with drugs. Integration of NPs with antigen or adjuvant or biomolecules ultimately leads to immune enrichment in the body. Silver nanoparticles with inactivated flu have reduced viral load and enhanced IgA Ab, sig­ nature molecules of mucosal immunity [60]. Moreover, nanoparticles as vaccine adjuvants more effectively cause an antigen-specific immune response and lower inflammation than aluminum adjuvants [61]. Lipid self-amplifying RNA, encoding proteins of SARS-CoV-2 inside lipid NP activate antibody of IgG concentration in sera. The amount of antibodies is proportionate to adequate protection against the virus [62]. Fc-conjugated receptor-binding domain (RBD) mRNA vaccine with lipid nanoparticles has shown 70% survivability when encountering SARS CoV-2 [63]. LNP also acts as an adjuvant similar to aluminum salts when split influenza vaccines enclosed within lipid nanoparticles, without any inflam­ mation, evidently have shown an increase in the IgG1 and IgG2 response [64]. The mesoporous silica nanoparticles with HSP70 antigen, a surface polypeptide of Mycoplasma hyponeumoniae, produce an immune response increasing total IgG level as an adjuvant compared to alum [65]. Furthermore, the large pores of mesoporous nanoparticles have been increased to carry the effective amount of TLR-9 and antigen protein, ovalbumin. The resulted NPs activate the dendritic cell in vitro as well as prevent tumor growth when challenged with the tumor in mice [66].

1.4 NANOPARTICLES TOXICITY Nanoparticles of distinctive physicochemical properties, production, and application are increased in different sectors like medicine, agriculture, mining, combustion, and automobiles, creating an intoxi­ cating environment. Nanoparticles are small in size with a larger surface area, resulting in a higher penetration rate that makes them more hazardous [67]. The release of NPs in the environment leads to a challenging and alarming situation, including bioaccumulation. The magnitude of toxicity is governed by the NP type: SLNs exhibit lower toxicity contrary to metallic NP [68].

8 Nanomaterials in Healthcare

1.4.1 Metallic-Based Nanoparticles Some physiologically important monometallic particles are copper, magnesium, chromium, iron, man­ ganese, cobalt, copper, zinc, selenium, and molybdenum. These nanoparticles show different toxicities in the body using different mechanisms (Figure 1.4). Gold nanoparticle toxicity is still debated, as it is a noble inert metal that accommodates some therapeutic values. The toxicity of metallic particles is also fluctuated by some parameters like particle size, surface chemistry, and their releasing capability [69]. Metallic nanoparticles enter the human body in several ways, like inhalation, ingestion, skin exposure, and injection [70]. The heart, liver, spleen, kidney, and brain are organs sensitive to nano­ particles. The perspective of silver nanoparticle toxicity was seen in Prochilodus lineatus fish, and their analysis was performed in four distinct tissues. Silver (Ag) accumulation in liver tissue was higher than in the intestine, followed by brain tissue, blood, and plasma parameters with rising concentration after 5 days of exposure [71]. Titanium dioxide nanoparticles (TiO2 NPs) toxicity was monitored in zebrafish embryos at different concentrations and exposure times. TiO2 NPs toxic effect was observed deterio­ rating the egg surface, indicating their lethal impact on living organisms [72]. Nanoparticles through the food chain uptake from contaminated soils set foot in living organisms leading to health risks [73]. Inside the body, nanoparticles hinder the globular proteins of the gastrointestinal tract, accumulate in cells, produce cytotoxicity, and hamper gut microbiota.

1.4.2 Lipid-Based Nanoparticles Lipid nanoparticles are efficient and specific technology for drug delivery. Their toxicity varies according to their size, charges, hydrophobic nature, lipid composition, and uptake way. Toxicity of supersaturation of lipids nanoparticles or disruption of the lipid bilayer structure of the cellular mem­ brane is induced by cationic or short-chain lipids nanoparticles [74]. Lipid nanoparticles demonstrated hepatotoxicity after giving an overdose of LNPs injection [75]. Accumulation of LNPs was identified in liver cells, causing neutrophilic inflammations and the release of cytokines from it. However, NPs toxicity was overcome by adding N-acetyl-D-galactosamine with LNPs. In other cases, the toxicity of LNPs occurs because of their surface charge [76]. Also, lipid-based nanomedicines toxicity was observed in the induction of acute hypersensitive reactions and fast phagocytic responses. The extensive

FIGURE 1.4 The toxicity mechanisms and their resulting effect on human blood exposed to metallic based nanoparticles.

1 • Introduction to Nanomaterials and Their Scope in Drug Delivery 9 use of nanoagrochemicals in the soil is a significant source of health risks causing environmental pol­ lution. As justified by [77], lipid nanoparticles are the sole members that are toxic to roundworm Caenorhabditis elegans rather than pesticide molecules. These NLPs need more scientific improvements to use as an agrochemical. Contrary to chitosan/tripolyphosphate (CS/TPP) polymeric nanoparticles, the phytotoxicity of solid lipid nanoparticles was relatively less. Therefore, there is a need to reformulate the chemical composition before adding pesticides [78].

1.4.3 Silica-Based Nanoparticles Toxicity assessment of silica-based nanoparticles (SiNPs) depends on different parameters like size, dose, cell type, and route of entry [79]. The hazardous effects of SiNPs should not be ignored, concerning their increased use in the biomedical, cosmetics, and food industries. Silica nanoparticles induce immunotoxicity by communicating with immunocompetent cells. Examining immunotoxicity relates to cell dysfunctions, cytotoxicity, and genotoxicity. The mechanisms underlined are proinflammatory responses, ROS-induced oxidative stress, and autophagocytosis showing toxicology issues with SiNPs. These toxic studies are becoming concerned, and there is a need to modify their shapes and structure to overcome this problem [80]. Synthetic amorphous silica nanoparticles (SASNs) impart toxicity by the identified geminal silanols that interact with cell membranes and induce oxidative stress. SASN toxicity is assessed by its tendency to lyse red blood cells (hemolytic potential), apoptosis (cytotoxicity), cause lung inflammation, and cause cancer in some when inhaled. The intensity of silica nanoparticle toxicity also varies by degree of exposure to the environment, irrespective of their synthesis history [81]. Mesoporous silica nanoparticles (MSNs) after 30 days of exposure demonstrated the induction of cardiotoxicity and pulmonary toxicity. The study also provides information of SiNPs’ related mechanisms of cardiotoxicity studied in cultured cardio­ myocytes indicating heart failure, arrhythmias, and sudden death in living organisms [82].

1.4.4 Carbon-Based Nanoparticles Despite having wide applications, they are studied for the toxicity they pose to the environment and human health if not handled correctly. Econanotoxicology is the information provided about nanoparticle syn­ thesis, its life cycle, and the significant impact of hazards and risks [83]. Carbon-based nanoparticles are synthesized in different shapes and sizes. Single-wall carbon nanotubes (SWCNTs) show genotoxicity by causing single- and double-stranded DNA break in fibroblast cells of the Chinese hamster. Carbon nanotubes (CNTs) were also found to generate toxicity via oxidative stress in living organisms [84]. In humans, several toxicities are associated with CBNs including hepatotoxicity, immunotoxicity, neurotoxicity, cardiotoxicity, nephrotoxicity, skin toxicity, carcinogenicity, and genotoxicity [85]. Many studies demonstrated the toxic effects of CBNs on the respiratory tract in the form of particulates after combustion. Aerosol CNPs are assessed on chronic bronchitis-like mucosa of primary bronchial epi­ thelial cells (PBEC). A noticeable effect of proinflammatory, tissue injury response, and oxidative stress show higher susceptibility towards exposed carbon nanoparticles [86]. Carbon black nanoparticles triggered oxidative stress on the brain of chicken embryos and catalase activity in brain tissues. Even differential expression of apoptotic markers was observed. So, CBNPs enact in neurotoxicity [87]. Oil industries often use black carbon nanoparticles modified with ethylenediamine, referred to as NP CB-EDA. Their tendency of toxicity was evaluated on fibroblasts LA-9 at various concentrations at 24 and 48 hours’ duration. The results showed that NP CB-EDA particles aggregate at higher con­ centrations to form a bulk. Though, at lower concentrations and because of its small size and larger surface area, capable of absorbing and disrupting cell viability, damaging cell membrane, induction of apoptosis and proinflammatory responses (interleukins TNF-α and IL-6 increases). The abundance of triggered reactive oxygen species (ROX) and nitrogen species (NOX) triggers oxidative stress. Hence, NP CB-EDA carries potential cytotoxic effects showing high damage to health.

10 Nanomaterials in Healthcare

1.4.5 Polymer-Based Nanoparticles The extensive use of polymer-based nanoparticles in the drug delivery zone raises the issues of the safety and the toxicity they inherit. The study of their synthesis, structure, and designs could reveal their potential for toxicity. Magnetic chitosan/graphene oxide (MCGO) composite materials evaluated have shown a decreased cell viability with increased concentration [88]. Moreover, cationic surface-charged polymers like polypropylenimine dendrimers are more toxic, showing reduced survival of zebrafish embryos and weakening cardiovascular function compared to the toxicity of anionic polyamidoamine dendrimers [89]. Other studies also suggest the toxicity of both natural and synthetic polymer-based nanoparticles, which leads to in vivo toxicity like a proinflammatory response, oxidative stress, DNA damage, and impaired cardiovascular functions [90]. The toxic level was reduced by new formulations of polymer-based nanoparticles having natural biodegradable characteristics used in the drug delivery. A comparative approach for toxicity analysis of polymer-based and lipid-based nanoparticles was studied on their potential use in chronic disease treatment. An investigation showed their accumulation in different tissues, but no toxicity or disease signs and no fluctuations in their body mass were observed, even when exposed to high doses [91].

1.5 PROSPECTS AND CHALLENGES Drug discovery to availability in the market undergoes successive steps that initiate the fundamental molecular discovery. It takes 10 to 15 years and investing lots of money to get final regulatory approval for new medicine [92]. Nanoparticles have become an emerging platform focusing on therapeutics possessing efficient diagnostics using unique properties at the nanoscale. In this era, researchers initiate to explore nanotechnology in drug discovery and development. Nanocrystals quantum dots (QDs) and other nanoparticles like nanoshells, fullerenes, dendrimers, nanobodies, nanobarcodes, and magnetic and gold colloids have demonstrated unique advantages in the drug discovery and drug development. QDs are a special class of fluorescent labels with diverse optical properties like optical and long-term colloidal stability and higher brightness [93]. Ideal nano-based therapeutics have demonstrated tissue-specific targeted delivery to avoid off-target effects of active molecules on healthy tissues. More focused drug delivery can be achieved by conju­ gating targeting ligands on the surface of nanoparticles resulting in more focused cell surface compo­ nents of pathologic tissues. These targeting ligands can be categorized into different classes: nucleicacid-based aptamers, antibodies, protein domain, peptides, and small molecules. Folic acid (vitamin B9) is a small molecule targeting ligand used frequently in clinical applications as they have a high affinity to bind with endogenous folate receptors (upregulated in cancer). This folate conjugation is also helpful for delivering genes, oligonucleotides, radiotherapeutic agents, chemotherapeutics agents, protein toxins, and other therapeutics [94]. Advances in nano-based drug delivery open a new direction in the medical field, including drug delivery. This technology brings hopes to increase efficiency and bioavailability with reduced toxicity. Several nanomedicines have already reached the marketplace, and many more are in clinical trials. Surace conjugation with antibody or ligand on nanoparticles is a newer approach to deliver drugs to target tissue, enhancing permeability and retention. Due to nanosize, these nanoparticles are successfully used in detecting disease, imaging specific biological systems, and employed in developing medical devices. These nanomedicines have no specific regulatory framework as they follow the same regulation procedure as all medical products based on the International Council for Harmonisation (ICH) for human use [92]. Despite successive advancements in nanotechnology, very few nanoformulation are being

1 • Introduction to Nanomaterials and Their Scope in Drug Delivery 11 approved by the U.S. Food and Drug Administration (U.S. FDA). The primary concerns are the toxi­ cological and regulatory aspects. The utmost priority is given to the cytotoxicity of nanomaterials inside the body. Full proof methods should be performed to evaluate nanoparticle’s long-term and short-term toxicity before being used for human administration. The advanced, sophisticated analytical techniques help understand nanomaterials’ hidden mechanism and interaction with biological systems. This understanding allows researchers to develop novel nanoformulations for effective outcomes specifically to host targeted or host-responsive drug delivery. The future scope should focus on low-cost, safer nanomedicines with higher drug loading and a controlled release profile for better management of diseases.

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Application of Nanomaterials in Medicine

2

A Clinical Perspective Arnab Ghosh and Alyssa Gomes Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 2.1

2.2

2.3

2.4 2.5 2.6 2.7

2.8

Introduction 2.1.1 History of Nanomedicine 2.1.2 Advantages of Nanomaterials in Clinical Medicine 2.1.3 Disadvantages of Nanomaterials in Clinical Medicine 2.1.4 Latest Trends in Nanomedicine Nanomaterials Used in Communicable Diseases 2.2.1 Tuberculosis 2.2.2 HIV/AIDS 2.2.3 Influenza Nanomaterials Used in Non-Communicable Diseases 2.3.1 Cardiovascular Diseases 2.3.2 Diabetes 2.3.3 Neurodegenerative Diseases 2.3.4 Autoimmune Disorders Nanomaterials Used in Cancer Nanomaterials for Imaging, Screening, and Diagnosis Tackling COVID-19 Using Nanotechnology Nanomedicine and Associative Technologies 2.7.1 Additive Manufacturing 2.7.2 Artificial Intelligence, Machine Learning, and Bioinformatics 2.7.3 Robotics, Automation, and IoT Ethical Concerns about Nanomedicines

DOI: 10.1201/9781003322368-2

18 18 18 19 20 20 21 22 22 23 23 23 23 24 24 25 26 27 27 27 27 28

17

18 Nanomaterials in Healthcare 2.9 Clinical Trials and Approvals of Nanomedicine 2.10 Conclusion References

29 30 30

2.1 INTRODUCTION Over the past ten years, disease-focused theranostic modalities have undergone fast development, and several are currently on the path to medical translation for successful clinical applications. In recent years, advances in non-invasive theranostic techniques, such as the use of light, sound, and electricity, have provided a new avenue for the development of cutting-edge functional nanomaterials that can be used in conjunction with commercially available clinical devices to provide precision and visualization in the treatment of new-generation diseases. The term ‘nanotechnology’ coined by Professor Taniguchi in 1974 refers to the field of science that deals with structures and molecules at the nanoscale, typically ranging between 1 to 100 nm. Richard Feynman, popularly known as the father of nanotechnology, introduced the concept of nanotechnology back in 1959 during his talk at the American Physical Society, where he suggested the manipulation of matter at the atomic level [1]. Nevertheless, research in this field only gained serious attention in the early 1980s with new technologies like the scanning tunneling microscope and the discovery of ful­ lerenes. Particles at the nanoscale have vastly different optical, electrical, magnetic, mechanical, and thermal properties compared to their bulk counterparts, thus facilitating their use across a wide range of disciplines, from pure sciences like physics, chemistry, and biology, to application areas such as elec­ tronics, medicine, energy, and many more. Due to this, nanotechnology has gained much importance and is among the most promising domains of research in the 21st century [2].

2.1.1 History of Nanomedicine One of the key application areas of nanotechnology in medicine is ‘nanomedicine.’ The application of nanotechnology in the medical field can be traced back to the early 19th century Nobel Prize (Physiology/Medicine, 1908) winners, Metchnikov and Ehrlich. They have since been referred to as the modern pioneers of nanomedicine for their work on immunity [3]. [4] delves into the journey of these two scientists and their key findings and developments in detail. Since then, there has been a steady increase in the application of nanotechnology to DNA-drug complexes, antibody-drug conjugates, polymeric, liposomal, and metallic drug-carrier molecules, antimicrobial nanoparticles, and polymerprotein conjugates, and many more nanoformulations [3]. Nanotechnology has been adopted to solve many of the world’s leading healthcare problems, including cancer, microbial infections, neuro­ degenerative and autoimmune disorders, diabetes, reproductive issues, and cardiovascular diseases. The fact is supported by the growing number of nano-related products seeking FDA approval. Table 2.1 lists a few FDA-approved nanomedical products and their applications.

2.1.2 Advantages of Nanomaterials in Clinical Medicine Nanomaterials have several advantages in clinical applications, as listed below: • Effective uptake of nanoparticles across cell membranes due to their small size. • Since many biological entities like antibodies, receptors, proteins, enzymes, and hemoglobin are within the nano-size range, nanomedicine is a highly beneficial option [22]. For

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 19 TABLE 2.1

FDA approved nanomedical products

TRADE NAME/COMPANY Doxil/Janssen

ACTIVE INGREDIENT(S)

APPLICATION(S)

Kaposi’s sarcoma, multiple myeloma, ovarian cancer Cimzia/UCB IgG Fab’ fragment that binds to TNF-α Crohn’s disease psoriatic arthritis [ 6] Ankylosing spondylitis Rheumatoid arthritis Avinza/Pfizer Morphine sulfate Psychostimulant Onpattro/Alnylam Patisiran Transthyretin (TTR)‐mediated amyloidosis DaunoXome/Galen Ltd. Liposomal daunorubicin Cancers, HIV‐associated (non‐PEGylated) Kaposi’s sarcoma (primary) Lipodox/‘Sun Pharma Doxorubicin-hydrochloride Metastatic ovarian cancer, Global FZE’ HIV-associated KS Vyxeos/Jazz Cytarabine and Daunorubicin Acute myeloid Pharmaceutics leukemia (AML) Hensify/Nanobiotix Hafnium-oxide nanoparticles Locally advanced squamous cell carcinoma Inflexal/Crucell Berna Inactivated influenza virus vaccine Influenza infection Biotech prevention Moderna COVID-19 mRNA vaccine COVID-19 infection Vaccine/ ModernaTX Inc. prevention Pfizer-BioNTech Vaccine/ mRNA vaccine COVID-19 infection Pfizer prevention Mircera/Vifor Epoetin β (EPO) (a genetically Anemia (associated with recombinant form of erythropoietin) chronic kidney disease)

• • • • • •

REFERENCES

doxorubicin (adriamycin)

[ 5] [ 7] [ 8] [ 9] [ 10] [ 11] [ 11] [ 12] [ 13] [ 14] [ 15, 16] [ 17, 18] [ 19, 20] [ 21]

example, nanopores can be used to study the properties of nucleic acids at the molecular level [23]. Less invasive diagnostic and treatment options. Ability to serve the dual purpose of a diagnostic and therapeutic agent referred to as ‘theranostics.’ Nanoparticles can be employed as carriers for highly specific drug targeting. Lower drug dosage requirements, thus reducing the chances of multi-drug resistance (MDR). Improved functionality, efficacy, and cost-effectiveness of existing products like drugs once encapsulated or conjugated and delivered at the target site [2]. Nanomedicine can meet unmet clinical needs by providing personalized treatment options.

2.1.3 Disadvantages of Nanomaterials in Clinical Medicine The following are the difficulties faced in utilizing nanomaterials in medicine: • The ineffectiveness of existing in vitro nanotoxicology tests in gauging the toxicity of nanoparticles within the human body is due to their complex nature [22]. • Lack of proper knowledge of the long-term effect of the accumulation of nanoparticles within different biological tissues, their pharmacokinetics and pharmacodynamics, and possible environmental effects [22]. Because nanoparticles are rapidly cleared from the bloodstream by RES and mononuclear cells, they are a key downside of nanoparticle use (MPS). In some imaging applications, however, this quick clearance could be useful [24].

20 Nanomaterials in Healthcare

FIGURE 2.1 Trends in publications using ‘nanomedicine’ in their title, abstract, or as a keyword. Data Source: Obtained from Scopus and PubMed for the search query “nanomedicine.”

• Once scaled up for industrial manufacturing, challenges in maintaining the same character­ istics and stability of nano-formulations and nanomaterials [22]. • The FDA delays clinical trial approval due to a lack of specific regulatory guidelines [25].

2.1.4 Latest Trends in Nanomedicine Based on a brief bibliometric analysis of the publications recorded in the PubMed and Scopus databases, it is evident that the research output of nanomedicine has increased drastically over the years (Figure 2.1). Figure 2.2 shows the geographical distribution of the research published in the past decade. Based on the keywords used in these publications, the most common nanomedical research topics include drug delivery systems, controlled studies, theranostic nanomedicines, antineoplastic agents, neoplasms, biocompatibility, drug carriers, pathology, liposomes, cancer therapy, nanocarrier, controlled drug delivery, metal nanoparticles, imaging, diagnosis, theranostics, tumor microenvironment, photo­ thermal therapy, photodynamic therapy, chitosan, personalized medicine, molecularly targeted therapy, micelles, drug cytotoxicity, and immunotherapy.

2.2 NANOMATERIALS USED IN COMMUNICABLE DISEASES Communicable diseases are caused by various microorganisms like bacteria, fungi, and viruses and are characterized by their highly infectious nature. They spread from person to person through contact with

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 21

FIGURE 2.2 Geographical distribution of publications between 2011–2021. Data Source: Obtained from Scopus and PubMed for the search query “nanomedicine.” Created using Tableau Public.

bodily fluids, blood, fecal matter, contaminated fomites, or via animal and environmental vectors such as food, water, air, soil, insects, etc. [26]. Some of the most common examples of communicable diseases are influenza; hepatitis A, B, and C; measles; HIV/AIDS; tuberculosis (TB); shigellosis; and malaria, among others. The widespread emergence of multidrug resistance (MDR) among viruses and bacterial strains has rendered conventional antimicrobial drugs ineffective. Such ‘super bugs’ are the cause of common healthcare-associated infections (HAI), also known as nosocomial infections, such as respi­ ratory tract, urinary tract, and bloodstream infections (BSI). This is especially concerning in the case of MDR gram-negative pathogens. MDR is a consequence of mutations, gene transfer, inappropriate use of antimicrobials (incomplete or higher dosage), and inadequate diagnostics, among others. Sections 2.1 to 2.3 highlight the use of nanomaterials and nanoparticles in combating such diseases.

2.2.1 Tuberculosis Tuberculosis, or TB, is a leading healthcare concern across the globe, especially in developing countries. It is caused by Mycobacterium tuberculosis (MTB), a bacterium that primarily infects its hosts via the respiratory tract [27]. Because of the prevalence of drug-resistant tuberculosis (MDR TB) and latent tuberculosis infection (LTBI), new approaches to TB diagnosis and treatment are required. For the treatment of tuberculosis, nanotechnology can provide several advantages, such as improved drug bio­ availability, longer half-lives and controlled release, fewer side effects and the ability to administer the drug via a variety of routes, including oral, nasal, intravenous, and multiple drug encapsulation [28]. So far, published literature has reported the efficacy of liposomes, solid lipid nanoparticles (SLNPs), polymeric nanoparticles, and micelles in delivering TB drugs [29]. To treat intracellular MTB, [27] developed pH-sensitive inhalable macrophage-targeting SLNPs using the amphiphilic compound 6-octadecylimino-hexane-1,2,3,4,5-pentanol (MAN-SA), and a

22 Nanomaterials in Healthcare lipophilic isonicotinic acid octylidene-hydrazide (INH-CHO). The result was an SLNP (MAN-IC-SLN) that could trigger the degradation based on pH sensitivity and INH release inside the macrophages and provide a promising solution for treating LTBI. [30] fluorescent assays for detecting methyl nicotinate (MN) in vapor samples for diagnosing TB. MN is a volatile organic marker of MTB, typically found in TB patients’ breath. They used a probe consisting of synthesized CdTe quantum dots (QD) and CoTCPP nanosheets. The QDs act as fluorescent units, while the CoTCPP nanosheets act as fluorescent quenchers and MN screening agents.

2.2.2 HIV/AIDS AIDS, also known as acquired immune deficiency syndrome, is a global health crisis that has claimed the lives of over 36 million people. The human immunodeficiency virus (HIV) is responsible for the infection (HIV). It is passed from mother to child during pregnancy or delivery by exchanging bodily fluids such as blood, breast milk, semen, and vaginal secretions. HIV weakens the immune system and increases the susceptibility of the infected persons to different infections and certain types of cancers. Most people with HIV remain undetected until the later stages and experience little to no influenza-like symptoms such as fever, sore throat, headache, or rashes. As the infection progresses, it manifests through other signs such as weight loss, cough, diarrhea, swollen lymph nodes, etc. Since the immune system is seriously weakened, persons infected with HIV also contract illnesses like meningitis, tuberculosis, and cancers. Polymeric NPs, solid lipid NPs, nanostructured lipid carriers (NLCs), and inorganic NPs are the three primary types of NPs explored for their potential in anti-HIV therapy. These are conjugated with entities like antibodies and peptides, increasing their functionality and targeting abilities. When com­ bined with gp120 or CD4, dendrimers (highly branched macromolecules) reduce HIV’s capacity to attach to host cells, making them a promising therapeutic agent and non-viral vector [31]. [32] developed PLGA-PEG nanoparticles conjugated to the Tm cell-specific CD45Ro antibody to bind to Tm cells. The NPs developed were then used to deliver SAHA (histone deacetylase inhibitor suberoylanilide hydro­ xamic acid and Nel (nelfinavir – a protease inhibitor), which increased the reactivation of latent HIV and inhibited its spread, indicating the efficacy of these agents in eliminating latent HIV reservoirs [32]. [33] synthesized 2 nm gold NPs and conjugated them with a fragment of the TAK-779 inhibitor and SDC1721, a TAK-779 homolog, which served as effective HIV fusion inhibitors.

2.2.3 Influenza Influenza, or the flu, is a highly contagious viral illness that affects the respiratory system – nose, throat, and lungs. According to the NIH, influenza is responsible for around 0.29–0.65 million deaths worldwide [34]. The most common flu symptoms are cough, sneezing, fever, runny nose, sore throat, fatigue, headaches, etc. These can sometimes lead to other complications, such as sinus and ear infections and pneumonia, and can worsen the patient’s condition, such as congestive heart failure, diabetes, or asthma. The most cost-effective countermeasure against influenza is vaccination. However, the current vaccines are strain-specific, highlighting the need for broadly protective flu vaccines. This gap has been attempted to be bridged using nanotechnology. [35] developed a polyethyleneimine (PEI)functionalized graphene oxide (GO) flu vaccine nanoplatform using recombinant influenza hemag­ glutinin (HA) for intranasal vaccination. The nanoparticles could boost the breadth of the immune response, providing both homologous and heterologous protection against flu viruses, highlighting the potential uses of them against respiratory pathogens. [36] computationally designed nanoparticlebased vaccines that demonstrated hemagglutination inhibition (HAI) activity and protective stemdirected antibodies in animal models.

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 23

2.3 NANOMATERIALS USED IN NON-COMMUNICABLE DISEASES Non-communicable diseases (NCDs) or chronic diseases are the most common cause of death and disability globally. They are long-term health conditions that result from genetic, environmental, physiological, and behavioral factors. Common NCDs include cancer, diabetes, cardiovascular diseases (CVDs), strokes, etc. Sections 2.3.1 to 2.3.4 discuss how nanomaterials and nanoparticles have been leveraged to improve therapeutics for such diseases.

2.3.1 Cardiovascular Diseases Cardiovascular diseases (CVDs) are characterized by destitute blood perfusion in the body and refer to various diseases such as hypertension (high blood pressure), stroke, coronary artery disease, myocardial infarction, heart failure, restenosis, and aneurysm. With 0.659 million annual deaths annually, CVDs are responsible for every 1 in 4 deaths in the United States [37]. CVDs are usually a consequence of other medical conditions and poor lifestyle choices such as obesity, unhealthy diet, smoking, alcoholism, diabetes, physical inactivity, etc. Despite the various diagnostic and therapeutic options currently available, CVDs continue to be a leading cause of mortality worldwide. This prompted the emergence of nanomedicine for dealing with CVDs due to its various advantages over conventional methods. Non-invasive theranostics for CVDs using HDL-like magnetic nanostructures (HDL-MNS), phos­ pholipids, and HDL-defining apolipoprotein A1 [38]. The HDL-MNS would act as cholesterol efflux agents and serve as a detector via magnetic resonance imaging. In another study, S-nitrosylated phos­ pholipid (DPPNOTE) was incorporated with HDL NPs to develop a nanoplatform via self-assembly. The developed NPs were able to deliver an effective dose of nitric oxide and show immense potential for treating vascular diseases [39].

2.3.2 Diabetes Diabetes is a chronic disease wherein a person’s pancreas is unable to produce sufficient insulin or when the insulin produced cannot effectively regulate the glucose levels in the body. Since the 1980s, the number of diabetes patients rose from 108 million to 422 million in 2014 [40], with 37.3 million Americans alone diagnosed with the disease in 2019 [41]. One of the main difficulties faced in diabetes care is maintaining glucose levels within ideal limits. Hence, much research is dedicated to optimizing insulin pharmacokinetics and administration and developing novel diagnostic and therapeutic agents. [42] synthesized dextran-coated gold NPs bound with insulin and acted as insulin carriers. The insulin released from these Dextran-AuNPs could maintain kinetic equilibrium and bind with insulin receptors, facilitating longer-lasting insulin activity. Another study tested a gliclazide-loaded PLGA second-generation formulation in a type-2 diabetes mouse model. The PLGA formulation improved gliclazide’s dissolution rate, solubility, and bioavailability which would be highly advantageous in improving patient compliance and therapy management [43].

2.3.3 Neurodegenerative Diseases Two of the most common neurodegenerative diseases are Parkinson’s disease (PD) and Alzheimer’s disease (AD). PD and AD affect millions of people worldwide. They are the result of loss of the function of the nerve cells in the brain and the peripheral system, which worsens over time. The available treatment

24 Nanomaterials in Healthcare options can only alleviate certain physical and mental symptoms, but there are no existing cures for these diseases. Nanotechnology has been used for early imaging and diagnosing neural loss in PD patients. Several nanodevices have also been developed to detect amyloid peptides, characteristic of pathogenesis in AD [44]. Oxidative stress is a key player in the degeneration of dopaminergic neurons in PD patients [45]. Metal nanomaterials have been employed to reduce oxidative stress in PD and AD by mimicking anti­ oxidant nanoenzymes. Metallic nanomaterials are especially suited for this since they can be used to develop bimetallic nanoparticles like PtCu that can mimic multiple enzymes simultaneously [46].

2.3.4 Autoimmune Disorders An overly active or dormant immune system is the hallmark of autoimmune illnesses. In response to an unknown trigger, the immune system attacks and damages healthy tissues within the body. Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes mellitus (T1DM), Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Grave’s disease, Hashimoto’s thyroiditis, and myasthenia gravis. These conditions are also known as autoimmune reactions. [47] encapsulated interleukin-2 (IL-2) and transforming growth factor β (TGFβ) using PLGA NPs, which could expand the CD4+ and CD8+ Treg cells and suppress autoimmune responses in lupus. Apart from these, many NPs composed of liposomes, micelles, dendrimers, etc., have been investigated to treat chronic inflammation. For example, [48] nanostructured lipid carriers loaded with moxifloxacin (MFX) and dexamethasone (DEX) dem­ onstrate antibacterial and anti-inflammatory actions at the same time.

2.4 NANOMATERIALS USED IN CANCER Cancer diagnosis and treatment are currently two of the most important nanomedicine applications. According to the World Health Organization (WHO), nearly 10 million people worldwide will die from cancer in 2020, making it one of the top causes of mortality [49]. The most common cancers reported were lung, colorectal, liver, stomach, and breast. The global age-standardized rate (ASR) of incidence and mortality of various cancer sites according to the WHO in 2020 are depicted in Figure 2.3. Lung, breast, and colorectum cancers are responsible for the highest incidence of cancer mortality [50]. Early diagnosis and screening of cancers can avoid a majority of cancer-related fatalities. Hence, there is a colossal amount of scope in research and development for a new diagnosis, screening, and treatment options in this area. Cancer is caused by the unregulated growth of cells, which can potentially metastasize to other parts of the body. The success of nanotechnology in oncology (termed ‘na­ nooncology’) is linked to the tumor microenvironment (TME). The TME is characterized by poor lymphatic drainage and a leaky vasculature, which allows nanoparticles to accumulate in the tumor interstices, a phenomenon popularly known as the cornerstone of nanooncology – the enhanced per­ meability and retention (EPR) effect [51]. A wide range of cancer treatment options are currently available, including surgery, chemotherapy, radiation therapy, immunotherapy, or a combination of these. Chemotherapy is the treatment of choice for malignant malignancies. Chemotherapy interferes with the DNA synthesis and mitosis of the rapidly dividing cancer cells, leading to cell death. However, some of the major drawbacks of chemotherapy include poor targeting and penetrability of antineoplastic agents into the cancer site, which in turn increases the dosage needed for treatment and sometimes even results in residual tumor cells even after prolonged periods of treatment. This causes drug resistance and death of healthy, non-cancerous cells, leading to adverse side effects. The severe side effects of chemotherapy are responsible for the high

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 25

FIGURE 2.3 Incidence and mortality rates for both sexes and all age groups, based on age-standard esti­ mates for 2020 (Worldwide). Data Source: GLOBOCAN 2020, International Agency for Research on Cancer, World Health Organization.

mortality rate of cancer patients. Further, many anticancer drugs have low solubility levels, limiting the drug’s diffusion level into the desired tissue. Targeted drug delivery using nanocarriers provides a means of selectively delivering a controlled concentration of drugs directly at the tumor site using passive or active targeting methods. The devel­ opment of anticancer drugs at the nanoscale has also made it possible to modify properties like solubility, diffusivity, drug release profiles, bioavailability, etc., which contribute to lower levels of toxicity, lesser side effects, and improved drug life in the body. Nanooncology is not just limited to therapeutics; nanoparticles have also been employed for cancer diagnosis and imaging. Besides, nanoparticles are used in biomarker research, cancer protein detection, metastatic tumor cells, single-molecule assays, and tracking apoptosis [52]. Some nanoparticles also serve the dual purpose of diagnosis and therapy and are called theranostic nanoparticles. Although there are many advantages of using nanotechnology in oncology, there are still concerns regarding their safety and toxicity for clinical translation. Hence, an important research area in nanooncology is nanoparticle toxicity, which deals with studying the interactions of engineered nanoparticles with various biological systems and molecules and the subsequent effects.

2.5 NANOMATERIALS FOR IMAGING, SCREENING, AND DIAGNOSIS Nanoparticle contrast agents have been developed for imaging and visualisation of sick biological tis­ sues. This is done by allowing the nanoparticles to concentrate in specific tissues. MRI, CT, radioactive imaging, fluorescence imaging, photothermal imaging, and photoacoustic imaging are some methods used to see them [53].

26 Nanomaterials in Healthcare Modified nanoparticles have also been used as transducers for in vitro sensing of biomolecules, cells, and tissues. This screening is also facilitated by the surface plasmon resonance effect, surfaceenhanced Raman scattering (SERS), and electronic read-out of detected disease markers in biological samples like blood, urine, and saliva via the use of suitable nanobiosensors. The biomechanical prop­ erties of cells can also be used to indicate cell physiology and pathology [53].

2.6 TACKLING COVID-19 USING NANOTECHNOLOGY The COVID-19 outbreak of 2019 left a lasting mark on the world, with its colossal infection rates and the subsequent changes it brought to the way we live. Since the initial detection in late 2019, the SARSCoV-2 has taken an enormous toll on the global healthcare sector, resulting in approximately 6.13 million deaths worldwide as of March 2022 [54]. Coughing, fever, exhaustion, headache, sore throat, and a loss of taste and smell were among the first symptoms of the coronavirus sickness. If the condition is severe, individuals may have trouble breathing and have chest pain. The disease is trans­ mitted via direct contact or exposure to droplets expelled from the mouth or nose of an infected person. Hence, one of the first precautions imposed was personal protective equipment (PPE) like masks, gloves, face shields, and bodysuits/aprons. Apart from the standard N95 masks widely used during the pandemic, various masks and gloves made of antimicrobial nanomaterials were introduced. These include: • • • • • • •

Self-cleaning MVX Nano Mask containing titanium and silver zeolite NPs Reusable Guardian masks containing a blend of silver and copper NPs Defense Series-Respirator masks containing a silver-copper NP blend Surgical masks by ESpin containing nanofibers for particle screening Everyday Protect Gloves containing silver NPs, thiabendazole, and zinc pyrithione Chlorhexidine wash gloves containing silver NPs and 2% chlorhexidine PADYCARE gloves coated with silver NPs – commercialized by [55,56]

Various nano-formulations containing silver NPs suspended in some matrix-like polyvinyl alcohol (PVA), cellulose, titanium dioxide, etc., were used to disinfect surfaces. Nanotechnology was also used to develop fabrics for PPE kits to make them hydrophobic, antimicrobial, breathable, and facilitate the blocking or filtering of pathogens [55]. SARS-CoV-2 POC screening techniques were also the subject of extensive investigation. Examples include the use of gold nanoparticles (NPs) to develop lateral flow assays (LFAs) for the detection of immunoglobin G (IgG) and immunoglobin M (IgM) antibodies. These results can be analyzed by the naked eye or scanned by a smartphone to determine the target protein concentration. Some COVID-19 vaccines, like the Pfizer and Moderna vaccines, were developed using mRNA with lipid NP. (LNP) carriers. In contrast, others like the AstraZeneca and Johnson & Johnson vaccines use a type of adenovirus vector to deliver RNA, which subsequently triggers an immune response to produce antibodies [57,58]. During the pandemic, poor nutrition was linked to the inadequate response of the immune system against the coronavirus. This is because vitamins and minerals are essential to the different stages of the body’s immune response, owing to their antioxidant and anti-inflammatory properties. Hence, artificial nutritional intake was recommended to aid the recovery of hospitalized COVID-19 patients. For example, [59] developed multidrug anti-inflammatory and antioxidant nanoparticles to mitigate inflammation by delivering adenosine and vitamin E (alpha-tocopherol). In the past, small organic molecules like biotin (vitamin H) [60] and folic acid (vitamin B9) [61] have been used as targeting agents for the treatment of cancers due to their stability and ease of preparation. A similar targeting

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 27 strategy could be utilized for high-efficiency uptake of essential micronutrients compared to conven­ tional vitamin and mineral supplements in infected COVID-19 patients. The advantages of immune-enhancing supplements also led to integrating nanomedicine with nat­ ural compounds and products rich in micronutrients. Some of the herbs employed include Ashwagandha (Withania somnifera), Guduchi Ghanavati (Tinospora cordifolia), Tulsi (Ocimum sanctum L.), and Mulethi (Glycyrrhiza glabra). These were delivered using nanocarriers such as liposomes, SLNPs, metallic and magnetic NPs, polymeric NPs, quantum dots, dendrimers, etc. For example, due to their mucoadhesive properties, chitosan NPs treat digestive system responses induced by COVID-19 [62].

2.7 NANOMEDICINE AND ASSOCIATIVE TECHNOLOGIES Machine learning (ML) and artificial neural networks (ANNs) have taken center stage in analyzing large data sets due to advances in technology and computing. Industry 4.0 has also emphasized the advantages of robots, automation, additive manufacturing (AM), and the Internet of Things (IoT) in current R&D. The development of these associative technologies in conjunction with one another is at the forefront of every industry.

2.7.1 Additive Manufacturing Additive manufacturing or 3D printing is developing a product by depositing material layer by layer. AM is more cost-efficient than conventional manufacturing, where the material is removed from a block to develop the product, resulting in unnecessary wastage. AM can be used to develop products made of different polymers, metals, and graphene-based materials.

2.7.2 Artificial Intelligence, Machine Learning, and Bioinformatics AI and ML have been utilized as important tools to retrieve, compile, and organize the large volumes of experimental data generated over the years. This data can then be leveraged to optimize nanoformulations by understanding the nanoparticle structure and its characteristics. Synthesized nano­ particles can be virtually simulated using ML models to study their interaction with the target environment, its cellular uptake, adherence, release, and subsequent activities [63]. For example, [64] used multiple regression models and a genetic algorithm (GA) to predict the cytotoxicity of different metal oxide particles against E. coli. Apart from this, AI and ML are actively used in imaging to quantify results effectively. Applying bioinformatics to nanomedicine (called nanoinformatics) involves mana­ ging nanomedical information. This helps mine large volumes of nanomedicine and related research publications, standardize nanomaterials’ non-uniform classifications, and analyze complex biochemical and physical process data [65].

2.7.3 Robotics, Automation, and IoT The coronavirus pandemic catalyzed the shift from manual and offline work to an online workspace. This was not easy in laboratory settings requiring the operator’s physical presence to perform everyday wet lab tasks. This hurdle can be avoided using robotics, automation, and IoT. Common lab techniques to

28 Nanomaterials in Healthcare synthesize nanoparticles can be automated or operated remotely using smart devices and robotics with AI integration [66]. For example, microfluidic chips can facilitate the processing and visualization of minute amounts of liquids containing cells or nanoparticles using micro-pumps and other micro-peripherals like valves and fluid channels [67]. Robotic liquid handlers (LiHas) have also been developed to reduce human error during manual pipetting. They have been adopted for common lab techniques, including PCR, liquid-liquid extracting, ELISA, genomics, solid-phase extraction (SPE), and cell-nanoparticle tests. Robotic arms also speed up and increase the efficiency of producing multiple batches of formu­ lations, characterizing them, and their subsequent automated optimization. Once the nano-formulation has been optimized, it is analyzed and tested using several assays and microscopic techniques. Automating microscopy and well-plate readings further increase the results’ reliability and efficiency. IoT can further aid the large-scale production of nanomaterials by ensuring efficient monitoring, transfer, and analysis of data between processes and instruments at every stage of the protocol. Such automation brings us closer to achieving more robust disease models and improving personalized medicine [66]. Hence, automation of lab procedures reduces human errors and ensures reproducibility of results every time the procedure is repeated. It can also be developed as a ‘closed-loop feedback system,’ which would help correct errors on the go.

2.8 ETHICAL CONCERNS ABOUT NANOMEDICINES Beauchamp and Childress’ Principles of Biomedical Ethics, published in 1979, outlined the most important ethical principles for medical practice, patient care, and treatment [68]. When dealing with medical difficulties, it provided a framework for doing so. Everyone in the medical field, from medical students and residents in training to members of hospital ethics committees, agree that the four pillars of beneficence, non-malfeasance, respect for autonomy, and fairness should serve as a basis. All medical operations and scientific research are based on these four guiding principles. People and other emotive beings like “humans” may believe that the only ethical and moral value in a person’s life is what they value right now [69]. In medical ethics, the notion of autonomy (the ability to make one’s own choices) is critical; patients (as rational beings) can act and make moral decisions of their own free will and with full knowledge of the consequences [70]. Patients should make any competing requirements clear to them so they can make informed decisions about the device’s use and purpose. The individual or patient receiving them must sign a written declaration attesting to their understanding of this need. If a procedure is permissible by law and the operation is not plainly against the patient’s interests, they should be offered to see if the advantages exceed the risks. As part of the autonomy principle, patients have a right to know all the details of their treatment and what they can expect. Based on the above concept, we can imagine how nanotechnology could affect or even violate the autonomy principle. This poses serious ethical problems since nanomedicine and brain implant tech­ nologies threaten the medical profession’s moral norms. Respecting the patients’ autonomy, for example, means allowing them to live, following their values and views. Non-voluntary manipulation of patients for experiential and mind control reasons raises ethical questions, not the use of neural dust therapies in therapy with the patient’s knowledge and permission of the effects of this approach. The result of nanomedicine’s ability to detect even the tiniest changes in an organ’s chemical composition challenges our understanding of what it means to be healthy. Another advantage of nanotechnology for scientists in the medical area is the ability to wirelessly collect data on interior body states like temperature, pulse, and blood glucose, as well as fix organ dysfunctions, including vision loss and hearing dysfunctions. When nanotechnology is used to diagnose or treat patients, the biomedical ethics notions that Beauchamp and Childress emphasize may collide. These standards may be legally binding. It is called prima facie when a context justifies a concept or behavior as prescriptive without absolute (meaning on

2 • Application of Nanomaterials in Medicine: A Clinical Perspective 29 the first appearance but subject to further evidence or information). This supports a course of action until it is overridden by another prima facie that is thought to be more accurate [71]. All prerequisites must be completed for the four biomedical principles to be binding in theory. Although there are times when they conflict, the agent must decide what to do by identifying the prima facie right that overrides or outweighs all other principles, acting impartially toward all of the parties affected, and making the decision without weighing morally irrelevant information into the decision [72]. Regarding diagnosis and therapy, biomedical ethics can be utilized to evaluate the dangers and advantages of nanotechnology. The benefits and cons of the technology can be analyzed. One strategy to achieve this goal is to consider both probable outcomes.

2.9 CLINICAL TRIALS AND APPROVALS OF NANOMEDICINE Any new drugs or therapies must undergo clinical testing before they can be approved. The medicines are put through clinical trials, assessing the new treatments’ efficacy and the hazards. Preclinical animal research is the first step, followed by clinical trials utilizing a double-blind approach. For new drugs to be approved by the FDA, they must undergo extensive clinical trials. FDA approval for nanomedicine devices is tough, according to Glenn and Boyce. These standards and requirements for approval apply to this technology since it falls into numerous categories under the FDA, each of which has its own set of regulations [73]. The clinical tests associated with nanomedicine have been criticized for having many limiting factors. Experiments on animals, which are necessary for the preclinical testing that precedes clinical trials and aims to minimize the hazards of nanomedicine, have considerable limitations. The first of these limits is that people and animals may react differently to the same object or chemical. This is an example of a possible deviation. At low exposure levels, a substance that is not harmful to animals may be harmful to humans, and the other way around. This is since a drug or material may be absorbed, dis­ tributed, metabolized or eliminated differently by animals and humans. Few long-term implications of novel drugs, biologics, or medical technologies are studied in animal research. The second limitation of preclinical medical research is this. It is possible, however, that some of the detrimental effects of a substance may not become apparent for some time. All three stages of the clinical investigation yielded conflicting outcomes. Because the process only takes seven years, and post-marketing studies typically only take a few more years, it is much easier to get a product to market. It is impossible to predict the long-term consequences of these findings. For example, thirty years after cigarette smoking, genetic or tissue damage may result in disease, such as lung cancer. In order to properly manage clinical trial risks, it is necessary to find and evaluate potential risks and benefits and maintain an acceptable and justifiable equilibrium between the two. As a last consideration, both sides of the issue must be well informed. Potential risks are not insignificant, so the benefits of an experiment must be substantial. Research involving more than a trivial degree of risk is subject to additional precautions for vulnerable populations such as newborns, fetuses, and inmates. You must discern between studies that provide medical benefits to the subjects (diagnosis and therapy, for example) and studies that do not benefit the participants (information about their ailment). Putting people in danger that is more than what is considered “modest” is only acceptable if the subjects are expected to receive some medical benefit. For example, chemotherapy can lead to side effects like nausea, loss of weight and nerve damage, anemia, neutropenia, thrombocytopenia, weariness, hair loss, infections, dizziness, migraines, mental disorders, and even death. If the benefits to participants (such as therapy) and society (such as new knowledge) are substantial, running a phase clinical trial to examine a novel chemotherapeutic drug may be justifiable. If the risks are minor and the benefits to society are substantial, the risks are not reasonable.

30 Nanomaterials in Healthcare Even if the risks are substantial, and the patients are unlikely to gain any direct medical advantages due to participating, the risks may be justified. Phase I drug trials, for example, frequently involve volunteers subjected to more than minimal risk. These investigations, known as Phase I trials, are designed to look into the potential side effects and dangers of various human medications. Suppose the risks of a Phase I clinical trial for a new treatment are minimal, and the benefits to society (such as new medical knowledge and the creation of new pharmaceuticals) are large. In that case, the experiment may be justified. The experiment’s dangers can be defended [74].

2.10 CONCLUSION Although much research has been dedicated to nanomedicine over the years, only a fraction has been translated into effective clinical trials and subsequent clinical use. This is due to the various toxicity and safety concerns revolving around using nanomaterials and nanoparticles. Hence, proper analysis and understanding of the current scenario of nanomedicine and its regulatory measures are crucial to speeding up development in this area. Leveraging associative technologies like AI, ML, automation, and robotics can further increase the efficiency of nanomedicine by reducing the processing time, on-site personnel requirements, and errors generated while simultaneously raising the accuracy and throughput.

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Advancement of Polymer-Based Nanocarrier System in Drug Delivery

3

Pallavi Kiran, Abhinanda Kar, Arpita Banerjee, and Arzoo Puri Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 3.1 3.2

Introduction Types of Polymer-Based Nanocarriers 3.2.1 Classification of Polymer Nanocarriers Based on Affinity to Water 3.2.1.1 Hydrophilic 3.2.1.2 Hydrophobic 3.2.1.3 Amphiphilic 3.2.2 Classification of Polymer Nanocarriers Based on Source 3.2.2.1 Natural 3.2.2.2 Synthetic 3.2.3 Classification of Polymer Nanocarriers Based on Charge 3.2.3.1 Cationic 3.2.3.2 Anionic 3.2.3.3 Charge reversible polymers 3.3 Application of Polymeric Nanoparticles in Drug Delivery 3.3.1 Oral Drug Delivery 3.3.2 Vaginal Drug Delivery 3.3.3 For Cancer Therapy 3.3.4 Ocular Delivery 3.4 Conclusion and Future Prospect References

36 38 38 39 39 40 40 40 41 42 42 42 42 44 44 45 45 46 48 49

DOI: 10.1201/9781003322368-3

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36 Nanomaterials in Healthcare

3.1 INTRODUCTION The intricacy of medical illnesses and associated toxicity experienced due to certain treatment therapies demands the exploration of novel drug delivery methods. Any device or formulation that allows active compounds to be introduced into the human body, enhancing their efficacy and safety, by regulating the time, amount, and controlled drug release at the targeted site, passing biologic membranes to reach the therapeutic target is known as a drug-delivery system (DDS) [1]. This encompasses not just the drug delivery strategies, but also the employment of vectors to aid in their application and dispersion into the body. Depending upon medical conditions and patients, various combinations of active compounds and vectors potentially will allow for a wider range of treatment personalization options. When addressing a disease with a drug-delivery system for treatment, the administration routes employed to inject and deliver active chemicals to their target area are important vectors [2]. Given the severity of the condition or the drug’s underlying toxicity, it may be necessary to introduce it straight to the affected organ. The possible toxicity of active substances or the heavy dosage required to produce pharmacological effect seems to be a challenge that many administration routes, particularly systemic approaches, face. Another issue associated with administration routes like the oral route has been that they restrict the use of extremely hydrophilic or pH-resistant medicines to ensure adequate absorption by intestinal epithelial cells [2,3]. The conventional methods of drug delivery also possess other complications such as deficient solubility, poor bioavailability, in-vivo instability, and adverse side effects. Hence, a new drug delivery system that targets drugs to specific regions of the body can solve the above-mentioned complications [3]. Nanotechnology is the study of matter at the atomic, supra-molecular, and molecular levels, on scales ranging from a few to hundred nanometres (nm) in size [3]. Over the last two decades, this field has been used extensively for the development of nano-based drug delivery systems overcoming the shortcomings of traditional drug carriers. In general, a nanocarrier is a colloidal drug carrier system with submicron particle sizes, typically less than 500 nm [4]. They have the potential to manipulate bioactivity and basic characteristics of drugs due to their large surface-area-to-volume ratio. Some of the advantages that nanocarriers can produce in drug delivery systems include enhanced bio-distribution and pharma­ cokinetics, lower toxicities, better solubility and stability, controlled release, and location-specific delivery of therapeutic drugs [5]. Furthermore, nanocarrier’s physiochemical properties can be tailored by varying their compositions (inorganic, organic, or hybrid), forms (rod, sphere, or cube), sizes (big or tiny), and surface attributes (functional groups, surface charge, attachment of targeting moieties) [4,5]. The ultimate goal of using a nanocarrier system in drug delivery is to successfully treat an illness with minimal side effects [5]. Investigation into the utilization of various compounds as nanocarrier pre­ cursors becomes a necessity for improving the outcomes and applicability of these systems. Biodegradability, biocompatibility, and non-immunogenicity are some of the prerequisites for these precursors. Polymers, in particular, have attracted enormous interest in creating nanocarriers, and their potential in medical applications is immense [6]. They are molecules made up of one or more types of components (units) called monomers that are covalently joined to form a linear or branching chain. These monomers can have any structure as long as they have at least two or more functional groups which can interact with another monomer. Polymers are not just a unique type of material that can have all of the aforementioned desirable features of a nanocarrier precursor, but their high synthetic versatility also allows researchers to tailor them to specific requirements or end goals [1]. Polymeric tailoring can be done simply on biopolymers via chemical derivatization to achieve certain characteristics [7]. Another alternative is to make synthetic polymers using monomers, which can result in a wide variety of con­ figurations and applications [8]. Polymers in a nanocarrier system are usually amphiphilic, meaning they include both hydrophilic and hydrophobic moieties. Polymer-based nanocarriers can bind a wide range of pharmacological compounds because of their hydrophobic moiety. Polymeric nanocarriers with a diameter of several

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 37

FIGURE 3.1 Schematic representation of polymeric nanocarriers.

hundred nanometers may boost the bioavailability and expand the blood circulation duration of insoluble pharmaceuticals (drugs) due to the presence of a hydrophilic moiety like chitosan or polyethylene glycol. These are some of the aspects due to which polymeric nanomaterials are becoming incredibly valuable in nanotechnology and are now being employed in drug delivery nanocarrier systems. Figure 3.1 shows the type of polymeric nanocarriers (PNCs) that include liposomes, micelles, dendrimers, and biocompatible and biodegradable polymer-based nanoparticles [9]. The diameter (size) of a polymeric nanocarrier plays a key role in defining the in-vivo fate of the particles in order to fabricate an optimal nanocarrier system. To portray the bio-distribution of nano­ particulates in the body, the size is relatively within 10 nm–200 nm. PNC stability is also critical, and changes in ionic strength, pH, polarity, or temperature in an in-vivo or physiological environment must not impact it. In addition to these factors, surface functionalization is an important characteristic in therapeutic applications. Polymer-based nanocarrier systems must carry cargo molecules at the correct location, at the correct time, and in the correct dose. The dispersibility and stability of PNCs should not

38 Nanomaterials in Healthcare be affected by the changes in pH, ionic strength, polarity, and temperature in a physiological or in vivo environment. In this book chapter, we focus on the current developments in polymer-based nanocarrier systems and their potential use in drug delivery.

3.2 TYPES OF POLYMER-BASED NANOCARRIERS 3.2.1 Classification of Polymer Nanocarriers Based on Affinity to Water Polymers that have applications in drug delivery can be classified broadly based on their interaction with an aqueous environment. This property of a polymer is an important consideration while designing nanocarriers because it not only decides the fate of the particles under physiological conditions, but also it directs the type of cargo that can be encapsulated within the polymer core/matrix. The subsequent sections will broadly highlight polymeric nanoparticles of varying affinity to water and how this property has been leveraged largely (Figure 3.2).

FIGURE 3.2 Types of polymer-based nanocarriers based on affinity to water, source, and charge.

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 39

3.2.1.1 Hydrophilic Hydrophilic or “water-loving” polymers are relatively low in cost, easy to use in nanoparticle synthesis, and have better in vitro-in vivo correlation. However, due to the poor interaction of such polymers with water-soluble drugs that result in poor drug dispersion, uncontrolled pharmacokinetics, and “burst effect”, only a few studies are reported on exclusively hydrophilic polymer nanoparticles in drug delivery. In one such study, the high hydrophilic property of the polymer polyvinyl alcohol (PVA) has been combined with the mechanical properties of silk fibroin protein to obtain core-shell nanoparticles in cancer drug delivery. The strong affinity of PVA to water allowed penetration of water molecules into the core which dissolved the PVA matrix, creating holes for the drug to diffuse out into the aqueous milieu [10]. Doxorubicin base (DOX) being essentially a hydrophobic anti-cancer agent, attempts have been made to modify hydrophilic polymers that can effectively entrap and deliver this therapeutic agent. A highly hydrophilic polymer called poly(agmatine) (PCA), has been modified by adding guanidinium groups that helped in Π- Π interactions with aromatic ring-containing compound DOX. On the other hand, for the delivery of Doxorubicin hydrochloride, which is water soluble, a synthetic hydrophilic copolymer, poly(hydroxyethyl methacrylate-glycidyl methacrylate) or p(HEMA-ran-GMA) has been used to make a water-in-oil reverse nanoemulsion. The polymer hydrophilicity was intended to enable longer circulation times, thus facilitating the EPR effect at tumorigenic target sites [11]. Therapeutic proteins and peptide drugs are majorly water soluble. Hence, biodegradable hydrophilic polymers like gelatin have been used for nano-delivery of water-soluble protein drugs. To prevent rapid diffusion of an encapsulated model protein drug, lysozyme which does not crosslink with the gelatin matrix, hydro­ phobic crosslinker diiso-propylcarbodiimide has been employed that generated 200–300 nm carriers [12]. For the peptide hormone insulin delivery, glucose-responsive, hydrophilic polymeric nanosystems have been explored. For example, phenylboronic acid (PBA) modified with electron withdrawing groups can give rise to hydrophilic structures that can swell and release entrapped insulin. A study in 2015 reported such a double-layered nanogel composed of glycol chitosan (GC), sodium alginate (SA), and a PBA derivative poly(L-glutamate-co-N-3-L-glutamylphenyemPNP’slboronic acid) (PGGA) which suc­ cessfully showed controlled, stimuli-responsive insulin delivery in mice [13]. Overall, hydrophilic polymers have been studied mostly due to their biocompatibility and closeness to physiological conditions, but research is underway to modify such hydrophilic polymer nanocarriers to achieve optimum therapeutic efficacy.

3.2.1.2 Hydrophobic Many of the therapeutic agents administered are hydrophobic or lipophilic in nature, so they require delivery vehicles that can encapsulate such moieties within a hydrophobic core, away from the aqueous physiological microenvironment. One common polymer under this category that has widespread applications in the field of drug delivery is the synthetic polymer poly-lactide-co-glycolide or PLGA. For the non-toxic delivery of a poorly water-soluble anti-inflammatory agent, curcumin, PLGA nanoparticles have been synthesized using a microfluidic-assisted nanoprecipitation method. These hydrophobic, surfactant-free polymer nanoparticles have reported high colloidal stability, preventing curcumin deg­ radation and better uptake of curcumin in leukemia Jurkat cells, thus enhancing the anti-cancer activity of curcumin in nano-form. To address the issue of drug resistance in cancer and minimize systemic toxicity, PLGA nanocarriers with dual hydrophobic drug-loading feature has been used. In one study, PLGA nanoparticles decorated with transferrin, targeted to the transferrin receptors on breast cancer cells, were co-loaded with paclitaxel and elacridar which helped overcome the resistance to the free drugs and facilitated cancer cell cytotoxicity [14]. Next to PLGA, polylactic acid (PLA) nanoparticles have also gained familiarity in the domain of drug delivery. For example, 200 nm PLA nanoparticles loaded with Tamoxifen have been studied for breast cancer therapy [15]. Further, hydrophobic polymer nanoparticles have also been used in combination with platforms like patches or lenses to improve the efficacy of topical administration. V. Vijayan et al. reported in vivo prolonged delivery of

40 Nanomaterials in Healthcare hypoglycaemic agent Repaglinide from degradable hydrophobic polymer nanoparticles PLA and poly ε-caprolactone (PCL) that were incorporated into Methocel transdermal patches [16]. Cross-linked hydrophobic nanoparticles made of PCL, 2-hydroxy ethylmethacrylate (HEMA), and polyethylene glycol diacrylate (PEGDA) loaded into polymeric contact lenses have been used for prolonged ocular delivery of loteprednol etabonate up to 12 days. Drug-free polymeric nanoparticles, hydrophobic in nature, have also been engineered for their inherent antibacterial properties. Single-chain polymeric nanoparticles containing synthetic hydrophobic monomers have found potential applications in treating multidrug-resistant bacterial infections. Polymer hydrophobicity could be varied systematically causing tunable cell membrane/wall disruption in gramnegative Pseudomonas aeruginosa, thus governing the rate at which bacteria acquires resistance [17].

3.2.1.3 Amphiphilic In several drug delivery carriers, a combination of hydrophilic and hydrophobic properties are desirable in order to overcome the hurdles faced by each type of polymer alone. Hydrophilic polymer particles often face the issue of solubilization and burst drug release, whereas hydrophobic carriers often suffer from very slow degradability or the formation of bulkier particles. To address these issues, amphiphilic polymer nanosystems have gained popularity in achieving optimum parameters suitable for drug delivery. Polyethylene glycol (PEG) and its derivatives are common hydrophilic polymers of ethylene oxide that are often used in modifying/coating hydrophobic polymer nanoparticles. For example, D. Saha et al. studied the differences in physical parameters (especially the size and mechanism of particle formation) between hydrophobic nanoparticles (PLGA and PCL) and amphiphilic PEG-conjugated PLGA/PCL particles. The latter showed limited particle growth with random PEG chains imparting steric stability, making the amphiphilic system better suitable for drug delivery applications [18]. This PEG-PLGA copolymer system has also found applications in cancer drug delivery where these amphiphilic particles, the surface decorated with folic acid were designed for active targeting of cisplatin and paclitaxel to cancer cells [19]. Amphiphilic polymeric nanosystems are mostly capable of self-assembly into supramolecular micelles that are reportedly highly stable in solution. One such system is comprised of a hydrophobic micellar core made with linear poly(DL-lactide) and a hydro­ philic micellar shell made of poly(2-methyl-2-oxazoline). The shell assumed a seven-armed star structure via ring polymerization and the micelles loaded with cabazitaxel prolonged survival in mice with tumors by enhancing the antitumor effect of the micellar drug formulation [20]. Such amphiphilic polymeric nanocarriers thus offer several advantages over simply hydrophilic or hydrophobic nano­ particles in drug delivery.

3.2.2 Classification of Polymer Nanocarriers Based on Source Based on the source from which polymers are obtained, they can be natural (found within nature) or synthetic (synthesized by chemical or physical crosslinking of monomers). The source affects several properties of the polymers that in turn affect their usage as drug carriers, which are discussed in detail below (Figure 3.3).

3.2.2.1 Natural Natural polymers include proteins or polysaccharides that are abundantly available, highly bio­ compatible, and biodegradable. One of the most common polysaccharides in the field of drug delivery is chitosan which has been extensively used alone or in combination with other polymers as nanocarriers. Chitosan being a linear polysaccharide of repeating D-glucosamine and N-acetyl-D-glucosamine shows easy uptake by cells due to the presence of the surface amine groups. This has been leveraged in cancer

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 41

FIGURE 3.3 Polymer nanocarriers based on source: (left) natural and (right) synthetic.

drug delivery where dual drugs, quercetin, and 5-fluorouracil were encapsulated in chitosan nano­ particles which showed significant toxicity to pancreatic cells, both in 2D and 3D cell culture systems [21]. Further, chitosan has been blended with other natural polymers to yield nanosystems with improved properties. For example, chitosan grafted with -D-α-tocopheryl polyethylene glycol 1000 (TPGS) have been applied in synthesis of ~150 nm particles capable of loading anticancer drug DOX and showing efficacy against multidrug-resistant cancer cells and potent in vivo antitumor activity [22]. Another environment-friendly polymer starch has found application in nanotechnology due to its mechanical properties and ease of preparation of nanoparticles from starch granules. The physicochemical properties of starch NPs, their digestibility in simulated gastric fluid in vitro, and the release behavior of loaded drug captopril has been evaluated by Y. Ding et al. [23]. A broad class of polysaccharide natural polymers includes gums such as guar gum. Guar gum is known to target certain mannose-like receptors on the surface of macrophage cells. Guar gum nanoparticles coated with eudragit polymer to protect from the acidic pH of the GI tract, have been loaded with amphotericin B and bioenhancer piperine, which has shown enhanced bioavailability and therapeutic activity in vivo in the leishmaniasis model of golden hamster [24]. Natural protein polymer nanoparticles have been designed such as albumin NPs cross-linked with glutaraldehyde [25], and such albumin NPs have also been used to deliver model proteins like lysozyme. Gelatin is another natural protein polymer that has been used for nano-delivery of ocular drugs like timolol maleate.

3.2.2.2 Synthetic Synthetic polymers refer to polymers synthesized in the laboratory by combining two or more, similar or different monomers by chemical cross-linking. One of the most common polymers in this category is poly-lactide-co-glycolide (PLGA) synthesized from lactic acid and glycolic acid monomers, and it has been explored widely in the field of drug delivery. PLGA NPs prepared by microfluidic nanoprecipi­ tation have been tested for curcumin delivery to leukemia Jurkat cells [26]. Further, for dual drug delivery in cancer nanotherapy, PLGA NPs have proved beneficial. For example, DOX-Verapamilloaded PLGA NPs have been designed to overcome P-glycoprotein-mediated drug resistance in cancer [27]. PLGA is a very versatile synthetic polymer that has been used for drug delivery to various organs of the body such as lungs, across the blood-brain barrier, to the eyes for topical delivery as well as across the barrier of the inner ear. PCL is another hydrophobic polymer that is synthetic in nature and has widespread applications in anticancer drug delivery. PEG-modified PCL NPs have been synthesized for this purpose due to high drug loading capacity of hydrophobic drugs, increased bioavailability, evading phagocytes, and increased drug scattering time in blood circulation. PEGylated PCL NP has also been used for pHsensitive release of MTX in MCF-7 cancer cells [28]. Acrylamide-based synthetic polymers have also been explored for such applications, though limited due to their comparatively low biocompatibility. Some examples in this direction include

42 Nanomaterials in Healthcare N-Isopropylacrylamide based NPs for siRNA delivery [29] and oligo(ethylene glycol) methacrylate (OEGMA) NPs for therapeutic delivery to ovarian cancer and lung epithelial cells [30].

3.2.3 Classification of Polymer Nanocarriers Based on Charge 3.2.3.1 Cationic Cationic polymeric nanosystems bear a positive charge or zeta potential that helps in optimum encapsulation of negatively charged genetic material (gene delivery) or several drug moieties. Positively charged chitosan nanoparticles, surface modified with T-cell specific antibodies have been used to encapsulate and deliver siRNA in RNA interference based therapies [31]. Beta-cyclodextrin modified cationic chitosan nanoparticles have been used for various applications such as delivery of naringenin to aqueous humor of eye, delivering antibiotic salazosulfapyridine [32]. Further, cationic nanoparticles have proved beneficial for co-delivery of drug and gene. For example, T. Liu et al. reported the design of star-shaped cyclodextrin-poly (L-lysine) nanoparticles that efficiently delivered the drug docetaxel and MMP-9 siRNA plasmid for potential nasopharyngeal cancer therapy [33]. The cationic side chains of several polymers are so designed that they are pH sensitive, releasing drug in response to pH triggers. For instance, a novel cationic ortho ester-based homopolymer, poly(N((2-(2-(dimethylamino)ethoxy)-1,3-dioxolan-4-yl)methyl)methacrylamide (PMAOE) having acid-labile cationic side chains has been synthesized as a gene delivery vector [34]. Acid sensitive cationic nano­ particles of acetylated dextran modified with spermine have shown efficacious encapsulation of siRNA, comparable to commercial Lipofectamine transfection agent [35]. Cationic polymers have gained widespread acceptance in drug delivery due to enhanced permeation and cellular uptake, while providing colloidal stability and pH-responsive cargo release.

3.2.3.2 Anionic Although cationic polymer NPs have been explored extensively for their several advantages, their positively charged nature sometimes gives rise to increased cytotoxicity. In such cases, anionic or negatively charged polymers find applications, though limited examples exist. Anionic core-shell NPs made of acrylic/methacrylic acid and poly (N-Isopropylacrylamide) have been synthesized which were reportedly responsive to multiple factors like temperature, pH and ionic strength [36]. Anionic hyal­ uronic acid polymer has been used to make polyelectrolyte complexes with cationic drugs like pent­ amidine for cancer drug delivery [37]. Finally, for gene delivery, usually cationic NPs are preferred which can effectively bind the negatively charged genetic material; but a recent study has reported the coating of anionic shielding polymer on micelles via RAFT polymerization for efficient gene delivery. Cationic micelles of the copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide) were applied with negatively charged shielding polymers like poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) for reduction in cytotoxicity and enhanced transfection effi­ ciency (Figure 3.4) [38].

3.2.3.3 Charge reversible polymers As discussed before, cationic polymers are preferred as drug delivery vehicles owing to their enhanced permeability, better cellular uptake, and ability to encapsulate therapeutic nucleic acids which are negatively charged. However, they also suffer from issues of greater cytotoxicity and likelihood of getting cleared from blood circulation. To solve this challenge in nano-drug delivery, an advanced strategy is to use charge-switchable polymers that will remain negatively charged in circulation but reverse its charge in response to triggers (like pH and ionic strength) at the target sites such as in the tumor microenvironment. Many examples exist that have leveraged the advantages of

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 43

FIGURE 3.4 Mechanism of action of one of the anionic polymeric nanocarrier; HA-azo/PDADMAC nano­ capsule loaded with doxorubicin (DOX) drug.

charge-reversible polymers in cancer and other types of drug delivery. PEG-b-PCL copolymer micelles with acid labile beta-carboxylic amide functionalization displayed a negative charge while in circulation but converted to a positively charged system at pH 6 due to hydrolysis of the acid labile moiety [39]. Charge reversible core-shell nanoparticles bearing a pullulan-based shell and poly-beta amino ester/PLGA core have shown stepwise charge switching in the tumor microenvironment and endo-lysosome respectively and have been used for dual drug delivery of paclitaxel (anticancer drug) and combretastatin A4 (antiangiogenic drug) [40]. In addition to anticancer drug delivery using charge reversible polymers, this nanoplatform has also been explored for non-cancer therapies. One such study by D.H. Kim et al. highlights the use of a pH-sensitive polymer poly (urethane amino sulfamethazine) (PUASM) for delivery of a chemoattractant stromal cell-derived factor-1α (SDF-1α) to a cerebral infarct region post-stroke condition. The sequential charge reversal of this polymeric nanosystem is convenient for the formulation, assembly of the polymer with the therapeutic protein as well as the release of the cargo at target site: the polymer was water soluble at pH 8.5 while at pH 7.4 it could effectively form a micelle for protein encapsulation and at pH 5.5 there was micellar structure disassembly due to ionization of tertiary amines in the copolymer, thus releasing the protein [41].

44 Nanomaterials in Healthcare

3.3 APPLICATION OF POLYMERIC NANOPARTICLES IN DRUG DELIVERY 3.3.1 Oral Drug Delivery The FDA has deemed biodegradable and biocompatible polymers that are commonly used in NPs are safe for use in oral drug delivery technology [42]. Attaching several biologics to the polymeric nano­ particles’ (PNPs) surface can enable meticulous interactions with cellular receptors or tissue components [43]. PNPs have been proved effective and safe in distributing medications with limited bioavailability or are otherwise found to be injurious in animal model studies. Trigger responsive PNPs hold a lot of potential when it comes to delivery of proteins/peptides or poorly water-soluble medications. The other advantage of such PNPs is that they can be fine-tuned for controlling the release to control the release as well as the dosage frequency of the medication. A list of the most explored polymeric nanoparticles used for oral drug delivery has been tabulated below in Table 3.1.

TABLE 3.1

Different polymeric nanoparticles (PNPs) used in oral drug delivery

CONSTITUENTS OF POLYMERIC NANOPARTICLES

ACTIVE PHARMACEUTICAL INGREDIENT

PLGA

Epirubicin

Chitosan-coated PLGA

Fluorescent yeast extract

PLGA

Paclitaxel

PLA-chitosan

Lamivudine

PLGA-alginate-chitosan

Clotrimazole Econazole

Polyacrylate

N-thiolated b-lactam antibiotics

PLGA

Estradiol

Poly (acrylic acid)-gcysteine (PAA-Cys)

Insulin

EFFECTIVENESS OF ORAL DRUG DELIVERY A process for increasing epirubicin’s biodistribution such that it can take the place of an intravenous treatment. Enhancing the mucoadhesive properties facilitating cellular uptake in the gut. Increased the bioavailability of oral paclitaxel. Anti-HIV drugs can be entrapped and protected in the stomach environment (acidic pH), and then releases are sustained at neutral pH (intestine). In both absolute and relative terms, each drug’s bioavailability improved. It appears that nanobiotics, or polyacrylate nanoparticle antibiotics, enhance water-insoluble antibiotics’ bioactivity against MRSA In addition to slow oral bioavailability, narrow therapeutic indices, and toxicity issues related to PNPs can all be addressed by their formulations In this study, a promising method for delivering insulin orally was revealed, as well as for using peptide drugs in general

REFERENCES [ 44]

[ 45]

[ 46] [ 47]

[ 48] [ 49]

[ 50]

[ 51]

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 45

3.3.2 Vaginal Drug Delivery Certain diseases can be treated by administering drugs through the vagina to achieve a local or systemic effect, thus bypassing the liver’s metabolism. This approach has been mostly applied to treat infections or diseases that are spread sexually. The vaginal route of drug administration, however, is difficult and has several drawbacks. The main hurdle to the typical dose form is the significant amount of mucus produced by the vagina, and as a result, the conventional medicine does not exhibit prolonged and focused activity. These flaws led to the development of the modified release dosage form for the vagina. To overcome the physiological barrier, polymeric nanoparticles have been developed, and they have a number of advantages, including mucoadhesiveness, ease of mucosal penetration, and sustained and targeted effects. There has been a surge in demand for polymers with natural and synthetic origins that are both biodegradable and non-biodegradable [52]. For drug delivery, polymeric nanoparticles are utilized, both natural and synthetic, however, the natural ones are usually avoided due to their preparation issues, antigenicity, fluctuations in source, and complex preparation procedures. As a result, the usage of biodegradable synthetic polymeric nano­ particles in vaginal medication delivery systems is increasing. The polymeric nanoparticle needs to fulfil a number of criteria, including being mucoadhesive, stable at vaginal pH, and simple to penetrate the mucous. There are four generations of the polymeric nanoparticle used for vaginal medication admin­ istration. The first-generation nanoparticle is captured by the reticuloendothelial system (RES); the second generation is coated with a water-soluble polymer such as PEG to make it stealthy. A ligandbased nanoparticle belongs to the third generation of nanoparticles, which selectively targets a particular receptor with a ligand. In this way, the nanoparticle is circulated more widely and passively targeted. Finally, multifunctional nanoparticles make up the fourth generation of nanoparticles. This nanoparticle combines a therapeutic agent, a diagnostic agent, and a reporter of treatment efficacy into one unit. Alginate and chitosan are two examples of natural polymers that are frequently utilized to create nanoparticles for vaginal medication delivery formulations. Chitosan polymer acts as a mucoadhesive in a vaginal medication delivery system, prolonging drug retention and improving drug action. Some of the routinely used synthetic polymers for vaginal drug delivery include poly(DL-lactic acid), poly­ caprolactone, poly(lactic-co-glycolic acid), polyacrylates, and PLGA [52].

3.3.3 For Cancer Therapy The use of nanocomposites (NCs) in conventional chemotherapy is acknowledged, and the FDA has approved many NCs for use in other contexts. Since their discovery and use in cancer therapy, liposomes have been one of the most researched and used NCs-drug delivery systems. Meanwhile, PNPs were grouped with those made characteristically for anticancer drug delivery [53]. Apparently, PNPs’ targeted delivery can overcome issues with traditional anticancer treatments, such as their poor wettability, rapid clearance, and lack of specificity, which not only results in a wide range of side effects for healthy tissues but also reduction in the bioavailability of the drugs in cancer tissues. As anticancer medications are administered orally, PNPs have become increasingly popular. It is important to note that despite the fact that the polymeric coating is designed to prevent degradation of active pharmaceutical ingredients (API’s) by bodily enzymes, the major route for the administration of the APIs is through enzymatic degradation, diffusion, and hydrolysis. As a result of the PNPs, medi­ cations have been shown to be more stable, remediation results to be prolonged, cellular uptake to be modified, degradation to be reduced, and metabolism to be inhibited [54]. Due to the dysfunctional permeable tumor vessels and the weakened lymphatic network, PNPs influence tumors during their passive targeting. Nanotechnology has paved ways for specific targeting of nanocomposites to cancer tissue sites. This prevents unwanted toxicity in healthy tissues. Because of the enhanced permeability and retention effect (EPR), drug-PNPs will accumulate in tumor cells’ interstices [55]. The active tumor targeting method, on the other hand, involves the delivery of ternary-constructed

46 Nanomaterials in Healthcare TABLE 3.2

Polymeric nanoparticles (PNPs) used in cancer therapy

PNP USED

(APIS)

Bovine serum albumin

Gefitinib

ENHANCING EFFECTS OF THE PNPS

Targeting over expression of folate receptors. PLGA-PLA 3-bis (2For treating glioma in vivo, the chloroethyl)-1combination chemotherapy of nitrosourea (BCNU) implantation of a graft with (BCNU)loaded nanoparticles was more effective in inhibiting the tumor growth than the standard chemotherapy alone (164%). Poly-butylcyanoacrylate with Doxorubicin Enhanced the anti-tumor properties of polysorbate 80 Doxorubicin Poly-butylcyanoacrylate with Gemcitabine Enhanced antitumor activity both in-vitro polysorbate 80 and in vivo Poly (ε-caprolactone-bMannose PNPs with mannose functionality may be ethylene glycol) (PEGdirected towards antigen-presenting b-PCL) cells’ mannose receptors to stimulate the immune system to fight tumors. Poly (d,l-lactide)-graft-poly 5-fluorouracil (5-FU) Enhanced intracellular delivery (N-isopropyl acrylamide-comethacrylic acid) Hyaluronic acid Paclitaxel Enhancing the biodistribution and therapeutic effectiveness of drug-loaded PNPs PLGA-PVP Tamoxifen Increased sustained release and targeted delivery PLGA-PEG Vinorelbine PNPs improved the therapeutic index of drugs by delivering them to the body’s intended location

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[ 61] [ 62] [ 63]

[ 64]

[ 65]

[ 66] [ 67]

particles to the tumor vasculature and/or tissues via receptor-mediated endocytosis. Among the com­ ponents of this assembly are an anticancer moiety, an importer polymer, and a target ligand [56]. These specific ligands are used to provide the drug-loaded PNPs with a particular functionalization. Molecular ligands, such as proteins, aptamers, small compounds, peptides, and modified antibodies, have been used to identify PNPs [57]. Poly (3-caprolactone) (PCL), a polymer considered to be biodegradable has also been used to capture hydrophobic medications [58]. PCL-PNPs were created by solvent displacement in an acetone-water system. This method uses aqueous solutions containing these stabilizers to adsorb PPOPEO groups to polymeric organic solutions. Moreover, its properties allow it to entrap hydrophobic medicines such as tamoxifen, which is easily incorporated into the polymer during organic synthesis. Studies have shown that in MCF-7 estrogen receptor-positive and MDA-MB231 human breast adeno­ carcinoma cell lines, most of the PNPs were absorbed into the cells and internalized within 30 minutes of incubation. A list comprising the commonly used PNPs used for treating cancer is summarized in Table 3.2.

3.3.4 Ocular Delivery Because of the delicate and pharmacokinetically unique environment found in the eye, ocular drug distribution is one of the most intriguing and difficult tasks addressed by pharmaceutical scientists. The eye’s architecture, physiology, and biochemistry make it extraordinarily resistant to foreign objects.

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 47 Nearly 2.2 billion people worldwide had some sort of near or far vision impairment or were blind as of 2021, according to the World Health Organization (WHO), and at least 1 billion instances may have been avoided with the right care [68]. The statistics show that with the right care, over half of blindness or vision impairment might be prevented worldwide. The current state of ocular therapy calls for the creation of innovative and effective mechanisms. For drug delivery in general or ocular delivery in particular, poly-lactic-co glycolic acid is most frequently used due to its tested biodegradable and biocompatible properties. The U.S. Food and Drug Administration (FDA) gave it the go-ahead for clinical usage in 1989. It has undergone testing to see if it can distribute various medications manufactured with polyvinyl alcohol over time (PVA). The PLGA/ PVA system is well established for the production of bevacizumab-loaded micelles for treating ocular neovascularization. The effectiveness of this chemical in treating retinal and choroidal neovasculariza­ tion has been well documented; however, numerous intravitreal injections are necessary due to its brief half-life in vitreous fluid. Interestingly, this drug loaded micelle system provided sustained release, allowing the drug concentration in vitreous humor to remain above 500 ng/mL for up to two months. This amount of drug was found sufficient to prevent the formation of new blood vessels aided by vascular endothelial growth factor. This kind of NP also included the potent anti-inflammatory dexa­ methasone. This potent anti-inflammatory concoction of dexamethasone loaded micelles has been commercialized in the form of dry tablets, and commercially marketed in the form of eye drops called Maxidex®, which increases the bioavailability of the ocular drug by several folds. Another ocular comorbidity is myosis which often comes as an add-on during intraocular surgery and can be prevented by using Eudragit RS 100® and RL 100® resins infused with the drug flurbiprofen. When used during intraocular surgery, FLU, a non-steroidal anti-inflammatory medicine (NSAID), inhibits cyclooxygenase and counteracts papillary constriction. Additionally, it lessens polymorphonuclear leukocyte (PMN) infiltration in the aqueous humour, which dramatically lessens post-operative oedema after intraocular surgery. They have a lot of potential for ocular application because the FLU-loaded nanosuspensions are made using the quasi-emulsion solvent diffusion (QESD) approach, which typically avoids the harmful chemicals employed in solvent evaporation techniques. Further, the presence of positive charges on its surface aids in better corneal adhesion. In light of this, it can be said that the use of nanosuspensions in ophthalmic pharmaceutical formulations is a promising field, providing a significant opportunity to get beyond the inherent challenges of ocular drug delivery. Chitosan is another polysaccharide that is commonly used for ocular delivery due to its excellent mucoadhesion and penetration making it the best choice for medication delivery in mucosa and ocular regions. CH-based micelles are often fabricated using sodium tripolyphosphate (TPP) as a cross-linker for the administration of levofloxacin for treating ocular infections [69]. This technology demonstrated biocompatibility for topical ophthalmic application, demonstrated a longer retention duration in the ocular area compared to levofloxacin solution, demonstrated a reduction in corneal clearance, and demonstrated a reduction in naso-lachrymal drainage. A hyaluronic acid ocular implant was later im­ planted with drug-loaded CH NPs. In vitro tests showed a sustained drug release over a two-month period, despite the fact that in vivo experiments were not published. A similar DDS fabricated with hyaluronic acid has been fabricated that utilizes the mucoadhesive properties of hyaluronic acid for better adhesion over biological membranes. Several eye infections, such as keratitis, were treated with cefta­ zidime, a highly unstable antibiotic. Physicochemical properties and pharmacological properties of this nano-formulation were found to be suitable for topical administration to the eyes in their research. Additionally, mucin, which increases ocular globe residence time, also interacts with this substance able to maintain the antibacterial activity while possessing pertinent mucoadhesive qualities. Instead of TPP, other authors have employed sodium deoxycholate as a crosslinking agent. An embedded CH/PVA system with sodium deoxycholate was used to treat illnesses involving ocular inflammation [70]. In vivo experiments using a guinea pig model showed that at 24 hours, the optimized NPs formulation produced twofold greater prednisolone release than the commercial micronized drug loaded gel. The best ways to acquire CH-based micelles have been investigated. One of these is the functionalization of the CH chain’s main amino groups with lipophilic derivatives. In keeping with this idea, Xu et al. created a new

48 Nanomaterials in Healthcare branching CH by introducing stearic acid and valylvaline in varying amounts to the polymer’s main chain. Dexamethasone was encapsulated by these polymeric polymers, which were able to self-assemble. As a result, NPs showed sustained release and improved penetrating qualities while demonstrating access to the posterior segment via the conjunctival channel. Male rats and male New Zealand albino rabbits used in in vivo experiments showed equivalent amounts of dexamethasone to dexamethasone-loaded hydrogenated castor oil-40/octoxynol-40 NP, a product like Cequa that has been approved by the FDA. Another possibility is to create block copolymers with a CH section that are hydrophilic and hydro­ phobic. Research groups have also created a cationic CH grafted methoxy poly (ethylene glycol)-poly (-caprolactone) (PEG-PCL) in this line for the purpose of encasing diclofenac. The polymer’s amphi­ philic nature allows it to self-assemble into micelles while its positive charges interact with the mucin’s negatively charged surface to prolong the duration that NP is retained there. Compared to the drug’s commercial eye drops, this formulation demonstrated improved diclofenac penetration and retention while being benign (1.4-fold higher). Diclofenac concentration in rabbit aqueous humour was 2.3 times higher than it was when the commercial medication formulation was used in the eyes. Dendrimeric structures have also been researched, even though micelle-based NP systems have received the majority of attention in studies on polymeric nanocarriers. For the creation of nano systems for the transport of drugs to the posterior portion of the eye, polyamidoamines serve as the prototypical dendritic polymer. Yang et al. investigated the potential of penetration and cyclic arginine-glycineaspartate hexapeptide-modified polyamidoamine dendrimers as DDS [71]. A non-invasive administra­ tion approach for more than 12 hours showed that these functionalized NPs were still present in the posterior portion of the eye. Lancina et al. also researched a polyamidoamine-based dendrimer [72]. In this instance, a timolol analogue, a widely used medication for the treatment of ocular hypertension, was employed to derivate the dendrimeric core. Their findings showed no irritation or toxicity after a week of daily dosing, as well as a 30% drop in intraocular pressure in adult male Brown Norway rats with normotensive after 30 minutes of topical application. Tai et al. looked at how hyaluronic acid and polyamidoamine dendrimer combine to produce a complex [73]. By controlling the expression of target proteins and genes in cells, this combination was functionalized with penetration and loaded with an­ tisense oligonucleotides for the treatment of eye disorders. This system demonstrated improved distri­ bution to the ocular posterior portion and eye permeability, making it a suitable formulation for topical ocular delivery. Some additional dendrimer structures, such as polylysine (PLL) or phosphorus den­ drimers, can be discovered in literature despite the fact that the majority of contemporary scientific research with dendrimer NPs has been centered on the usage of a specific form of polyamidoamine polymer (Figure 3.5) [74].

3.4 CONCLUSION AND FUTURE PROSPECT Today, every researcher’s main objective is to employ newly developed technology to improve human existence and do things that were previously perceived as unattainable. Goals which include improving the efficacy to deliver drugs in innovative ways (administration routes) to treat severe illnesses and developing vaccines to help defend against specific diseases by replicating an infection, can now be achieved using novel paths opened by nanotechnology and nanomedicine. Though developing new drug delivery systems is never straightforward, especially when dealing with poorly soluble molecules (drugs), most obstacles can be handled with the use of nanomaterials as drug delivery carriers. They can direct drug compounds to their desired locations, where they can work on the infected tissues without doing too much damage to healthy cells. Recently, in the area of drug delivery, polymeric nanocarriers such as micelles, capsules, vesicles, capsules, nanogels, nanofibers, and nanospheres have received an exponentially growing amount of interest. The diverse sources (natural or synthetic), techniques of

3 • Advancement of Polymer-Based Nanocarrier System in Drug Delivery 49

FIGURE 3.5 Application of polymeric nanoparticles in drug delivery.

synthesis, and charges of the nanosized polymers allow them to provide a regulated release of drugs and a specific potential effect. Their use in medical treatment therapy (for various diseases) has made it feasible to resolve problems with drug toxicity and patient safety. In particular, the emergence of advanced smart-polymeric nanocarriers enables tailored and on-demand therapies (vaccines/drug delivery systems) extremely possible. The successful innovations of polymeric nanocarriers not only gives them the ability to recognize damaged tissue or cells and start a cell’s positive response to treatment with drug, but also to act as a switch to accomplish directed drug delivery within lesion locations. The potential, recent advances of polymeric nanocarriers brings chal­ lenges alongside great opportunities for researchers to create novel, more desirable polymer-based nanocarrier drug delivery system by exploiting current knowledge of variety of polymeric nanocarriers for controlled drug release and its (nanocarrier) utilization in other applications such as in cancer therapy, gene delivery, and as vaccines. Keeping in mind the opportunities and great possibilities, a new paradigm for the development of gene and polymeric drug delivery systems may soon be possible in the near future by fusion of viewpoints from the biological and synthetic sciences.

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Liposomes and Lipid Structures Classification, Characterization, and Nanotechnology-Based Clinical Applications

4

Suditi Neekhra, Priyanka Maske, and Roshan Keshari Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 4.1 4.2 4.3

4.4

4.5

Introduction Need for Liposomes Evolution through Advancements 4.3.1 Solid Lipid Nanoparticles (SLNs) 4.3.2 Nano Lipid Carriers (NLCs) Conventional Methods for Liposome Preparation 4.4.1 Film-Hydration Method 4.4.2 Double-Emulsification Method 4.4.3 Reverse Phase Evaporation Method 4.4.4 Solvent Injection Method 4.4.5 Detergent Dialysis Method Novel Methods for Liposome Preparation 4.5.1 Supercritical Fluid (SCF) Technology 4.5.2 Dual Asymmetric Centrifugation Techniques (DAC) 4.5.3 Membrane Contactor Technology 4.5.4 Microfluidic Technique

DOI: 10.1201/9781003322368-4

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56 Nanomaterials in Healthcare 4.6

Characterization of Liposomes 4.6.1 Morphological Characterization 4.6.2 Stability Study 4.6.3 Encapsulation Efficiency 4.6.4 In Vitro Drug Release 4.6.5 Freeze-Drying 4.6.6 Miscellaneous Methods 4.7 Liposomes for Treatment of Various Diseases 4.7.1 Cancer 4.7.2 Reproductive Organs 4.7.3 Developmental Disorders 4.7.4 Arthritis and Bone Abnormalities 4.7.5 Wound Healing 4.7.6 Antimicrobial Diseases 4.7.7 Nervous System Disorders 4.8 Clinical Applications of Liposomes Concerning Nanotechnology 4.9 Future Prospective 4.10 Conclusion References

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4.1 INTRODUCTION Liposomes are minuscule vesicles made of similar substances to the cell membranes. ‘Lipos’ denotes fat and ‘Soma’ denotes body as derived from two Greek words; thus, liposomes are fat-derived small bubbles resembling cell membranes. The cell membranes are made of phospholipids consisting of a polar head group and a non-polar tail, similarly, the liposomes are formed from their structural building blocks i.e., the phospholipids. Phospholipids are molecules made of a polar head that is attracted to water and a non-polar tail that has a hydrocarbon long-chain serving as a hydrophobic tail, thus making a lipid bilayer. The liposomes based on their structural construct can be formed of various sizes as unilamellar or multilamellar structures. The testing of an electron microscope using a phospholipids sample stained with a negative stain lead to the discovery of liposomes by A. D. Bangham and R. W. Horne in 1964 at the Babraham Institute in Cambridge. Initially, the liposome-based formulations were composed of natural lipids only, however, currently natural and/or synthetic lipids along with surfactants are frequently used. As suggested, liposomes are tiny bubbles and can be used to load drugs into the vesicles. These drugs otherwise delivered by conventional routes can impose high toxicity, hence, drug-loaded liposomes can be applicable in diseases where systemic toxicity and site-specific action are of high concern. These vesicles range from nanometers to micrometres; however, liposomes used for medical applications are in the range of 50–450 nm [1].

4.2 NEED FOR LIPOSOMES Liposomes, since their discovery has been used in many areas, including cosmetics, pharmaceutical, food, and farming industries as well. Liposomes with enhanced encapsulation of unstable compounds to

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FIGURE 4.1 Schematic representation of liposome with various surface modification and pay-loading strategies.

improve their shelf life and stability, including antimicrobials, antioxidants, flavours, and bioactive elements have been used. As they are made of amphipathic phospholipids, hence they can entrap or encapsulate hydrophobic as well as hydrophilic molecules. [2]. They are cell-mimicking biocompatible, biodegradable, possess low toxicity and can trap a versatile range of drugs in the core or the layer. These lipophilic moieties have not only solved the problem to inject hydrophobic drug compounds for treating various diseases but have also penetrated through diverse barriers, thus have applications in site-specific drug delivery for many diseases, including bacterial infections, nervous diseases, cancer and tumour tissues, and many more [3,4]. Recent developments in technology and understanding of the biology of diseases, both have led to the growth of liposome-based platforms to deal with critical diseases. Providing safe shells and sta­ bility to drugs, liposomes have proven to be the best nanocarriers available for being used via various routes of administration in the body. They have significantly increased the circulation time in the blood, therefore improving the bioavailability and higher working concentrations of drugs in the body for longer time durations. Depending on the desired requirements, liposomes can be easily built for controlled or shattering release, pH or temperature, targeted or systemic [5]. Therefore, liposomes can be used as an investigational tool in research and as site-specific or systemic drug delivery systems (Figure 4.1).

4.3 EVOLUTION THROUGH ADVANCEMENTS Lipid-based nanoparticles have attracted great attention in the past few decades with advancements in the understanding and requirements like targeted drug delivery, high physical and chemical stability, and increased payload. This progress of liposomes also initiated a crucial toward the development of solid lipid nanoparticles (SLNs) and nano lipid carriers (NLCs); thus, they are briefly discussed in the chapter further.

58 Nanomaterials in Healthcare

4.3.1 Solid Lipid Nanoparticles (SLNs) SLNs are sub-micron colloidal carriers, which are composed of physiological lipids, dispersed in water or aqueous surfactant solution. SLNs have gained considerable attention, due to their ability to incor­ porate a high percentage of drugs and ease of synthesis particularly to minimize the usage of organic solvents, compared to liposomes. Surfactant type and lipid concentrations play an important role to stabilize the surface charge of SLN and controlling drug loading along with its size; SLN was first synthesized in the 1990s by Muller and Gasco [6]. The most commonly used lipids include Compritol 888 ATO, Precirol ATO5, Stearic acid, and Glyceryl Monostearate, while surfactants include Tween 80, Kolliphor P188. These have less/negli­ gible toxicity and have high encapsulation efficiency for lipophilic drugs. The lipids used are solid at body temperature and room temperature; thus, they show prolonged and sustained release of the drug inside the body, also preventing the drug degradation in this duration. SLN achieved huge success since then and was well established as they provide better stability than liposomes along with the sustained release.

4.3.2 Nano Lipid Carriers (NLCs) NLCs emerged a decade later after the establishment of SLN, portraying the second generation of liposomal structures that possess some major advantages over the previous generation structures. NLCs are composed of liquid lipids, solid lipids, aqueous phase and surfactants, and their ratios are heavily dependent upon solid lipid/liquid lipid miscibility and the drug that is to be loaded inside the less ordered core [7]. Surfactant type and concentration also play an important role to stabilize the surface of NLC and control its size (Figure 4.2). Most commonly employed liquid lipids include oleic acid, Labrafil, olive oil, and miglyol and solid lipids generally used are Compritol 888 ATO, Precirol ATO5, Stearic acid, and Glyceryl Monostearate, while surfactants include Tween 80, lecithin, and Poloxomer188 [8]. Lower drug payload was the major challenge to be dealt with the SLN that NLC overcame due to their composition and improved availability to load drugs inside their loose core. Having higher loading capacity and improved bioavailability in comparison to SLN, these NLCs are proving to be a great success [7]. Different types of NLCs can be obtained including amorphous, imperfect, and mixture, depending upon the composition of solid/liquid lipid and surfactant, and also the method employed for the synthesis [9].

FIGURE 4.2 Structural differentiation in various lipid carriers is depicted herewith: (a) Liposomes, (b) solid lipid nanoparticles (SLN), (c) nano lipid carriers (NLCs).

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4.4 CONVENTIONAL METHODS FOR LIPOSOME PREPARATION Numerous methods have been developed for the preparation of liposomes, which include both advanced and traditional techniques. All properties of the liposome are widely dependent upon the techniques and composition of materials required like phospholipids, drugs, as well as other excipients. Care should be taken while selecting materials because each material can change both physical and chemical properties of liposomes like size, drug loading, drug release, interference in the targeted site, etc. (Figure 4.3). The traditional methods of preparation of liposomes include the thin-film hydration method, reverse phase evaporation, detergent analysis etc. This method requires numerous toxic organic solvents to dissolve the lipids. After careful analysis of all physical parameters and chemical compatibility, lipids that are compatible with the organic solvents and temperature can be chosen else these might hinder reproducibility. Some of these traditional techniques are detailed below.

4.4.1 Film-Hydration Method Thin-film hydration techniques were one of the earliest techniques that have been developed for the preparation of liposomes and it is beneficial for the loading of mostly lipophilic drugs [10]. In this method usually, phospholipids and cholesterol were dissolved in the organic solvent and the thin film will be created by evaporating the organic solvent in flask rotation under a complete vacuum. It gives the dried lipid membrane which can be hydrated by using an aqueous solvent like buffer or water. The resultant solution gives the multilamellar vesicles which can be reduced to small lamellar vesicles via

FIGURE 4.3 A schematic representation of the classification of liposomes.

60 Nanomaterials in Healthcare sonication, a high-speed homogenisation technique [11]. The major advantage of this technique is the straightforward approach while the disadvantages are low encapsulation efficiency, difficulty in removal of organic solvent, small-scale production, and large vesicle size without particle size control.

4.4.2 Double-Emulsification Method In this method, liposomes are prepared by dissolving lipid and water-soluble carriers in the cryoprotectant-like tert-butyl alcohol/water co-solvent systems, which results in the formation of cakelike materials of an isotropic monophasic solution [12]. Upon the addition of water, it gives the water in oil-water (W/O/W) types of emulsion, the freeze-dried product forms a homogeneous solution of multilamellar vesicles, the size of which can be reduced by the extrusion/homogenization. The particle size is additionally dependent upon the cryoprotectant used for having better encapsulation efficiency, high stability, and reproducibility. Furthermore, by using this method, sterile liposomes can also be produced with better storage stability. It removes the water from the liposome from the frozen at extremely low pressure in the presence of some sugars like sucrose or trehalose to prevent the leakage of the encapsulated drug but simultaneously increases the liposome size upon rehydration [13]. The major advantage of this method is it can store heat-sensitive materials for a longer period while a few dis­ advantages are limited usage of cryoprotectants for diabetic patients and removal of organic solvent; otherwise, it may cause some toxic effects.

4.4.3 Reverse Phase Evaporation Method The further development of the preparation of liposomes was significantly improved by this method. The basis of this design is the creation of inverted micelles, which are shaped by sonication of the lipid formulation. The slow removal of organic solvents converts inverted micelles into a viscous gel state. At the critical point, these viscous gels collapse, which leads to the disruption of the inverted micelles and excess phospholipid donates for the formation of bilayer around the residual micelles and resulting in the creation of liposomes. Liposomes prepared by this method allow the accommodation of a high aqueous space to lipid ratio and enhance the capability to encapsulate a substantial percentage of aqueous materials. The ratio of lipids to water should be approximately four times more than the hand-shaken liposome or multi-lamellar liposomes. In this method, water-in-oil emulsion forms and after the eva­ poration of the organic phase, an aqueous suspension containing large lamellar vesicles [14] as well as multilamellar vesicles is formed. The major benefit of this method is for encapsulating both small and large macromolecules with suitable encapsulation efficiency. The drawbacks of this method are the contact of materials that need to be encapsulated in the organic solvent along with the short period of sonication may lead to the denaturation of protein or breakage of DNA strands or the denaturation of protein along with it may not be suitable for encapsulation of the organic solvent [14].

4.4.4 Solvent Injection Method In this method, lipids are dissolved in the organic solvent and this solution is further mixed in the aqueous medium to form unilamellar liposomes. Ethanol is the organic solvent that is widely used for this method because of its safety reasons. The nanoparticles prepared by this method usually range between 80–300 nm depending on the preparative condition [15]. The organic solvent used can be removed by using different techniques like evaporation, lyophilization, dialysis, or diafiltration, and the final formulation can be concentrated to the required volume. Extreme care should be taken while formulating the liposome like flow rate, temperature, lipid selection and concentration, and stirring, which widely affect the property of liposomes like stability, size, and encapsulation of the drug. The major benefit of this method is that

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additional methods like sonication or extrusion are not required for the reduction of particle size. Moreover, small liposomes with narrow particle size distribution can be obtained by this method without the utili­ zation of further aid like extrusion or ultrasound. Major drawbacks are heterogeneous population, i.e., 30–110 nm, which leads to the dilution of liposomes. It also encounters the difficulties in complete removal of the ethanol because of the formation of an azeotrope with water and high possibilities of inactivation of bioactive macromolecules even in low concentrations of ethanol.

4.4.5 Detergent Dialysis Method Using this method, liposomes are formed by dissolving phospholipids with the detergents to produce phospholipid-rich micelles in the size range of 40–180 nm, which leads to the formation of LUVs [16,17]. The detergents used in this method are removed by either the chromatographic method or dialysis. Benefits of this method are that the liposomes of unilamellar vesicles are formed with a better size distribution while drawbacks are difficulty in removal of organic solvent and detergent residues, poor encapsulation efficiency, and time-consuming method [18]. The major hurdle associated with this process is to remove the organic solvent from the end products if not completely removed, they may encapsulate into the liposome along with the drug and show toxic effects along with interference in the stability of vehicles. Some other problems associated with tradi­ tional methods are initially the particle size is large and high polydispersity so it requires an additional process to get the desired size. Henceforth, other novel methods are also being worked upon to overcome the above-mentioned issues and get desired results.

4.5 NOVEL METHODS FOR LIPOSOME PREPARATION 4.5.1 Supercritical Fluid (SCF) Technology This method utilizes both the desirable property of liquid and gas. Fluids used for this method are noncondensable and very dense at a certain temperature and pressure, beyond a certain value. When the line between the gas and liquid disappears, this supercritical fluid possesses many characteristics when compared with conventional fluids. The major advantage of using this method is the use of carbon dioxide, which is an excellent substitute for the organic solvent. Additionally, the low preparatory cost, non-toxic, and non-inflammatory nature of this method is one of the most attractive parameters, along with the very low critical temperature and pressure i.e., 31°C and 73.8 bar with better dissolution properties compared to non-polar organic solvents [19]. The supercritical anti-solvent (SAS) method has been also reported in which the organic co-solvent containing phospholipid is sprayed into the SCF, which acts as an anti-solvent and results in the preparation of micronized particles of lipids. Particle size is widely dependent upon the spray droplet and the concentration of phospholipid in the co-solvent and the particles are hydrated with the aqueous buffer to obtain liposomes [20].

4.5.2 Dual Asymmetric Centrifugation Techniques (DAC) A liposome prepared by this method requires homogenization of the highly concentrated lipids in a vial. In this method, homogenization takes place by centrifuging the dispersed viscous liquid in a central axis (axis A) and similarly a vial used is turned around its vertical axis (axis B). The concentrated viscous

62 Nanomaterials in Healthcare liquid is accelerated away from its axis A due to the centripetal in the outward direction; similarly, materials moved towards the inward direction due to the friction of the materials to the wall of constantly turning vials. Both the overlying movement of the lipid results in homogenization and leads to the formation of a highly viscous liposome known as vesicular phospholipid gel, which can be further diluted in an aqueous solution to obtain the final liposome [21]. Major advantages of this method include small equipment that is easy to operate and has good reproducibility, so liposomes with a small particle size can be prepared without any homogenization or granulation technique. As no organic solvent is used for dissolving lipids, it also imparts better encapsulation efficiency of water-soluble drugs. But the drawbacks are it requires a high amount of lipid to form sufficient viscosity.

4.5.3 Membrane Contactor Technology This is one of the novel methods based on the principle of the ethanol injection method. As this method was previously applied for the preparation of emulsion, precipitates, and polymeric lipid nanoparticles. Membrane contractors are mainly used to mix two materials efficiently and many different types of dispersion systems can be formulated via the utilization of membrane contactor technology. In this method, homogenization of both the lipid and water phase is required, which is a major procedure for the preparation of liposomes. By utilizing the principle of ethanol injection technique, Laouini and their co-worker combined this technology with the hollow fibre module; this combination leads to an increase in the membrane surface area and thus offers better efficiency [22]. This method can be directly scaled up to produce large-scale production of homogenous vesicles, and/or multilamellar vesicles having high encapsulation efficiency for lipophilic drugs.

4.5.4 Microfluidic Technique In this method, fluids flow in a channel having cross-sectional dimensions, usually in the range of 5–500 μm [23]. Some of the types of microfluidic techniques are micro hydrodynamic techniques, which are used for generating small vesicles and giving the monodisperse population of both small lamellar and large lamellar vesicles with control over particle size also. Similarly, another method is microfluidic droplet formation of giant vesicles of two immiscible phases such as oil and water are allowed to flow in a microchannel, where small droplets of one phase result in the formation of uniform size particles under certain conditions [24]. The major advantage of this technique is that it can precisely deliver nanolitre volumes along with precise control at the position of the interface.

4.6 CHARACTERIZATION OF LIPOSOMES Since the final therapeutic effect of the drug-loaded liposome is entirely dependent upon some of the parameters like size, shape, lamellarity and surface charge, drug loading efficiency, etc. and all the parameters must be carefully monitored. Some of the major characteristics of liposomes that should be strictly evaluated are as follows.

4.6.1 Morphological Characterization The size and distribution of the liposome can be characterized by the static and dynamic light scattering (DLS) method, also known as photon correlation spectroscopy, which is one of the most efficient

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methods for size estimation and distribution, as size characteristic is closely parallel with the lipid bilayer structure [25,26]. When the particles are highly polydisperse, fractions collected after the separation by the field flow fractionation and high-pressure liquid chromatography can be analyzed by DLS or multiangle light scattering (MALS) to obtain more accurate particle size. Since signals produced from it are dependent upon the molecular weight and particles size concentration, the concentration of the liposome can be analyzed at the time of separation, but a major limitation is that it does not provide an accurate estimation of size distribution when the liposome has a highly polydisperse population [27]. As the exact size of the liposome cannot be estimated by the DLS, so it can also be analyzed by a scanning electron microscope (SEM), transmission electron microscope (TEM), or environmental scanning microscope (ESEM), to understand the exact radius, size, and morphology [28]. Significant drawbacks of microscopic techniques are that they are time-consuming, and sample preparation processes like staining and drying may alter liposome morphology. Another substitute for this technique is atomic force microscopy which gives data on the three-dimensional shape of liposomes without any sample modification. The nanometre-sized probe on a fixed glass or a mica surface is utilized for scanning which doesn’t cause any damage or alteration in the morphology of the liposome. After drop-casting the liposome, a quick analysis should be done because the evaporation of the aqueous medium may lead to vacuum rearrangement. The drawbacks are that fragile liposomes may get altered by interacting with the mica surface or the AFM probe.

4.6.2 Stability Study The stability study of the liposome is one of the most critical parameters that should be studied. It can be all physical, chemical, and biological stability studies as they may influence the size, zeta, encapsulation, release study, etc. The physical stability of liposomes can be studied by parameters like odour, colour, sedimentation ratio, size and distribution, and microscopic observation with the SEM, TEM, AFM, etc. to prevent the aggregation of the particles and enhance the shelf life; similarly, for chemical stability of liposomes, by analyzing the composition of phospholipid and drug degradation via liquid chromatog­ raphy and spectroscopic techniques. Moreover, phospholipids can be protected from oxidation by using the lipids of pure form of high quality, and also by using inert gas like argon or nitrogen during the preparation. Storage of liposomes should be at lower temperatures, away from light sources along with some anti-oxidant materials like butylhydroxytoluene (BHT) and α-tocopherol or by removal of water through freeze-drying with suitable cryoprotectants [29,30].

4.6.3 Encapsulation Efficiency This is the method that gives the estimation of how much drug has been encapsulated inside the liposome. This is the principal characteristic that should be determined to get the required bioavailability or therapeutic efficacy. Initially, the final formulation should be separated from the unentrapped or non-encapsulated drug, which can be achieved by centrifugation or dialysis techniques. Then the amount of drug encapsulated inside the liposome can be estimated by disrupting the lipid bilayer with any organic solvent in which the drug is soluble; for example, methanol, chloroform, or triton-x etc., followed by filtration and measurement by HPLC, UV, or LCMS, which is known as direct technique, etc. In the indirect technique, the amount of unencapsulated drug is determined, and subtraction is done from the total amount of drug used [31].

4.6.4 In Vitro Drug Release This technique gives information about how much drug can be released from the liposomes at a specific temperature and sink medium. It is usually performed at 37°C because it resembles the body temperature

64 Nanomaterials in Healthcare under a sink medium that can be buffer or a mixture of buffer and organic solvent, dependent upon the solubility of the drug. In this method, a dialysis membrane has a specific molecular weight cut-off in which the drug is filled and soaked in the receiver compartment at 37°C. It is always suggested that the release medium should be a buffer having a pH of 7.4 because it mimics the in-vivo condition. After a specific interval of time, like 1, 2, 4, 6, 8, 12, 24, 72 hrs and so on, certain aliquots of the medium should be taken out, and replaced by the fresh receiver medium, which can be adjusted during calculation, while aliquots can be analyzed by HPLC, UV, or LCMS.

4.6.5 Freeze-Drying Freeze-drying of liposomal preparation is mainly used when liposomes are required to be stored for a more extended period or if the liposome is unstable after a certain period in an aqueous solution. When stored, the active ingredients will leak out from the lipid bilayer and form aggregates and so, it’s suggested that the liposome should be freeze-dried, increasing the shelf life and stability. During freeze-drying, there are several steps involved like freezing, ice sublimation, and desorption of unfrozen water [32]. During these all-process number of physical changes occur, which lead to alteration in size, surface charge, morphology, and loss of incorporated drug. To overcome these problems, sugars like sucrose, glucose, trehalose, mannitol, and lactose can be used [33]. All sugars can interact with the head groups of phospholipids via forming an H-bond, leading to the depression of phase transition temperature.

4.6.6 Miscellaneous Methods Zeta potentials give information about the particle’s surface charge, which provides information about the stability of the prepared liposome. It is considered that the liposome having a zeta potential of ±30 mV is electrostatically stabilized. An electric field is applied to measure the zeta potentials, and thus the particles’ electrophoretic ability is measured. It is precisely dependent upon the scattering of an incident laser by moving particles while the factors affecting the zeta potentials are pH, ionic strength, and the concentration of the particles. The lamellarity of liposomes is mainly analyzed by the 31P NMR in which the signal ratio determines the lamellarity of liposomes before and after Mn2+ addition [34]. This is a widely used technique but the concentration of Mn2+ and buffer and type of liposome used must be selected sensitively.

4.7 LIPOSOMES FOR TREATMENT OF VARIOUS DISEASES A wide range of antimicrobial drugs in combination and individually as well has been loaded onto liposomes for antimicrobial, antiseptic, anti-inflammatory, and other various purposes. Anionic lipo­ somes are prone to faster reticuloendothelial system (RES) clearance and less stability in blood circu­ lation due to their charge in comparison to cationic or neutral liposomes. Cationic liposomes instead have better cellular interaction and gets easily internalized via blood circulation, and thus are preferred for drug delivery. Therefore, depending upon the requirements, nature of depot and target tissues, the lipids are chosen.

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4.7.1 Cancer Over the past few decades, various liposome-based formulations have been developed by loading anticancer agents, having better therapeutic effects, targeted efficacy, and being non-toxic than conventional or free drugs, both in the conventional form of the liposomes or using pegylated liposomes. A recent lipo­ somal formulation was prepared by conjugating folic acid with the dual drug, i.e., celastrol and irinotecan. Phospholipids like DPPC (Dipalmitoyl phosphatidylcholine) and DSPC (Distearoylphosphatidylcholine)PEG (Polyethylene glycol)-NH2 were used to prepare liposomes which showed the particle size of 190 nm having narrow size distribution 0.1. It showed a better drug release profile at pH 5 for both drugs. Furthermore, its cellular studies were carried out in (MCF-7 and MDA-MB-231, which is breast cancer cell lines which showed better cellular uptake and improved apoptosis [35]. Similarly, the pharmacokinetic property of doxorubicin was enhanced by encapsulating it in the liposome for treating human lung carcinoma. Since its cellular study was carried out in A549, it also showed good efficacy when it was encapsulated with selenium-coated doxorubicin. Lecithin S 10, DOTAP (1,2-Dioleoyl-3trimethylammonium propane) and cholesterol are the phospholipids used to prepare liposomes by using ammonium sulphate as a gradient method. The final prepared liposome showed better cell cytotoxicity on A549 with significantly low IC 50 i.e., 0.92 ± 0.16 μg/mL than that of free i.e., 4.40 ± 0.58 μg/ mL) while the liposomal doxorubicin has 5.68 ± 0.73 μg/mL) [36].

4.7.2 Reproductive Organs The principal applications of liposomes in the male reproductive system include i) protective effect on sperm function post cryopreservation; ii) as vehicles for the incorporation of foreign material (DNA or ATP); iii) regulation of sperm function at steps of capacitation, gamete interaction, and/or fusion and acrosome reaction; and iv) the study of membrane ion channels by patch-clamp methodology. Cryoconservation of sperm is a routine procedure used for the preservation of human fertility as well as for agricultural purposes, primarily to promote reproductive efficiency in reproductive species. However, the difficulty associated with this is the freeze–thawing-induced damage which may cause impaired sperm viability and functions. A great proportion of the damage is related to membraneassociated loss of function. This damage is due to the relative resistance of the cellular structure to temperature and physical state changes. Initially, egg yolk because of its phospholipid moieties, was being used as a cryo-protective agent, however, because of sanitary and reproducibility issues, it could not be used further. To study the effects on sperm functions, after the freezing and thawing, artificial liposomes were designed as a replacement for egg yolk using bull spermatozoa [37]. The data docu­ ments, in preserving the percentage of progressively motile spermatozoa after freeze-thawing solely the phosphatidylserine/cholesterol liposomes were efficiently protective similar to egg yolk. This result was further comforted showing the use of dioleoylphosphatidylcholine (DOPC) vesicles combined with BSA could also protect sperm membrane integrity at a level similar to egg yolk [38]. It is also documented that membrane-stabilizing agents were more efficient than ice-preventing agents (such as glycerol or sucrose) in sperm cryoprotection. Liposomes have also been employed in the treatment of vaginal infections. However, due to challenges in oral antibiotics along with treatment of antimicrobial resistance, alternative treatment lines need an hour. In the same line, using polyphenol resveratrol (RES) comprising liposomes was incor­ porated in chitosan hydrogel in the treatment of C. trachomatis infection [39].

4.7.3 Developmental Disorders The prenatal and/or perinatal period is highly critical in terms of foetal development as the foetus can get exposed to various drugs via maternal circulation. Due to a poorly developed metabolic system, the

66 Nanomaterials in Healthcare embryo or the neonates cannot metabolize these xenobiotics and thus cause embryotoxicity. Hence, approaches have been made for drug delivery systems using liposomes for foetuses or during the critical window of development. Vancomycin hydrochloride can cross the maternal-foetal barrier and thus if a higher concentration reaches the foetus, it can cause fetotoxicity. Hence, to overcome the same, PEGylated liposomes were constructed for a known nephrotoxic agent, i.e., vancomycin hydrochloride for drug delivery during foetal development [40]. Antibody conjugated indomethacinloaded liposomes targeted for uterine oxytocin receptor have been reported to induce preterm birth in mice [41].

4.7.4 Arthritis and Bone Abnormalities Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder that affects bones and multiple joints of the body. RA is an autoimmune disease that leads to chronic inflammation in the synovial membrane. Arthritis affects females more compared to males due to hormonal and physiological differences. The treatment lines available for RA are combinations with nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), corticosteroids, biological agents, and natural agents [42,43]. Along with nonconventional therapies, newer approaches have been introduced using liposomes. The diclofenac sodium an NSAID was incorporated in liposome, noisome, lipogelosome, and niogelosome formulations. The lipogelosome formulation loaded with diclofenac sodium showed enhanced anti-inflammatory action in a single dose in comparison with other commercial formulations [44]. Encapsulation of hydrocortisone acetate in liposomes has been reported to be highly efficient for the treatment of arthritis [45]. Several preclinical trials on different drug formulations loaded with liposomes have been well studied and documented. Prednisolone phosphate-loaded liposomes have also been reported to inhibit osteoclast activity and thus aid in the ease of arthritis anomalies. Dexamethasone palmitate-loaded liposomes in rabbits have been shown to have higher retention in the synovial fluid [46].

4.7.5 Wound Healing Wound healing has always been a major clinical concern affecting people worldwide, and increasing multidrug resistance has worsened the situation. Hence, this has been a huge genre for loading drugs onto liposome-based formulations for quicker absorption, healing, high cellular compatibility, and preventing microbial infections. Burns and deep wounds quickly become the home to microbes in the surrounding environment and open the door for them to enter inside the body as well. Therefore, these formulations necessarily need to incorporate antibiotics with them to repair damaged tissues and promote their growth. Antimicrobial agents play a crucial role in wound healing formulations to prevent infections and biofilm formation that increase inflammation and hinder the healing process. Chronic wounds remain a concern as the tissues are permanently damaged many times or repaired extremely slow. Liposomes with different surface charge properties are being used in combination with nanofibres, meshes, and hydrogels for improved efficacy and healing. Gallic acid-loaded lipo­ somes and gallic acid powder gel have been analyzed for wound healing and to reduce periodontal inflammation [47]. A nanofiber mesh of chitosan loaded with gentamicin liposomes has been reported for slow and sustained release of gentamicin for avoiding infection and wound healing [48]. In a similar study, non-woven gauges and functionalized with liposomes loaded with anti-inflammatory Piroxicam for effective wound healing [49]. For burn healing, usnic acid-loaded liposomes have also been developed and incorporated into collagen-based films that will reduce inflammation and improve the burn healing process [50]. Curcumin, being a well-known antioxidant and antibacterial compound, is anti-inflammatory as well, which makes it appropriate for use in burns and wound healing. Chemical

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instability and poor solubility of curcumin emphasize exploring novel delivery methods to convey the depot at the desired location and effective concentration. Herein, deformable liposomes containing curcumin have been designed that were loaded on chitosan hydrogel targeting chronic wound treat­ ment [51]. Diabetes is another major concern to deal with wound healing and active microbial attack in open wounds. Elwakil et al. designed chitosan and liposome nanoparticles loaded with cinnamon extracts to provide an anti-proteolytic effect and heal the wound effectively. Similarly, insulin-loaded liposomes have been explored in blended-in hydrogel-controlled release and safe delivery of insulin for wound healing [52]. Chitosan-coated liposomes loaded with Substance P (SP) neuropeptide have been studied for wound healing, thus approaching novel nanoformulations for treating difficult wounds [53]. Liposomal film formulation encapsulating D-panthenyl triacetate (PTA) and coenzyme Q10 (CoQ10) was standardized for wound healing and is intended for topical use [54]. Liposomal for­ mulations have also been used for film formation and thin coatings, like Umar et al. developed a filmforming spray using chitosan with liposomes incorporated into it. The liposomes are loaded with human epidermal growth factor (hEGF) and have been studied for direct spray on wounds with sustained release of liposomes and effective wound healing [55].

4.7.6 Antimicrobial Diseases Despite huge success at the early stages, antibiotics still face significant challenges to treat bacterial infections. The growing complexity of antibacterial resistance due to overuse and misuse of anti­ biotics, and the spread of resistant bacteria is aggravating the situation. This is further worsened by the declining interest of pharma giants in searching for new antibiotics. With the development of nano­ technology in medicine, new ways to deliver these drugs have been worked upon that are providing promising solutions to deal with the above issues. As an assured drug delivery system, liposomes have been heavily explored to deal with antimicrobial resistance by releasing a sufficient amount of anti­ biotics directly inside the bacteria with long-term stability and enhanced permeability. Encapsulation in liposomes and their loading onto hydrogel has been the most studied work design for antimicrobial drug delivery. Recently, the therapeutic efficacy has also been greatly improved by incorporating metallic nanoparticles, polymeric nanoparticles, dendrimers, and carbon tubes. Here in this section, we’ll only cover the liposome-based formulations designed for dealing with advanced resistance mechanisms of bacteria. Polymyxin B-loaded liposomes have been developed against the resistant strain of Pseudomonas aeruginosa against the infection caused by gram-negative bacteria [56]. Similarly, azithromycin-loaded liposomes have been synthesized against P. aeruginosa for its biofilmforming activity that affects cystic fibrosis patients [57]. The antimicrobial activity of lauric acid liposomes against inflammatory acne is caused by Propionibacterium acnes (P. acnes) [58]. Liposomes encapsulating tea tree oil and silver nanoparticles against bacteria lead to chronic infections at wound sites that delay healing as well as lead to septic conditions [59]. Electrospun nanofiber mats made of chitosan-containing liposomes have been designed that are loaded with tea tree oil for longterm antibacterial efficacy with controlled drug release for excellent antibacterial effects [60]. However co-encapsulated liposomes loaded with gentamicin and gallium also have been developed for strong antibacterial efficacy against resistant strains of P. pseudomonas aeruginosa isolated from clinics [61]. Various other materials and bioactive agents have been tested after incorporating them into liposomes.

4.7.7 Nervous System Disorders The biggest challenge faced while treating any nervous system and associated disorders is the accessibility to that system guarded by the blood-brain barrier (BBB). Therefore, either to get through or surpass the barriers, liposomes have been explored the most, especially due to their high

68 Nanomaterials in Healthcare biocompatibility and good permeation capability. PEGylated immunoliposomes also have been reported in this study that cross the BBB by using anti-α-synuclein LB509 and anti-transferrin receptor OX26 antibodies for treating Parkinson’s disease [62] while dual functionalized liposomes using transferrin and cell-penetrating peptide for permeating the brain by crossing the BBB can also be useful for studying gene delivery or drug delivery to the brain [63]. Additionally, transferrin and penetration protein-coated liposomes encapsulating plasmid DNA to transfect neuronal cells via endocytosis and to develop efficient strategies to treat neurodegenerative diseases are also under investigation [64]. SiRNA-liposome (cationic/anionic) based on various formulations for analyzing the successful delivery of siRNA to the CNS by crossing BBB have been reported [65]. Bi-functionalized liposomes that target BBB for entry into the brain and then promote the disaggregation of amyloid plaques for treating Alzheimer’s disease have been promising in several other studies.

4.8 CLINICAL APPLICATIONS OF LIPOSOMES CONCERNING NANOTECHNOLOGY The recent advancements in the field of drug delivery have led to a significant understanding of the interaction of drugs with specific tissue or cell type, thus adding more knowledge on targeted drug delivery. This fundamental understanding of the interaction of drugs with tissues imparts great impor­ tance to chemotherapeutic agents, as these chemotherapeutic drugs or gene therapies possess toxicity and non-specificity. Liposome-based drug-delivery methods have been well employed in clinical applications since 1995. Out of various approved liposome-based drugs, DoxilTM, AmBisome®, and DepoDur are a few known examples [66]. The fundamental advantage of using liposomes in drug delivery is an enhance­ ment in the pharmacokinetics and pharmacodynamics of the drug. Hence, liposomes are used in a variety of drug formulations intended for human use and further ongoing research is continued. Doxil™, the first liposomal doxorubicin-based formulation, was used in the treatment of ovarian carcinoma and AIDSrelated Kaposi’s sarcoma in the United States in 1995 [67]. Furthermore, in 1996, liposomes have also been developed by NeXstar Pharmaceuticals for deli­ vering daunorubicin (DaunoXome®) in the management of advanced HIV-associated Kaposi’s sarcoma. In the same line, other formulations in the treatment of cancer have also been introduced, including Mepact®, DepoCyt®, Marqibo®, and Myocet®. Liposome-based products have also been developed as anti-fungal agents, like Amphotec® and AmBisome®. Interestingly liposomes have also been used as a vaccine carrier, e.g., Epaxal® for hepatitis and Inflexal V® for influenza. Furthermore, research and development in the field of liposome-based drug delivery has some new formulations that are under clinical trial. The following products are under clinical trial, including Grab-2 liposomes for tumour cell suppression [68]; LEM-ETU liposomes for leukaemia, breast, stomach, liver, and ovarian cancers [69]; INX-0125 for Hodgkin’s and non-Hodgkin’s lymphoma; and INX-0076 protect the drug from degradation in in-vivo system. OSI-211 liposomes consisting of lurtotecan in the treatment of ovarian cancer, along with next-generation OSI-211 and NX 211 formulations are used for the treatment of ovarian and neck cancer [70]. Cisplatin-based liposomes are used in a few cancer applications, like Lipoplatine, a cytotoxic formulation used in pancreatic cancer, and also Nanoplatin is used in lung cancer [71]. The following table gives us an overview of a few lipid-based vaccines that are FDA-approved (Table 4.1).

4 • Liposomes and Lipid Structures

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TABLE 4.1 Selected liposomal formulations and lipid-based vaccines approved for human use for the treatment of various diseases that are approved by the FDA are listed COMMERCIAL NAME ®

COMPANY

ACTIVE COMPOUND

Ambisome

Astellas Pharma

Amphotericin B

Amphotec®/ Amphocil® Abelcet® Epaxal®

Ben Venue Laboratories Sigma-Tau Pharmaceuticals Crucell

Inflexal®

Crucell

Arikayce®

LIPID COMPOSITION

INDICATION Fungal infections

Amphotericin B

HSPC:DSPG: Cholesterol Cholesteryl sulfate

Amphotericin B

DMPC:DMPG DOPC:DOPE

DOPC:DOPE

Influenza

Insmed, Inc.

Formalininactivated Hepatitis A virus Inactivated hemagglutinin of Influenza virus Amikacin

Invasive severe Fungal infections Hepatitis A

DPPC:Cholesterol

Transave, Inc.

Amikacin

DPPC:Cholesterol

RTS, S/AS01

GlaxoSmithKline

ALIS

Insmed, Inc.

Recombinant fusion MPL:DOPC: of P. falciparum Cholesterol circumsporozoite protein and Hepatitis B surface antigen Amikacin DPPC:Cholesterol

Mycobacterium avium complex (MAC) lung disease Pseudomonas aeruginosa infections (cystic fibrosis) Malaria

Vaxisome

NasVax

JVRS-100

Juvaris BioTherapeutics Aronex Pharmaceuticals Statens Serum Institut

Arikace

TM

Nyotran CAF01

Vaxfectin

Vical

MPER-656 Liposome Vaccine

National Institute of Allergy and Infectious Diseases (NIAID)

Inactivated Influenza virus Inactivated Influenza virus Nystatin Subunit protein antigen Ag85BESAT, DDA, TDB Plasmid DNAencoded influenza proteins Immunogenicity of an HIV-1 gp41 MPER-656

Fungal infections

CCS

Nontuberculous Mycobacterial lung infection Influenza

CLDC:Cholesterol

Influenza

DMPC:DMPG: Cholesterol DODAB:TDB

Fungal infections Tuberculosis

VC1052:DpyPE

Influenza

DOPC:DOPG

HIV infections

(Continued)

70 Nanomaterials in Healthcare TABLE 4.1 (Continued) Selected liposomal formulations and lipid-based vaccines approved for human use for the treatment of various diseases that are approved by the FDA are listed COMMERCIAL NAME Duanoxome®

COMPANY

ACTIVE COMPOUND Daunorubicin

LIPID COMPOSITION

Depocyt®

NeXstar Pharmaceuticals SkyPharma Inc.

DSPC and Cholesterol (2:1) DOPC, DPPG, Cholesterol and Triolein Verteporphin:DMPC and EPG (1:8) EPC: Cholesterol (55:45)

Cytarabine/Ara-C

Visudyne®

Novartis

Verteporfin

Myocet®

Elan Pharmaceuticals

Doxorubicin

Marqibo®

Vincristine

SM:Cholesterol (60:40)

Onivyde®

Talon Therapeutics Inc. Merrimack Pharmaceuticals Inc.

Irinotecan

DSPC:MPEG-2000:DSPE (3:2:0.015 molar ratio)

Depodur®

SkyPharma Inc.

Morphine sulfate

DOPC, DPPG, cholesterol, and Triolein

INDICATION AIDS’s-related Kaposi’s sarcoma Neoplastic meningitis

Choroidal neovascularisation Combination therapy with cyclophosphamide in metastatic breast cancer Acute lymphoblastic leukaemia Combination therapy with fluorouracil and leucovorin in metastatic adenocarcinoma of the pancreas Pain management

4.9 FUTURE PROSPECTIVE With various types of systems explored, liposomes still hold promising solutions. Liposomes customized with biological signatures can also impact the drug delivery system and lead to more effective treatment. A novel drug delivery system (NDDS) is a combination of interdisciplinary approaches useful for treating chronic and fatal diseases. Trigger-dependent liposomes, multiple active agent-loaded lipo­ somes, combinational theranostic-based liposomes, and immunoliposomes are hot areas for research around the globe. Although, heavy production costs, large-scale manufacturing, stability, sterilization, and multiprocess systems are still a few hurdles that need to be overcome to get more lipid nanoparticles in the commercial domain.

4.10 CONCLUSION Liposomes are applicable in several therapeutic applications due to their biocompatibility and reduced toxicity have been used in the treatment of various diseases from cancer to fungal to pain management. Liposomes are highly advantageous as they can be used for controlled drug release for highly toxic drugs

4 • Liposomes and Lipid Structures

71

and can be synthesized in varying sizes and shapes that can be modulated based on different medical uses such as temperature-sensitive, anionic/cationic liposomes, and liposomal-based vaccines. Preclinical and clinical trials have shown greater efficacy and improved target specificity and thus, liposome-based formulations are available commercially with extensive research. Expanding capabilities of lipid nanoparticles like these are driving researchers to invent and explore more novel approaches to drug delivery, and disease diagnosis showing the deep establishment of their position in the medicinal systems.

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formulation enhances the in vitro wound healing efficacy of substance P neuropeptide. Pharmaceutics. 2017 Dec 6;9(4): 56. doi: 10.3390/pharmaceutics9040056. PMID: 29211047; PMCID: PMC5750662. Sağıroğlu AA, Çelik B, Güler EM, Koçyiğit A, Özer Ö. Evaluation of wound healing potential of new composite liposomal films containing coenzyme Q10 and d-panthenyl triacetate as combinational treatment. Pharm Dev Technol. 2021;26(4):444–454. Umar AK, Sriwidodo S, Maksum IP, Wathoni N. Film-forming spray of water-soluble chitosan containing liposome-coated human epidermal growth factor for wound healing. Molecules. 2021 Sep 1;26(17). Alipour M, Halwani M, Omri A, Suntres ZE. Antimicrobial effectiveness of liposomal polymyxin B against resistant Gram-negative bacterial strains. Int J Pharm. 2008 May 1;355(1–2):293–298. Solleti VS, Alhariri M, Halwani M, Omri A. Antimicrobial properties of liposomal azithromycin for Pseudomonas infections in cystic fibrosis patients. Journal of Antimicrobial Chemotherapy. 2015 Mar 1;70(3):784–796. Yang D, Pornpattananangkul D, Nakatsuji T, Chan M, Carson D, Huang CM, et al. The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes. Biomaterials. 2009 Oct;30(30):6035–6040. Low WL, Martin C, Hill DJ, Kenward MA. Antimicrobial efficacy of liposome-encapsulated silver ions and tea tree oil against pseudomonas aeruginosa, staphylococcus aureus and candida albicans. Lett Appl Microbiol. 2013;57(1):33–39. Ge Y, Tang J, Fu H, Fu Y, Wu Y. Characteristics, controlled-release and antimicrobial properties of tea tree oil liposomes-incorporated chitosan-based electrospun nanofiber mats. Fibers and Polymers. 2019 Apr 1;20(4):698–708. Halwani M, Yebio B, Suntres ZE, Alipour M, Azghani AO, Omri A. Co-encapsulation of gallium with gentamicin in liposomes enhances antimicrobial activity of gentamicin against Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy. 2008;62(6):1291–1297. Loureiro JA, Gomes B, Coelho MA, do Carmo Pereira M, Rocha S. Immunoliposomes doubly targeted to transferrin receptor and to α-synuclein. Future Sci OA. 2015 Sep 10;1(4). FSO71. doi: 10.4155/fso.15.71. PMID: 28031922; PMCID: PMC5137902. dos Santos Rodrigues B, Lakkadwala S, Kanekiyo T, Singh J. Dual-modified liposome for targeted and enhanced gene delivery into mice brain. Journal of Pharmacology and Experimental Therapeutics. 2020 Sep 1;374(3):354–365. dos Santos Rodrigues B, Banerjee A, Kanekiyo T, Singh J. Functionalized liposomal nanoparticles for efficient gene delivery system to neuronal cell transfection. Int J Pharm. 2019 Jul 20;566:717–730. Bender HR, Kane S, Zabel MD. Delivery of therapeutic siRNA to the CNS using cationic and anionic liposomes. Journal of Visualized Experiments. 2016 Jul 23;2016(113):54106. doi: 10.3791/54106. PMID: 27501362; PMCID: PMC5091666. Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: An updated review. Pharmaceutics [Internet]. 2017 Jun 1 [cited 2022 Jun 29];9(2). Available from: https://pubmed. ncbi.nlm.nih.gov/28346375/ Barenholz Y. Doxil®– The first FDA-approved nano-drug: Lessons learned. J Control Release [Internet]. 2012 Jun 10 [cited 2022 Jun 29];160(2):117–134. Available from: https://pubmed.ncbi.nlm.nih.gov/ 22484195/ Ashizawa AT, Cortes J. Liposomal delivery of nucleic acid-based anticancer therapeutics: BP-100-1.01. Expert Opin Drug Deliv [Internet]. 2015 Jul 1 [cited 2022 Jun 29];12(7):1107–1120. Available from: https://pubmed.ncbi.nlm.nih.gov/25539721/ Wicki A, Witzigmann D, Balasubramanian V, Huwyler J. Nanomedicine in cancer therapy: Challenges, opportunities, and clinical applications. J Control Release [Internet]. 2015 Feb 28 [cited 2022 Jun 29];200:138–157. Available from: https://pubmed.ncbi.nlm.nih.gov/25545217/ Tomkinson B, Bendele R, Giles FJ, Brown E, Gray A, Hart K, et al. OSI-211, a novel liposomal topo­ isomerase I inhibitor, is active in SCID mouse models of human AML and ALL. Leuk Res. 2003 Nov 1;27(11):1039–1050. Boulikas T. Clinical overview on Lipoplatin: aA successful liposomal formulation of cisplatin. Expert Opin Investig Drugs [Internet]. 2009 Aug [cited 2022 Jun 29];18(8):1197–1218. Available from: https://pubmed. ncbi.nlm.nih.gov/19604121/

Functionalized CarbonBased Nanoparticles for Biomedical Application

5

Monalisha Debnath1, Swati Patil2, and Sujit Kumar Debnath1 1

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 2 Department of Pharmacology, Lokmanya Tilak Municipal General Hospital and Lokmanya Tilak Municipal Medical College, Mumbai, Maharashtra, India

Contents 5.1 5.2

Introduction Types and Properties of Carbon-Based Nanomaterials 5.2.1 Fullerenes 5.2.1.1 Structural dimension 5.2.1.2 Physical property 5.2.1.3 Mechanical property 5.2.1.4 Electrical property 5.2.1.5 Thermal property 5.2.1.6 Optical property 5.2.1.7 Chemical property 5.2.2 Carbon Nanotubes 5.2.2.1 Structural dimension 5.2.2.2 Physical property 5.2.2.3 Mechanical property 5.2.2.4 Electrical property 5.2.2.5 Thermal property 5.2.2.6 Optical property 5.2.2.7 Chemical property 5.2.3 Graphene and Its Derivatives 5.2.3.1 Structural dimension 5.2.3.2 Physical property 5.2.3.3 Mechanical property 5.2.3.4 Electrical property

DOI: 10.1201/9781003322368-5

76 77 77 77 78 79 80 80 80 80 80 81 81 81 81 82 82 82 82 82 82 82 83 75

76 Nanomaterials in Healthcare 5.2.3.5 Thermal property 5.2.3.6 Optical property 5.2.3.7 Chemical property 5.2.4 Nanodiamond 5.2.4.1 Structural dimension 5.2.4.2 Physical property 5.2.4.3 Mechanical property 5.2.4.4 Electrical property 5.2.4.5 Thermal property 5.2.4.6 Optical property 5.2.4.7 Chemical property 5.2.5 Carbon Dots 5.2.5.1 Structural dimension 5.2.5.2 Physical property 5.2.5.3 Mechanical property 5.2.5.4 Electrical property 5.2.5.5 Thermal property 5.2.5.6 Optical property 5.2.5.7 Chemical property 5.3 Synthesis of Functionalized Carbon-Based Nanoparticles 5.3.1 Synthesis 5.3.2 Exohedral Functionalization 5.3.2.1 Covalent functionalization 5.3.2.2 Non-covalent functionalization 5.3.3 Endohedral Functionalization 5.4 Risk-Assessment of Functionalized Carbon-Based Nanoparticles 5.5 Application of Functionalized CBNs 5.5.1 Biosensors 5.5.2 Drug Delivery 5.5.3 Therapy 5.5.3.1 Role in tissue engineering and regenerative medicine 5.5.3.2 Role as free radical scavengers 5.5.3.3 Role as an antimicrobial 5.5.3.4 Role in cancer therapy 5.6 Future Prospects and Challenges References

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5.1 INTRODUCTION Carbon can exist in different amorphous and crystalline forms due to the unique configuration of electrons (1s2, 2s2, 2p2). These materials are incredibly flexible and present in other allotropes. The persistence of allotrope depends on the atomic bonding and hybridization of carbon (sp3, sp2, and sp1), resulting in different physical and chemical properties. In sp3 hybridization, it forms “hard,” e.g., dia­ mond, whereas it forms “soft,” in sp2 hybridization, e.g., graphite. The usage of carbon is increasing as it can create a linkage with almost all materials. The expansion of carbon allotropes was observed in the 20th century after the consecutive discovery of different low-dimensional carbon forms [1]. Carbonbased nanomaterials (CBNs) are a growing interest nanomaterial in the biomedical field. The subsequent

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discovery of fullerenes, carbon nanotubes, graphene, etc., with unique properties cannot be completed by non-carbon-based nanomaterials. However, these CBNs possess few limitations, including large-scale manufacturing feasibility. CBNs can be of different types: graphene, carbon nanotubes, mesoporous carbon, fullerenes, carbon dots, nanodiamonds, etc. [2]. CBNs availability is not dependent on nature reserves due to negligible quantities. Most CBNs are synthesized artificially in bulk due to unlimited access to raw materials. These CBNs have some toxic side effects that render their further application in drug delivery and the medical field. Functionalization or modification of specific moieties of CBNs help to overcome these problems. Surface modification with amide and amine groups helps to increase positive charge density that binds with the negatively charged bacterial envelope using electrostatic interactions. Additionally, surface modification with metal oxide nanoparticles augments antibacterial activities by releasing metal ions and enhancing the reactive oxygen species (ROS) generation in the bacterial milieu [3]. Carbon nanotubes’ inner and outer sides can be functionalized with antibacterial agents that serve sustained release to improve the antibacterial effects. Drug-loaded CBNs work differently against gram-positive and gram-negative bacteria depending on the type of functionalization. These surfacemodified CBNs are also effective against antibiotic-resistant bacteria.

5.2 TYPES AND PROPERTIES OF CARBON-BASED NANOMATERIALS Carbon-based nanomaterials are emerging promising nanocarriers and striking nanoparticles in bio­ medical engineering. Carbon is the tetravalent naturally occurring element capable of forming several allotropes (crystalline and amorphous forms) [4]. These are fullerenes, carbon nanotubes (CNT), gra­ phene, graphite, diamond, amorphous carbon, and lonsdaleite (Figure 5.1). Each has different physical, chemical, thermal, electrical, optical, and chemical properties (Table 5.1). Depending on versatile properties, diversified structures, and abundancy, these CBNs are explored for several biomedical applications.

5.2.1 Fullerenes The molecules containing interconnected carbon atoms arranged in hexagonal and pentagonal ring structures are termed fullerenes. It was named fullerenes by architect Buckminster Fuller, who fabricated the cage in the 1960s [6]. They can be a sphere or ellipsoidal or tube in shape. Based on their structure, they can be classified as buckyball clusters (spherical form), nanotubes, megatubes, polymers, linked ball and chain dimers, and nanoonions [7].

5.2.1.1 Structural dimension Fullerenes are zero-dimensional structures, closely packed carbon cages where each carbon atom is attached to three carbon atoms through covalent bonds. These carbon atoms are sp2 hybridized in nature. Typically, fullerenes contain various numbers of hexagons and 12 pentagons [8]. Sixty closely packed carbon atoms usually form the structure of first fullerenes, i.e., Buckminsterfullerene (C60). It is composed of 20 hexagons and 12 pentagons. The most stable fullerenes follow the isolated-pentagon rule (IPR): no two pentagons share an edge, and each pentagon is surrounded by a hexagon [9]. This pentagonal adja­ cency causes an increase in strain energy and a higher local curvature of the molecular surface. All fullerenes follow this IPR rule except C20 [10]. The pyramidalization angle determines the spherical

78 Nanomaterials in Healthcare

FIGURE 5.1 Classification of CBNs based on their size [ 5].

geometry of carbon atoms. Depending on the number of the carbon atom, spherical fullerenes have a pyramidalization angle of θσπ-90 [11]. That indicates an angle of 101.6° lies between the π and σ orbitals of the carbon atom. The pyramidalization angle of C60 fullerenes is observed as 11.6°. Buckyballs contain hollow structures hence capable of trapping other particles within. They are trigonally spheroids having a C-C bond [12]. Linked ball and chain dimers are the forms of buckyballs. These are composed of two buckyballs connected by the carbon chain. Nanotubes are hollow structures with single or multiple walls. Compared to nanotubes, megatubes demonstrate larger tube diameters with various wall thicknesses. Polymers are chain-like structures formed under high temperatures and pressure. Nanoonions are spherical particles of 3–5 nm diameter and contain several layers around the buckyball core.

5.2.1.2 Physical property Pure fullerenes are insoluble in water. C60 fullerenes show a density of 1.65 g/cc with a molar mass of 720.66 g.mol−1 [6]. However, fullerenes functionalized with -OH and -COOH can dissolve in water and

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79

Comparison between carbon-based nano-materials

TYPES

HYBRIDIZATION

Fullerenes

sp2

Carbon nanotubes

sp2

Graphene and its derivatives

sp2

Nanodiamonds

sp3

Carbon dots

Sp2

STRUCTURE 0D, Cluster

PROPERTIES

Water-insoluble, capable of accepting and donating electrons, stable can resist high pressure and temperature, electrical and thermal conductor, photoluminescence, and corrosion resistance. 1D, Single, Stable, chemically inert, double, or elasticity, semiconductor and multiple walls metallic, thermal insulator, electroluminescence, and photoluminescence. 2D, Hexagonal, The high surface area, larger crystal length to diameter ratio, mechanical stiffness, high stability, chemical inertness, easier surface grafting with receptors, more substantial thermal and electrical conductivity, photoluminescence, and low intrinsic toxicity. 3D, cubic, Nanosize, larger surface area, crystal high adsorption capacity, high Young’s modulus, fluorescence, biocompatible, and chemical inert core. 0D, QuasiChemically inert, highly waterspherical soluble, facile modification, nanoparticles biocompatible, photoluminescence, and high resistance to photobleaching.

EXISTENCE REFERENCE Synthetic

[ 6– 8]

Synthetic

[ 22]

Natural or synthetic

[ 32]

Natural or synthetic

[ 38]

Synthetic

[ 44]

show a tendency of aggregations. The fullerenes are functionalized with more than one moiety to prevent these agglomerations. However, the separation of individual fullerene isomers is difficult from their regioisomers. An alternative way to solubilize fullerene is the incorporation of them into polymers. Several polymers have been synthesized to solubilize fullerenes like the pearl necklace and charm bracelet [13]. In pearl necklaces, fullerenes are attached with polymer using a short bridging group. The charm bracelet structure hangs the fullerene molecules from the polymer backbone. Among all fuller­ enes, C20 is the smallest and contains high surface areas. C60 fullerenes sublimate at ~600 °C.

5.2.1.3 Mechanical property Fullerenes as nanofillers reinforce the mechanical properties of several polymer composites. Dispersing fullerenes in the epoxy system enhances the epoxy system’s Young’s modulus and tensile strength [14]. Different mechanical strength was observed when fullerenes and oxidized fullerenes were incorporated separately into the epoxy system. The oxidized fullerenes incorporation showed higher toughness and

80 Nanomaterials in Healthcare strain energy release rate. However, these oxidized fullerenes decreased in toughness and percentage elongation of the epoxy system compared to fullerenes incorporated in an epoxy system.

5.2.1.4 Electrical property C60 has shown exceptional electron acceptor properties and is considered an organic electronic building block [12]. In electrochemical reduction, the triple degenerated lowest unoccupied molecular orbital (LUMO) of C60 accepts six electrons. At the same time, the doubly degenerated LUMO orbitals of C70 can carry up to four electrons. Fullerenes have relatively low electrical conductivity, i.e., 10−5 S/cm [15]. Polymer materials embedded with fullerenes has high resistivity and suitable dielectric property. C20 contains a high source of π electrons. As a result, it shows an extensive direct band gap of 2.89 eV [16]. This feature indicates the potential of C20 to fabricate high voltage and high-power devices.

5.2.1.5 Thermal property A C60 molecule has 30 C=C bonds capable of capturing more than 34 free radicals [17]. C60 is a radical sponge due to its super high radical capture capacity. Fullerenes can increase the thermal property of polymers by trapping the free radicals. It is necessary to choose the appropriate polymers-additives combination to get the increased thermal stability of polymers. The fragmentation temperature of C20-C90 fullerenes is approximately constant for more giant fullerenes (> C58). However, the frag­ mentation temperature is sensitive to the cluster size for smaller fullerenes [18]. Hence, giant fullerenes are found to be more stable than smaller fullerenes. A thermal stability study of some quasi-fullerenes shows a time frame of t = 1 ps, at T = 800–1000 K, without breaking the interatomic bonds [19]. C60 offers the lowest thermal stability among all the fullerenes.

5.2.1.6 Optical property Spherical fullerenes can produce photoluminescence (PL) in the visible-near infrared region (NIR). The energy transformation from the lowest energy singlet-electronic state to the ground state results in photoluminescence. As a suitable electrophile, the fullerenes can produce both excited and ground state complexes with several aromatic solvents [11]. This phenomenon is known as the solvatochromism phenomenon of fullerenes. The polarity of the solvents and the intermolecular interaction between chemical structures are the two major determinants of the optical property of fullerenes. The temperature, the solvent, and the environment strongly influence the PL phenomena. PL of fullerenes is weak at low temperatures.

5.2.1.7 Chemical property Fullerenes (also known as electron-deficient polyalkenes) show aggregation’s tendency based on their chemical structure [20]. The disruption of the π-system and the higher degree of functionalization can reduce the antioxidant activity of fullerene derivatives. Fullerenes undergo various chemical reactions such as nucleophilic, halogenation, oxidation, reduction, radical, hydrogenation, regioselective, transi­ tion metal complex, etc. [6].

5.2.2 Carbon Nanotubes Carbon nanotubes are the most approaching among the carbon-based nanomaterials due to their exceptional optical, electrical, mechanical, and thermodynamic properties. CNTs are also known as buckytubes. CNTs are one-dimensional hollow structures having a high aspect ratio. However, these materials are stable and difficult to degrade. They are degraded by combustion or with strong oxidants

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[21]. These are reported as derivatives of both fullerenes and carbon fibers. This unique physicochemical nature pertains to the concern for the biosafety of CNTs.

5.2.2.1 Structural dimension Single-walled carbon nanotubes (SWCNTs) have an exceptional honeycomb structure comprised of the sp2 carbon where the hexagonal arrangements are designated by a pair of chiral indices [22]. SWCNTs are made of a monolayer of graphene sheets having diameter ranges between 0.4 and 2 nm. While the multiwalled carbon nanotubes (MWCNTs) are composed of multilayer graphene sheets having diameters ranging from 1–3 nm [23]. Each carbon atom is sp2 hybridized and arranged in the hexagon. In a 2D structure of CNTs, three σ orbitals are configured at three 120° angles. Mainly graphene is the single layer of the carbon atom. This graphene layer must be rolled up to produce either SWCNTs or MWCNTs. Based on the rolling direction, CNTs are further categorized as armchair, chiral, and zigzag [24]. The hexagons are lined up parallel to the nanotube axis in the armchair configuration. The hexagons are oriented in a circle around the nanotube in a zigzag design. While in a chiral structure, the hexagons do not form any line arrangement and are twisted to the nanotubes. Sometimes CNTs appear in the form of nanofibers and nanohorns [25]. These nanohorns are the SWCNTs with a structure similar to nanotube caps. The structural dimensions of nanofibers are reported as the width varies from 10–200 nm with a few microns in length.

5.2.2.2 Physical property Some physical properties of CNTs vary depending upon their synthesis mode [26]. SWCNTs synthe­ sized by laser ablation produce CNTs with an average of 1.3 nm and 1.5 µm diameter and length, respectively. CNTs are synthesized by arc discharge, resulting in SWCNTs with 0.6–1.4 nm in diameter and MWCNTs with 10 nm in diameter. On the other side, chemical vapor deposition results in CNTs of variable length, diameter, and chirality. The interlayer distance in zig-zag, chiral, and armchair con­ figurations was found as 3.40 Å, 3.38 Å, and 3.37 Å, respectively [27]. However, the lattice parameter for zig-zag and chiral was reported as 16.53 nm, while it was 16.55 nm for the armchair. Density also varied for each configuration of zig-zag (1.33 g/cm3), armchair (1.32 g/cm3), and chiral (1.41 g/cm3).

5.2.2.3 Mechanical property Carbon nanotubes are an excellent composite material in the mechanical industry and aerospace due to their poor density and high strength. The mechanical property is improved due to their Young’s modulus and high intrinsic tensile strength. The Young’s modulus range for CNTs was reported as 1.0–1.27 TPa with a tensile strength of about 100 GPa [27]. Based on their outstanding elasticity, they can twist, bend, and eventually buckle without damaging configuration under high pressure and the compressive force imposed on their axial direction.

5.2.2.4 Electrical property In the field of material science, CNTs are well demonstrated as the enhancer of the electrical conductivity of materials. The electrical property of CNTs is varied depending upon the orientation of the hexagons along the tube axis in their structure. SWCNTs are further categorized as metallic SWCNTs (m-SWCNTs) and semiconducting SWCNTs (s-SWCNTs) based on their electrical property [26]. The armchair CNTs act as conducting metal, whereas the zig-zag configuration of CNTs behaves like semiconductors. Free energy in the form of a light or electrical field is imposed on the free electron of the carbon atoms of the chiral and zig-zag CNTs resulting in the conduction of an electrical current. The current density and conductance for CNTs are observed as 1,015 A/m2 and 13.0 (K.ohms)−1, respectively [27].

82 Nanomaterials in Healthcare

5.2.2.5 Thermal property The thermal conductivity of CNTs varies significantly based on their synthesis mode, forms, and structure. Individual SWCNTs showed a thermal conductivity value was 6,600 W/mK, indicating that they are thermal insulators. The thermal conductivity of MWCNTs was less than 0.1 W/mK [28]. The length of the CNTs also impacts their thermal conductivity. Usually, thermal conductivity enhances with the length up to the mean free path of a phonon (around 500 nm for MWCNTs and even longer for SWCNTs). The thermal conductivity of MWCNTs varies from 650–3,000 W/mK at room temperature [29]. The thermal conductivity of MWCNTs bundles and sheets decreases to two orders due to their mutual interaction. CNTs are found to withstand temperatures up to 750°C in normal conditions and 2800°C under atmospheric vacuum pressure [25].

5.2.2.6 Optical property CNTs have photoluminescence and electroluminescence properties that change with chiral angle and tube diameter. The tuning of emission wavelength (between 850 nm and 2 μm) is another desirable feature of SWCNT [30]. The optical property of CNTs’ network depends on its thickness and density [31]. Apart from these, the arrangements of CNTs affect the optical property of the CNT network.

5.2.2.7 Chemical property CNTs are chemically inert and hydrophobic. During the covalent attachment of functional groups, they tend to agglomerate onto the sidewalls of CNTs with fully bonded sp2 hybridization [27]. However, the non-covalent functionalization is the facile one that may eventually affect their physical properties. They are dispersed in some solvents upon sonication, whereas they immediately precipitate if any interruption occurs. SWCNTs form suspension in solvents like tetrahydrofuran (THF), N‫׳‬, N-Dimethyl formamide (DMF), and toluene [25].

5.2.3 Graphene and Its Derivatives Graphene is the two-dimensional (2D) planar structure where a single layer of carbon atoms is connected in a honeycomb lattice configuration. Graphene derivatization has broadened its application and enhanced its functionalization ability. Some common graphene derivatives are graphene oxide (GO), reduced graphene oxide (rGO), etc. Profuse oxygen-containing functional moieties observe in the honeycomb lattice of GO.

5.2.3.1 Structural dimension The sp2 hybridized carbon atoms in graphene are interconnected by neighboring three carbon atoms through σ bonds. These planar σ bonds show a length of about 1.42 Å. Conversely, π bonds are formed with un-hybridized supplementary Pz-orbitals upright to the planar structure [32]. The specific theoretical area reported for graphene is 2,630 m2g−1 [33].

5.2.3.2 Physical property Vital properties of graphene are vast surface area (2,630 m2.g−1), high intrinsic mobility, outstanding heat, and electricity conductor make it ideal for biomedical applications [34].

5.2.3.3 Mechanical property Graphene shows extremely high stiffness with superior tensile strength of 130 GPa [34]. The Young’s modulus observed for single-layer graphene is 2.4 ± 0.4 TPa, and for double-layer graphene is 2 ± 0.5 TPa.

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5.2.3.4 Electrical property Graphene’s electronic mobility and electrical conductivity were reported as 2.105 cm2 V−1 s−1 and up to 2.104 S cm−1, respectively [33]. Based upon the adsorption of the donor (like CO, NH3) and acceptor compound (like H2O or NO2), the electrical conductivity of graphene varies [34]. The adsorption of donor compounds on the surface of graphene reduces its electrical conductivity. The adsorption of acceptor compounds on its surface increases its electrical conductivity.

5.2.3.5 Thermal property Graphene is a highly thermally conductive material. The thermal conductivity of graphene was found as 5,000 W m−1 K−1 [33]. Graphene can convert the electrical current into heat and exhibits thermoelectric power (TEP) ~50–100 μV K−1 [35].

5.2.3.6 Optical property Graphene and its derivatives have outstanding optical properties like broadband light absorption in the visible and near-infrared range, polarization-dependent solid effect, and the capability to quench fluo­ rescence [36]. Electronic energy transitions of GO result in its fluorescence. Sometimes overlapping many fluorescence peaks indicates the presence of various oxygen-containing groups (like hydroxyl, epoxy, carboxyl, and aromatic rings) in their structure. GO shows pH-dependent fluorescence property due to the excited state proton transfer. It shows a broad fluorescence peak at ~680 nm in acidic pH. However, this peak gradually decreases or disappears with the increasing pH.

5.2.3.7 Chemical property Graphene has high chemical stability and the capacity to absorb biomolecules through π-π stacking [36]. These features make graphene and its derivatives suitable for fabricating optical/ electrochemical/elec­ tromechanical/pH sensors. GO is converted into rGO by reducing its oxygen content using chemical, photochemical, thermal, and microwave methods [33]. The significant differences between rGO and GO are lower oxygen content in reduced form, an abundance of carboxyl groups, and many other surface defects. The hydrophilicity of both forms depends on the presence of functional oxygen groups such as hydroxide, carboxyl, epoxide, etc.

5.2.4 Nanodiamond Nanodiamonds (NDs) are another type of carbon nanostructures that demonstrate excellent biocompatibility.

5.2.4.1 Structural dimension NDs have three considerable fractions of facet on their surfaces such as carbene like {100}, {110} and hexagonal {111) [37]. The carbene-like surface dimerizes into rows between the radical orbitals of the dimer with a considerable amount of π bonding. The facet {110} contains the zigzag chains of carbon atoms and unpaired antibonding electrons. With a smaller orbital overlap, these unpaired antibonding electrons are hybridized away from each other. These unpaired antibonding electrons are well separated on a hexagonal surface without interacting with adjacent radicals. The carbon atoms are sp3 hybridized to form cubic diamond lattices in nanodiamonds. This hybridization generates four strong covalent bonds oriented along the tetrahedron axis. NDs can be as small as 3–5 nm if synthesized by the detonation method [38].

84 Nanomaterials in Healthcare

5.2.4.2 Physical property The nanodiamonds have a size range of 5–100 nm, depending upon their mode of synthesis [39]. NDs are found poorly dispersed in aqueous solutions and highly agglomerates, limiting their biomedical applications. The NDs shows the characteristics of both nanomaterials and diamond [40]. The NDs are proven a good reinforcement material for many nanocomposites for their excellent chemical stability, low friction coefficient, wear resistance, etc.

5.2.4.3 Mechanical property As a nanofiller, incorporating nanodiamonds on different composites improves the mechanical strength in terms of flexural modulus, flexural stress, and hardness.

5.2.4.4 Electrical property The sp2 carbon atom in the structural configuration of NDs is accountable for its electrical conduc­ tivity [41]. Additionally, surface modification and edge defects also can tune their electrical conductivity.

5.2.4.5 Thermal property NDs show high thermal conductivity up to 2,300 W m−1 K−1. Stable hydrosol of NDs can increase the heat transport efficiency of the solution [42].

5.2.4.6 Optical property Nanodiamonds are broadly transmitted due to their optical band gap as high as 5.5 eV [39]. Depending on multiphoton processes, nanodiamonds’ transmission spectrum showed moderate absorption in the 2.6–6.2 μm range and a flat featureless window for wavelengths longer than ~225 nm. NDs generate red photoluminescence under excitation by external light sources. NDs show temperature-dependent fluorescence [38]. The fluorescence intensity decreased with increasing temperature due to nonradiative tapping in NDs occurring by thermal activation. They act as color centers due to the abundance of the large variety of lattice defects in the structure of NDs [43]. The most common defect is nitrogen vacancy (NV). These NDs possess two optical activated states depending on the surface charges on the substituting nitrogen atom near the vacancy defect. These are the neutral charge state NV0 center with a zero-phonon line (ZPL) emission at 575 nm and negative charge NV- center with ZPL emission at 638 nm.

5.2.4.7 Chemical property A variety of functional groups are present in NDs. These are hydroxyl, carboxyl, sulfur, ester, anhydride, and amino groups. These NDs are thermodynamically unstable due to high surface energy.

5.2.5 Carbon Dots Carbon dots (CDs) are the newly developed highly luminescent carbon-based nanomaterials growing tremendous attention for researchers in various fields. Their unique photophysical property, facile synthesis, biocompatibility, and low cost make them suitable for several applications.

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5.2.5.1 Structural dimension Transmission electron microscopy (TEM) reveals that CDs synthesized by ultra-sonication are well crystalline and have the lattice spacing of 0.21 nm with {100} graphene plane. The sp2 hybridized carbon core is generally conjugated with oxygen-containing groups.

5.2.5.2 Physical property CDs are zero-dimensional carbon-based nanomaterials having a size less than 10 nm. Usually, CDs are spherical. Based on their crystal lattice, they are further categorized as carbon quantum dots (CQDs) and carbon nanoparticles. CDs are often classified as CQDs, graphene quantum dots (GQDs), and carbonized polymer dots (CPDs) based on their synthesis techniques, changing the degree of carbonization and graphene layer, nano/microstructures, and properties [44]. CQDs are multiple-layer graphitic structures with oxygen-containing functional groups. At the same time, GQDs exhibit one/multiple layer graphitic structures with functional groups on their edges/surfaces or within the interlayer defects. CPDs are carbonized polymer hybrid “core-shell” nanostructures. Their carbon core is less than 20 nm containing intensely hydrated cross-linking polymers, and the shell comprises either polymer chains or functional groups. CPDs and CQDs are spherical, while GQDs are anisotropic with lateral dimensions larger than their height. The physical state of CDs is characterized by X-ray diffraction (XRD), such as if the broad hump is centered in and around 2θ = 26° indicates the amorphous nature of CDs. One of the attractive physical properties of CD is their easy dissolution in water owing to their hydrophilicity.

5.2.5.3 Mechanical property CDs improve the nanocomposite’s durability, strength, and flexibility in several polymer-CD composites.

5.2.5.4 Electrical property CDs are semiconductor materials. They act as both electron donors and electron acceptors. This feature aids favorable charge transfers during electro-catalytic reactions like oxygen reduction reaction (ORR), oxygen evolution reaction (OER), hydrogen evolution reaction (HER), and carbon dioxide reduction reaction (CO2RR).

5.2.5.5 Thermal property CDs exhibit excellent thermal stability. Due to their temperature-dependent multi-emission property, CDs have been used as temperature-sensing fluorophores. They are also considered suitable nanomaterial for nanothermometers. CDs exhibit thermal sensitivity of 1.48% °C−1 and thermal linearity in a wide temperature range (5–85°C) [45].

5.2.5.6 Optical property CDs are well known for their colorful and bright fluorescence emission due to the electronic transition of their core carbon atoms. It shows a broad absorption spectrum from UV to the visible range that can be extended up to near-infrared (NIR). In the NIR, CDs are identified as the most refined two-photon absorbers [46]. The quantitative optical absorptivity of CDs at 400–450 nm is 100 MC atoms, the per molar concentration of carbon atoms in the core of the nanoparticles. The fluorescence property of CDs can be fine-tuned by changing the excitation wavelength and pH of the dispersing solution. pH changes result in protonation/deprotonation of the functional moieties on the surface of CDs. Depending on their UV-visible spectra, the basic structure of CDs can be elucidated [47]. Such as the sp2 hybridization

86 Nanomaterials in Healthcare of n-π* transition at 342 nm indicates the presence of functional groups like C-N, C-S, and C=O. However, at 273 nm, sp2 hybridization of π-π* transition represents C=C functional group. Additionally, absorption shoulder at 298 nm and 354 nm attributed to n-π* transitions of the nitrogen atom and sulfur atom, respectively, that are doped on the surface of CDs.

5.2.5.7 Chemical property Some outstanding chemical properties of CQDs are good dispersibility, high crystallization, super­ conductivity, rapid electron transfer with enhanced electrical conductivity, photoluminescence, photo­ bleaching resistance, and catalytic activity.

5.3 SYNTHESIS OF FUNCTIONALIZED CARBON-BASED NANOPARTICLES Although carbon-based nanoparticles have gained remarkable attention in the scientific community, most of its popular forms like carbon dots, graphene, and fullerenes are less soluble in common solvents. In addition, these particles have a strong propensity to aggregate, which is the major hurdle in their practical applications, principally when we aim their administration in biological systems. Resolve this issue by exploiting tailored approaches of organic chemistry, which have proven successful in several medical applications.

5.3.1 Synthesis The mode of synthesis and the selection of precursors are the crucial factors that can influence several physicochemical properties of carbon-based nanomaterials like size, morphology, crystallinity, degree of carbonization, and photoluminescence property. Additively, in the case of CDs, the choice of molecular precursors influences their environmental behaviour and degradation process. There are mainly two synthesis strategies for carbon-based nano-materials top-down and bottom-up approaches (Figure 5.2). In top-down approaches, the larger or complex carbon structures are broken down into nano­ particles. Arc discharge, laser ablation, and chemical/ electrochemical oxidation are the most frequently

FIGURE 5.2 Strategies to synthesis of CBNs and functionalizing.

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used methods by which the large starting carbon materials like graphitic layers, graphene oxide, carbon nanotubes, and activated carbons are degraded into carbon nanoparticles [48]. Among these, the chemical oxidation is superior in producing the high emissive and hydrophilic carbon nanoparticles by functionalization with oxygen-containing groups like hydroxyl, carboxyl, etc. The top-down approaches to synthesizing graphene are chemical exfoliation, mechanical exfoliation, and GO reduction methods [32]. In bottom-up approaches, the smaller carbon structures are converted into the desired size carbon nanoparticles. Hydrothermal and solvothermal reactions, microwave-assisted pyrolysis, and ultra­ sonication are the most common bottom-up approaches used in synthesizing CBNs [49]. Among these, hydrothermal and microwave pyrolysis are the most used methods to produce carbon nanoparticles. Three main bottom-up approaches have been established for graphene synthesis are the chemical vapor deposition (CVD) method, plasma-enhanced chemical vapor deposition (PECVD) method, and epitaxial growth on silicon carbide (SiC) [32]. CNTs are mainly synthesized by laser ablation, arc discharge (using high temperature >3000°C), and CVD methods [23]. The nanohorn form of SWCNTs is synthesized by treating fullerene soot under high temperatures [25]. Some standard methods for synthesizing nanodiamonds are CVD, laser ablation, detonation, high pressure, and high temperature (HPHT), and ball milling [38]. In the detonation tech­ nique, graphite precursors transform into nanodiamonds through phase transition under high pressure and temperature generated by the explosion. In the high pressure and temperature method, about 5–6 G Pa and 1300–1500°C were applied to graphite precursors to produce the nanodiamond. The most common top-down approach to synthesizing graphene is exfoliation (such as mechanical, liquid-phase), and the bottom-up approach is CVD [33,34]. Other top-down approaches to synthesizing graphene are unzipping CNTs, arc discharge, and oxidative exfoliation reduction. Some different bottom-up approaches in graphene synthesis are organic synthesis, epitaxial growth, template route, and substrate-free gas-phase synthesis (SFGP). Additionally, methods demonstrated for the synthesis of GO are Hofmann (HOGO), Hummers (HUGO), Brodie (BRGO), and Staudanmaier (STGO). A strongly acidic environment influences the degree of oxidation in the presence of a potent oxidizing agent such as potassium perchlorate or potassium permanganate in all these methods. Pristine graphene is another derivative of graphene usually synthesized by the reduction of GO. Additionally, the liquid phase method enables a high content yield of pristine graphene bio-hybrids. Chemical/electrochemical oxidation, ultrasonic treatment, and laser ablation commonly use top-down approaches to synthesizing CDs. Recently, top-down approaches are becoming less popular due to drawbacks like harsh reactions involving strong acids, difficulty controlling the response, long processing time, expensive materials, large nanoparticle distribution, and low quantum yields (QY). Although pyrolysis and hydrothermal reactions are predominantly used nowadays to produce carbon nanomaterials, several studies demonstrated that it results in a high diversity of carbon nanoparticles. Centrifugation and filtration are widely used to separate and purify the carbon nanoparticles from their heterogeneous mixture. Common bottom-up approaches for synthesizing CDs are microwave-assisted pyrolysis, carbonization, hydrothermal/solvothermal technique, ultrasonic treatments, and thermal decomposition. Compared to pyrolysis, the hydrothermal method is strident. The predominancy of yielding the graphitic core structure is more in pyrolysis. At the same time, hydrothermal processes produce amorphous CDs with incomplete carbonization. However, the pure carbon-based nanomaterials synthesized using the above-mentioned approaches persist with shortcomings, such as poor solubility, agglomerate formation, non-biodegradability, toxicity, etc. [50]. Specifically, to address this issue, chemical surface functionalization was demonstrated as a crucial step for improving the therapeutic effectiveness of carbon-based nanomaterials. Additionally, the functionalization showed improved tumor accumulation and longer blood circulation time for the carbon nanoparticles and their selectivity towards target tissue and their cellular uptake. Passivation and surface functionalization improved CDs’ stability and their PL intensity. Generally, two methods are employed in the chemical functionalization of carbon-based nanomaterials. These are exohedral and endohedral functionalization. Another promising approach to modifying the surface of carbon quantum dots is the sol-gel technique.

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5.3.2 Exohedral Functionalization Two techniques are involved in exohedral functionalization, such as covalent and non-covalent func­ tionalization. In CNTs, exohedral functionalization refers to functionalization in both the side walls and terminals.

5.3.2.1 Covalent functionalization Covalent functionalization is also known as chemical functionalization. The desired functional groups are attached by covalent bonding to the surface of nanomaterials in covalent functionalization. Such bonding results in sp2 to sp3 switching of carbon nanoparticles by distortion of conjugated π electron framework [50]. Covalent techniques involve dehydronation, ozonolysis, oxidation, and plasma treat­ ments [32]. The surface modification of carbon nanomaterials with covalent functionalization usually overcomes their stability-related issues in aqueous suspension. Additionally, covalent functionalization includes radical addition, cycloaddition, electrophilic addition, nucleophilic addition reaction and hal­ ogenation [25]. In covalent functionalization, several heteroatoms, such as boron, nitrogen, phosphorus, etc., were chemically attached to the backbone of sp2 hybridized carbon atoms. A common technique used in the chemical functionalization of CNTs is treatment with strong acids to attach groups like hydroxyl, carbonyl, and carboxylic acids to the end of the tubes and the site of the defect [51]. Such treatments with strong acids on graphene and its derivatives shorten the length of CNTs by removing their end caps. Further, chemical reactions to functionalize with groups like thiols and amides also can be done on those oxide groups containing CNTs. In this context, CNTs having a car­ boxyl group allow further molecules’ covalent couplings through ester and amide bonds. Photochemical and electrochemical modification and thermally activated chemistry are some methods of covalent functionalization of SWCNTs. Amidation, esterification, and silanization are the common organic reactions employed for the covalent functionalization of graphene and its derivatives like GO and rGO [33]. In amidation, the nucleophilic addition reaction involves the incorporation of an amino group (R2-NH2) in the structure of GO by activating the carboxylic acid group (R-COOH) present on the surface of GO using coupling agents like carbodiimide derivatives (DDC or EDC). In silanization, organosilane is mainly used to promote the covalent binding between GO and silicon. Covalent functionalization of carboxylated NDs with lysine amino acids enables NDs to maintain their stability and enhances their interactions with the biological system. Reactions involved in the covalent modification of CDs are esterification, sulfony­ lation, silylation, copolymerization, and amide coupling. Often, urea, polyethyleneimine (PEI), poly­ ethylene glycol (PEG), ethylenediamine, etc., are used as a precursor for producing biocompatible, rich amino acids group-containing CDs with higher quantum yields.

5.3.2.2 Non-covalent functionalization Non-covalent functionalization is also known as physical functionalization. This non-invasive mode of functionalization depends on the hydrophobic or π-π interaction and van der Waals force. Such inter­ actions involving intermolecular forces define the nano-materials physical state, boiling point, melting point, and viscosity [33]. This functionalization is problematic to control, even tricky in their charac­ terization as weekly bound molecules result in desorption. These may lead to partial functionalization, further allowing the carbon nanomaterials to aggregate again. However, compared to the covalent method, it is an easy process to perform and doesn’t make any changes to the chemical structure of the nanomaterials. Polymer wrapping mechanism, micellar force, and π-π stacking interactions are the non-covalent approaches employed for the physisorption of functional moieties on the surface of CNTs [52]. Nanocomposite/exfoliated graphene (EG) composite was prepared based on electrostatic interaction where negatively charged nanoparticles (MoS4− and Cl−) were reported assembled at the positively

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charged edges of exfoliated graphene [53]. Hydrogen bonding and π-π or n-π interactions are the sig­ nificant intermolecular interactions reported for the non-covalent modification of graphene. Electrostatic interactions, chelation, or complexation are some methods for the non-covalent modification of CDs.

5.3.3 Endohedral Functionalization In CNTs, endohedral functionalization refers to nanotubes filling at the defect site with atoms or molecules. Hence, endohedral functionalization exploits the inner cavity of CNTs. In such cases, the non-covalent endohedral encapsulation of molecules/atoms depends on the tube diameter. The first endohedral fullerenes were synthesized by laser vaporization of graphite [54]. The endohedral fullerenes are further categorized into two types according to the IPR rule: IPR fullerenes and non-IPR fullerenes. The non-IPR fullerenes are stabilized by the endohedral inclusion of metal or metal clusters. The metals usually entrapped are Hf, Ce, Y, La, Ba, etc. [55] Non-metal doped endohedral fullerenes are synthesized by exposing C60 at a pressure of around 3 bar in the presence of helium and neon gas.

5.4 RISK-ASSESSMENT OF FUNCTIONALIZED CARBON-BASED NANOPARTICLES CBNs have a wide array of biomedical applications but also have lots of safety concerns. The CBNs’ safety is determined by their biocompatibility issues which arise due to their in vivo degradation and distribution in various organs [56]. In-vivo degradation of CBNs depends on their type and physico­ chemical properties; most of them are eliminated by the renal route without any noticeable degradation [57]. This toxicity arises mainly from trace elements incorporated or doped during their synthesis. The biocompatibility is governed by the surface chemistry, which regulates their dispersibility and is responsible for the aggregation and accumulation of CBNs in the body [56]. These issues can be tackled to some extent by increasing purity, solubility, and polarity by chemical transformation. Different analytical techniques have been developed to capture and understand the in-vivo behaviors of CBNs, such as absorption, distribution, metabolism, and excretion (ADME). Primarily, in-vivo techniques like neutron activation study and isotopic tracing to counting isotope-labeled-CBNs with considerable sen­ sitivity are used to execute this analysis [58]. Several cytotoxicity studies on macrophages revealed lethal actions of carbon nanomaterials, like mitochondrial dysfunction, lysosomal damage, DNA damage, ROS generation, and ultimate cell death through necrosis or programmed cell death. Most CBNs comprise heterogeneous mixtures of various carbon forms and catalytic metal residues. This composition determines the biocompatibility, making it imperative to characterize them in detail during their toxicity assessment for data’s reproducibility, reliability, and comparability [59]. Studies on in-vitro and in-vivo models showed that CBNs could produce direct and indirect genotoxicity, cause oxidative damage and inflammation, and trigger diverse cell signaling pathways resulting in diverse cellular responses. While taking the molecule ahead in Phase I/II clinical trials, it is essential to ascertain new substances’ carcinogenic or mutagenic potential as part of preclinical safety testing. The DNA damage potential of CBNs impacts fertility and may initiate and promote carcinogenesis or subsidize ecogenotoxicity. Thus, a genotoxicological assessment is crucial. Fullerene possesses antioxidant activity and is part of many skin care products. They have the potential to display a range of actions resulting in cell death or dysfunction, which would be evident in chronic use only [60]. Bone mesenchymal stem cells (BMSCs) in rats showed acid oxidation with MWCNTs. RawMWCNTs showed significant cytotoxicity on BMSCs, whereas PEGylated (PEG)-MWCNTs and hydroxyapatite (HA)-MWCNTs had a minimal toxic effect on BMSCs and also demonstrated better

90 Nanomaterials in Healthcare biocompatibility. The cytotoxicity can be attributed the mechanisms like mitochondrial and DNA damage, increased oxidative stress, and damage to cellular membranes [61]. Functionalization of carbon-based nanomaterial is an attempt to reduce the toxicity implications. In this contrast, an in-vivo study was conducted in mice, where functionalized CNTs were found as less toxic to mice lungs. The cytotoxicity of CNTs can be neutralized by covalent and non-covalent func­ tionalization. Functionalization increases the compatibility when pristine CNTs are compared to their functionalized counterparts. The catalyst used throughout the synthesis of the nanotubes, such as iron or platinum, may cause cytotoxicity, which needs to be differentiated from the toxic potential of CBNs. Effectively functionalized CNTs are biocompatible and exhibit favorable pharmacokinetic parameters like prompt elimination through urine or biliary pathway when given intravenously and significantly minimal to negligible uptake of CNTs in reticuloendothelial systems, including the liver, lung, and spleen [62]. The cytotoxicity of varied functionalized CBNs like hydroxyl, carboxylic acid, and epoxide dem­ onstrated high metabolic activity and had no enhanced cytotoxic effects on macrophages except oxidized CBNs, which still had a cytotoxic impact on membrane integrity and DNA synthesis. These toxicity profiles were observed with a short exposure of high particle concentrated CBNs. The same effects must be studied for long exposure and low particle concentration. The interaction between particles and biological entities such as proteins or cells must also be assessed [63].

5.5 APPLICATION OF FUNCTIONALIZED CBNs CBNs have unique physicochemical characteristics, making them suitable for various applications from diagnosis to drug delivery and therapeutics (Figure 5.3). A brief overview of these biomedical appli­ cations is shown in Table 5.2.

5.5.1 Biosensors Biosensors provide user-friendly, economical, reliable, and a rapid sensing platform over the conven­ tional diagnostic techniques, which require skilled personnel for diagnosis using techniques like spec­ troscopy or chromatography. Nanomaterials fit the bill as they are miniature, have an extensive surface

FIGURE 5.3 The risk-benefit profile of functionalized carbon-based nanomaterials (CBNs).

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Summary of biomedical applications of functionalized carbon-based nanomaterials (CBNs)

APPLICATION AREA Biosensor Electrochemical sensor Optical sensor Electronic sensor

Drug delivery

Tissue engineering and regenerative medicine Free radical scavengers Antimicrobial activity Cancer therapy

MEDICAL USE • Communicable disease detection • Detection of hepatitis B virus DNA • Detection of HIV-1 Protease • Detection of mycobacterium tuberculosis • Non-communicable disease detection • Estimation of glycosylated haemoglobin in diabetes • Analysis of biomarkers in cancer care • Oncology • Target specific delivery of oncological drugs like doxorubicin, methotrexate, and paclitaxel • Carriers for gene therapy • Vectors for vaccine antigens As a scaffold for bone and tissue engineering for non-union fractures A possible role in decreasing reactive oxygen species-induced damage in neurodegenerative and inflammatory disorders Antibacterial action against various bacteria, so disinfecting property • A combined approach to provide chemotherapy and photothermal therapy • PH sensitive delivery of drugs explicitly targeting the tumor microenvironment • Theranostic-combining diagnosis and therapeutic approach

area, and can be used with minimally invasive procedures for diagnostic purposes. Functionalized immobilization can be done for biomolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), microRNA, peptide nucleic acid (PNA), aptamers, enzymes, viruses, antigens, and antibodies. A carbon allotrope-based nanomaterials in the above situation can act as transducers by offering appropriate interfaces for deciphering bio-recognition inputs to vastly sensitive and assessable outputs [64]. A biosensor based on the carbon-based material could be an electrochemical biosensor determining the electrical response of an analyte arising from the electrochemical reaction between the analyte and the surface of the functional electrode. Another approach would be exploring the optical arena using optical biosensors like graphene-derived products, which effectively quench the fluorescence by using CNTs and CDs having optical specifications such as fluorescence chemiluminescence, electrochemiluminescence, phosphorescence, up-conversion photoluminescence, and photo-stimulated electron transfer activity. The recent ones like the field-effect transistor with CNT or graphene-based FET bioanalytical sensors have been of great interest as they are susceptible to electric disturbances and have high carrier mobility with ease of surface functionalization [65]. Hepatitis B-DNA detection is an essential step for recognizing active viral replication and the efficacy of antiviral therapy. But current HBV-DNA detection has drawbacks like low sensitivity and reliability and is time-consuming. Based on the robust interplay between single-stranded DNA and graphene material, a simplistic but intelligent electrochemical instrument using GQD modified glassy carbon electrode connected to a specific sequence of DNA molecules as probes can detect HBV-DNA with high sensitivity and specificity [66]. Similarly, to shorten and simplify the dengue virus detection, biomarkers like NS1 antigen can be sensed using a CNT-based immune sensor [67]. A graphene oxide (GO)-based fluorescence biosensor can be used to sense HIV-1 protease, an essential component of the human immunodeficiency virus (HIV) life cycle. This biosensor has HIV-1 protease that fluorescentlabeled HIV-1 protease covalently linked to GO. This sensor could detect HIV 1 protease in human

92 Nanomaterials in Healthcare serum at as low as 1.18 ng/mL with reasonable rapidity, sensitivity, and accuracy. In the presence of HIV-1 protease, the substrate peptide will be cleaved into short fragments to produce fluorescence which GO effectively quenches in the absence of HIV-1 protease [68]. Not only virus but Mycobacterium tuberculosis detection for the H37Rv strain can also be done rapidly and precisely with the help of a biosensor constructed by a single-strand deoxyribonucleic acid (ssDNA) aptamer. The SWCNTs are bonded to the DNA aptamers by π-π stacking. H37Rv replaces the SWCNTs due to a stronger affinity with aptamers than SWCNTs. This replacement causes a significant change in the electrical properties that are detected. This nano complex provides a linear signal in the concentration range of 1 × 103 to 1 × 107 CFU mL−1 with a LOD of 100 CFU mL−1. It thus helps in rapid, sensitive, and early detection of H37Rv in clinical diagnosis [69]. For non-communicable diseases, a label-free chemiresistor-type FET affinity sensor for hemoglobin A1c (HbA1c) estimation for people with diabetes using SWCNT as a transducing element and a bacterial periplasmic protein (SocA) as a receptor was also studied [70]. Carbon dots are employed to detect cancer-specific biomarkers for early diagnosis and monitor tumor cell proliferation. Gold doped CD–cytosensor are used to recognize metallic ions, such as Fe3+, in cancer cells [71]. Also, nitrogen and sulfur co-doped CD (NS-CD), as well as undoped CD, can be used for the detection of carcinoembryonic antigen (CEA), a biomarker for ovarian carcinoma [72]. Chemiresistive paper-based MWCNT biosensor detects a prostate-specific antigen, a biomarker for prostatic cancer, and quantifies the biomarker down to 1.18 ng mL−1 [73].

5.5.2 Drug Delivery Functionalized carbon-based nanomaterial promotes efficient intracellular delivery of biomolecules like nucleic acids and proteins in a specific and nonspecific manner. Such nucleic acids like small interfering RNA (siRNA), and ribonuclease (RNase), show potential in the treatment of various diseases [20]. Recently, CNTs have appeared as a promising candidate for delivering pharmacological and genetic compounds into the cells owing to their vast surface area and outstanding ability to permeate the cell wall. Drug delivery of anti-cancer drugs using CNTs offers a promising aspect by lowering their overall toxicity and enhancing selective local accumulation at the desired site. Critical efforts are made toward conjugating anti-cancer drugs like doxorubicin, flutamide, cisplatin, methotrexate, paclitaxel, etc., with CNTs to increase their wider application in cancer treatment [74]. Folic acid-modified pH-responsive delivery of MWCNTs can be used to target doxorubicin to tumor sites actively. The MWCNTs not only reduced the side effects of doxorubicin but also suppressed the progression of tumor growth. Through extended endo-lysosome delivery, this nano-system prevents the efflux of doxorubicin from the cancer cells and releases doxorubicin in the acidic hydrolase tumor microenvironment resulting in a reduction in the effective dose of doxorubicin needed for chemotherapy [75]. Herbal anti-cancer drugs like curcumin have been formulated as-fabricated polysaccharides with SWCNTs to serve as carriers for delivery of curcumin in human lung adenocarcinoma (A549) cells. The chitosan and alginate functionalized curcumin-loaded SWCNTs were designed to achieve the pH-sensitive release of the drug at the target site [76]. CNTs are used to transfer genetic materials like DNA, where they boost the transfection by releasing DNA before the cellular defense mechanism destroys it. CNTs combined with gene sequence slicing effectively treated asthma and bronchitis caused by a respiratory syncytial virus (RSV). To hinder the further growth of the virus, the RNA fragments capable of inhibiting target proteins are encapsulated in CNTs and applied in the form of nasal drops or spray. Similarly, various vaccine antigens can be immobilized to the surface of CNTs. Immobilization of streptavidin on CNTs was carried out with 1-pyrene saturated fatty acid and succinimidyl ester. This CNT-based delivery of vaccine antigens will avoid the conventional approach of exploiting dead microorganisms as antigen sources and ensure better antigen processing and a more robust immunogenic response [77].

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5.5.3 Therapy 5.5.3.1 Role in tissue engineering and regenerative medicine Tissue engineering is an expanding field for repairing damaged tissue that conventional repair measures cannot fix. Tissue engineering approaches like attempts at bone replacement by using scaffolds can offer a promise for treating skeletal defects that fail to heal spontaneously or by conventional repair strategies. Nanotechnology and tissue engineering are being explored to create such a biocompatible scaffold. Among the diversified cells of the physique, bone cells are the critical ones that urge a well-designed scaffold to regenerate bony tissues. Non-union resulting from complex fractures and significant bone defects are challenging to treat with existing treatments and will thus need these approaches. Regenerative medicine relies on cellular growth factors and scaffolds for success. All carbon nanoma­ terials like fullerenes, CNTs, GO, nanodiamonds, and CDs are bioactive and highly capable for bone tissue engineering, owing to their excellent mechanical properties. Additionally, these are non-toxic toward osteoblasts and possess an intrinsic antibacterial activity that doesn’t need further antibiotic treatment [78].

5.5.3.2 Role as free radical scavengers The pathogenesis of neurodegenerative diseases and various inflammatory disorders suggests that oxi­ dative damage is caused by dysfunctional reactive oxygen species (ROS) and free radicals. Considering structural peculiarities of CBNs like fullerenes, they can adsorb unpaired electrons from various free radicals and scavenge them. Also, fullerenes can eliminate free radicals generated during cancer pho­ todynamic therapy [20].

5.5.3.3 Role as an antimicrobial The antimicrobial action of CBNs is exerted by oxidative damage mediated by bacterial membrane disruption. CNTs show antimicrobial activity against E. coli, where SWCNTs were much more toxic to bacteria than MWCNTs because of the larger surface area, whereas fullerenes owe their antibacterial action to the cationic nature, which shows strong interaction with negatively charged bacteria like E. coli and Shewanella oneidensis. Other mechanisms for antimicrobial activity of CBNs are impairing the respiratory chain, inhibiting energy metabolism, physical interaction with the plasma membrane, forming of cell-CNTs/cell-GO aggregates, and inducing cell membrane disruption. The use of CBNs for dis­ infection also can be considered a promising approach [79].

5.5.3.4 Role in cancer therapy Anti-cancer therapy has multiple challenges like access to the dynamic tumor microenvironments (TME), comprising complex structures and numerous components, including immune cells, circuitous blood vessels, and fibrous collagen structures. Because of unique physicochemical features, the diverse range of CBNs can be modified to fulfill the exact needs of TME to potentiate anti-cancer therapy [80]. CNTs have exceptional optical properties, thermal properties, electronic conductivity, easy functiona­ lization ability, and higher drug loading ability, making them a promising candidate for cancer treatment and diagnosis, i.e., theragnostic. CNTs can be used for intracellular targeted delivery of chemotherapy agents, imaging, and tumor cell killing by the encroachment of the tumor microenvironment. The intracellular drug targeting can deliver chemotherapy to the nucleus, cytoplasm, or mitochondria [81]. Doxorubicin, a commonly used anti-cancer drug for breast cancer, limits its use due to significant toxicity. A nano complex combining chemotherapy and NIR-irradiated photothermal therapy (PTT) using doxorubicin (DOX) with PEGylated SWCNTs has shown effective in killing breast cancer cells compared to the treatment alone with DOX or PTT [82]. Oxidized-MWCNTs encapsulated with cisplatin

94 Nanomaterials in Healthcare in their inner structure, and DOX loaded on the external surface were fabricated for anti-tumor activity. PEG and folic acid were also used to prevent the release of cisplatin from the inner aspect of the MWCNT. This TME-responsive MWCNT-based nano-system showed a pH-sensitive release of drugs at pH below 6.5, which depicts the acidic TME seen in cancer cells. The tumor cell killing in breast cancer cells was more efficient at pH 6.5 than at pH 7.4 [83]. A fullerene C60 and docetaxel (C60/DXT) nanosystem also achieved higher bioavailability and enhanced cytotoxicity in human breast cancer cell lines [84]. Thus, the potential of these functionalized CBNs in cancer is being explored enormously. Still, all these applications must stand the test of short-term and long-term toxicity.

5.6 FUTURE PROSPECTS AND CHALLENGES Carbon-based nanomaterials draw significant research interest in bioimaging, drug delivery, biosensing, catalysis, and electronic devices. The exciting features like optical and electrical properties, chemical stability, structural dimension, and surface tunability of CBNs represent these materials as the most competitive candidate in the biomedical field. Due to these reasons, CBNs usage is increasing gradually to solve problems faced in therapeutic and diagnostic applications. The promising application of CBNs has been observed in diagnosis, light-mediated therapy, phosphorescence, luminescence, and fluores­ cence imaging. The additional surface modification of CBNs with metallic substances, polymers, pep­ tides, or functional groups serves high loading efficiency, the sustained-controlled release of drugs, and high-resolution imaging contrast. Stimuli-responsive materials are also used to functionalize CBNs to facilitate temperature, enzyme, pH, and light-responsive release of therapeutic molecules. This surface functionalization approach also helps target-specific delivery to stem cells, inflam­ matory cells, and cancer cells. The long residency of therapeutic molecules is possible with polyethylene glycol conjugation by reducing nonspecific cellular uptake and organ toxicity. The target specificity can be extended to intracellular components like nuclei and mitochondria. Numerous disease eradication and tissue repairing happen through the activities of cellular organelles. Therefore, targeting these areas using CBNs can be an emerging area of research. CBNs are also explored in different types of disease treatment. CBNs help in light-induced therapies (photothermal and photodynamic) in cancer treatment without drugs. This strategy can nullify side effects and tissue toxicity associated with chemotherapeutics. Nitrogen doping, surface chemistry, size, and shape of CBNs influence the generation of ROS (e.g., PDT) and represent opto-thermal properties (e.g., PTT). Combining PTT and PDT in chemotherapy synergizes therapeutic efficacy with controlled drug delivery. Additionally, this combination system can also be helpful in theranostic purposes like imaging cancer areas and chemotherapy. The theranostic ability of CBNs can be improved with different functional nanomaterials like polymer, gadolinium, iron oxide, and cobalt NPs, improving drug loading, therapeutic efficacy, and multifunctional theranostic using bi-/tri-model imaging. Another potential application of CBNs is their outstanding biocompatibility and biodegradability. Few in-vivo studies reported variable toxicity profiles of CBNs depending on surface chemistry, size, and shape [56]. Investigating the organ/tissue toxicity profile and interaction of CBNs with cells/tissue is essential for long-term use. Therefore, controlling particle size/shape and suitable surface functionali­ zation can improve the stability and body clearance of CBNs. The fabrication of multifunctional syn­ thetic CBNs can be an intelligent approach for advancing the theranostic field. In this contrast, costeffectiveness is a key concern where sensitivity and accuracy should not be compromised [85]. Cellular uptake and organ accumulation of these materials should be analyzed carefully. The mechanism of CBNs’ clearance is not well established. That’s why future studies should be focused on these unrevealed issues, specifically long-term exposure. Nano-bio sensors have expanded significantly due to the demand for analytical techniques in health care sectors and industries. However, the complexity of the synthesis method of CBNs pulls away from the competitive environment of different sectors.

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Engineered Magnetic Nanoparticles Challenges and Prospects

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Roshan Keshari and Bhingaradiya Nutan Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 6.1 6.2

Introduction Synthesis of Magnetic Nanoparticles (MNPs) 6.2.1 Physical Method 6.2.2 Chemical Method 6.2.3 Biological Synthesis Method 6.3 Properties and Application 6.3.1 Characteristics of Magnetic Particles 6.3.1.1 Particle size 6.3.1.2 Particle density 6.3.1.3 Particle shape 6.3.1.4 Magnetic property 6.3.2 Application of Magnetic Nanoparticles 6.3.2.1 Hyperthermia 6.3.2.2 Photothermal therapy 6.3.2.3 Drug delivery 6.3.2.4 Infection treatments 6.3.2.5 Magnetic resonance imaging 6.4 Conclusion References

102 104 104 105 106 106 107 107 107 107 107 108 108 109 109 110 110 110 111

DOI: 10.1201/9781003322368-6

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102 Nanomaterials in Healthcare

6.1 INTRODUCTION In all medical fields, innovative diagnostic and therapeutic concepts are quickly advancing, and nanotechnology plays an important role in this development. Due to distinct size and physical and chemical characteristics, using nanoparticle (NP) materials have several implications. A class of nanoparticles known as magnetic nanoparticles (MNPs) are designed of particulate magnetic material such as iron, nickel, and cobalt, and a chemical component that has functionality and are less than 100 nm in size and may be controlled by an external magnetic field. Numerous studies have been conducted on the synthesis of various MNPs types due to their extensive utilization in the fields of biotechnology, healthcare, material science, engineering, and environmental conservation [1]. Because of the advantages they offer, magnetic nanoparticles have found various promising applications in biomedicine. First, they can be tuned to be at dimensions from a few nano meters to tens of nanometers that are similar to biological entity of interest such as cell, a virus, a protein, or a gene. Secondly, they can be “tagged” with biological molecules offering easy interaction or binding to a biological entity. In addition, these are manipulated by an external magnetic field gradient which leads to numerous applications involving the transportation and immobilisation of magnetic nanoparticles or of magnetically tagged biological entities into human tissue. A time-varying magnetic field can be used to have the magnetic nanoparticles respond resonantly [2]. Due to their several functions, including their small size, superparamagnetism, low toxicity, and other characteristics, MNPs have drawn the attention of researchers from a variety of disciplines, including biology, medicine, and physics [3,4]. Additionally, magnetic nanoparticles are now being used in MRI, gene therapy, cancer therapy, microwave devices, targeted medication delivery, tissue engineering, cell tracking, and bio-separation [5–10]. Various NPs, like superparamagnetic NP are guided to the target tissue by an external magnetic field to deliver medications and improve stability against destruction by enzymes [11] (Figure 6.1). By how electrons orbit and spin defines the magnetic properties of a substance. These bulk materials can be classified as ferromagnetism and ferrimagnetism on the basis of these interactions and their behaviour in response to magnetic fields at different temperatures. MNPs usually demonstrate superparamagnetic behaviour due change in the polarisation caused by thermal energy to their limited volume. There are numerous different compositions and states used to create magnetic nanoparticles. MNPs are

Magnetic Nanoparticle

Physico-chemical properties · Size and shape · Composition · Surface charge · Hydrophobicity

Synthesis routes · Top-down · Bottom-up · Biogenic · Microfluids

Functional components · Ligands · Imaging modalities · Therapeutics

Coating · Polymers · Co-polymers · Silica · Gold

FIGURE 6.1 Flow chart showing general consideration taken into account for magnetic nanoparticles.

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generally ferrites or oxides and be may be structurally complete or hollow nature depending on the type of material and mode of application. These MNPs contain different shapes and sizes. Hollow structure can be multi-chambered, microporous, or have multi-shell structure and rattle-type structure and have a potential application in MRI, drug delivery, catalysis [12]. Maghemite or magnetite MNPs’ inert surfaces prevent the formation of potent covalent interactions with molecules that function as functionalizers. However, the surface of the magnetic nanoparticles can be coated using silica, to which different functional groups and fluorescent dyes can be linked, to increase their reactivity. Some MNPs may be made up if metallic nanoparticles are due to their higher magnetic moment [13]. Some metallic MNPs can also be coated with a material forming a shell. For instance, non-reactive materials like graphene or gold can surround a core of iron and cobalt to be employed in biochemistry, radiography, and other medical treatments [14]. Nanoparticles can be prepared by using a variety of techniques. These are classified into two main categories termed the top-down method and the bottom-up method. The grinding procedure is one of the straightforward ways to create magnetic nanoparticle dispersion. The method’s biggest flaw is the contamination and irregularity of nanomaterials. For use in healthcare, MNP are often created by arc discharge, co precipitation, thermal decomposition, laser ablation, microemulsion, and chemical vapor deposition other processes [15,16]. Spherical and narrowly scattered MNPs are produced by these techniques. Iron, cobalt, and nickel have greater saturation magnetization are hazardous therefore iron oxide in the forms of magnetite (Fe3O4) or maghemite (Fe2O3) is typically employed in MNPs for therapeutic diagnostics [17]. Green nanotechnology has attracted a lot of attention in the synthesis of metal NPs using inactivated plant tissue, plant extracts, exudates, and other parts of living plants in order to decrease or eliminate toxic substances to restore the environment. Alternatives to chemical and physical techniques of making nanoparticles that are more environmentally friendly include biological methods that make use of bacteria, enzymes, fungi, plants, or plant extracts. By removing the laborious labour required to maintain microbial cultures, the creation of nanoparticles utilizing plants or portions of plants can occasionally be advantageous over other biological procedures [18]. Co-precipitation is the most popular and effective approach for creating MNPs with regulated sizes and magnetic characteristics. This technique involves adding a base into aqueous salt solutions in an inert atmosphere at low or high temperatures to create MNPs. Partially oxidizing ferrous hydroxide suspensions with various oxidizing agents, or by aging stoichiometric mixtures of ferrous and ferric hydroxides in aqueous environment led to spherical MNPs in a solution [19]. Microemulsion can also be used to produce MNPs. Comparatively speaking, this method has a number of benefits, including the ability to synthesize a wide range of materials with high levels of control over particle size and composition, the ability to prepare nanoparticles (NPs) with crystalline structures and high specific surface areas, and the use of simple conditions of synthesis with ambient temperature and pressure [20]. For instance, the size of ferrous dodecyl sulfate (Fe(DS)2)) is used for synthesizing magnetic nanoparticles regulated by the concentration and temperature of the surfactant. Industrial and biomedical sectors make up the majority of application for magnetic nanoparticles. For industrial application, synthetic pigments in ceramics, paints, and porcelain are made of magnetic iron oxides nanomaterials. High surface area, large surface-to-volume ratio and easy separation under external magnetic fields offer advantages for MNPs to be used industries. Hematite and magnetite act as an industrial catalysts for preparation of ammonia, the oxidation of alcohols and other processes [21]. New catalytic systems that are immobilized on magnetic nanocarriers have achieved significant advancements. One such example is enzyme and protein immobilization, modification, and engineering which had a result in improving the activity, stability, and substrate specificity. Biomolecules that have been magnetically immobilized are employed in bioassays or as affinity ligands and alter target molecules or cells. Skin penetration for immobilized enzymes on MNPs exhibit low allergy and low penetration. The enhanced stability enzymes provide advantage for both storing and operational conditions under high temperature [22].

104 Nanomaterials in Healthcare Use of magnetic nanoparticle in biomedicine can be categorized based on whether they are used within or outside of the body (in vivo, in vitro). In vivo applications include the use of superparamagnetic iron oxide, magnetite cationic liposomal nanoparticles and dextran-coated magnetite as therapeutics, where it increases the temperature of tumor tissues and kills the cancerous cells by hyperthermia. The MNPs’ ability to target certain tumour tissues is made possible by their customization with cancer-specific binding agents [23]. In a study by Y. Krupskaya, the use of carbon nanotubes (CNTs) containing iron (Fe-CNT) for treatment of hyperthermic cancer was discussed. MNPs along with external magnetic field had made possible for drug delivery and their fixation at the targeted site for releasing drug with advantage of lowering drug usage. NMR (nuclear magnetic resonance) imaging and MRI (magnetic resonance imaging) of diseased and normal tissues using MNPs have made diagnosis easier. In-vitro assays include the use of MNPs in immunoassays particularly known as magnetorelaxometry where the magnetic viscosity of the system [24]. A difficult undertaking is to isolate and purify a variety of biomolecules, including proteins, DNA, antibodies, nucleic acids, and antigens, in a highly pure form. Therefore, use of MNPs with targeted molecules and their bio-separation by applying magnetic field is one excessively used in life sciences. Super-paramagnetic Fe3O4 nanoparticles can be directly used or coated with biocompatible molecules such as polymers, organic and inorganic materials to isolated DNA and proteins. Additionally, coating of MNPS with cell specific antibodies are used to separate red blood cells, bacteria, lung cancer cells and breast cancer cells. Environmental application of Iron nanoparticles with modifications, such as catalysation and support are used in remediation of pollutants in air, soil, and groundwater. One of the finest descriptions of a nuclear accident in Fukushima, Japan, in 2011 is the simple and quick cleanup of radioactive caesium through MNPs complexed with Prussian blue [25]. Although MNPs offer several advantages, there are still some challenges that are needed to be overcome. One of the main challenges is the passive approach for synthesis of MNPs where the reactants are improperly mixed and the unreacted compounds affects the reproducibility of using MNPs. Therefor immediate purification is necessary to prevent undesirable reaction. Active and complete mixing approaches can be employed for avoid getting unreacted reagents [26]. Certain MNPs composed of nickel ferrites have demonstrated toxic effect and compromised cell viability and cell proliferation. The production of reactive oxygen species (ROS) occurs during MNP cell uptake. These species have the potential to either trigger apoptosis or initially act as a barrier against invading foreign species. Due to impropriate size of MNPs, they may accumulate inside cells and show negative impact on the intracellular signaling pathway in cellular cytoskeleton. Investigation is still needed to determine whether the cytoskeletal disruption by MNPs results in functional implications such apoptosis, impaired cell proliferation, cell division [27–29].

6.2 SYNTHESIS OF MAGNETIC NANOPARTICLES (MNPs) There are generally three methods for the synthesis of magnetic nanoparticles (MNPs) viz. physical, chemical, and biological. These methods produce different MNPs of required morphology, stability, biocompatibility, etc. (Figure 6.2).

6.2.1 Physical Method This method consists of top-down and bottom-up approaches. In the top-down approach, bulk materials are broken down into nano-size and in bottom-up approach; the fine nano-sized particles are obtained from a top-down method.

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FIGURE 6.2 Flow chart showing methods for the synthesis of magnetic nanoparticles.

a. Ball milling method - This is a top-down approach of synthesis. The process involves mechanical grinding of coarse textured particles into fine particles. The samples are prepared in a steel vial where all the raw materials are put and they are crushed using strong forces of steel balls. The nano-sized magnetic particles are formed due to the collisions between them. The speed, vibration, ratio of raw materials to balls, size of the balls, and time are the important factors that define the type and size of the magnetic particles [30]. b. Wire explosion method - The electrical wire explosion method is a simple and clean process that was first developed to synthesize iron oxide magnetic nanoparticles for the removal of arsenic content from water. This method employs electrical wire explosion in the oxygen/ nitrogen atmosphere. The iron wire is put into the reaction chamber through an optimized hole of metal contact plate which acts as an upper electrode. The wire is continuously moved at the bottom of the chamber that works as a lower electrode. Both this upper and lower electrode are connected to high voltage current, which results in explosion that results in the formation of MNPs [31]. c. Laser evaporation method - This is a bottom-up approach where a high energy laser is applied on the raw material to produce MNPs. The process is simple where raw materials are submerged in a liquid solution and evaporated using high beam of laser. The vapours are then cooled in a gas phase like CO2 that results in condensation and nucleation for the formation of MNPs. Under stable conditions, there is continuous production of MNPs. The raw materials and parameters of the laser evaporation process influence the properties of nanoparticles [32–34].

6.2.2 Chemical Method a. Thermal decomposition method - MNPs of high monodispersity and size control are formed in this method. Organometallic precursors are decomposed in the presence of organic surfactants and stabilizing agents like fatty acids, oleic acid, and hexadecylamine. The role of stabilizers is to slow down the nucleation process of nanoparticles which in turn will control the growth of MNPs resulting in spherical shape and preferable size of less than 30 nm [35]. Depending upon the precursor, temperature is required. Reaction time, type of solvents and surfactants, and their aging period are maintained to obtain the desired shape and size of MNPs [30,36]. This method produces MNPs in uniform size and homogenous shape in large scale and therefore reported as one of the best methods. This method produces toxic organic soluble solvents, which is a limitation in the application in the biomedical field [19].

106 Nanomaterials in Healthcare b. Co-precipitation method - This is the most commonly and widely used method for the synthesis of controlled size and magnetic properties. The MNPs produced by this method are widely used in biomedical applications due to use of less harmful materials and procedures [37,38]. Different nanocrystals are formed using various metal ions like Fe3+ and Fe2+ where they are coprecipitated by addition of NaOH [39]. Factors like pH, metal ions, and their concentration, reaction temperature, etc. can change the composition of MNPs. c. Microemulsion synthesis method - In this method, there is emulsion of oil/water using lipophilic and hydrophilic phases of surfactants and co-surfactants. The process involves mixing of oil in a surfactant and water is gently added at ambient temperature. There are three kinds of microemulsion viz. oil in water, water in oil, and oil in water where both are used in approximately equal quantities. The type of surfactant used determines the size and shape of MNPs. For the treatment of tumor cell separation, these MNPs were synthesized using a silica coating and further modified with amino [35]. d. Sol-gel method - This process involves formation of gel at room temperature by hydrolysis and polycondensation reactions of metal alkoxides. This is one of the most widely used methods for the synthesis of MNPs. In this method, metallic salts are dissolved in water or solvents and homogeneously dispersed to produce colloidal solution or sol [40]. Generally iron oxide and silica coated MNPs are formed using this method in large quantities with controlled size and shape [30]. The principle behind this method is van der Waals forces between the particles and the interaction of the particles increases by stirring and increasing the temperature. Until the solvent is removed, the mixture is heated and thus the solution is dried to form a gel. The main advantage of this method is that it can be done at room temperature and controlling the size and shape of MNPs. One of the disadvantage is that it requires lengthened reaction time and toxic organic solvents [41]. e. Hydrothermal synthesis method - This is one of the most successful solution reaction based approaches where MNPs are formed in an aqueous solution under high pressure and temperature by hydrolysis and an oxidation reaction [42,43]. Mixing of solvent, pressure, temperature, and solvent is important for the morphology of MNPs to be formed. The crystals are formed, depending upon the solubility of minerals in water [44]. The main disadvantage of this method is slow-reaction kinetics at any temperature. This problem can be solved using microwave heating during hydrothermal synthesis [19,45].

6.2.3 Biological Synthesis Method Biological synthesis is a popular method to synthesize MNPs where living organisms like plants and microorganisms (fungi, bacteria, viruses, etc.) are used [46]. The MNPs produced by this method are very biocompatible and thus widely used in biomedical application. The main advantages are that these are eco-friendly, efficient, and clean process. The disadvantage is poor dispersion [47]. The formation of NPs with a biological method is still under investigation. Plant exudates, inactivated plant tissues, and extracts are used for the formation of NPs.

6.3 PROPERTIES AND APPLICATION Magnetic nanoparticles have different chemical, electrical, magnetic, and structural properties. Depending on properties, they have selective applications [48]. Because of selective magnetic properties, magnetic nanoparticles allow surface modification and attachment of functional groups. It is also applicable for target-specific delivery manipulation and transportation at the desired location through the control of a magnetic field [49]. Nanoparticles have different layers from the inside core and outer coated surface and

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functionalized surface. A core with magnetic properties allows manipulation and modulation of particles in an external magnetic field. But it also has some toxicity. So currently less toxic magnetic materials with high oxidative stability like maghemite (Fe2O3) and magnetite (Fe3O4) have been accepted for biomedical application [50]. Magnetic particles with other metals with magnetic properties like nickel, cobalt, boron, and neodymium have also been used for biomedical applications. But these are more toxic because of more susceptibility to oxidation [51]. Properties also depend on the method of synthesis of magnetic nanoparticles. The properties like magnetic property, size, magnetic agglomeration, and influence of gravitational force depend on the method of synthesis of particles [52]. Agglomeration, interaction, and stability of particles are affected by van der Waals forces between particles. Surface coating and functionalization is done to prevent agglomeration by providing steric repulsion. Surface functionalization can be done by specific polymer, functional tag molecules or antibodies, and drug molecules.

6.3.1 Characteristics of Magnetic Particles 6.3.1.1 Particle size Particle size, the physical property of a particle, affects the magnetic and other properties of particles. The size of particles directly affects mobility, magnetic property and density. Dry powder method used for particle size measurements gives more idea about particle size distribution rather than size. The shape of the particles like round or rod shape affects particle size and size distribution. Magnetic particles with high residual core magnetism and coercivity cause aggregation of particles, which affects the size of the particle. Surface coating or non-magnetic encapsulation prevent this aggregation.

6.3.1.2 Particle density Commercially available particles with a density close to 4.5 depend on the form of iron, either iron powder or oxides. Particle size and shape are interconnected and affect the overall property of the particle. Particles encapsulated or coated using polymer may have lower density compared to iron oxide particles and affects attractive force. The density and coating decide whether the particle will sink or form a suspension in media. Manipulation of particle size, shape, and density is required to maintain suspensibility.

6.3.1.3 Particle shape The shape of the particle affects the magnetic property, fluorescence property, and colour of the particle. The particles may be of different shapes like a sphere, toroid, rod shape, or ovoid shape depending on the method of preparation. The magnetized surface tends to align in the applied magnetic field when an external field is applied. This property is higher in rod shape of particles or particles with a high length to width ratio compared to spheroid-shaped particles.

6.3.1.4 Magnetic property Magnetic particles should have low retentivity and high permeability. Particles with high permeability and low retentivity respond rapidly toward leakage fields. There will be ease of removal of particles after application. But particle size, shape, and density also affect it. There are five types of magnetism • Diamagnetism • Paramagnetism

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FIGURE 6.3 Magnetic property of superparamagnetic ferromagnetic and NPs under an external magnetic field.

• Ferromagnetism • Antiferromagnetism • Ferrimagnetisms The orbital motion of electrons opposes the external applied magnetic field. Diamagnetism is a weak repulsion to the applied field. Materials having filled electronic subshells with paired moments show diamagnetism. Diamagnetic materials weakly oppose the applied magnetic field and have negative susceptibility like quartz [53]. Other types of magnetic properties are seen in unpaired moments, commonly in 3d or 4f shells. Paramagnetic materials are having positive magnetic susceptibility like pyrite [54]. Ferromagnetism havs equal magnitude moments, crystalline structures, and aligned magnetic moments like in cobalt, nickel, and iron. Hard magnets maintain their magnetization in the absence of an applied magnetic field. Antiferromagnetism is the property of materials that arranges their magnetic moments antiparallel with zero net magnetization; for example, FeS [55] Paramagnetic behavior is observed at Neel temperature where thermal energy causes random fluctuation in equal and oppositely aligned atomic moments. Materials like Fe3O4 and Fe3S4 show ferrimagnetism. The ions or atoms are in non-parallel arrangement below Neel temperature. Material becomes paramagnetic above the Neel temperature [56] (Figure 6.3).

6.3.2 Application of Magnetic Nanoparticles Magnetic nanoparticles have different biomedical applications like in diagnosis and theragnostic. Sunnano-sized particles like superparamagnetic iron oxide NP [SPIOs] have been used for targeting by binding with special ligands. It also provides prolonged blood circulation time [30] (Figure 6.4).

6.3.2.1 Hyperthermia Paramagnetic nanoparticles flip the magnetization directions between antiparallel and parallel orientation in presence of an applied AC magnetic field. This allows the transfer of magnetic energy in the form of heat to the particles. This phenomenon is helpful to increase tumor tissue temperature and causes cancer cell death [23]. Dextran coated and cationic liposomal magnetic particles have been reported to cause effective hyperthermia in tumor cells making a new way to treat cancer [11]. This will provide heating in

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42°C

Magnetic hyperthermia

Photothermal therapy

Magnetic nanoparticles

Chemotherapy

Antibacterial MRI

Target specific

FIGURE 6.4 Biomedical application of magnetic nanoparticles.

the local area of the tumor. Nanosized particles absorbs more power compared to micronsised particles [57]. Thus, therapy depends on shape, size and way of synthesis of particles. Fe3O3 and Fe3O4 coated with surface active agents, polymer, ceramics, or silica are currently used as hyperthermic agents. It is difficult to control the Curie temperature in the desired range during the therapy. The uncontrolled rise in temperature may affect the normal cells. Here, perovkite oxides with controlled heating ability is useful [58]. Sr doping composition has provided formulation with selfcontrolled heating for hyperthermia treatment with improved biocompatibility [59]. Gadolinium doped NiFe2O4 has been reported for relaxometry/magnetic hyperthermia studies [60]. Co-doping was used to modify structural and magnetic property of nanoparticles in presence of AC magnetic field using iron carbide and oleic acid. Sub-nano-sized co-doped CoFe2O4 synthesized using solvothermal reflux method has proven a good hyperthermic effect.

6.3.2.2 Photothermal therapy MNPs are having low molar absorption coefficient in the near IR region and so combined with other photothermal agents for photothermal application [61]. This provides a new route for cancer treatment by site-specific action [62]. SPIONs coated using poly (acrylic acid) have been reported. The particles have shown good stability before and after irradiation without significant loss of coating and change in size with variable photothermal performance at a different wavelength. Hyaluronic acid and poly(dopamine) coated sphere-shaped magnetic particles have been reported for their good photothermal therapy [63]. The particles have displayed a significant reduction in cell proliferation and auspicious necrosis at the tumor site. Fe3O4@Au MNPs have been reported for its high potential and effectiveness in cervical tumor [64].

6.3.2.3 Drug delivery Target-specific delivery is a more promising way to deliver drugs without causing any drug loss or systemic side effects. In recent years, magnetic nanoparticles are used widely for target-specific theragnostic applications [65]. Magnetizable implants loaded with magnetic nanoparticles allow target-specific

110 Nanomaterials in Healthcare desired release of a drug at the local site after applying an external magnetic field [66]. This reduces side effects and dose requirement. The biocompatibility of particles is improved by surface modification using polymers, bioactive molecules, or metal oxides [67]. Target-specific delivery is affected by the force generated external magnetic field and the force generated by blood on the particles. Doxorubicinloaded CoFe2O4 nanoparticles with surface modification using meso-2,3-dimercaptosuccinic acid (DMSA) has been reported, where the drug has been loaded through electrostatic interaction or hydrogen bonding. Theragnostic applications i.e., chemotherapy and thermal therapy, were done by using CoFe2O4 particles.

6.3.2.4 Infection treatments Magnetic nanoparticles have also shown significant applications as an antiviral and antibacterial agent to treat the infection. Ag@Fe3O4−PEI MNP have been reported for its fast and effective photothermal activity against bacterial infection. [68] The particles have displayed antibacterial activity within 10 min. SARS-CoV-2 detection and control can be done by using Fe3O4 nanoparticles [69]. Antibody-coated liposomes encapsulated with calcein were reported for site specific delivery. The particles have shown fast and easy detection of H1N1 influenza, Zika, and hepatitis E virus [70]. Anisotropic particles with magnetic moments compared to spherical particles are useful as the contrast agent [71]. Surface functionalization is more feasible in nanorods. The anisotropic shape also reduces the total dose and side effects in cancer therapy [72]. Progress in the antibacterial and antiviral application can be achieved by taking the size and shape of the particles into consideration.

6.3.2.5 Magnetic resonance imaging Imaging is a powerful technique to visualize and differentiate tissue. In-vitro and in-vivo molecular and cellular imaging can be done using superparamagnetic iron oxide NPs as contrast generating diagnostic agents by enhancing proton relaxation in MRI. Gadolinium-based contrast agents are generally used in MRI. But they are required in large quantities and may cause nephrotic damage and other side effects. So that SPIO agents are more suitable as contrast agents as it requires less quantity. The compatibility can be improved by applying a biocompatible coating. The particles were prepared with singular biodistribution, high safety margin and high relaxivity. SPIONs coated with zwitterion has been reported with high T1 contrast power [73]. Folic acid-coated Fe3O4 MNPs loaded with paclitaxel has been reported for its effectiveness as to treat cancer [74]. It is reported to lower the tumor volume by 40%. MNPs along with boron-dipyrromethene (BIODIPY) has displayed good fluorescent imaging of photoinactive pathogens and bacterial cells.

6.4 CONCLUSION Magnetic nanoparticles possess great properties and can be used in diverse range like biomedical, diagnostics, drug-delivery, etc. These particles can be synthesized and designed based on the specific application and purpose. Different types of magnetic nanoparticles acquire different properties and can be specifically targeted which will have potential cure. The ions used for preparation imbibes its own properties and the process of developing these MNPs is greatly evolved. The size, shape, and composition of the MNPs can be easily modified. MNPs now are being used in nanomedicine as well as overcoming several problems like target-site delivery, localization, biological barriers, etc. Recent discovery shows these particles have antibacterial properties and this property can be combined in therapies to improve the efficacy of antimicrobial drugs. Currently there are lots of challenges and novel research

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ideas needs developed to overcome the limitations for several diseases. Technology to develop and fabricate better MNPs is developing enormously using multidisciplinary approach which shows promising results for future.

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Nano Metal-Organic Frameworks as a Promising Candidate for Biomedical Applications

7

Dhruv Menon1, Swaroop Chakraborty2, Prateek Goyal1, Eugenia Valsami-Jones2, and Superb K. Misra1 1

Materials Engineering, Indian Institute of Technology Gandhinagar, Gujarat, India 2 Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, UK

Contents 7.1 7.2

7.3 7.4

7.5

Introduction Synthesis of NMOFs 7.2.1 Solvothermal Synthesis 7.2.2 Microemulsion Synthesis 7.2.3 Microwave-Assisted Synthesis 7.2.4 Ultrasound/Sonochemical Synthesis 7.2.5 Electrochemical Synthesis 7.2.6 Mechanochemical Synthesis Biofunctionalization of NMOFs NMOFs for Drug Delivery and Targeted Tumor Therapy 7.4.1 pH-Responsive Drug Delivery 7.4.2 Temperature-Responsive Drug Delivery 7.4.3 Ion-Responsive Drug Delivery 7.4.4 ATP-Responsive Drug Delivery 7.4.5 Redox-Responsive Drug Delivery NMOFs for Bio-Imaging

DOI: 10.1201/9781003322368-7

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7.1 INTRODUCTION Due to a great control over their porous space, in terms of uniformity and size, metal-organic frameworks (MOFs) have surfaced as a unique class of materials for a wide range of domains, from environmental remediation to biomedical applications [1,2]. While porous materials are conventionally either inorganic or organic, MOFs leverage the unique advantages of each of these classes into a single entity, with a whole new set of physicochemical properties [3]. Best described as hybrid inorganic-organic crystalline materials, they are characterized by huge internal surface areas (of the order of 1,000–10,000 m2/g) and very high porosity (~90% free volume) [4]. Composed of metal ions or clusters, linked via special organic functional groups such as carboxyl or heterocycle groups, these hybrid complex materials have networked periodic structures (refer to Figure 7.1a) [5]. The geometric size and underlying morphology of these materials are such that they lead to the generation of large pores, which can be exploited for the storage of large molecules [4]. Additionally, these frameworks can be modulated through post-synthetic modifications, where either useful guest molecules can be trapped within the large pores of the framework or can be covalently or noncovalently bound to their outer surface to enhance their properties [6]. This is particularly useful for nanomedicine applications, as MOFs can be employed to store and deliver therapeutic biomolecules. The nano-sized counterparts of these bulk MOFs (nano-MOFs or NMOFs) have recently gained extensive interest because of their ability to enhance the existing physicochemical properties of their bulk variants [9], which coupled with good biocompatibility, make them promising candidates for biological applications pertaining to drug delivery, bio-imaging, bio-sensing, biopreservation, and cell and virus manipulation (refer to Figure 7.1b). NMOFs typically having dimensions within 5–500 nm, have shown to retain the porosity of their bulk counterparts [9], facilitating high bio-active molecule loading capacities for diagnostic and therapeutic applications. NMOFs are often functionalized with biomole­ cules such as DNA or enzymes to improve their biocompatibility and stability in biological media and enhance cellular uptake for targeted delivery applications [6,10]. For drug delivery and other related applications, the pre-requisites of ideal nano-sized carriers include (A) a high drug-loading capacity, (B) biocompatibility and bio-clearance (the material precursors should not accumulate in the body after degradation), (C) an outer surface that can be easily modified using covalent or noncovalent interactions, (D) the ability to target diseased cells with high specificity and selectivity, (E) the potential to facilitate a controlled release of the encapsulated drug, and (F) the ability to be detected by imaging techniques [11,12]. The innate properties of most NMOFs enable them to satisfy criteria A-C, while functionalization with appropriate biomolecules enables them to satisfy criteria D-F (Figure 7.2 highlights the structural morphology of commonly used MOFs, that endow these materials with innate properties that make them useful for nanomedicine applications). Recently, there have been reports of NMOFs being used at a preclinical stage for applications such as tumor therapy and drug delivery. For example, Chen and colleagues developed novel nucleic acid biofunctionalized zirconium MOF nanoparticles, where the nucleic acids had sequences complementary to the VEGF aptamer. The NMOF was loaded with an anti-cancer drug post-synthesis and used for targeted tumor therapy. The NMOF synthesis, functionalization and characterization strategies, and drug loading and release experiments carried out by them have been discussed in detail in the subsequent sections. In this chapter, we discuss the synthesis and functionalization strategies for developing NMOFs that make them fit to serve as carriers for the targeted delivery of bioactive molecules. Following this, we

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FIGURE 7.1 (a) SEM micrographs of the networked structures and periodic nature of common MOFs such as MOF-5, HKUST-1, MOF-177, MIL-101, UiO-66, and ZIF-8. Reprinted (adapted) with permission from ref. [ 7]. Copyright 2022 Elsevier. (b) A schematic illustration of the applications of NMOFs for biomedical applications pertaining to drug delivery, biosensing, biopreservation and cell & virus manipulation. Reprinted (adapted) from ref. [ 8] under Creative Commons CC-BY license.

discuss the unique properties of NMOFs in detail that make them ideal materials for therapeutic and diagnostic applications. Next, we discuss the applications of these materials in drug delivery, tumor therapy, and bio-imaging. We conclude with insights into the major obstacles that lie ahead in the widespread adoption of NMOF-based technologies for addressing biomedical problems and shed light on possible ways to overcome these obstacles, for improved drug delivery, tumor therapy, and bio-imaging based applications, making NMOFs the next generation of materials for advanced therapeutic and diagnostic applications.

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FIGURE 7.2 Examples of metallic secondary building units and corresponding organic ligands that serve as precursors for common MOFs and their reported morphologies/topologies. Reprinted with permission from ref. [ 13]. Copyright 2022 International Union of Crystallography.

7.2 SYNTHESIS OF NMOFs For nano-sized materials, the synthesis protocols employed should be such that they lead to the formation of uniform and monodisperse particles. This is still a challenge for NMOFs owing to the hybrid inorganic-organic nature of these materials, which warrants an in-depth understanding of the initiation, growth, and termination phases of crystallization [14]. In the case of NMOFs, proper control over the reaction conditions is essential to ensure that the organic linker does not decompose or degrade, and concurrently, the kinetics should be favorable to ensure that the nucleation and growth of the desired phase occurs at an acceptable rate [6,15]. Two synthesis strategies that have achieved acceptable

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FIGURE 7.3 A schematic illustration of the most commonly used techniques for the synthesis of MOF nanoparticles (or NMOFs), along with the major advantages they offer over their counterparts. Reproduced from ref. [ 6] with permission from the Royal Society of Chemistry.

uniformity and monodispersity, with moderate reaction conditions and acceptable kinetics, are the sol­ vothermal and microemulsion methods. Additionally, methods such as microwave-assisted, ultrasound, electrochemical, and mechanochemical synthesis have reported varying degrees of success [14]. Here, we will briefly discuss each of these methods (Figure 7.3 provides a brief overview of the advantages of each of these methods).

7.2.1 Solvothermal Synthesis Solvothermal synthesis is the most effective and universal strategy for synthesising NMOFs [14]. Here, solutions of the MOF precursors (the metal salt and the organic ligand) are mixed in appropriate solvents, and the resulting mixture is maintained at a relatively high temperature (usually over 100°C) for a specific time [16]. This method is referred to as ‘hydrothermal synthesis’, when the solvent used is water. For example, let us consider the synthesis of a Cu-centered MOF (known as HKUST-1) using the solvothermal method employed by Goyal and colleagues [17]. Cupric nitrate hemi­ pentahydrate (metal salt) was added to deionised water, while benzene-1,3,5-tricarboxylic acid (organic ligand) was added to a 1:1 ethanol-water mixture in suitable concentrations (calculated stoichiometrically), and both mixtures were vigorously stirred till complete dissolution. Next, both mixtures were transferred to a stainless steel autoclave and heated at 1100C for 18 h in an oven. After heating, the resulting product was washed several times using ethanol and deionised water and underwent filtration to extract HKUST-1 (detailed discussions on the nomenclature and terminology of MOFs have been provided by Vittal et al. [18], Batten et al. [19], and Reedijk et al. [20]) in powder form [17]. This method modulates the crystal size and properties by altering the ratio at which the organic ligand and metal ions are mixed, the type of solvent used, the pH, and the reaction temperature [5].

7.2.2 Microemulsion Synthesis The microemulsion method is another effective route to synthesize uniform and monodisperse NMOFs. In this method, microemulsions are formed through interactions between incompatible solvents. These interactions are stabilized by surfactants (or emulsifiers), making the entire system thermodynamically stable and monodisperse [13,14]. Here, nano-sized monodisperse droplets are formed during the solvent

120 Nanomaterials in Healthcare mixing process, and the size of these droplets can be controlled based on the emulsifier concentration. As a representative example, Lin and colleagues [21] used a hexadecyl trimethyl ammonium bromide (CTAB)/water/isooctane/1-hexanol micro-emulsion system to form Gd2(BDC)1.5(H2O)2 nanorods. They found that by changing the ratio of concentrations of water and CTAB, they could alter the dimensions of the nanorods formed [14,21].

7.2.3 Microwave-Assisted Synthesis The microwave-assisted synthesis is a method for the rapid synthesis of NMOFs. In the case of MOFs, this method is also called the ‘microwave-assisted solvothermal synthesis’, since most of the steps involved closely resemble the solvothermal process (refer to Section 7.2.1), except for the heating stage. The solution obtained after mixing the metal salt and the organic ligand is heated for about an hour in a microwave. This method has been shown to produce crystals having the same quality as the solvothermal method, but at a fraction of the time [22]. Due to their strong penetration ability, relatively high effi­ ciency, and rapid response, microwave-assisted methods are becoming increasingly popular for rapid NMOF synthesis. This method has been used for the rapid synthesis of NMOFs such as MIL-140, a zirconium-based MOF [5,22,23].

7.2.4 Ultrasound/Sonochemical Synthesis In sonochemistry, through the application of ultrasound radiation in the frequency range of 20 kHz to 100 MHz, molecules undergo chemical changes. These changes occur due to the formation of highly localized regions of high temperature and pressure, resulting from the formation, growth, and collapse of bubbles in a solution induced by the ultrasound radiation [22]. These chemical changes facilitate the rapid formation of NMOF crystals that are homogeneous. This method has shown to have a good NMOF yield, in a fast reaction time, at reduced costs, as has been demonstrated for HKUST-1 and MIL-88, an iron-based MOF [5,24].

7.2.5 Electrochemical Synthesis Regarding the large-scale bulk production of materials, continuous processes are preferred over batch processes. In the context of NMOF synthesis; solvothermal, microemulsion, microwave-assisted, and ultrasound methods of syntheses are batch processes. This has motivated research into potential continuous processes such as the electrochemical synthesis method for the large-scale industrial production of MOFs. Following the basic principles of electrochemistry, NMOFs are formed through chemical reactions facilitated by a gain and loss of electrons at the cathode and anode of an elec­ trochemical cell. Interestingly, this method does not rely on metal salts and works on releasing metal ions upon anodic dissolution, which reacts with the organic linkers and electrolytes, leading to the formation of NMOFs [5,22]. Although this method has its obstacles since the electrochemistry of organic linkers is less investigated, it has been successfully used for the large-scale production of HKUST-1 [6,25].

7.2.6 Mechanochemical Synthesis Mechanochemistry involves mechanical force (such as tension, shearing, extrusion, or friction) mediated chemical reactions. In the context of NMOF synthesis, it involves the construction of bonds between the metallic center and organic linker through mechanical routes such as neat grind (NG), liquid-assisted

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grinding (LAG), and ion-and-liquid assisted grinding (ILAG). In most cases, this route is solvent-free, involving reactions between solid phases without extreme conditions such as high temperatures or pressures, making them scalable techniques for mass production [5,10,22,26]. This technique has been used for the synthesis of several NMOFs, most notably, nickel-based MOFs [27].

7.3 BIOFUNCTIONALIZATION OF NMOFS Despite the promise that NMOFs hold, there are some concerns about their rate of degradation, and the rate at which their precursors accumulate in tissues and organs, which limits their applications at clinical levels [28]. As stated earlier, these concerns can be alleviated to certain extents, by ‘functionalization’, where different functional groups are introduced via covalent or noncovalent means to enhance the existing properties of these nanomaterials, and possibly introduce a new set of properties useful for the application at hand. Particularly for nanomedicine-based applications, functionalization is carried out using special classes of biomolecules (known as biofunctionalization) such as DNA, lipids, aptamers, and enzymes [6,29–32]. Biofunctionalization helps incorporate each biomolecule’s unique properties in the modified NMOF, thus enhancing its performance for the intended therapeutic or diagnostic appli­ cation [6]. While conventional functionalization can be carried out during NMOF synthesis or postsynthesis, the reaction conditions are usually too extreme for biomolecules such as enzymes and DNA, making post-synthetic methods more viable. Post-synthetic methods can further be broken down into two categories depending upon the type of interactions that are exploited: covalent functionalization and noncovalent functionalization (refer to Figure 7.4).[6] In this section, we will discuss these two methods in brief. For a more detailed discussion, readers may refer to review articles by Menon and Bhatia [6] and Cohen [33]. Covalent functionalization or post-synthetic modification involves using reagents to facilitate the modification of certain components of the NMOF (typically the organic linker), leading to the formation of a covalent bond [33]. The chemistry of covalent bonding is such that it works to stabilize the structure of the NMOF, increasing its applicability for drug delivery applications [31]. The concept of covalent functionalization was derived from that of post-translational modifications of proteins [34], and initially involved tagging MOFs with aldehydes, amines, azides, or alkynes, but has since been expanded to other handles such as thioethers and carboxylate groups [33]. For example, Mirkin and colleagues used click chemistry [35] to develop NMOF-nucleic acid conjugates by reacting dibenzylcyclooctyne (DBCO)functionalized DNA with UiO-66-N3 MOF nanoparticles. These covalently functionalized NMOFs showed improved colloidal stability and cellular transfection capabilities, creating a new class of nanomaterials for intracellular delivery applications [36]. In the event the reagent forms a dative bond (such as a hydrogen bond, π-π bond or electrostatic bond) as opposed to a covalent bond, the functionalization strategy is termed noncovalent (or dative) functionalization [6,31,33]. Owing to the innate chemistry of these bonds, the strength of interactions is much weaker than their covalent counterparts; thus, they play a smaller role in the improvement of stability of the NMOF. That being said, this strategy is much less time-consuming, has a high efficiency, and does not alter the structure of the NMOF [6]. For example, Farha et al. functionalized insulin loaded NU-100 and MOF-545 (zirconium-based MOFs) with phosphate-modified DNA. This helped them achieve a tenfold increase in cellular uptake for protein delivery and improved the stability of MOF nanoparticles in high dielectric media [37]. The major advantage of the functionalization strategy they employed was that it was not host and guest material chemistry specific, and could easily be generalized to be independent of the biomolecule that needs to be loaded and delivered. This strategy is also the preferred route for functionalizing NMOFs with enzymes since it does not alter the individual chemical properties of the entities involved.

122 Nanomaterials in Healthcare

FIGURE 7.4 General schematics for the functionalization of NMOFs post-synthetically. The first method as described is by the formation of a covalent bond between the NMOF and biomolecule under consideration. In the second method, the functional group is attached via noncovalent interactions. The third method as shown is through post-synthesis deprotection where chemical bonds are broken after synthesis to achieve functionali­ zation. In this chapter, the two more commonly used methods, covalent and noncovalent functionalization have been discussed. Reprinted (adapted) with permission from ref. [ 33]. Copyright 2022 American Chemical Society.

7.4 NMOFS FOR DRUG DELIVERY AND TARGETED TUMOR THERAPY The biggest challenges for efficient drug delivery and targeted tumor therapy are the barriers to transport that drugs encounter from the site at which they are introduced into the body, to the site of molecular action. These barriers include clearance through the reticulo-endothelial system, crossing the plasma membrane, escaping endo-lysosome acidic environment and other multiple drug resistance mechanisms [38]. This has motivated a venture into using nanomaterials as carriers for efficient drug delivery. Here, drugs or other bioactive molecules are encapsulated in nanomaterials that are essentially used as delivery vehicles to transport biological payloads by overcoming the aforementioned barriers. However, conventionally ex­ plored nanomaterials have a poor ability to specifically and selectively target the desired molecular site (such as tumor), leading to a poor biodistribution. This warrants a larger drug dosage, leading to unwanted immunogenic side effects [39]. Over the past decade, MOF nanocarriers have emerged as efficient drug delivery systems, owing to increased biocompatibility, cellular uptake, and controlled drug release, par­ ticularly upon biofunctionalization. They have been actively explored for the delivery of anticancer drugs,

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antimicrobial agents and metabolic labeling molecules among others [40–43]. Similar to the functionali­ zation strategies discussed in Section 7.3, the drug loading in NMOFs can be carried out using covalent or noncovalent routes. The strategy for encapsulation of drugs depends upon the required release type. Covalent drug encapsulation would correspond to a slower (and more controlled) drug release due to a stronger binding to the NMOF matrix, while a noncovalent drug encapsulation would correspond to a relatively faster drug release [3]. Depending upon the microenvironment at the site of molecular action, and the response of the NMOF to the microenvironment, NMOF based drug delivery can be broadly classified as: (A) pH-responsive, (B) temperature responsive, (C) ion responsive, (D) ATP responsive, and (E) redox responsive, among several other possible stimuli [5]. In this section, we will provide a brief overview of each of these drug delivery mechanisms in the context of NMOFs.

7.4.1 pH-Responsive Drug Delivery In various segments, organs, and tissues of the body, local pH values tend to differ. For example, in the stomach it ranges from 1.5–3.5, while in the small intestine it ranges from 5.5–6.8 and in the colon, it ranges from 6.4–7.0. In the case of tumors and other inflamed tissues, it has been found that the pH values are generally lower than those of the blood and healthy tissues. These pH differences serve as good physiological stimuli for the release of drugs, and nanocarriers that can respond to such stimuli are ideal for efficient drug delivery [44,45]. A NMOF can degrade upon pH-based stimuli by controlling the constituent organic linker. With the aim of developing a drug delivery system that does not release the biological payload under physiological conditions, and only releases it upon encapsulation in the target cells in response to pH changes, Zou et al. developed a nontoxic and biocompatible ZIF-8 NMOF composed of zinc as the metallic center and 2-methylimidazolate as the organic ligand. They reported a one-pot synthesis of doxorubicin (DOX) encapsulated ZIF (DOX@ZIF-8), and observed no drug release at physiological conditions (pH = 7.4) and a controlled drug release over 7–9 days over a pH range of 5–6 (refer to Figure 7.5a), which is observed to be within the range of the lysosome and endosome. They further observed the efficacy of this system in vitro on breast cancer cell lines and primary macrophages and measured cellular uptake via confocal microscopy techniques [46]. Paul et al. synthesized iron-based MIL-101 MOF nanoparticles having an average particle size of 480 nm using the solvothermal method. To facilitate increased drug loading via covalent interactions, the NMOF was functionalized using an amine group. The outer surface was coated with polyethylene glycol (PEG) to act as a barrier against pH mediated biodegradation, facilitating a more controlled release of the encapsulated drug. In vitro drug release experiments were carried out for pH 7.4, pH 6, and pH 5, where the highest drug release was observed for pH 5, for non-PEG-coated MOF nanoparticles [47]. NMOFs, as drug delivery systems are particularly useful when the drug to be delivered, could have extremely toxic effects if its distribution is not carefully controlled. One such example is that of arsenic trioxide (As2O3), which has shown great success in the treatment of acute promyelocytic leukaemia (APL) [51]. Since then, there has been a lot of research into the potential use of this drug to treat solid tumors such as brain tumors, cervical cancer, and breast cancer [52]. That being said, the usage of As2O3 for the treatment of solid tumors is incredibly challenging due to its toxic effect on healthy tissues and organs. To mitigate this challenge, Bunzen et al. reported a Zn-MOF-74, a zinc-based MOF as a drug nanocarrier of As2O3. Upon loading with high capacities (~153 mg of drug per 1 g of NMOF), they observed a faster release at pH 6 compared to pH 7.4, hinting towards a pH-triggered release of the drug is a favorable characteristic for tumor therapy [52].

7.4.2 Temperature-Responsive Drug Delivery Among the various stimuli that can trigger drug release, temperature stands out as it can act as an internal trigger (diseased tissues and/or tumors generally have elevated temperatures) or an external trigger

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FIGURE 7.5 (a) The controlled release profile of DOX@ZIF-8 triggered by pH changes. Reprinted (adapted) with permission from ref. [ 46]. Copyright 2022 American Chemical Society. (b) The controlled release profile of zirconium based NMOFs that were capped with CP5-based pseudoro [ 2] taxanes triggered by thermal therapy. Reprinted with permission from ref. [ 48]. Copyright 2022 Royal Society of Chemistry. (c) A schematic illustration of ion-responsive drug delivery, where a drug-loaded bio-MOF releases the encapsulated drug due to cationic interactions. Reprinted (adapted) with permission from ref. [ 49]. Copyright 2022 American Chemical Society. (d) A schematic illustration of an ATP-responsive system for the delivery and release of large protein payloads into the cytosol due to the formation of ATP/ion complexes. Reprinted (adapted) with per­ mission from ref. [ 50]. Copyright 2022 American Chemical Society.

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(a high temperature can be applied outside the body to elicit a drug release). As an example of these external triggers, hydrogels have been shown to expand upon response to temperature changes, leading to a sudden drug release [53,54]. Over the recent years, researchers have observed drug release even on relatively small variations in temperatures [53,55]. In the case of NMOFs, it has been observed that by increasing the temperature, the host-guest inter­ actions (in this case, the interactions between the NMOF matrix and the encapsulated drug) tend to weaken, eliciting a faster release [56,57]. As an example of this temperature-responsive release of encapsulated drugs, Qian et al. developed a novel zirconium-based NMOF ZJU-801, having (2E,2’E)-3,3’-(naphthalene1,4-diyl)diacrylic acid (H2NPDA) as the organic linker. The organic linker had a naphthalene moiety, which was introduced to prevent premature drug release, preventing harmful side effects to healthy tissues. Postsynthesis, they obtained a high drug loading, of the order of ~42% for the model drug diclofenac sodium (DS) and observed a temperature-responsive drug release. While the release was negligible for temperatures of 25°C and 37°C, they observed accelerated release at 45°C and 60°C [58]. Along similar lines of ther­ motherapy (temperature mediated therapy), Yang et al. prepared multi-stimuli responsive ‘gated scaffolds’ through the synthesis of zirconium-based NMOFs that were capped with CP5-based pseudoro [2] taxanes. These systems remained stable at the normal Ca+2 concentration, temperature, and pH conditions of the body, but underwent degradation, facilitating controlled drug release around the site of action (such as bone tumor cells) upon thermal therapy (refer to Figure 7.5b), and/or pH or Ca+2 concentration change [48].

7.4.3 Ion-Responsive Drug Delivery Just as there are pH gradients in the internal organs of the human body such as stomach and intestines, there are also gradients in the concentrations of certain ions. These ionic gradients could potentially be exploited to serve as an appropriate stimuli for releasing the bioactive molecule encapsulated in the nanomaterial [59]. In the case of NMOFs, over the past few years, several metallic-ion gradients have served as switches for drug release, such as Mg+2, Ca+2, and Pb+2, among others [57,60]. This particular type of drug delivery works typically in three ways, (A) through the formation of a metal ion/NMOF complex, (B) through anionic exchange in the event an anion is used as the trigger, and (C) competitive binding, where the ion binds with the open sites of the NMOF which were initially bound to the encapsulated drug, thus facilitating a controlled release. An in-depth discussion of these mechanisms has been discussed by Yang and colleagues [57]. One of the earliest reported examples of ion-responsive drug delivery using NMOFs was by Rosi and coworkers, who developed a permanently porous ‘bioMOF’ with zinc as the metallic center and adenine as the organic ligand. Here, adenine was particularly chosen because of its rigidity, welldeveloped molecular coordination chemistry and multiple sites for metal attachment. The anionic nature of this NMOF was of great applicability as it was used for the storage and release of procainamide, a cationic drug. Having observed a high drug loading (~220 mg drug/1 g of NMOF), cationic interactions with the NMOF triggered the release of procainamide (refer to Figure 7.5c) [49]. Along similar lines of ion-responsive drug release, Yang et al. developed a novel theranostic platform composed of zirconiumbased NMOFs that were activated using carboxylatopillar [5] arene-based supramolecular switches as nanovalves. The release of the loaded Fu drug was mediated by the Zn+2 concentration, which acted as a trigger. The advantage of this novel system was the negligible premature release, high biocompatibility, low cytotoxicity, high drug loading capacity, a potential for imaging, and the ability to regulate the drug release through external heat treatment, making this a viable option for brain disease therapy [61].

7.4.4 ATP-Responsive Drug Delivery Owing to a very sharp contrast in concentrations between the extracellular and intracellular environ­ ments, ATP can act as a highly efficient trigger for releasing bioactive molecules encapsulated in drug

126 Nanomaterials in Healthcare delivery systems. In order to trigger a release in response to ATP concentrations, the nanocarrier must be modified with special functional groups that are able to recognise, interact with and differentiate between ATP and other molecular entities. This is usually achieved by functionalization with single-stranded DNA aptamers that can selectively and specifically bind to ATP and enzymes that consume ATP for energy [62]. In the context of NMOFs, release triggered by ATP, is usually facilitated by the formation of either ATP/aptamer complexes or ATP/ion complexes. A detailed overview of these mechanisms is discussed in Wang et al. [57]. As an example of the ATP/aptamer complex driven mechanism, Willner and coworkers developed novel doxorubicin loaded MOF nanoparticles that were coated with a polyacrylamide hydrogel. This hydrogel assisted in locking the encapsulated drug, preventing its premature release, and was functio­ nalized with nucleic acid hairpins that contained an anti-ATP aptamer sequence. Since ATP is over­ expressed in tumor cells, when the NMOFs came in contact with them, an ATP-aptamer complex was formed, leading to the degradation of the hydrogel, triggering a release of the encapsulated drug [63]. Coming to the ATP/ion complex formation driven mechanism, Mao and colleagues developed protein encapsulated ZIF-90 (zinc-based) NMOFs to deliver large proteins payloads into the cytosol. The release of the encapsulated proteins was triggered by ATP, which competitively forms ATP/Zn+2 complexes, leading to the degradation of the NMOF matrix (refer to Figure 7.5d) [50].

7.4.5 Redox-Responsive Drug Delivery Tumor tissues have shown to have a different microenvironment as compared to healthy tissues. As a result, systems that are able to sense this difference and use it as a stimuli to trigger the release of bioactive molecules would emerge as promising drug delivery systems. For tumor cells, it has been observed that the environment is reducing in nature, and is maintained by the oxidation and reduction states of glutathione (GSH) and NADPH/NADP+, with GSH playing a pivotal role in regulating the tumor microenvironment [64]. Therefore, if NMOFs are synthesized as oxygen-reducing materials, their properties would be such that they could trigger a release of encapsulated bioactive molecules in response to the reducing tumor microenvironment. To illustrate this, Liu et. al. developed manganesebased NMOFs composed of a disulphide containing organic ligand. The NMOF was loaded with dox­ orubicin and coated with polydopamine (PDA) and polyethylene glycol (PEG). In the tumor micro­ environment, the excess GSH facilitated a reduction of the disulphide bond, leading to a degradation of the NMOF matrix, resulting in the release of the encapsulated doxorubicin. The PDA and PEG coatings helped prevent a premature release of the drug [65].

7.5 NMOFS FOR BIO-IMAGING With the advent of research into the development of nanoparticle systems for biomedical applications, the domain of bio-imaging has benefited tremendously. Several favourable properties of nanoparticles such as their particle size, shape, charge, and hydrophilicity make them promising candidates for bioimaging related applications [66]. The use of nanoparticles for bio-imaging facilitates the early detection and diagnosis of diseases and helps to understand the pathological characteristics of biological tissues [6]. The secondary building units of MOFs are such that they can individually contribute to bio-imaging, the metal ions through photoluminescence and the organic ligands through fluorescent emission upon irradiation. Additionally, MOFs offer the prospect of being loaded with ‘guest’ molecules, which also can be used for fluorescence based applications. The flexibility MOFs offer with respect to their size, shape, and porosity further compounds their applicability to bio-imaging [67].

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As an interesting example of NMOFs being used for bio-imaging, Mao and colleagues [68] developed a fluorescent nanoprobe wherein a Zn-based MOF (ZIF-90) encapsulated a fluorescent dye, Rhodamine-B (RhB). The resulting RhB/ZIF-90 probe underwent decomposition triggered by ATP, leading to the release of RhB which facilitated sensing and live imaging of ATP. This probe allowed the researchers to efficiently image mitochondrial ATP in live cells and monitor its fluctuation in biological events such as apoptosis and glycolysis, making it a useful tool for studying cellular metabolism [68].

7.6 CONCLUSIONS AND OUTLOOK In this chapter, we have provided a brief overview of the applicability of NMOFs for therapeutic and diagnostic applications, particularly for drug delivery and tumor therapy. Along the way, we shed light on the most commonly used techniques for synthesising NMOFs and discussed routes through which they can be functionalized with biomolecules to enhance their applicability. Lastly, we discussed the applications of NMOFs for pH responsive, temperature responsive, ion responsive, ATP responsive, and redox responsive drug delivery and tumor therapy. Despite the promise of these nanomaterials, we still foresee some challenges that limit their widespread application. NMOFs are plagued with poor material design, metal ion toxicity, a poor understanding of the structural mechanisms, and host-guest interactions. Since the organic ligand is one of the fundamental building blocks of NMOFs, the viable chemical space that can be explored is almost infinite. Therefore, purely relying on experimental techniques for finding optimal organic ligands is not the best way to design efficient NMOFs for cutting-edge appli­ cations. While other areas of materials science have evolved in this aspect with an increasing usage of computational techniques such as molecular dynamics (MD) and machine learning (ML) [69,70], MOFrelated research is lagging. While this could be because of challenges pertaining to finding appropriate datasets, and accurately featurizing data, [71] an increased focus on developing computational tools for probing MOF systems would improve the ability to design more efficient systems for drug delivery and other biomedical applications. A more obvious limitation of NMOFs is the toxicity of the metal ions used, and the potential adverse immune responses that they could elicit. It is because of this toxicity that several NMOFs with excellent properties such as Gadolinium-based MOFs, cannot be used for biomedical applications. Additionally, the manner in which the NMOF matrix interacts with its surroundings in biological media and the interactions between the host matrix and encapsulated guests are largely unknown, making it difficult to predict how the NMOF will react in the diseased tissue or tumor microenvironment. A focused effort towards answering these questions could hold promise in the ever-evolving field of optimized NMOFs for biomedical applications.

ACKNOWLEDGMENTS SC acknowledges the School of Geography, Earth and Environmental Sciences, University of Birmingham, UK for GEES Research Support fundand NERC Discovery Sciences Discipline Hopping Award (Grant number: NE/X017559/1), EV-J acknowledges a Royal Society Wolfson Fellowship (RSWF\R2\192007).

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Porous Silica Nanoparticles for Targeted Bio-Imaging and Drug Delivery Applications

8

Dhwani Rana1, Raghav Gupta1, Bharathi K.1, Rupali Pardhe1, Nishant Kumar Jain3, Sagar Salave1, Rajendra Prasad4,5, Derajram Benival1, and Nagavendra Kommineni2 1

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gujarat, India 2 Center for Biomedical Research, Population Council, New York, New York, USA 3 Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 4 Department of Mechanical Engineering, Tufts University, Medford, USA 5 School of Biochemical Engineering, Indian Institute of Technology‐BHU, Varanasi, India Contents 8.1 8.2 8.3

8.4 8.5 8.6 8.7

Introduction Strategies for Functionalization of Silica Hybrid Nanocarriers Drug Delivery Applications of Silica Nanohybrid 8.3.1 Silica Polymer Nanohybrid for Drug Delivery 8.3.2 Silica Nucleic Acid Nanohybrid for Drug Delivery Silica Protein Nanohybrid for Drug Delivery Silica Peptide Nanohybrid for Drug Delivery Silica Quantum Dot for Drug Delivery Silica Magnetic Nanohybrids for Drug Delivery

DOI: 10.1201/9781003322368-8

134 135 138 138 139 139 144 145 146 133

134 Nanomaterials in Healthcare 8.8 Clinical Trials for Silica-Based Nanoformulations 8.9 Conclusion References

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8.1 INTRODUCTION Drug delivery is an enthralling area of a comprehensive study that entails the site-specific release of a therapeutically active drug at a predetermined rate and efficacy. Administering drugs without carriers at times poses several concerns about biocompatibility, solubility, permeability, and clearance [1–3]. The concept of developing drug carriers that are capable of incorporating both hydrophilic and lipophilic drugs, which in turn limits premature drug release, thereby allowing controlled release and attainment of the desired drug concentration for a prolonged time, has long been considered [2,4]. The utility of nanomaterials as potential drug carriers is widely explored owing to their superiority with respect to structural integrity, smaller particle size, release profiles, improved pharmacokinetic and pharmaco­ kinetic profiles of drugs, improved drug loading, and the potential to cross the cell membranes as well as the leaky vasculature that occurs in cancer cells. Moreover, the nanoparticulate systems can be altered to impart desirable therapeutic properties such as enhancement of the stability of the system, improved retention time, targeted drug delivery, stimuli-responsive release, simultaneous administration of dual or multiple agents, and multifunctionality [1,5–9]. The inorganic carriers are known to impart these properties owing to their exceptional mechanical, optical, and electrical properties. However, in order to enhance stability and dispersibility, these carriers are often subjected to functionalization. The surface functionalization through organic molecules increases biodegradability and biocompatibility, minimizes side effects, and triggers site-specific drug release without damaging the normal tissues [2,10,11]. Thus, the “nanohybrids” combine the physicochemical features of their constituents in a synergistic manner to achieve the desired properties, that make this system a prospective alternative for the design of novel multifunctional systems with a wide range of applications [4]. Further, advances in drug delivery technologies and material sciences have accelerated the development of organic-inorganic nanohybrids, combining their favourable attributes to address the shortcomings of both. This has ultimately led to a good progress in fulfilling the unmet needs in drug delivery. Mesoporous silica materials have gained wide attention over the past decade in the fabrication of nanoparticles, due to their promising characteristics such as tailorable surface properties, uniform pore size, and high chemical stabilities. There exist different classes of mesoporous silica nanoparticles, differentiated based on their unique pore sizes and pore volumes, such as the Molecular 41 Sieves (M41S) family of nanoparticles, Santa Barbara amorphous (SBA), organically modified silica (ORMOSIL), periodic-mesoporous organosilica (PMO), and hollow-type mesoporous silica nano­ particles. The M41S class of nanoparticles is characterized by the arrangement of well-ordered pore structures with a diameter ranging from 2 to 10 nm. It can further be classified based on their structural geometries into three types, namely MCM-50 which has a lamellar structure and MCM-41, MCM-48 which possess a three-dimensional cubic pore structure. The MCM-41 type is regarded as the most widely used material in drug delivery applications, owing to its high specific surface area, thermal stability, hydrophobicity, and tunable pore size. The shape of MCM-41 largely dictates its drug delivery applications, for example, spherical-shaped nanoparticles are used to achieve higher drug loading due to their large surface area. The complexation ability of MCM-41 nanoparticles with metal ions confers it the ability to be used in magnetic resonance imaging. The MCM-50 type of silica nanoparticles comprises porous-aluminosilicate layers that are interspersed by layers of surfactants that possess catalytic and sorbent activities, making it widely applicable in biosensing and other biomedical purposes. SBA mesoporous silica nanoparticles are synthesized under acidic conditions

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 135 using amphiphilic block co-polymers that possess a thickness of around 3.1–6.4 nm, making them more mechanically stable in comparison to M41S. Further, in order to overcome the issues regarding the colloidal instabilities of silica nanoparticles, organically modified silica nanoparticles have been researched extensively. ORMOSIL are inert and optically transparent materials, synthesized in the form of an oil-in-water microemulsion that possesses a high storage stability and an increased shelflife. The hollow-type mesoporous silica-nanoparticles possess a tremendously high drug-loading capacity of approximately 1 g of drug per gram of silica. Further, the ease in its surface functiona­ lization and ability to target cells, makes it ideal for cancer therapy as it can overcome the limitations of conventional formulations [12]. Silica-based drug delivery systems have demonstrated effectiveness over conventional delivery systems owing to their potential to maintain therapeutic drug concentrations with only minor varia­ tions; to prevent systemic and local adverse effects; to increase therapeutic plasma levels of drugs having a short half-life; and to reduce the frequency of administration leading to patient compliance. The chemical, physical, and optical properties of silica impart a vast range of applications in drug delivery [4]. Silica nanohybrids have been extensively researched for their potential use in targeted drug delivery. In an active targeting strategy, silica organic nanohybrid containing cancer-specific ligands has shown potential to target receptors whose expression is upregulated in cancer cells (like folic acid, human epidermal growth factor receptor-2 (HER2), transferrin, and epidermal growth factor receptor (EGFR)) [2]. The functionalization of mesoporous silica nanoparticles with various organic and inorganic molecules, polymers, surface-attachment of other nanoparticles, loading and entrapping cargo molecules with on-demand release capabilities, opens up limitless possibilities for designing advanced nanoconstructs with multiple functions, like simultaneous cancer-targeting, imaging, and therapy [13]. Likewise, silica-polymer nanohybrid, silica-protein nanohybrid, silica-nucleic acid nanohybrid, silica peptide nanohybrid, and silica magnetic nanohybrid are broadly used for drug delivery applications. This chapter provides a detailed understanding of these systems and their role in drug delivery.

8.2 STRATEGIES FOR FUNCTIONALIZATION OF SILICA HYBRID NANOCARRIERS Functionalization of nanocarriers enables to achieve control over the interactions between the nano­ carrier and the biological system for effective utilization of inorganic materials in drug delivery. Functionalization improves the payload binding capacity and enhances cellular internalization which are essential for effective intracellular drug delivery [14]. Moreover, silica hybrids have also proved effective for diagnosis. Prasad et al. reported a thorough in vivo evaluation of folic acid functionalized gold nanorods embedded mesoporous silica nanohybrids (GNR-MS-FA) for localized 4T1 breast tumor diagnosis, organ safety, and excretion following a single dose administration of nanohybrid [15]. Multifunctional cellular targeting, drug delivery, and molecular imaging with spatiotemporal resolution can also be achieved using integrated mesoporous silica systems. The nanostructure of a mesoporous-silica nanosphere with an optical gold nanocrescent antenna (MONA) has been investi­ gated for this purpose [16]. A pH-responsive aspartic acid functionalized hybrid nanocarrier composed of PEGylated mesoporous silica nanoparticle and graphene oxide was investigated for the in-vitro curcumin delivery to MCF-7 breast cancer cells. The high pore volume and hydrothermal stability of silica when combined with the inherent characteristics of graphene oxide due to its carboxyl, epoxy, and hydroxyl functional groups confer it the ability to be functionalized with drugs for biological applications. Further, the end-groups of silica are tailorable to achieve fine-tuning of drug encapsulation and targeted release characteristics. The size of the curcumin-loaded nanohybrid was

136 Nanomaterials in Healthcare

FIGURE 8.1 Schematic representation for the development of curcumin-loaded aspartic acid functionalized nanohybrid.

found to be 75.8 nm and the loading efficiency was 67±1.5%. A higher drug release was achieved at low acidic pH (90% after 100 h) than in normal pH (56% after 100 h), making the nanohybrid highly suitable for cancer therapy [14]. Figure 8.1 represents the development of curcumin loaded aspartic acid functionalized nanohybrid. Functionalization may result in alteration of the surface characteristics, such as hydrophilic or hydrophobic properties, and can also influence the chemical affinities towards drug, which in turn will affect drug adsorption and release. An attempt towards loading of the anti-inflammatory drug bude­ sonide, onto amino-functionalized mesoporous silica nanoparticles coated with the bio-adhesive polymer carbopol, resulted in an enhancement of the drug loading efficiency with a sustained release profile [17]. Various strategies have been adopted for the functionalization of silica hybrid nanocarriers. Solidphase peptide synthesis (SPPS) is one of the strategies that has been employed for the functionalization of porous silica hybrids with amino acids. SPPS allows specific functionalization of the surface corre­ sponding to the inner pores that enable tuning of surface polarity. The mesoporous SBA-15 silica surface contains an NH2 or COOH group that allows step-by-step covalent coupling of each amino acid to give the desired peptide sequence. Coupling takes place through a reaction between the C-terminus of the amino acid with the NH2 functional group located on the surface. The N-terminus of amino acid is capped with a Fmoc protection group to avoid multiple coupling reactions. Once the amino acid gets attached to the silica support, cleavage of the N-terminal Fmoc group takes place, facilitating the cou­ pling of the next amino acid to the NH2 group of the amino acid that was attached initially. The monitoring of each reaction can be achieved by 13C solid-state NMR spectroscopy. Thus, SPPS acts as a powerful and significant tool for the functionalization of silica nanohybrids [18]. Functionalization of silica nanohybrids can also be achieved through an in-situ co-condensation approach. The high density of silanol groups located on the surface of silica can be covalently modified

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 137 with organic groups such as the silanes ((RO)3SiR’). In this approach, the organic groups are added during the synthesis of mesoporous silica, such that these groups can be grafted to both the inner and outer surfaces of the silica walls [19]. The co-condensation approach provides a uniform and homog­ enous surface coverage of the organic groups without blockage of the mesopores. It is characterized by a low consumption of organic precursors and a simplicity in approach. The functionalization of 3-aminopropyltrimethoxy-silane to SBA-15 by co-condensation has been explored for use as a Cu2+ ion adsorbent in aqueous solutions. The mesoporous adsorbent exhibited good efficiency and sensitivity for the removal of copper from dilute solutions, that in turn finds numerous practical applications in the prevention of copper toxicity [20]. Further, the co-condensation of various molar ratios of tetra­ ethoxysilane (TEOS) with aminopropyltriethoxysilane (APTES) was achieved under an acidic environment, in the presence of a triblock copolymer P123 for the functionalization of mesoporous SBA15. A molar ratio of TEOS: APTES in the range of 15:1 and 20:1 yielded a highly ordered pore structure, hexagonal in shape, with a uniform size of about 90 Å [21]. The amine-functionalized mesoporous silica solid support finds a vast range of applications in immobilization of enzymes, DNA, antibodies, and cells. The huge specific surface area (around 1,000 m2/g), highly defined pore structure and controllable pore dimensions (20–500 Å) make it ideal for immobilization. In fact, immobilization of the biocatalyst lipases on n-propyl group and methyl group modified silica matrices have been attempted by researchers. The immobilized enzyme exhibited a higher activity by a factor of 88 when compared to commercially available enzyme powders. High retention of the enzymatic activity was concluded based on stability studies of the immobilized lipases in both reaction conditions (aqueous or organic medium) as well as storage conditions (dry) [22]. Post-grafting is one of the functionalization approaches in which organic functional groups like organosilanes are introduced to the surface of preformed silica. The functional group distribution on the pore surface is heterogenous as the accessibility of organosilanes to surface silanols is influenced by steric factors. However, well-defined structures are obtained and, since the functional groups are located only on the outer surface of mesoporous silica, a high level of functionalization can be obtained com­ pared to the co-condensation approach [23]. Novel luminescent (3-aminopropyl) triethoxysilane func­ tionalized dense nano-silica hybrid materials (DNSS) loaded with 1,8-napthalic anhydride have been fabricated by the post-grafting approach. Luminescent materials are widely employed to track the efficiency of systems to deliver the drugs to the targeted site of action. Self-quenching generally hinders the luminescence of organic-inorganic hybrids. However, amino-functionalization of the nano-silica spheres have resulted in the successful loading of luminescent molecules. Modification with APTES was found to exert a significant influence on the luminescence properties, paving the way for their potential use as novel-tracers [24]. Synthetic equivalents that can serve as new alternatives to the conventional trialkoxysilanes to achieve post-grafting of silica surfaces are emerging rapidly. These synthetic equivalents overcome the limitations associated with trialkoxysilanes such as slow reaction rates and sensitivity to hydrolysis. It is also difficult to attain the high-purity alkoxysilanes that are a prerequisite to controlling the surface functionalization. Large-sized alkoxysilanes cannot be purified by distillation. Hence, synthetic equivalents such as silazanes, allylsilane, arylsilane, vinylsilane, and methallyl have been employed to achieve silylation of silica surfaces that promotes new opportunities in the area of organic-inorganic hybrid materials [25]. One of the unique approaches to achieve modification of mesoporous organic-inorganic hybrids is the preparation of periodic mesoporous organosilicates (PMO). It entails the use of bridged silse­ quioxanes such as bis(triethoxysilyl)ethene as silica precursors, allowing the addition of organic func­ tional groups without any possibility of pore blocking. Chemical modification has been demonstrated through the bromination of ethene groups present in the pore wall. Thus, by utilizing the abovementioned techniques, complicated functional groups can be immobilized in the mesoporous environ­ ment for numerous uses in the ever-growing domain of nanosciences and nanotechnology [25]. Figure 8.2 summarizes the functionalization approaches of silica nanohybrids and their benefits.

138 Nanomaterials in Healthcare

FIGURE 8.2 Functionalization approaches of silica nanohybrids and their benefits.

8.3 DRUG DELIVERY APPLICATIONS OF SILICA NANOHYBRID 8.3.1 Silica Polymer Nanohybrid for Drug Delivery Mesoporous silica nanohybrids (MSNs) have attracted a lot of interest because of their exceptional characteristics like stable mesostructured and tailorable pore sizes, facile synthesis, thermal stability, biocompatibility, high load capacity, and ease of functionalization of its surface. In this technique, targeted and controlled drug delivery is achieved via surface modification of MSNs by the coating of various functional polymers which may show responses concerning pH, temperature, photoirradiation, redox changes, competitive binding, and enzyme [26–28]. These coated polymers also reduce toxicity due to the silanol group present in the MSNs [29]. Doxorubicin-loaded MSNs with a coating of gelatin have been explored for treating cancer as gelatin is a natural polymer that undergoes cleavage when exposed to the matrix metalloproteinase expressed by cancer cells. To hold the gelatin layer over doxorubicin-loaded MSNs, cross-linking was achieved through N, N’-bis(acryloyl)cystamine, a disulfide cross-linker. The accumulation of intra­ cellular doxorubicin released from MSNs increased by 1.5-fold in comparison to free doxorubicin, as concluded from the cellular uptake studies in A549 cells [26]. Niclosamide is a drug with multiple therapeutic applications, also used in the treatment of COVID-19, but has limitations due to its low bioavailability. Here, the MSNs approach can be utilized for controlled drug delivery. Niclosamide has been loaded into ordered mesoporous silica like PG (geopolymer), SBA-15 (Santa Barbara Amorphous-15), and MCM-41 (Mobil Composition of Matter No. 41) to form nanohybrids that were further coated with tween 60. Prepared silica nanohybrids showed improved bioavailability and controlled niclosamide release [30]. In another study, MSNs showed dual functionality of intracellular

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 139 doxorubicin release and subsequent internalization providing site-specific delivery of doxorubicin. To achieve this, pH-responsive polymer morpholin-4-yl-acetyl-poly (ethylene glycol)-b-poly (lactic acid) (MOP) was used along with capping by grafting the surface of MSNs using acid-labile hydrazone bonds. The prepared MSN-hyd-MOP@Dox showed less drug leakage in a normal cellular environment and also improved drug release in tumor cell environments [31]. The MSNs have been widely studied in recent times, which are listed in Table 8.1.

8.3.2 Silica Nucleic Acid Nanohybrid for Drug Delivery To treat genetic diseases, MSNs can be used as a carrier for nucleic acid–based reagents [32]. MSNs conjugated with nucleic acids are gaining attention due to their targeted delivery and absence of pre­ mature drug release [33,34]. MSNs with nucleic acids show high specificity towards complementary strands, robustness, and as well chemically stable, therefore they have various applications when combined with advanced development processes like aptamer-mediated active vectorization, advanced sensor development, and building stimuli-responsive molecular gates [35]. NIR-responsive DNA-hybrid-gated nanocarrier composed of Cu1.8S coated mesoporous silica was prepared by Zhang et al. Mesoporous silica structure loaded with curcumin (hydrophobic drug) and modified-aptamer two-stranded DNA (GC rich aptamer), capable of holding doxorubicin (hydrophilic drug). These two kinds of drugs with synergistic effects were formulated as a nanohybrid structure and exposure to photothermal agents resulted in the denaturation of strands of DNA, ultimately leading to the release of doxorubicin and curcumin [34]. Aptamer-based ATP responsive MSNs hold a promising potential in targeted delivery. In a study, a model dye Ru(bipy)32+ was incorporated into the MSNs and further surface coating of ATP aptamer was achieved to prevent the leakage of the model dye. The ATP aptamer was obtained from two ssDNA arms that were cross-bred for producing a double-stranded DNA structure. The author concluded that these drug delivery systems can be utilized for the successful delivery of anticancer agents because of the higher ATP concentration in the tumor environment [33]. Folic acid-functionalized umbelliferone MSNs coated with polyacrylic acid (Umbe@MSN-PAAFA) developed MSNs showed targeted drug delivery with controlled release and reduced systemic toxicity. Here, folic acid grafting provides targeted delivery by binding to folate receptors in cancerous cells, and polyacrylic acid, which is a pH-responsive polymer, provides controlled release of umbelli­ ferone and also reduces systemic toxicity [36].

8.4 SILICA PROTEIN NANOHYBRID FOR DRUG DELIVERY The phenomenon of protein adsorption on solid surfaces is widely investigated for its use as bio­ composite materials in the area of nano-technology and material sciences. The interactions between the protein and solid surface confer biocompatibility to the nanocarrier that can be utilized in drug delivery applications. Proper control of the support surface is highly essential in order to enhance the selectivity and efficiency of protein adsorption. The chemical nature of the biomaterial, surface morphology, and texture are some of the important characteristics that influence the stability and adhesion of the protein layer. Mesoporous silica due to their unique surface properties like high specific surface area, porosity, as well as functionalization capacity hold a promising potential for adsorption of proteins to form hybrid nanocarriers [42]. The applicability of silica-protein hybrids as drug-delivery nanocarriers have been investigated in various studies. Silica-coated human serum albumin nanoparticles have been explored for achieving the

3.

2.

1.

SR. NO. TARGET SITE

STRATEGY

REMARKS

Doxorubicin (Anticancer) MSNs

A. Silica polymer nanohybrid for drug delivery Tumor GSH and temperature are Folic acid helps in MSN@PBLGF (MSNs coated with poly(gtargeted the two stimuli that targeted delivery, and benzyl-L-glutamate), before cause controlled and also makes the system targeted drug release more biocompatible coating folic acid is attached with PBLG. MSNs Surface treated MSNs with Tumor Gelatin cleaved by matrix – cross-linked gelatin and N, targeted metalloproteinase 2 N′-bis(acryloyl) enzyme in the tumor cystamine (BAC) environment and BAC provides enzymatic and redox sensitivity for release of DOX MSNs Perylene-functionalized Tumor pH and visible lightDual stimuli result into poly(dimethylaminoethyl targeted sensitive polymers increased anticancer methacrylates) MSNs activity by increasing the concentration of doxorubicin in cancer cells Doxorubicin (Anticancer) Dox-loaded Tumor pH-dependent controlled High drug loading PNS (Porous Nanosilica nanohybrids) PNS@Chitosan-PEG targeted drug delivery capacity and also releases drug in controlled manner Ibuprofen Chitosan-coated Ibuprofen pH dependent Ibuprofen release in mild – (As Model Drug) laden MSNs drug release acidic condition as of ibuprofen chitosan dissolves and tailored drug release profile

NANOHYBRID SYSTEM

Applications of MSNs in various delivery system

DRUG NAME WITH CATEGORY

TABLE 8.1

[ 38]

[ 27]

[ 28]

[ 26]

[ 37]

REFERENCE

140 Nanomaterials in Healthcare

Zinc (Antibacterial Agent)

Immunoadjuvants drug delivery CpG ODN’s (CytosinephosphodiesterguanineOligodeoxynucleotides)

4.

1.

–

Antibacterial activity is Silica and HPC were [ 39] due to the generation of used to form sol-to-gel oxygen reactive species system and prepared nanohybrid system showed antibacterial activity and improved biocompatibility [ 40] Zinc oxide–silica nanohybrid – Antibacterial activity is ZnOSiO2 Geopolymer based sustainable due to the generation of nanohybrid system geopolymer (GMZnO–Si) oxygen reactive species provides resistance against biodeterioration and also good mechanical and structural performance B. Silica nucleic acid nanohybrids Chitosan–silica/CpG Immune The nanohybrid system Sustained release of [ 41] oligodeoxynucleotide system shows good CpG ODN’s nanohybrid activator biocompatibility, cellular uptake capacity, and induction of interleukin6 mediated via toll like receptor 9

Zinc-doped silica and hydroxypropyl cellulose (HPC) nanohybrids

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 141

142 Nanomaterials in Healthcare

FIGURE 8.3 Demonstration of silica coated human serum albumin nanoparticles for the sustained release of ruthenium polypyridyl complexes.

sustained release of ruthenium polypyridyl complexes (Figure 8.3). The loading efficiency of the drug in human serum albumin nanoparticles (HSANP) increased from 52% to about 90% upon coating with a small quantity of silica, and about 100% of loading was achieved with increasing amounts of silica. Generally, hydrophilic drugs that are incorporated in HSA nanoparticles tend to undergo burst release. However, silica-coated HSANP was able to prevent the initial burst release of the ruthenium complexes, despite its hydrophilic nature. Further, a controlled release rate was achieved by varying the amount of silica coating. Also, encapsulation of the photosensitive ruthenium complexes in silica-coated nano­ particles protected them from light-induced degradation. The Ru-HSA/SiO2 nanosystem, internalized by clathrin-mediated endocytosis, was able to successfully deliver the complex into cells, making it an ideal and versatile nanocarrier for bio-imaging and photodynamic therapy [43]. Multidrug resistance associated with cancer can be overcome by encapsulation of the drug in silica nanoparticles. Sericin-coated MSNs loaded with doxorubicin have been explored for lysosome-targeted delivery. Sericin, a protein obtained from cocoons of silkworms can be coated to MSNs (SMSNs) through pH labile imine linkages. Sericin coating avoids the leakage of drugs in the extracellular environment and, owing to its cell-adhesive property, it promotes the cellular uptake of SMSNs on reaching drug-resistant tumors. Then, the SMSNs are further transported into perinuclear lysosomes, whose acidic pH initiates the imine linkage cleavage between sericin and MSNs. In addition, lysosomal proteases cause the degradation of the sericin shell. Thus, the pH/protease dual responsiveness is responsible for the burst release of doxorubicin into the nuclei of the cell. The in-vivo efficacy and robustness of the SMSNs is evident by a significant reduction in tumor growth that has been achieved in an animal model expressing drug-resistant breast cancer. Further, no signs of cardiotoxicity have been observed on systemic administration of SMSNs, thus overcoming the major limitation of doxorubicinmediated chemotherapy [44]. MSNs functionalized with protease-sensitive avidin caps were looked into for their capability to be used as drug cargoes in the therapy of chronic lung diseases such as cancer. The high protease

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 143 concentration in lung tumor areas causes cleavage of the avidin caps that facilitates the delivery of chemotherapeutic agents with high tumor-selectivity [45]. Further, controlled drug release from the nanocarriers can be achieved in a spatiotemporal manner based on the responsiveness towards diseasespecific enzymes. For example, an upregulation in the expression of the enzyme matrix metalloproteinase-9 occurs in tumor cells which enhances its metastatic potency. Targeted drug delivery in such areas can be attained by capping avidin molecules to MSNs through MMP-9 sequence-specific linkers. MMP-9 trig­ gered delivery of cisplatin from MSNs to ex-vivo 3D lung tumors has been successfully achieved in Kras mutant mice with no potential toxicity being observed, thus demonstrating a site-specific drug delivery [46]. Biotin-avidin capped MSNs have been explored for site-specific delivery of the immunostimulant drug resiquimod to specialized antigen-presenting cells. Stimuli-responsive drug delivery occurred at a pH of 5.5 or below as a consequence of lysosomal uptake that subsequently resulted in the activation of an antigen-specific T-cell response. A sixfold improvement in the half-life of resiquimod was achieved. Hence, these nanocarriers aid as a promising tool for the delivery of immunostimulants, including vaccines [47]. Bilayer-coated MSNs find their application in the controlled delivery of anti-cancer drugs such as gemcitabine. Bilayer coating using a pH-responsive polymer named poly (acrylic acid-co-itaconic acid) as the inner shell, and human serum albumin as the outer shell has been designed for gemcitabine-loaded nanoparticles. HSA confers colloidal stability and biocompatibility to the nanocarrier and also facilitates the uptake of nanoparticles that can be accredited to enhanced permeation and retention effect. Maximum release of the drug from the pores of mesoporous silica was achievable at endosomal pH [48]. Further, pH-responsive casein-based silica nano-composite films loaded with the drug ibuprofen have also demonstrated an enhancement in the drug loading efficiency, depicting enhanced adsorption of drugs onto silica within the porous casein matrix [49]. Functionalized silica nanoparticles are being extensively explored for targeted intracellular protein delivery. Aldehyde-functionalized MSNs loaded with the protein arginase via imine linkages were developed. The silica-protein nanocomposites were uptaken site-specifically into the lysosomes of various cells, such as HeLa, HepG2, and L929 cells. Due to the pH-responsiveness of the nano­ composite, the release of the adsorbed protein was triggered by the hydrolysis of imine linkages under the influence of the acidic environment of lysosomes. Further, cytosolic delivery of the protein was attained as a consequence of endolysosomal escape. Thus, aldehyde functionalized MSNs hold good potential for their applicability as a vector in the cytosolic delivery of proteins [50]. However, in a majority of the studies that have been done so far, the proteins are adsorbed on the surface due to the small size of the pores that prevent protein diffusion and, in turn, limit the efficiency of drug loading. To surpass this limitation, dendritic MSNs (DMSNs) have received a lot of focus recently, which because of their center-radial pore structures can accommodate large molecular-sized proteins inside them. pH-responsive aldehyde functionalized-DMSNs with a mean particle size of 174±17 nm and a mesopore size of 7.7 nm were developed. Loading of bovine serum albumin (BSA) into the aldehyde functiona­ lized DMSNs is achievable through the imine bond formation between the aldehyde groups of the nanocarrier and the primary amine group of BSA. The loading efficiency of the protein was found to be around 136 µg/mg and an acidic medium triggered the release of the protein. Further, no cytotoxicity was seen, making them promising nanocarriers for pH-sensitive protein drug delivery [51]. Native proteins, particularly negatively charged large-sized proteins are not permeable to the cell membrane, which necessitates the need for efficient delivery platforms for intracellular delivery. Novel hollow dendritic MSNs (A-HDMSN) functionalized with amine groups have been explored using the vesicle supraassembly approach, for loading β-galactosidase, in which composite vesicles acted as building blocks for further assembly into the final product. The developed A-HDMSN possessed a cavity core of around 170 nm with a characteristic mesopore size of around 20.7 nm and a high pore volume of 2.63 cm3 gm−1. A-HDMSN demonstrated an enhanced loading capacity of the negatively charged protein, β-galactosidase and a 40-fold increase in the cellular uptake of β-galactosidase was observed in com­ parison to free-galactosidase making it a versatile hybrid nanocarrier for the intracellular delivery of crucial therapeutic proteins [52].

144 Nanomaterials in Healthcare

8.5 SILICA PEPTIDE NANOHYBRID FOR DRUG DELIVERY Peptide-functionalized mesoporous-silica nanohybrids have been extensively studied in numerous bio­ medical applications owing to their biocompatibility. Silica-peptide nanohybrids obtained by the click reaction between azido-MSNs and alkynyl peptide dendrons, possess no in-vitro cytotoxicity, as assessed against various cell lines using the CCK-8 assay. Further, the in-vivo safety is also well established. Thus, the good biocompatibility of the nanohybrids makes them a potential tool for future biomedical applications [53]. These nanohybrids have been investigated for use as imaging probes by labeling them with Cy5.5 dye. MSN-peptide dendron nanohybrids provide an enhanced sensitivity in near-infrared fluorescence imaging as it enables precise filtering and passage of selective fluorescent light to the detector. The organic dye Cy5.5 reduces background interference as it possesses a fluorescence excitation and emission wavelength of 673 nm and 692 nm, respectively, that bypasses the absorbance spectra of biological tissues, blood, and plasma. The nanohybrids possessed a diameter of 60 nm with a negative surface charge. The biodistribution of the MSN-peptide dendron-Cy5.5 based nanohybrid was studied through ex-vivo fluorescence imaging, which in turn proved its potential utility as a functional vehicle in diagnosis [54]. MSNs in combination with peptide dendrons have also been explored for use as MRI contrast agents. Gadolinium-loaded MSNs functionalized with peptide dendrons owing to its highly paramagnetic nature and long electron relaxation time, is a commonly used contrast agent. However, its small molecular size results in low sensitivity, poor image enhancement, and rapid excretion from the body. Hence, in order to surpass the aforementioned limitations, chelates of gadolinium with MSNs are emerging that increase the paramagnetic relaxation of surrounding water molecules, thus promoting image enhancement. In fact, a 11-fold increase in relaxivity has been observed due to the nanohybrid. Further, the biosafety of nanohybrids is established through in-vitro and in-vivo toxicity tests enabling them to be used as a potential MRI contrast agent [55]. Targeted drug delivery through silica-peptide nanohybrids has been achieved in various investi­ gations, where the release of the payload occurs under endogenous conditions like pH, temperature, ATP levels, hypoxia, etc. A novel type of MSN functionalized with a peptide comprising of a charge-reversal plug gate nanovalve has been explored for the site-specific delivery of the anticancer drug doxorubicin. K8 peptide with an octa-lysine sequence was initially functionalized on the outer shell of MSNs by click chemistry and subsequently allowed to react with citraconic anhydride to generate a negatively charged MSN-K8(Cit). Further, to the negatively charged surface, a cationic K8(RGD)2 peptide composed of two units of Arg-Gly-Asp (RGD) sequences was incorporated through electrostatic interactions to form the plug gate nanovalve. The obtained nanocarrier demonstrated optimum stability under physiological pH conditions as the plug gate nanovalves were closed. At pH 5 (endolysosomal conditions), the hydrolysis of acid-labile amides caused a charge reversal from -41 mV to +19 mV that induced the opening of plug gate nanovalves, resulting in the release of doxorubicin. At an acidic pH, 79% of the loaded drug was released within 48 h. In-vitro experiments confirmed the effectiveness of the nanocarrier through its inhibitory effect on αvβ3-positive U87 MG cancer cells [56]. Doxorubicin-loaded nano-donuts of organosilica particles functionalized with a ring-shaped short peptide sequence tri-L-lysine are found to exhibit a high cellular uptake and upon internalization into cancer cells. The structural breakdown of the nanocarriers is triggered due to the interaction of the peptide sequence with peptidases. The subsequent release of doxorubicin shows enhanced cytotoxicity against HeLa cells [56]. Hybrids of histone H2A peptide-functionalized MSNs have been explored for the co-delivery of bortezomib and p53 gene. The conjunction of the cationic peptide histone H2A creates a positive surface charge to absorb the negatively charged therapeutic gene. Further, in addition to the enhancement of gene encapsulation, the nanocarrier exhibited an increase in transfection efficiency. Generally, mutations in the endogenous p53 gene are associated with tumor progression, which in turn necessitates the need for exogenous p53 gene therapy for the treatment of cancer. The p53 gene causes

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 145 tumor regression through apoptosis induction, cell cycle arrest, and alternation of metabolism. Further, loading of the drug bortezomib, a specific inhibitor of 26S proteosome produces a synergistic effect in the induction of apoptosis. Thus, the co-delivery of the drug bortezomib and p53 gene holds a promising therapeutic value in clinical cancer therapy [57]. MSNs are emerging as successful carriers for the delivery of antimicrobial peptides. T7E21R-HD5, a human defensin bactericidal peptide, has been incorporated into succinylated casein-coated MSNs for treating bacterial infections of the intestine. The MSNs enhance the membrane permeabilization ability of T7E21R-HD5, thus intensifying its antibacterial action against multi-drug resistant E. coli. Modification with succinylated casein promotes specific degradation by intestinal proteases, that prevents the loss of the peptide in an extremely acidic environment of the stomach. The nanohybrids are non-toxic to the host cells, making them biocompatible in nature [58]. MSNs loaded with the anti-mycobacterial peptide NZX are found to promote the inhibition of M. tuberculosis which primarily resides in the alveolar macrophages. The susceptibility of peptides to undergo degradation can be overcome by encapsulating them in MSNs, which in turn results in their efficient delivery to the site of action. The MSNs were capable of gradually releasing the functional peptide into simulated lung fluid, from where it was taken up into the primary macrophages. Further, in a murine infection model, the peptide containing MSNs exhibited enhanced mycobacterial killing in comparison to the free peptide [59]. MSNs have also been explored for bone tissue engineering applications. Silica-based nanocarriers possessing a mesoporous structure have been explored for the incorporation of a bone morphogenetic protein-7 derived peptide known as bone-forming peptide (BFP). The nitrogen adsorption-desorption isotherms suggested that the incorporation of peptide does not affect the mesoporous structure of MSNs. In-vitro cytocompatibility tests suggest a higher proliferation rate of MG-63 cells and increased alkaline phosphatase activity with the peptide-laden MSNs in comparison to bare MSNs. Further, osteodifferentiation of human mesenchymal stem cells was promoted owing to the release of BFP from the MSNs at a concentration of 500 µg/ml. Thus, peptide-laden MSNs can serve as potential candidates for utilization in bone repair, regeneration, and implant-coating applications owing to their enhanced osteodifferentiation ability [60].

8.6 SILICA QUANTUM DOT FOR DRUG DELIVERY Quantum dots, QDs (also referred to as fluorescent nanocrystals (NCs), are fluorescence-type semi­ conductor nanoparticles in the size range of 2–10 nm. They are made up of either heavy metals or inorganic materials, including cadmium, lead, and mercury. Structurally, they comprise an inner core and an outer shell. The core is made up of semiconductor material and is surrounded by a shell in order to stabilize it. QDs have found significant biomedical and pharmaceutical applications including imaging, drug delivery, sensors, evaluation of biomarkers, and theragnostic [61–64]. However, QDs suffer from some major limitations, which include toxicity to healthy cells, low aqueous solubility, and therefore low compatibility with the physiological environment. Silica (Silicon dioxide, SiO2) can be utilized as an important coating material in this regard as it retards the coagulation of coated nanocrystals in aqueous dispersion [65]. Graphene-conjugated hybrid SiO2-coated quantum dots (HQDs) were developed for targeted cancer fluorescent imaging, drug delivery monitoring, and cancer therapy. The coating of SiO2 on QDs not only helps in reducing their toxicity but also protects their fluorescence from being reduced by graphene. The scientists functionalized the surface of graphene-HQDs with transferrin (Trf), which can be utilized as a targeted imaging system that is capable of differential uptake and imaging of tumor cells that express the Trf receptor. The antineoplastic drug, Doxorubicin was adsorbed on the surface of graphene and showed a higher loading capacity of 1.4 mg mg−1. The results of the study revealed that this nanoparticulate system

146 Nanomaterials in Healthcare can deliver doxorubicin to the targeted cancer cells and intracellular doxorubicin release can be monitored. In-vitro studies of nanoparticle conjugate demonstrated their specificity and safety in cancer imaging, monitoring, and therapy [66]. Likewise, Prasad et al. designed a novel bioresponsive dual functional (conjugated with cystamine and folic acid) green fluorescent carbon QDs functionalized mesoporous silica nanoparticles-based nano-theranostic agent for targeted bio-imaging, prevention of premature drug release, and bio-responsive drug release. The doxorubicin-loaded nanocarrier releases the drug on exposure to the mimicked intracellular cancerous environment i.e., acidic pH and elevated levels of glutathione [67]. Similarly, in another study, hybrid silica-coated Gd-Zn-Cu-In-S/ZnS bimodal QDs were developed as a nanocarrier to deliver doxorubicin to breast cancer T41 cells. They coated QDs with mesoporous silica and further functionalized its surface by amino groups. The antineoplastic drug doxorubicin was then incorporated into the silica pores, and further biheterofunctional PEG was covalently attached to the surface of the core-shell QD-MSNs. Epithelial cell adhesion molecule (EpCAM) DNA aptamer was attached to the surface of doxorubicin-loaded PEGylated QDs-MSN for targeting cancer cells. Their prepared nanocarrier systems were further analyzed for particle size, particle size distribution, zeta potential, surface morphology, and magnetic susceptibility. The results of the study revealed that nanocarriers were spherical in shape with an average particle size of 100 nm and showed an encapsulation efficiency of 25%. In vitro release studies demonstrated the pH-sensitive release of doxorubicin from the nanocarrier. In-vitro cytotoxicity data showed that aptamer-targeted nanoparticles showed greater cytotoxicity towards cancer cells in comparison to nanoparticles without aptamer and free doxorubicin. Also, doxorubicin-loaded nanoparticles showed reduced tumor growth and an increased survival rate of cells. Thus, from this study, they concluded that the developed nanocarrier can be utilized as a theragnostic agent for the treatment and diagnosis of breast cancer [68]. MSNs capped with graphene QDs was utilized as a novel photothermal and redox-responsive drug delivery carrier for treating cancer. Redox potential as a stimulus for stimuli-sensitive drug delivery system has been widely utilized for drug release in cancer cells because of the significant difference of glutathione (GSH) concentration between the intracellular (10 mM) and extracellular (2–10 μM) environment of the cancer cells. In a study, researchers initially modified MSNs to form aminated MSNs (MSN-NH2). They were further reacted with cysteine, which led to the introduction of disulfide bonds in aminated MSNs (MSNs-SS-NH2). Drug (Rhodamine B, a red fluorescent dye) was loaded into the pores of MSNs-SS-NH2 and capped with graphene QDs via amidation reaction, to prevent the drug release. The formed nanocomposite, i.e., MSNs capped with GQDs had photo-thermal properties of graphene QDs. The disulfide bonds present in the nanocomposite break down in the presence of a particular concentration of glutathione leading to the release of drug from the pores of MSNs. Hence, drug release from the nanocomposite can be controlled and it was concluded that this newly developed drug delivery system can be used as a potential candidate for stimuli-responsive drug delivery [69].

8.7 SILICA MAGNETIC NANOHYBRIDS FOR DRUG DELIVERY Magnetic hybrid nanostructures have recently gained significant attention as nanocarriers for drug delivery, contrast agents, and in theragnostic applications because of their unique physicochemical properties. They comprise of a mixture of two or more components that are incorporated together in a magnetic material to form a single system that has enhanced multifunctional properties. Designing of various hybrid nanostructures entirely depends on the physicochemical as well as internal and external interfacial properties of individual components. Various structural modifications are performed on the surfaces of magnetic nanohybrids by using biodegradable materials like silica, targeting ligands, etc., to enhance their biocompatibility [70].

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 147 Radial mesoporous silica hybridized with magnetic nanoparticles was developed as a novel drug delivery carrier for MRI-guided and alternative magnetic field (AMF) responsive chemotherapy for breast cancer treatment. Super-paramagnetic iron oxide nanoparticles (SPIONs) were prepared via hydrothermal and thermal decomposition methods. Further, mesoporous silica spheres were grown on their surface and doxorubicin was incorporated within the radially oriented mesochannels. For effective targeting, the sur­ face of doxorubicin-loaded nanohybrids was incorporated with folic acid (FA). The prepared nanohybrids were analyzed for their morphology, drug loading, in-vitro cytotoxicity, and in-vivo antineoplastic activity. The prepared nanocomposites demonstrated significantly higher drug loading with stimuli-responsive release properties. According to the findings of this study, it was concluded that the prepared nanohybrid be utilized as a potential theragnostic agent for the chemotherapy of cancer [71]. Yang et al. synthesized polyglycerol-mediated covalently constructed magnetic mesoporous silica nanohybrids (MMSNs), which consisted of MSNs as the core and many SPIONs attached to its outer surface, as a nanocarrier for drug delivery. The prepared nanohybrid possessed the intrinsic porosity of MSNs and the magnetic properties of SPIONs. The SPIONs were first grafted with polyglycerol, which enhanced the hydrophilicity of SPIONs and also provided a hydroxyl (-OH) group for further deriva­ tization. It also enhanced the colloidal stability of prepared nanohybrids. The synthesized nanohybrids showed good aqueous dispersibility. Chlorine-6 (Ce6) was loaded into the MMSN (Ce6@MMSNs) and was effectively delivered into target cells under the influence of the magnetic field, thus showing a significant improvement in photodynamic therapy (PDT) [72]. Modified MMSNPs encapsulated with quercetin (QC), as shown in Figure 8.4, were developed and investigated for their anti-amyloid and antioxidant activity. MMSNPs were prepared to employ the sol-gel-sol method and were analyzed for their size and shape, zeta potential, and entrapment efficiency. The nanocarrier showed significant stability in biological/physiological media and encapsulated QC showed higher solubility in comparison to the free flavonoid, as demonstrated by results of drug loading and entrapment efficiency. In-vitro release studies showed higher bioavailability of QC. Data obtained

FIGURE 8.4 Schematic illustration QC-loaded MMSNP.

148 Nanomaterials in Healthcare

FIGURE 8.5 Concepts of tumor cell-activated therapy using IONP@MSN/DOX-DNA.

from biological evaluation studies confirmed the feasibility of magnetically directed delivery of QC to show its various pharmacological activities. From this study, it was determined that MMSNPs can be used as an effective nanocarrier for bioactive flavonoids [73]. Liu et al. developed a magnetic-targeted nano platform by capping mesoporous silica-coated iron oxide nanoparticles (IONPs) with programmable DNA hairpin sensor “gates.” This nano platform has the capability to entrap the loaded drug in healthy cells (HL-7702, human liver cells) and the platform was considered to be in an “OFF state.” However, in the case of tumor cells ((HepG2, human liver tumor cells)44s, this nano platform gets activated due to the overexpression of miRNA-21 via hybridization with DNA hairpin, which results in rapid drug release, and the nano platform is in an “ON state,” as shown in Figure 8.5. IONPs have a comparatively low drug-loading capacity, so in order to enhance this, nanoparticles with a core made of IONP and a shell of MSNs were prepared. These developed nano­ particles had both higher drug loading and magnetic properties, which make them suitable nanocarriers for tumor targeting. They can be targeted to the tumor site via the application of an external magnetic field. The prepared nano platform had a particle size of around 170 nm and a zeta potential of -30mV. In vitro and in vivo studies showed high antitumor efficiency and low toxicity because of its magnetic targeting and tumor-cell activating properties. Thus, it can be concluded that this nano platform can be used as a potential nanocarrier for personalized and targeted anticancer therapy [74].

8.8 CLINICAL TRIALS FOR SILICA-BASED NANOFORMULATIONS Silica-based nanoparticles are now under clinical trials for a wide variety of biomedical applications including drug delivery, diagnosis, and photothermal ablation ability. The data obtained from

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 149 TABLE 8.2 SR. NO. 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13.

Clinical trials on silica-based formulation [ 75] TYPE OF FORMULATION

Silica-gold-iron bearing nanoparticles Aurolase Gold NPs with silica-iron oxide Cornell dots 124 I-labelled Lipoceramic hybrid silica NP for oral ibuprofen delivery Cornell dots cRGDY-PEG-Cy5.5 Auroshell Mesoporous silica NP for oral fenofibrate delivery (Clinical study) Cornell dots 89 Zr-Crgdy-Cy5 Cornell dots NH2-PEG-Cy5.5 Lipoceramic hybrid silica NP for oral simvastatin delivery Auroshell Expected results from phase II clinical trials on silica NPs

CLINICAL TRIAL IDENTIFIER NCT01270139 NCT00848042 NCT01436123 NCT01266096

YEAR

STATUS

2007 2008 2010 2011 –

NCT02106598

Completed Completed Terminated Active, not recruiting 2013 Completed 2014 Active, Recruiting

NCT02680535 –

2016 Completed Completed

NCT03465618

2018 Active, Recruiting

NCT04167969

2019 Active, Recruiting

ACTRN12618001929291

2019 Completed

NCT04240639, NCT04656678 2020 Active, Recruiting NCT02106598 2023 Active, Recruiting

preliminary studies confirm the safety and efficacy of these nanoparticles. When it comes to oral drug delivery, the silica-lipid hybrid formulation is investigated to deliver ibuprofen in healthy volunteers, demonstrating twofold enhancement in bioavailability. They have also been used for plasmonic reso­ nance therapy to diagnose cardiovascular diseases, cancerous cells, and in photothermal ablation therapy for cancer [75]. Table 8.2 enlists different types of silica nanoparticles that were/are investigated in clinical trials.

8.9 CONCLUSION The role of silica-hybrid biomaterials is emerging rapidly in the field of nanomedicine owing to their ability to facilitate the encapsulated drug to reach the target site at desirable therapeutic concentrations, thus minimizing off-target effects. By employing various strategies for the functionalization of silica, such as the post-grafting and co-condensation approaches, a high payload binding capacity can be achieved that promotes intracellular delivery of the drug molecules. Further, the fabrication of inorganic silica with organic materials results in the synergism of the inherent characteristics of both the materials that constitute the delivery system, which enables effective drug delivery for various biological appli­ cations. Currently, though a majority of the research focuses on employing silica-nanohybrids for cancer therapeutics, in the near future, extensive investigation needs to be performed for extending its appli­ cation in various therapeutic domains. Conflict of Interest: Authors declare there is no conflict of interest involved in publishing this book chapter.

150 Nanomaterials in Healthcare

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152 Nanomaterials in Healthcare [47] Wagner J, Gößl D, Ustyanovska N, Xiong M, Hauser D, Zhuzhgova O, et al. Mesoporous silica nano­ particles as pH-responsive carrier for the immune-activating drug resiquimod enhance the local immune response in mice. ACS Nano. 2021 Mar 23;15(3):4450–4466. [48] Pourjavadi A, Tehrani ZM. Mesoporous silica nanoparticles with bilayer coating of poly(acrylic acid-coitaconic acid) and human serum albumin (HSA): A pH-sensitive carrier for gemcitabine delivery. Mater Sci Eng C. 2016 Apr 1;61:782–790. [49] Ma J, Xu Q, Zhou J, Zhang J, Zhang L, Tang H, et al. Synthesis and biological response of casein-based silica nano-composite film for drug delivery system. Colloids Surfaces B Biointerfaces. 2013 Nov 1;111:257–263. [50] Wu X, Wu S, Yang L, Han J, Han S. Cytosolic delivery of proteins mediated by aldehyde-displaying silica nanoparticles with pH-responsive characteristics. J Mater Chem. 2012 Jul 31;22(33):17121–17127. [51] Tian Z, Xu Y, Zhu Y. Aldehyde-functionalized dendritic mesoporous silica nanoparticles as potential nanocarriers for pH-responsive protein drug delivery. Mater Sci Eng C. 2017 Feb 1;71:452–459. [52] Meka AK, Abbaraju PL, Song H, Xu C, Zhang J, Zhang H, et al. A Vesicle supra-assembly approach to synthesize amine-functionalized hollow dendritic mesoporous silica nanospheres for protein delivery. Small. 2016 Oct 1;12(37):5169–5177. [53] Pan D, Guo C, Luo K, Gu Z. Preparation and biosafety evaluation of the peptide dendron functionalized mesoporous silica nanohybrid. Chinese J Chem. 2014 Jan 1;32(1):27–36. [54] Guo C, Hu J, Kao L, Pan D, Luo K, Li N, et al. Pepetide dendron-functionalized mesoporous silica nanoparticle-based nanohybrid: Biocompatibility and its potential as imaging probe. ACS Biomater Sci Eng. 2016 May 9;2(5):860–870. [55] Guo C, Hu J, Bains A, Pan D, Luo K, Li N, et al. The potential of peptide dendron functionalized and gadolinium loaded mesoporous silica nanoparticles as magnetic resonance imaging contrast agents. J Mater Chem B. 2016 Mar 23;4(13):2322–2331. [56] Luo GF, Chen WH, Liu Y, Zhang J, Cheng SX, Zhuo RX, et al. Charge-reversal plug gate nanovalves on peptide-functionalized mesoporous silica nanoparticles for targeted drug delivery. J Mater Chem B. 2013 Oct 3;1(41):5723–5732. [57] Rong J, Li P, Ge Y, Chen H, Wu J, Zhang R, et al. Histone H2A-peptide-hybrided upconversion meso­ porous silica nanoparticles for bortezomib/p53 delivery and apoptosis induction. Colloids Surfaces B Biointerfaces. 2020 Feb 1;186:110674. [58] Zhao G, Chen Y, He Y, Chen F, Gong Y, Chen S, et al. Succinylated casein-coated peptide-mesoporous silica nanoparticles as an antibiotic against intestinal bacterial infection. Biomater Sci. 2019 May 28;7(6):2440–2451. [59] Tenland E, Pochert A, Krishnan N, Rao KU, Kalsum S, Braun K, et al. Effective delivery of the antimycobacterial peptide NZX in mesoporous silica nanoparticles. PLoS One. 2019 Feb 1;14(2):e0212858. [60] Luo Z, Deng Y, Zhang R, Wang M, Bai Y, Zhao Q, et al. Peptide-laden mesoporous silica nanoparticles with promoted bioactivity and osteo-differentiation ability for bone tissue engineering. Colloids Surfaces B Biointerfaces. 2015 Jul 1;131:73–82. [61] Bajwa N, Mehra NK, Jain K, Jain NK. Pharmaceutical and biomedical applications of quantum dots. Artif Cells, Nanomed Biotechnol. 2016 Apr 2;44(3):758–768. [62] Matea CT, Mocan T, Tabaran F, Pop T, Mosteanu O, Puia C, et al. Quantum dots in imaging, drug delivery and sensor applications. Int J Nanomed. 2017 Jul 28;12:5421–5431. [63] Gidwani B, Sahu V, Shukla SS, Pandey R, Joshi V, Jain VK, et al. Quantum dots: Prospectives, toxicity, advances and applications. J Drug Deliv Sci Technol. 2021 Feb 1;61:102308. [64] Jha S, Mathur P, Ramteke S, Jain NK. Pharmaceutical potential of quantum dots. Artif Cells Nanomed Biotechnol. 2018;46(sup1):57–65. [65] Bernik D. Silicon based materials for drug delivery devices and implants. Recent Pat Nanotechnol. 2008 May 13;1(3):186–192. [66] Chen ML, He YJ, Chen XW, Wang JH. Quantum-dot-conjugated graphene as a probe for simultaneous cancer-targeted fluorescent imaging, tracking, and monitoring drug delivery. Bioconjug Chem. 2013 Mar 20;24(3):387–397. [67] Prasad R, Aiyer S, Chauhan DS, Srivastava R, Selvaraj K. Bioresponsive carbon nano-gated multi­ functional mesoporous silica for cancer theranostics. Nanoscale. 2016 Feb 28;8(8):4537–4546. [68] Akbarzadeh M, Babaei M, Abnous K, Taghdisi SM, Peivandi MT, Ramezani M, et al. Hybrid silica-coated Gd-Zn-Cu-In-S/ZnS bimodal quantum dots as an epithelial cell adhesion molecule targeted drug delivery and imaging system. Int J Pharm. 2019 Oct 30;570:118645.

8 • Porous Silica Nanoparticles for Targeted Bio-Imaging 153 [69] Gao Y, Zhong S, Xu L, He S, Dou Y, Zhao S, et al. Mesoporous silica nanoparticles capped with graphene quantum dots as multifunctional drug carriers for photo-thermal and redox-responsive release. Microporous Mesoporous Mater. 2019 Apr 1;278:130–137. [70] Iqbal MZ, Wu A. Magnetic nanohybrids for magnetic resonance imaging and phototherapy applications. In: Tissue Eng Nanotheranostics. Ed: Shi D, Liu Q. Singapore: World Scientific; 2017. [71] Gao Q, Xie W, Wang Y, Wang D, Guo Z, Gao F, et al. A theranostic nanocomposite system based on radial mesoporous silica hybridized with Fe3O4 nanoparticles for targeted magnetic field responsive chemo­ therapy of breast cancer. RSC Adv. 2018;8(8):4321–4328. [72] Yang X, Wen Y, Wu A, Xu M, Amano T, Zheng L, et al. Polyglycerol mediated covalent construction of magnetic mesoporous silica nanohybrid with aqueous dispersibility for drug delivery. Mater Sci Eng C. 2017 Nov 1;80:517–525. [73] Halevas E, Mavroidi B, Nday CM, Tang J, Smith GC, Boukos N, et al. Modified magnetic core-shell mesoporous silica nano-formulations with encapsulated quercetin exhibit anti-amyloid and antioxidant activity. J Inorg Biochem. 2020 Dec 1;213:111271. [74] Liu J, Liu W, Zhang K, Shi J, Zhang Z. A magnetic drug delivery system with “OFF–ON” state via specific molecular recognition and conformational changes for precise tumor therapy. Adv Healthc Mater. 2020 Feb 1;9(3):1901316. [75] Janjua TI, Cao Y, Yu C, Popat A. Clinical translation of silica nanoparticles. Nat Rev Mater. 2021 Oct 7;6(12):1072–1074.

Recent Advancement of Multifunctional ZnO Quantum Dots in the Biomedicine Field

9

Sayoni Sarkar1,3, Jaisen Lokhande2, and Sujit Kumar Debnath3 1

Centre for Research in Nanotechnology and Science Department of Pharmacology, Tilak Municipal Medical College and Lokmanya Tilak Municipal General Hospital, Mumbai, Maharashtra, India 3 NanoBios Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 2

Contents 9.1 9.2

9.3

Introduction Structure, Properties, and Fabrication Methodologies 9.2.1 Importance of Structure-Property Synergism of ZnO QDs 9.2.1.1 Optical characteristics 9.2.1.2 Physiochemical properties and surface chemistry 9.2.1.3 Biological features 9.2.2 Synthesis Routes: Trade-Offs and Accomplishments 9.2.2.1 Wet-chemical approaches (hydrothermal, sol-gel, microwave-assisted synthesis, continuous flow synthesis) 9.2.2.2 Bio-synthesis: Green and sustainable synthetic scheme Advancements of ZnO QDs in Biomedical Domains 9.3.1 Targeted Drug Delivery and Point-of-Care Diagnostics 9.3.2 Treatment of ROS-Mediated Disorders 9.3.3 Wound Healing and Engineered Tissue Regeneration 9.3.4 ZnO QDs with Anti-Microbial Potential 9.3.5 Sensing and Imaging Applications in Biology

DOI: 10.1201/9781003322368-9

156 156 156 157 157 157 158 158 158 160 160 162 163 165 167

155

156 Nanomaterials in Healthcare 9.3.6 Cancer Theranostics 9.4 Future Prospects and Challenges References

168 170 170

9.1 INTRODUCTION Over the last 20 years or so, the field of nanotechnology has been realized to be a domain of great importance and potential. The main characteristic of nanotechnology is its capability to make a miniature system of sizes ranging from a few nanometers to a sub-nanometer scale. Apart from the other uses, nanotechnology has been envisioned to have a considerable impact on patient care, including clinical diagnostics and therapeutics, including conditions like cancers, which can be made available at a low cost. Nanotechnology products, either alone or with the help of various smart nanocarriers with welldefined shapes and sizes, are emerging as effective nanomedicines. A large number of materials have reached various stages of research in nanotechnology. In this chapter, we will be focusing on zinc oxide (ZnO) quantum dots (QDs). QDs are nanoparticles (NPs) with dimensions of 1–10 nm and possess inherent photophysical properties that may be useful for diagnostic imaging and for treatment. Specifically, QDs may be used for functional imaging, including live-cell and whole-animal imaging, blood cancer assay, and cancer detection and targeted drug delivery for treatment [1]. ZnO semiconductor QDs possess multiple properties, which make them attractive candidates for biological uses. These properties include luminescence; release of Zn2+ ions, especially in acidic con­ ditions; production of harmful radicals called reactive oxygen species (ROS), after being irradiated with UV which can be brought in use for treatment; versatile surface chemistry; and accurate control of the size and shape of the particles through easy and economical synthetic processes. Efforts are continuously ongoing to design more effective applications of ZnO QDs concerning drug delivery systems, antimicrobial, anticancer, anti-diabetic, and anti-inflammatory activities, as well as for bio-imaging activity. This is achieved through multiple mechanisms like inducing intracellular ROS generation and activating apoptotic signalling pathways, which make ZnO QDs potential anticancer and antibacterial agents. Especially in the era of increasing antimicrobial resistance worldwide, the high antibacterial potential of ZnO QDs in gram-negative as well as gram-positive bacteria, which is seen even in the resistant bacteria, hold great potential in the future [2]. Additionally, ZnO QDs have also been widely known to assist the bioavailability of drugs or biomolecules when formulated as drug carriers to achieve enhanced therapeutic efficacy. Because of these properties, ZnO is regarded as a benign material that has prominent applications in food additives, biomedicine, and cosmetics.

9.2 STRUCTURE, PROPERTIES, AND FABRICATION METHODOLOGIES 9.2.1 Importance of Structure-Property Synergism of ZnO QDs ZnO QDs have garnered immense interest among researchers and engineers alike in the domains of biomedicine and clinical science, because of their significantly broad bandgap of 3.37 eV and an exciton binding energy of 60 meV, which is prominently huge. Moreover, their hexagonal wurtzite crystal structure, flexible defect chemistry, unique morphologies, and design have underscored the potential of these ZnO QDs in easy processability. Under the ambient process environment, the crystalline structure

9 • Recent Advancement of Multifunctional ZnO Quantum Dots

157

of ZnO corresponds to the P63mc [3]. The unique arrangement of the interlinked sublattices of Zn2+ ions and O2− ions endow it with an ionic nature [4]. Additionally, ZnO, among the group III–IV semi­ conductors, have been tested in accordance with the Mohs scale to be a soft material with the hardness value typically around ~4.5 [5]. These structural intricacies of ZnO QDs have made it one of the most sought-after metal oxide quantum dots that have prospered in all walks of the healthcare industry. From the perspective of exploring the opportunities of ZnO QDs in biology, some of the key attributes that make this nanomaterial system one-of-a-kind have been delineated as follows.

9.2.1.1 Optical characteristics Intense research on the atypical optical characteristics of ZnO QDs has revealed the predominance of the size-dependent quantum confinement effect, mainly when the particle size is less than 10 nm. When photons of a particular wavelength irradiate these ZnO QDs, their extremely small size gives rise to the quantum confinement phenomena wherein the electron-hole pairs or excitons being generated are spatially confined in all three dimensions, thereby leading to the discretization of energy levels. In fact, with decrement in the QDs’ size, a hypsochromic shift (blue-shift) is observed, whereas increasing the size result in a bathochromic shift (red-shift) [6]. Besides, the defect-mediated visible photoluminescence which is otherwise not prevalent in conventional nanoparticulate ZnO, is quite an intriguing feature of these QDs. This luminescence is attributed to the transitions from sub-band states or defect levels and is commonly termed the “defect emission”; however, the band-to-band transition is responsible for the peak in the UV region in a photoluminescence spectrum, which is broadly known as “band-edge emission.” Tailoring the photoluminescence features of ZnO QDs is crucial for state-of-the-art bio-imaging applications [7].

9.2.1.2 Physiochemical properties and surface chemistry There is a myriad of factors that regulate the inherent physiochemical behaviour of ZnO QDs. First and foremost, the colloidal stability of the fabricated ZnO QDs is a paramount acumen to affirm the feasi­ bility of using these QDs as bioactive agents in theragnostics and allied domains. In most cases, to negate the tendency of agglomeration in nanoparticles, favourable organic surfactants or stabilizers are employed. These organic capping agents are adsorbed at the surface of the QDs and give rise to steric hindrance because of their long hydrophobic chains that extend into the solvent systems [8,9]. Thus, these surfactants serve as efficient candidates that render the ZnO QDs with good colloidal stability. The other inter-related parameter with great significance is the zeta potential (ζ). The higher the ζ of the colloid, the higher the resistance towards particle agglomeration [10]. Arguably, the size of the QDs also regulates the bio-functionality to a large extent. Although the reports on the size-dependent response of QDs in the biological micro-environment are often paradoxical, yet the fundamental concept of an increase in surface reactivity with decreasing size is pertinent in most studies. Notionally, with the size shrinkage, there might be a promotion of defects that may lead to modified electronic properties and therefore altered surface charge. Thus, a robust synthesis platform that yields non-agglomerated col­ loidally stable ZnO QDs is vital in driving the point-of-care diagnostics and treatment.

9.2.1.3 Biological features Assessment and co-relation of the safety with the physiological characteristics of the ZnO QDs are imperative to understanding the reactions at the nano-bio interface. It is then prudent to investigate the possibility of generation of reactive oxygen species (ROS), dissolution of Zn2+, and effect of dose both via in vitro and in vivo studies. Enhancing the zeta potential through effective capping of the QDs plays a critical part in preventing the rapid dissolution of Zn2+ ions. Moreover, the reaction kinetics of ROS formation can be tailored by the addition of dopants or deliberate incorporation of defects. The design of every ZnO QD is exclusive in connection with their morphology, size, and surface functionality; they manifest distinct biodistribution patterns, cellular uptake mechanisms, and propensity to produce ROS.

158 Nanomaterials in Healthcare No two of these ZnO QDs formulations have these biological traits in common [11,12]. Hence, for­ mulating newer in vitro strategies for speedy evaluation of all these determinants is indispensable before proceeding with the clinical trials and developing safer QDs. High throughput screening of all these essential attributes is key to paving the path of ZnO QDs-based smart technologies from the lab bench to the healthcare industries and beyond.

9.2.2 Synthesis Routes: Trade-Offs and Accomplishments In recent years, substantial efforts in devising novel synthesis protocols for ZnO QDs that align with the paradigm shift towards “green” approaches have escalated the necessity to adopt cutting-edge inter­ disciplinary methodologies that mitigate the drawbacks of the conventional fabrication schemes. The broadly employed techniques involve hydrothermal, sol-gel, flow chemistry-based and microwaveassisted wet chemical routes, laser ablation, physical vapour deposition, and biological synthesis schemes, some of which are elaborated in Table 9.1. Despite the spiraling innovations in ZnO QDs production, there still exist several bottlenecks in stringent process control that impede further advancements in application-driven promises of these QDs. In typical physical vapour deposition or laser ablation methods, although the scheme is benign, handy, and relatively fast, the synthesized QDs exhibit poor stability and surface defects that many times hamper the optical performance. Hence, wet-chemical processes and bio-synthesis for ZnO QDs have gained popularity for pristine materials discovery.

9.2.2.1 Wet-chemical approaches (hydrothermal, sol-gel, microwaveassisted synthesis, continuous flow synthesis) The cumulative endeavour of researchers over the last few years has led to remarkable advancements in these chemical processing routes facilitating the road towards ZnO QDs fabrication with enhanced properties. Further, researchers, by formulating systematic synthesis strategies, have strived to make the routes more facile, ensure a more regulated processing environment conducive to yielding mono­ dispersed QDs, and improvised material performance. In spite of the rigorous modulation of these batch chemistry protocols to adapt to environment-friendly conditions, the transition towards the “green chemistry” approach is challenging to accomplish. Usage of severe reaction conditions results in elevated energy consumption that poses a threat to the environment. In such circumstances, microwave-assisted synthesis has proved to be a game changer with its hallmarks of being a facile, eco-friendly, and rapid method. The interplay of the dielectric heating rate of microwave reactor and the nucleation-growth kinetics expedites the fabrication of luminescent ZnO QDs for potential biological utilities. The QDs’ size was a function of the high heating rate and was correlated to the intensification of the photo­ luminescence quantum yield [6]. In addition, microwave assisted synthesis conforms to “green chem­ istry” principles. Regardless of these benefits, a few stumbling blocks persist, such as scaling up the fabrication of these ZnO QDs to meet the surge in demand. Ground-breaking technological innovations have successfully merged flow chemistry with ubiquitous “green” synthesis protocols via robust microreactor platforms for pilot-scale ZnO QD production. In one such pioneering study, a new-fangled continuous flow hydrothermal route has been adopted to explore the synergism between process intensification and the effect of flowrate on the desirable characteristics and nano-architecture of the nanoparticulate ZnO. This serves as proof-of-concept for bridging the prevalent complexities between the lab-bench and industrial-scale development of ZnO QDs with assured high process efficiency [22].

9.2.2.2 Bio-synthesis: Green and sustainable synthetic scheme Designing and conceptualizing eco-friendly methodologies have ushered in a plethora of opportunities to implement fabrication routes that can be performed under a mild reaction milieu, while simultaneously

9 • Recent Advancement of Multifunctional ZnO Quantum Dots TABLE 9.1

159

Summary of the synthesis methodologies for ZnO QDs for biomedical applications

SYNTHESIS ROUTES Hydrothermal

Sol-gel

Solvothermal

PRECURSORS (P) AND STABILIZERS (S) USED

MORPHOLOGY AND PARTICLE SIZE

Zinc acetate dihydrate (P), 2–4 nm, diethanolamine (P), oleic monodispersed acid (S) spheroids Zinc nitrate hexahydrate (P), 1–3 nm, combination ethylene diamine (P), fatty of nanorods and acid (S) spherical Zinc nitrate hexahydrate (P), 5–6 nm, arbitrary dodecylamine (P), particle-shaped polyvinylpyrrolidone (S)

Green chemistry route from plant extract Microwaveassisted method

Zinc acetate dihydrate (P), Eclipta alba extract (P)

6 nm, monodispersed spheres

Zinc acetate dihydrate (P), lithium hydroxide (P)

Solvothermal

Zinc acetate dihydrate (P), diethylene glycol (P), polyvinylpyrrolidone (S) Zinc acetate dihydrate (P), sodium hydroxide (P)

Three different sizes of ~ 3.5 nm, 5.12 nm, 7.18 nm, nearspherical structures 15 nm

Microwaveassisted hydrothermal route Spiral microfluidic reactor-based routes

Annular microfluidic reactor-based routes-kilogram scale synthesis

Zinc nitrate (P), sodium hydroxide (P)

100 nm, agglomerated sheet-like structure

Nano to sub-micron sized particles (~150–600 nm), spheroidal, ellipsoid, rods, cubic, urchinshaped, plateletshaped Zinc nitrate hexahydrate (P), Quasi-sphere (~17±6 zinc sulphate heptahydrate nm), star-like (P), zinc chloride (P), zinc aggregates (> 1 μm), acetate (P), potassium nanostars (~100 nm), hydroxide (P), sodium rods (~180 nm) hydroxide (P)

APPLICATIONS

REFERENCES

Nano-carrier for anticancer drugs

[ 13]

Antimicrobial and anticancer agents Cancer diagnostics, fluorescent nanoprobe Antimicrobial agent

[ 14]

Anti-diabetic nanomedicine

[ 17]

Biolabeling and fluorescence imaging Antibacterial agent

[ 18]

Biochemical applications

[ 20]

Antibacterial agent

[ 21]

[ 15]

[ 16]

[ 19]

rendering the QDs biocompatible. The size-tailored luminescent efficacy of the bio-synthesized ZnO QDs has been acknowledged to be profound and consistent. Specifically, in the biomedical sector, the safe biological formulations of these ZnO QDs have unprecedented characteristics that encompass the use of organisms with intrinsic nature of converting the toxic heavy metals to biocompatible material systems consisting of low-valence metals and proteins. To date, several enzymatic reaction-driven biogenic-synthesis platforms have popularized the rationale of stabilizing the QDs via the formation of minuscule biomolecules that act as a surfactant, thereby monitoring the monodispersity and colloidal stability of the samples. Perhaps the recent surge in scientific vigour aligned with “green” processing has helped explore the possibilities of using plant extracts as ideal candidates for capping. Indeed, these strategies can aptly eradicate environmental risks through their one-stop, facile, low-cost protocols that

160 Nanomaterials in Healthcare primarily focus on circumventing high energy consumption, material wastage, use of toxic materials, and endorsing safe operations. In a compelling study, a plant-extract-based “green” route for fabricating luminescent ZnO QDs was performed. Room-temperature synthesis employing pomegranate peel extract yielded water-soluble smart ZnO QDs, demonstrating notable dispersibility. Effective monitoring of the growth conditions of these QDs was achieved by the biomolecules available in the fruit peel-derived extract [23]. In another work, the trade-off between the emerging ZnO quantum dots technology for smart healthcare and their toxicology was meticulously inspected. The synthesis assisted by easily degradable bio-friendly chitosan capping in ZnO QDs loaded with doxorubicin hydrochloride (DOX) showcased superior tumour-targeted drug delivery and biocompatibility. The presence of non-toxic chitosan heightened the colloidal stability of the QDs by the resulting interfacial forces arising from their hydrophilicity [13]. Thanks to this well-established fabrication scheme, the long-term photostability denoted by the photoluminescence quantum yield remained intact and thus testified to the merits of efficient surface-functionalization mediated by the process conditions.

9.3 ADVANCEMENTS OF ZNO QDS IN BIOMEDICAL DOMAINS ZnO QDs are among the most interesting inorganic nanomaterials that catch significant research interest due to their inherent physicochemical properties and low-cost manufacturing. The application of these nanoparticles is more in cosmetic preparation due to the protection ability against UV-induced skin damage. Additionally, their application is observed in packaging and food industries as additives with antimicrobial properties.

9.3.1 Targeted Drug Delivery and Point-of-Care Diagnostics Advanced ZnO QDs drug delivery system offers several advantages over the traditional drug delivery techniques. ZnO QDs are easy to fabricate and possess a low-cost method of preparation that have spiralled their demand in smart point-of-care diagnostics (Table 9.2). These types of QDs rapidly dissolve at pH < 5.5. Drug conjugated with ZnO QDs has demonstrated antitumor activity. It was observed that ZnO QDs showed cytotoxicity against cancer cells after dissolution. These types of QDs were adopted as gatekeepers in blocking nanopores of silica nanoparticles that were mesoporous. A receptor/ligand-targeting system was developed to conquer lung cancer by delivering the drug. CD44 (transmembrane glycoprotein) is an over-expressed marker in many cervical and lung tumours. Hyaluronic acid (HA) is a linear polysaccharide used as a promising moiety for binding to receptor CD44. With surface modification, amino group luminescent and water-dispersible ZnO QDs were prepared [24]. The introduction of PEG into the NH2-ZnO QDs retained its stability in physiological conditions and targeted the HA-receptor CD44. This system behaved as an acid trigger drug release in a tumour site to improve therapeutic index. These pH-sensitive ZnO QDs have drawn significant interest in drug delivery systems. To improve the pH responsivity-controlled release pattern, zwit­ terionic poly(2-(dimethylamino)ethyl methacrylate) and poly(carboxybetaine methacrylate) were introduced in the ZnO QDs [25]. This modified ZnO QD promoted endocytosis, increased blood circulation time, and enhanced water stability. With an optimum ratio of zwitterions, ZnO QDs showed strong adhesion to tumour cell membranes. An anticancer drug like doxorubicin was loaded effectively in these ZnO QDs with a loading efficiency of 24.6%. After endocytosis by tumour cells, these QDs were undergone lysosomal acid degradation and efficiently released DOX resulting in synergistic anticancer activities.

9 • Recent Advancement of Multifunctional ZnO Quantum Dots TABLE 9.2

161

Implementation of ZnO QDs in targeted drug delivery system

NANOMATERIALS NAME

DRUG USED

REASON BEHIND THE FORMULATION

NH2-ZnO QDs (PEG-ZnO)

Doxorubicin hydrochloride

Need to develop CD44 target-specific drug delivery

ZnO@P(CBMA-coDMAEMA)

Doxorubicin

Need to develop a pHresponsive controlled release drug.

ZnO-ADH-Hep-PTX

Heparin and Paclitaxel

Due to poor solubility, paclitaxel use is limited and showed unexpected pharmacokinetics and biodistribution

ZnO-QD-chitosanfolate loaded DOX

Doxorubicin hydrochloride

Need to develop cancer targeted drug delivery

CHARACTERISTICS

REFERENCE

1. pH-responsive 2. Acid decomposable 3. Luminescent aminated 4. Synergistic therapy 1. Promote endocytosis and gives sustained release of drug 2. Increased blood circulation time 3. Established synergistic anticancer activities. 1. pH-responsive delivery 2. Prevent the formation of clots in the blood 3. Improving the delivery of paclitaxel 1. High drug loading efficiency 2. Chitosan enhanced the stability of ZnO QDs

[ 24]

[ 25]

[ 26]

[ 13]

In a study, adipic dihydrazide was used to surface modify ZnO QDs, followed by conjugation of heparin and paclitaxel [26]. The prepared complex improved the solubility of poorly soluble drugs like paclitaxel through conjugation. At the same time, surface modification with heparin prevents the for­ mation of clots in the blood. This ZnO QDs modified nanocomplex system efficiently delivers drugs to cancer. Not only that, ZnO QDs inherently showed anticancer activities. In a study, HEK-293T human embryonic kidney cell and HeLa cervical cancer cell lines were used to examine the cytotoxicity of ZnO QDs [27]. In a dose-dependent manner, ZnO QDs decreased mitochondria membrane potential, and increased the level of ROS. These QDs also regulated many gene expressions, including the caspase and bcl-2 gene, which are involved in apoptosis. ZnO QDs initiated apoptosis in HeLa cells in both early and late, whereas late apoptosis was observed against HEK-293T cells. With chitosan (biodegradable polymer), the blue light-emitting ZnO QDs were successfully employed for targeting tumour cells [13]. Long-term fluorescence, non-toxic, and water dispersed ZnO QDs were prepared by incorporating folate-conjugated chitosan using a chemical hydrolysis method. This folate is responsible for receptor-specific delivery. Doxorubicin hydrochloride was used as a model drug and found to have a higher drug-loading efficiency of 75%. Additionally, ZnO QDs showed dose-dependent and broad-spectrum antimicrobial activity against multidrug resistance (MDR) strains, including bacterial and fungal pathogens. A study showed a sig­ nificant reduction of cell viability of MDR isolates of Escherichia coli and Candida albicans in a dosedependent manner [28]. ZnO inhibited E. coli growth by increasing membrane permeability and cell membrane disintegration. ZnO lyses food-borne bacteria Staphylococcus aureus and Salmonella typhi­ murium. Therefore, ZnO QDs have a promising role in inhibiting pathogenic bacterial growth in the food system.

162 Nanomaterials in Healthcare Nano-sized oxide has demonstrated excellent photocatalytic and adsorption properties. Eventually, it can reduce secondary pollution and convert harmful gases and pollutants into small inorganic molecules (CO2 and H2O). These properties are used in the energy environment to develop efficient, cost-reduced development of devices. ZnO also has gas-sensitive properties like the change of carrier density and conductivity. Therefore, the change of resistance can be used to detect sensitive gases [29]. Magnetic resonance imaging (MRI) is used in radiology to map internal structure. This imaging system has inherent limitations like capturing larger images (more than a few micrometers) and lower sensitivity. To overcome this limitation, MRI-FI (fluorescence imaging) nanoprobes were prepared by doping gado­ linium (Gd) ion in ZnO QDs [30]. This modification provides better reliability for clinical diagnosis, including chemical stability in the air, lower toxicity, cost-effectiveness, and high quantum yield. The prepared nanoprobes were relatively smaller in size and demonstrated enhanced fluorescence suitable for medial and biological fields. The isoelectric point of ZnO is around 9.5, which is ideal for biomolecules immobilization using electrostatic attraction. Additionally, high electrical and environmental stability make ZnO QDs a suitable candidate for bio-sensing [31]. Carbohydrate antigen 19-9 (CA 19-9) is a biomarker for pancreatic cancer, which is difficult to diagnose early. Different testing procedures are available, but none are efficient. A sandwich-type sensitive immunoassay was developed using ZnO QDs as fluorescent and electrochemical labels [32]. This immobilization process tool was performed at a higher isoelectric point, and conjugating ZnO QDs formed the sandwich structure with the antibody of CA 19-9. The resulting immunosensor demonstrated high selectivity and sensitivity to detect antigens. Additionally, a concanavalin A (Con A) based sensor was developed with ZnO QDs to direct allergy chicken ovomucoid (CHOM) [33]. Using electrostatic interaction between negatively charged protein and positively charged QDs, CHOM was self-assembled onto the surface of ZnO QDs. The prepared biosensor showed concentration-dependent square wave voltammetric by capturing CHOM-tagged ZnO QDs on concanavalin A-coated chips. ZnO QDs have a unique resonance multiple-phonon Raman lines with an excitation wavelength at 325 nm employed for fingerprint signals [34]. Using this feature, these QDs become critical elements for analyzing various biological macromolecules, including deoxy­ ribonucleic acid (DNA) and proteins. ZnO/Au nanocomposites probe functionalized with thiololigonucleotide was developed to identify DNA microarrays [35]. The probe was further hybridized with overhanging portions of the 15-nucleotide target sequence thiol oligonucleotide. DNA-labeled ZnO/Au nanocomposites showed a strong Raman signal.

9.3.2 Treatment of ROS-Mediated Disorders Oxidative stress is the process of overproduction of reactive oxygen species (ROS) in the body. Generally, the cells and tissues of our body have efficient antioxidative systems to neutralize these harmful entities. Enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione per­ oxidase (GSH-Px) provide efficient antioxidant protection. However, in several conditions, an imbalance between oxidative and antioxidative systems arises, leading to damage to our cells and tissues [36]. Excessive levels of ROS lead to altered cellular proteins and lipids, leading to cellular dysfunction, impaired energy metabolism, inflammation, and immune activation [36]. Chronic oxidative stress is a major factor that leads to the development of pathologies such as cardiovascular diseases, insulin re­ sistance, diabetes mellitus (DM), and its complications like retinopathy, nephropathy, neuropathy, etc [36]. Various antioxidants are available for the prevention and treatment of damages due to ROS. Synthetic antioxidants have shown to have certain adverse reactions because of which some research is also directed toward antioxidants of natural origin [37]. Consequently, since the past few years, number of studies have focused on using plant extracts to obtain metallic and metal oxide quantum dots with varied sizes and shapes through green synthesis methods [38]. Plant extracts contain different concen­ trations and combinations of biocompounds like steroidal glycosides, flavonoids, anthocyanins, essential

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oil, etc. These biocompounds possess antioxidant and therapeutic activities and are responsible for various biological activities of plant-derived nanoparticles [39]. Recently, there has been a lot of research on zinc oxide quantum dots (ZnO QDs), which can be a valuable agent as an antioxidant. It is known that zinc is an essential micronutrient that plays crucial role in enhancing antioxidant defence, growth, and supporting the immune system. Moreover, the free radical scavenging activity of spherical ZnO QDs has been extensively studied as a function of time and concentration [40]. ZnO QDs are synthesized by either chemical method (coprecipitation) or through biological method using aqueous extracts of various parts of plants or herbs, fungi, actinomycetes, bacteria, algae, etc. ZnO QDs synthesized from extracts of the selected plant species have been found to exhibit enhanced antibacterial and antioxidant activities and can be more cost-effective and eco-friendlier as compared to chemical ZnO QDs [41]. Many different plant materials have been exploited for the synthesis of quantum dots through ecofriendly techniques without involving toxic chemicals [41]. A large number of studies are available which has investigated the beneficial effects of ZnO QDs derived from Azadirachta indica, Hibiscus rosa-sinensis, Ceropegia candelabrum, Moringa oleifera, Tamarindus indica, root extract of Polygala tenuifolia [42] etc. to name a few. Various preclinical studies on ZnO QDs have demonstrated antioxidant activity by scavenging free radicals and revealed excellent anti-inflammatory activity through dose-dependent suppression of mRNA and inflammatory mediator proteins like iNOS, COX-2, IL-1β, IL-6, and TNF-α [41]. Animal experi­ ments on ZnO nanocrystals using animal model of type 2 diabetes in rats showed that they can improve many of indicators of diabetic dysfunction like glucose tolerance, weight loss, insulin levels, fructosa­ mine levels, pancreatic SOD activity, and pancreas histology. The addition of the standard antidiabetic agent Vildagliptin, an inhibitor of the enzyme dipeptidyl peptidase IV (DPP IV), further improved these indices [43]. Another study on ZnO nanoparticles produced from aqueous extract of Aquilegia pubiflora leaves showed high toxicity for the HepG2 cancer cell line. It showed excellent inhibition of the enzymes, acetylcholinesterase and butyrylcholinesterase, which are involved in Alzheimer’s disease, and showed moderate inhibitory effects on enzymes involved in diabetes, inflammatory conditions, and aging. The article concluded that as biosynthesized ZnO QDs possess strong anticancer and antioxidant properties, it could be a good option against conditions like cancer, diabetes, Alzheimer’s, and other inflammatory diseases [44]. Thus, these ZnO QDs have become prominent in critical applications, particularly antibacterial, antioxidant, antidiabetic, and tissue regeneration activities. These QDs synthesized through the bio­ logical method showed promising results and appeared to be scoring over the chemical method of production in terms of cost-effectiveness and safety. ZnO QDs exert its antioxidants activity through its efficient electron donation to the free radicals. The antioxidant property of ZnO QDs protects many vital organs against ROS-induced pathophysiology, as in diabetes, Alzheimer’s disease, etc.

9.3.3 Wound Healing and Engineered Tissue Regeneration Wounds, acute or chronic, pose significant health concerns, primarily due to their large number of cases. One study reported more than 8.2 million people suffered from a certain type of wound in the United States, and the cost of care ranged from 28.1 to 96.8 billion [45]. The greater health burden is due to the chronic wounds especially seen in the elderly population, which affected over 6.5 million people in the United States alone [46]. Chronic wounds (venous leg ulcers, diabetic foot, pressure ulcers, etc.) are considered to be a “silent epidemic” that leads to morbidity of patients, adversely affects quality of life (QoL) of patients and escalates the cost of treatment. Oftentimes, chronic ulcers in the leg and foot can be prolonged for 12 to 13 months which is indeed quite alarming and its recurrence rate in patients is 60% to 70% [47]. These extensive treatments for chronic wounds over long time intervals significantly increases the financial burden of healthcare. Moreover, diabetic foot ulcers have a five-year mortality ratio comparable to cancer, and the situation has not improved even in the last 10 years [48]. Bacterial

164 Nanomaterials in Healthcare infection is often seen with wounds. Bacterial contamination prolongs the wound healing process and may have been responsible for a large percentage of the burn injury-related mortalities [49]. When infected, even acute wounds can become chronic or non-healing if no effective treatment is provided. Various types of dressing are available, which are intended to target the pathologies involved in nonhealing wounds, control the infection, and accelerate wound healing. These wound dressings include films, hydrogel, electrospun scaffold, sponge, and foams, many of which are incorporated with anti­ biotics, quantum dots (metal and metallic oxides and direct and indirect antimicrobial properties), organic, natural, and other agents [50]. Here, the effects of nanotechnology using quantum dots (QDs) and its role in wound healing and regenerative medicine has been delineated. QDs can be fabricated with a broad-spectrum of materials such as metals and metal oxides, ceramic based materials, and natural and synthetic polymers. Amongst the metal oxide QDs, zinc oxide (ZnO) is the most investigated because of its versatility, multifunctionality, and safety. ZnO has been considered generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). Zinc oxide quantum dots’ favorable properties, like their antibacterial effect and better safety to mammalian cells than silver nanoparticles, have led to their use in several hydrogelbased and bio-nanocomposite-based wound dressings, as portrayed in Figure 9.1 [51,52]. Zinc is one of

FIGURE 9.1 (a) Synthesis protocol depicting the steps involved in fabrication of ZnO nanocrystals modified with chitosan. (b) Images revealing accelerated wound healing for castor oil films with modified ZnO nanocrystals (bionanocomposite polymeric film) when compared with only castor oil films and a control monitored over a time frame of 14 days (c) Plot of percentage scar healing with time for the ZnO and castor oil-based bionano­ composites, castor oil films, and gauze (control). (Reprinted from Biomacromolecules, 16(9), Diez-Pascual et al., Wound healing bionanocomposites based on castor oil polymeric films reinforced with chitosan-modified ZnO nanoparticles, 2631–2644, Copyright 2015, with permission from American Chemical Society.) [ 51].

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the essential trace elements, which is a cofactor for many enzymes and regulates wound healing pro­ cesses in burns and slow-healing wounds. Neovascularization is an essential issue in regenerative medicine, which depends on factors like vascular endothelial growth factor (VEGF), and angiopoietin activators of integrins. This is especially important in diabetic patients. It is also to be noted that reactive oxygen species play a prominent role in the angiogenesis process by activating key steps of vascular cell proliferation. Concerning this, one experimental study showed that ZnO QDs hydrogel not only cleared the local resistant bacteria, but also downregulated inflammatory factors like TNF-α and IL-6 by 46.9% and 57%, respectively, upregulated 1.7-fold VEGF and twofold epidermal growth factor (EGF), all of which are important for neovascu­ larization. After 15 days of treatment, the rate of skin lesion closure in rats was 96.3%, compared to 65.4% in the control group [53]. Another study showed ZnO QDs accelerate rapid healing in chronic venous leg ulcers [54]. The zinc ions, which are released from its hydrogel dressing, may also promote fibroblast growth, thus enhancing wound tissue repair [55]. Besides its role in wound healing, ZnO is a key player in the domain of regenerative medicine and tissue engineering, a field that is dramatically expanding over the years. This particular regime involves the manufacturing of artificial structures that are biocompatible and in good resemblance with the native tissue/organ to create a scaffold on which natural cells are made to grow to resemble human tissues. Quantum dots (QDs) can be used for such scaffolds providing mechanical strength to the tissues and providing controlled release of bioactive agents. As of now, most of the articles published are related to wound healing and bone tissue regeneration. Scaffolds of ZnO composite have been studied to enhance angiogenesis for better wound healing, whereas ZnO nanocrystals fabricated in electrospun polycaprolactone scaffolds were reported to induced angiogenesis by the expression of key proangiogenic factors VEGF and fibroblast growth factor (FGF) [56]. There are attempts to use ZnO QDs tissue engineering to correct the pathological conditions of bonelike osteoporosis which is a condition wherein the trabecular and cortical bones are reduced in density, and there is increased likelihood of fractures. One study evaluated the effect of ZnO hydroxyapatite (Hap) containing biocomposite poly (L-lactic acid)-co-poly (ε-caprolactone) and silk fibroin (PLACL/SF) nanofibrous scaffolds on regeneration of bone tissue. The in-vitro study showed that ZnO and Hap increased proliferation of osteoblasts for bone tissue regeneration, enhanced the secretion of the bone mineral matrix (98%), and increased potential tensile properties (322.4%) [57]. Several other studies showed beneficial effects of scaffolds incorporating ZnO quantum dots and various other composites, which have significantly decreased bacteria with increased osteoblast density and improved strength of the bone material [58]. It is apparent now that ZnO nanostructures alone or combined with hydrogel, hybrid composite, or some form of scaffold appears to be emerging and promising materials for tissue engineering. ZnO scaffolds are effective in forming new blood vessels necessary for wound healing. ZnO structures have demonstrated good antibacterial properties and support the growth and proliferation of several cellular lines like osteoblast. Large-scale clinical studies are needed to validate the extensive benefits derived from pre-clinical studies.

9.3.4 ZnO QDs with Anti-Microbial Potential Even with the advancement of scientific research, bacterial infections, be they either communityacquired or hospital-acquired, are one of the major causes of death even today. Countries with low- and lower-middle-income are affected more than the upper-middle and high-income countries [59]. Apart from the health and economic concerns due to these infections, the indiscriminate use of antimicrobial agents also poses significant health issues. Adverse drug reactions (ADRs) due to antibiotics and anti­ microbial resistance (AMR) are considered a primary global concern, increasing not only the costs to the patients and the healthcare systems but also influencing the mortality rate in the population. The WHO and numerous other research groups agree that it is imperative to address the spread of AMR urgently and needs a global coordinated action plan [60].

166 Nanomaterials in Healthcare It is predicted that if the AMR is left unchecked, it could make the bacterial pathogens much more lethal, causing more harm to humanity. One study estimated that in 2019 AMR was associated with 4·95 million (3·62–6·57) deaths, including 1·27 million deaths directly attributable to bacterial resistance. The leading pathogens with antibacterial-resistant causing human deaths include Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Other organisms include drug-resistant tuberculosis, third-generation cephalosporin-resistant E coli, carbapenem-resistant A baumannii, fluoroquinolone-resistant E coli, carbapenem-resistant K pneumoniae, and third-generation cephalosporin-resistant K pneumonia have also caused deaths in a large number of patients [61]. AMR also incurs a substantial economic burden on society. One study showed that the economic costs of AMR per antibiotic consumed were considerable, often exceeding their purchase cost [62]. As newer drugs are finding their place in the treatment of resistant infections, nanotechnology is also providing opportunities for developing antibacterial agents. Metal-based nanoparticles (NPs) and quantum dots (QDs), in particular, are showing promising results against many bacteria, including resistant ones. Some examples of metal-based quantum dots include zinc oxide (ZnO), titanium dioxide, silver, Fe2O3, and copper oxide quantum dots. This chapter will focus on the ZnO quantum dots. ZnO quantum dots have several advantages, including high antibacterial effectiveness compared to CuO and Fe2O3 nanoparticles at low concentrations and activity against a wide range of strains of Grampositive and Gram-negative bacteria [47]. It exhibits remarkable photocatalytic efficacy that surpasses other inorganic nanomaterials and is much more bio-safe than many other metal ions that often cause heavy metal poisoning. Additionally, ZnO QDs possess superior selectivity, has good resistance towards heat and are way more durable [63]. Antibacterial activities of nanostructured ZnO against Mycobacterium tuberculosis, C. jejuni, against resistant bacterial strains like carbapenem-resistant Acinetobacter baumannii, [2] against S. aureus, E. coli, C. albicans, etc are being thoroughly investi­ gated over the years [63]. Zinc oxide quantum dots (ZnO QDs) have been evaluated through in-vitro methods against Listeria monocytogenes, Salmonella enteritidis, and Escherichia coli [30]. The proposed mechanisms (Figure 9.2) for zinc oxide nanocrystals include the production of reactive oxygen species (ROS), which is considered as one of the most important mechanisms responsible for antimicrobial activity. The oxygen (O2) and hydroperoxyl radical (HO2) produced do not penetrate the cell but cause direct damage to the membrane, whereas hydrogen peroxide (H2O2) is internalized and causes the destruction of cellular components such as DNA, proteins, and lipids; (b) the loss of cellular integrity following contact between ZnO NPs and the bacterial cell wall; and (c) ZnO

FIGURE 9.2 Proposed antibacterial mechanisms of action of nanostructured ZnO [ 63].

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QDs internalization [63]. Protein denaturation and inhibition of cell viability occurs as a result of Zn2+ penetration through the cell membrane. Moreover, Zn2+ ions have the ability to disrupt the electron transport system causing what is known as the cellular respiratory disorder [64]. ZnO nanoparticles have also been shown to have inhibitory effects against many fungi, possibly by promoting ROS release in the fungal cell [63]. Apart from the direct actions of ZnO QDs, there has also been an attempt to increase the antimicrobial capacity by doping it with other metal compounds like CuO, Ag, Fe ions, TiO2, etc. Few studies have shown that by changing the physical and morphological characteristics of ZnO nanoparticles, higher inhibition of the growth of organisms has been achieved [2]. Over the years, a large amount of data has been generated on the antibacterial effects of metal-based nanoparticles. ZnO QDs, in particular, are showing promising results against many bacteria, including the resistant ones, and may prove to be a valuable asset in the scenario of bacterial resistance. ZnO nanocrystals have been extensively studied for their antibacterial activities through a large number of pre-clinical studies. They appear to have good biocompatibility, stability, and other characteristics, making them suitable for the antibacterial agent. There is a need to conduct large-scale clinical studies to evaluate the role of ZnO QDs in patients.

9.3.5 Sensing and Imaging Applications in Biology The unfathomable implications of bio-imaging and bio-sensing in precise diagnosis, treatment, and investigation of crucial biological mechanisms have necessitated the development of cutting-edge nanoscale sensors and probes. Luminescent ZnO quantum dots have appreciable benefits in the vital areas of bio-imaging and bio-sensors. The novel quantum confinement effects in ZnO QDs that propel their improved bioactivity, enriched physiochemical characteristics and surface chemistry, and flexible size-dependent optical properties have made them quite lucrative among researchers. Contrary to many of the stereotypical metal-based-semiconductor QDs and fluorescent organic dyes that are deemed to be highly toxic to the environment and biological entities, these ZnO QDs are much more benign and amenable to broad-spectrum applications [65,66]. These biofunctionalized ZnO QDs hold promises in bio-imaging because of their encouraging molar absorptivity, transmittance profile, narrow emission spectra, and size-tailored optical bandgap. From the application point of view, the enormous possibilities of affordable, non-toxic ZnO QDs-based bio-sensors and fluorescent probes or labels predominantly rely on their boosted stability in the biological media through adequate surface modification [67]. The tunable emission in the visible region is ascribed to the presence of defect states (trap levels) between the valence and conduction band. Thus, in UV-irradiated ZnO QDs, electrons from the band edge are excited to the intermediate defect states, and subsequently, their transitions are responsible for the exceptional photoluminescence (PL) features. It is noteworthy that the PL intensity increases with decreasing particle size because of the large surface-area-to-volume effect. Some of the frequently availed analytical and characterization operando techniques to understand the phenomena determining such aberrant optical transition profiles include UV-vis absorption, photoluminescence spectroscopy, and time-correlated single-photon counting technique. This inherent surface defect chemistry of ZnO QDs has furnished intense PL quantum efficiency, which has been exploited in multi-purpose biological fields. Nevertheless, these quintessential optical qualities are constantly being improved for bio-sensing and bio-imaging by the design, development, and implementation of neoteric fabrication platforms that have streamlined the commercial aptitude of ZnO QDs by leaps and bounds. A hassle-free sol-gel synthesis method was utilized for nanostructured ZnO possessing an unconventional core-shell architecture. The processing and optimization of ZnO QDs with considerable compatibility in biological fluids is quite challenging. Thus, the tactics of obtaining water-stable QDs employed in this study were to initially cap the synthesized ZnO QDs to ensure their stability in an anhydrous solvent system and then immediately subject them to a phase transfer into a water-based solution. The polymerization reaction in the presence of the precursor poly(ethylene) glycol methyl ether methacrylate yielded a ZnO core and a copolymer shell. What was more intriguing was that the

168 Nanomaterials in Healthcare copolymer shell consisted of the polymethacrylate groups at the interior, rendering it hydrophobic, while FTIR verified the presence of the functional groups of poly(ethylene) glycol methyl ether at the exterior that endowed it with hydrophilicity. The synergistic effect of these two polymer shells covalently bonded to the core (ZnO) aided in sustaining its stability in water, albeit maintaining the ZnO core intact. Estimation of the PL quantum yield revealed an enhanced value that remained unaltered for a long time. Further, the images recorded by confocal laser scanning microscopy to understand the penetration of these QDs into the cell, their cell-labeling activity, and toxicity depict their encouraging potential as biocompatible fluorescent nanotrackers [31]. In another study, ZnO QDs doped with gadolinium (Gd) were researched for their utility in magnetic resonance imaging (MRI) as fluorescent probes. Synthesis of the Gd-doped ZnO QDs was carried out by a simple wet-chemical route. To certify the favourable dispersion of the oleic acid-capped QDs in water, N-(2-aminoethyl)aminopropyltrimethoxysilane was employed as a surface modifier and promoted sur­ face functionalization. Thorough analysis and characterization of the synthesized samples explicitly show the improvised emission characteristics of the Gd-doped ZnO QDs. The in vitro cytotoxicity studies demonstrated remarkable cell viability at desirable doses. Such striking biocompatibility results gave the impetus to discern the fluorescent imaging features further. Near-real-time monitoring of the captured images of the HeLa cells after being incubated with the samples illustrated a time-dependent change in the fluorescence. Additionally, an investigation of the efficiency of these Gd-doped ZnO QDs as MRI nanoprobes divulged the flair to amplify contrasts in MRI [68]. Water-dispersible PEG-coated ZnO QDs were synthesized by forming intermediate ZnOhexahydro-4-methyl phthalic anhydride in a batch process. The subsequent fluorescence spectra and transmission electron microscopy images delineated the formation of ultrasmall QDs of 2 nm with narrow PL bands (green emission). The significant antibacterial activity and non-toxic nature denoted from the cytotoxicity studies against HepG2 (nontumorigenic cell line), and osteoblasts underlined their potency in biomedicine. Above all, the proficient cell labeling and benign nature of the ZnO QDs epitomize their prospects as leading-edge fluorescent probes and sensors [69].

9.3.6 Cancer Theranostics Cancer, undeniably, is the most lethal disease with far-reaching effects worldwide. With the rapidly increasing cancer related-mortality rate, formulation, and development of smart diagnostic and therapeutic tools have become imperative. It is perceptible from the subsisting cancer treatments that the hurdles of overcoming the side effects associated with the commonly employed techniques, chemotherapy and radiotherapy, are often perplexing. The dramatic advancements in recent years have opened up newer avenues to explore the modern cancer therapeutic techniques and understand the fundamental biological phenomena and technical “know-how” controlling them. The major challenges faced include site-selective interaction of the anticancer drugs, wherein the probability of destroying the healthy cells and tissues in the biological microenvironment during the drug administration, is relatively high. Thus, there is an everincreasing need for anticancer agents with enhanced selectivity and admirable efficacy for adopting intelligent means of targeted drug delivery. Recent accomplishments in the field of nano-oncology have offered multiple trajectories by the conjugation of contemporary design of quantum dots and favourable attributes like versatility, biocompatibility, specificity in drug release that can inhibit cancer cell prolifer­ ation and retains the healthy cells and cost-effectiveness. The recent technological breakthroughs have emphasized on the ZnO QDs as adept candidates for activating cytotoxicity on cancer cells and fast-track cell apoptosis. In the clinical world, a lot of attention has been paid to ZnO QDs since their inception because their multifunctionality and pH-responsive behaviour make them more advantageous. The tradeoffs between water-stable ZnO QDs synthesis and preservation of their appealing traits have been con­ siderably alleviated through surface functionalization methodologies. Moreover, the surface-modified ZnO QDs are functional even in the acidic pH of the biological milieu at the tumour site. Thus, holistic maximization of this potential of ZnO QDs is the need of the hour. Beyond any doubt, the cancer

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FIGURE 9.3 Schematic diagram describing the encouraging potentials of ZnO quantum dots in cancer theranostics.

treatment-centric synthesis protocols for pristine ZnO QDs that are elementary, low-cost, and require a mild processing environment is a dire necessity and have rapidly progressed over the years. Pioneering research in these areas has resulted in the fast growth of many futuristic ZnO QDs-based nanoplatforms for specific anticancer drug delivery that holds stupendous scope of flourishing in the years to come (Figure 9.3). The commonly used chemical precipitation method was modified suitably to fabricate ZnO QDs which were extensively characterized to study their structural features. The conducive reaction environ­ ment yielded QDs displaying narrow PL bands and broad-spectrum absorption with the band edge at 362 nm. The successive investigations on cell proliferation and cytotoxic behaviour of the as-prepared ZnO QDs against the C2C12 myoblast cell line (cancerous) divulged a dose-dependency trend. The interlink of rudimentary biological mechanistic pathways of ZnO QDs with that of cancer cell inhibition was probed by the DCF-DA assay to quantify the intracellular reactive oxygen species (ROS). The higher level of ROS generated was directly indicated by the DCF fluorescence intensity. This sufficed to identify that oxidative stress promoted the cancer cell apoptosis, as observed from the time-dependent confocal scanning laser microscopy. The findings explaining that lower concentrations of these ZnO QDs were more effective in killing the cancer cells by easy penetration into the cells than at higher concentrations were certainly inexplicable and are monumental in implementing appropriate therapeutic strategies [70]. The amalgamation of the anticancer merits of natural extracts like flavonoids with the ZnO quantum dot systems has enriched their biological potential abundantly. These opportunities for cancer treatment are being widely recognized. One of the studies, in particular, presented the significance of tangeretin loaded-ZnO QDs, wherein tangeretin is a natural extract (flavonoids) commonly obtained from citrus fruits. Preparation of these QDs was executed through a sol-gel process comprising initial aminefunctionalization by 3-Aminopropyltriethoxysilane, and eventually, tangerine was loaded upon 12 h of vigorous stirring. The in vitro cytotoxicity results unveiled that the tangeretin-loaded-ZnO QDs for­ mulation induced toxic effects on the H358 lung cancer cell line causing damage and apoptosis. In the age of dual-modal cancer therapy, these QDs emerged as a dynamic pH-responsive anticancer drug and efficacious luminescent nanoprobes with fine-tuned tracking ability at the time of chemotherapy [71]. ZnO QDs fabricated by an amidation reaction to affirm precise conjugation with folic acid emphasized the practicality of favourable doxorubicin loading. The synthesis involved magnesium

170 Nanomaterials in Healthcare doping by the standard wet-chemical route as a preliminary step. The obtained ZnO QDs, when irra­ diated by UV, showcased bright green emission. The surfaces of these QDs were modified by 3-Aminopropyltriethoxysilane for fostering folic acid conjugation, which was followed by loading of doxorubicin via a chelation reaction resulting in Zn2+-doxorubicin. The anticipated performance of these ZnO QD nanocarriers for pH-specific drug delivery to attack the tumour sites and release doxorubicin in the acidic biological milieu, along with their boosted anticancer activity, was ascertained through in vitro toxicity (MTT assay) and confocal scanning laser microscopy studies. Thus, these QDs proved not only worthy for targeted cancer therapy but also an ideal alternative to many of the costly drug delivery and therapeutic techniques employed to cure cancer [72]. A novel Gd3+-doped ZnO QD architecture was constructed to make it more competent with respect to the prevalent QD-based anticancer drug delivery platform. Doxorubicin was loaded in the Gd3+-doped ZnO QD architecture, and their site-selective drug release was evaluated both in vitro and in vivo. The lowered toxicity and efficient performance of these luminescent QDs in fluorescent cell labeling and MR imaging were demonstrated as if like a concomitant effect. These multiple advantages of Gd3+-doped ZnO QDs are a rarity and bestow them with prospects as drug delivery nanocarriers for easy release of anticancer drugs, diagnosis, and targeted treatment [73].

9.4 FUTURE PROSPECTS AND CHALLENGES Zinc oxide has drawn significant interest more than other metal oxides due to wider bandgap and higher binding energy. That is why its application has become more prompt in ultraviolet laser diodes, elec­ troluminescent devices, transparent electrodes, piezoelectric transducers, chemical sensors, solar cells, photo-catalysts, pigments, and medicines [36]. The application of QDs has increased significantly in the biomedical field due to their manipulated size and luminescence properties. ZnO QDs are well known for their excellent water solubility, fluorescence stability, and dispersion. The size of QDs is the critical determining factor for fluorescence color. A lot of research is going on the synthesis of ZnO QDs with controlled crystal size and surface structure to make them more palatable for biomedical applications [74]. Due to the large surface-area-to-volume ratio, these QDs conjugate to a wide range of biological molecules for targeting particular organs or tissues. This surface modification is also helpful in reducing nonspecific binding and preventing aggregation. Several metal and metal oxide (zinc, titanium, gold, and silver oxide) nanoparticles are known to have antimicrobial properties. Out of them, ZnO nanoparticles can be a better choice due to their comparative lower toxic profiles. Even studies showed broad-spectrum activities of ZnO QDs against MDR strains [29]. These QDs can be a next-generation and broadspectrum alternative to combat MDR organisms. ZnO QDs are inexpensive inorganic nanomaterials. Although these QDs show improved physicochemical properties, the potential concern is the toxicity that mainly depends on several factors, including inherent physicochemical properties and environmental conditions. Still, further research is needed to explore the influence of surface modification and conju­ gation in improving fluorescence stability and quantum efficiency of ZnO QDs in developing novel quantitative, qualitative, and sensitive tools in diagnosis, imaging, and drug delivery.

REFERENCES [1] Martínez-Carmona, M., Gun’Ko, Y. and Vallet-Regí, M. 2018. ZnO nanostructures for drug delivery and theranostic applications. Nanomaterials 8:268.

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Relevant Properties of Metallic and NonMetallic Nanomaterials in Biomedical Applications

10

Parisa Fatehbasharzad1,2, Pavlo Ivanchenko3,4, Ola El Samrout3,5, and Jaime Gómez Morales6 1

Molecular and Preclinical Imaging Centers, Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 2 Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany 3 Department of Chemistry and Interdepartmental Nanostructured Interfaces and Surfaces (NIS) Centre, University of Torino, Torino, Italy 4 Vrije Universiteit Brussel (VUB), ETEC Department, MOBI Research Group, Brussels, Belgium 5 Laboratoire de Reactivité de Surface LRS, Sorbonne Université, Paris, France 6 Laboratorio de Estudios Cristalográficos. Instituto Andaluz de Ciencias de la Tierra (CSIC-UGR). Granada, Spain

Contents 10.1 10.2

Introduction Structural Engineering of Nanoparticles 10.2.1 Size of Nanoparticles 10.2.2 Shape of Nanoparticles 10.2.3 Surface of Nanoparticles 10.2.4 Structural Tuning for Biomedical Applications 10.2.4.1 Magnetic resonance imaging (MRI)

DOI: 10.1201/9781003322368-10

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176 Nanomaterials in Healthcare 10.2.4.2 Surface-enhanced raman spectroscopy (SERS) Ceramic Biomaterials 10.3.1 Hydroxyapatites as Biomaterials 10.3.2 The Surface Features of Hydroxyapatites 10.3.3 The Effect of the Surface Structure of Hydroxyapatites on the Adsorbed Proteins Structure 10.3.4 Luminescent Lanthanide Hydroxyapatite-Based Nanomaterials 10.3.5 Silica-Based Nanomaterials 10.3.6 Silica Surface Structure 10.3.7 Silica in the Drug Delivery Field 10.4 Overview References 10.3

182 183 185 185 187 187 188 189 190 190 191

10.1 INTRODUCTION Artificial materials are in wide use in biomedical applications covering different aspects of the field, such as biosensing, bioimaging, medical implantation, tissue engineering, and bioelectronics to name a few. All of the mentioned applications are united by the function taken by the solid surface, being an interface between artificial matter and living tissue. The fine details of a surface structure affect the characteristics of the water layers formed at the first instance after the insertion of the artificial material into a living system as described in detail by Kasemo [1]. Upon the contact of a material with a living system, the first molecules to arrive at the surface are the small water molecules; the bigger molecules like proteins are to arrive later, substituting water molecules; the state of these molecules in the adsorbed state depends on the properties of the surface. Finally, the living cells interact with the surface through the layer of preadsorbed biomolecules causing either ‘positive’ or ‘negative’ response upon recognition/non-recognition of the adsorbed proteins. Thus, the fate of the interaction of the materials with the biological systems may originate from the characteristics of the chemistry, charge, and wettability of the surface [2] affecting the adsorption of small molecules on the surface acting as potential ‘probes’ allowing to predict the final response of the more complex living system. Furthermore, in light of the use of nanosized materials, the role of the surface grows immensely since the reduction of a solid matter mass unit to nano-dimensions drastically increases the surface area available for the contact with the (biological) environment. In addition, surface treatment/engineering is an important tool to gain control over the behavior of the nanomaterials in the biological environment. In particular, the proper surface design of a nanobio­ material may enhance its non-cytotoxicity, resistance to aggregation, provide a specific recognition for biosensing application, tune the ζ-potential of the surface, or even provide the ‘stealth’ properties for avoiding the capture by the immune system [3]. Apart from the significance of the fine details of a surface structure concerning biological appli­ cations, another important parameter to consider is the morphological aspect. In this respect, the surface plasmon peaks of metallic nanoparticles (NPs) of nano-sphere, rod, cubes, branch, and bipyramids are known to be sensitive to both shape and size of the nanoparticles of the same elemental composition [3]. Also, shape and size of NPs are the factors of concern in regard to the toxicity towards humans and environment, thus the research efforts in the direction towards the safer and more effective morphologies are of big importance [4]. To transcend the limitation of traditional medical diagnosis methods, newly developed nanoscale imaging contrast agents provide us with a specific and accurate medical detection. Narrow peak width of Raman signals (1–2 nm) makes SERS (surface-enhanced Raman spectroscopy) nanoparticles preferential

10 • Relevant Properties of Metallic and Non-Metallic Nanomaterials 177 candidates for multiplexed imaging. Besides, the SERS probe provides robust signals in the near-infrared region by employing 630 or 785 nm laser excitation. This range of excitation wavelength overcomes the limitation factor for many optical probes, which arise from considerable light scattering and auto­ fluorescence in the visible light range. Beyond this, in magnetic resonance imaging (MRI) unlike the optical techniques, signals are not restricted by tissue scattering and therefore it allows deep inside body imaging. For this reason, MRI is a widely used technique in the most clinics for high-resolution imaging and targeted MRI contrast agent were presented to obtain high sensitivity and molecular imaging [5]. Calcium apatite (hydroxyapatite, hereafter referred to as HA, general formula Ca10(PO4)6(OH)2) is a material of special interest in the biomedical field due to its abundance in the mineral part of bone tissue. The broad application of this class of the materials is the result of the vast research activities carried out over decades that allowed the production of HA particles in controlled shapes, sizes, surface structures, morphologies, and with added functionalities (i.e., ionic exchange for agricultural, catalytic, therapeutic, or imaging [6] applications). When using fluorescence-labeling agents such as fluorescent dyes, fluorescent proteins, quantum dots, or lanthanide-containing nanoparticles for biological or specific biomedical applications, the use of confocal fluorescence microscopy as an imaging technique is the best option. The technique has become an indispensable tool due to several attributes that are not available in other contrast modes with tra­ ditional optical microscopy. The phenomenon of fluorescence involves the absorption of light energy (a photon) by the label (normally in the UV range), followed by the emission of some of this light energy (as another photon) a few nanoseconds to milliseconds later, at a longer wavelength (single photon fluorescence). It is also possible the excitation of the label by simultaneous absorption of two photons with half the energy of the corresponding one-photon absorption process, following the emission of one photon with higher energy, and therefore incident wavelengths are located in the infrared (700–1,200 nm). This is the principle underlying “two-photon” or “multiphoton” microscopy. The advantage of this configuration is that a longer-wavelength light can penetrate deeper into tissues better than shorter wavelength light because it is scattered less. The absorption of high energy (i.e., using UV excitation wavelength) also has the disadvantage that is more likely to damage cells. The advantages of lanthanide luminescence (long lifetime, sharp emission bands, and insensitivity to oxygen) are better exploited when using confocal two-photon excitation whose main characteristics are near-IR excitation, 3D resolution, and reduced photodamage [7]. Another type of ceramic material of biomedical interest is silica (SiO2). Its interaction with the human body has been deeply investigated in the nanomedicine field starting with the use of SiO2 as a drug-delivery system in the first decade of the 21st century [8]. These SiO2 NPs of high importance in nanobiotechnology and nanomedicine fields are in amorphous allotropic form and can be classified into two main categories based on their method of preparation: nonporous (fumed silica and colloidal silica) and porous (typically mesoporous silica) [9]. Since the surface of SiO2 NPs represents the interface between these inorganic materials and the biomolecules, understanding the surface properties that characterize the surface of SiO2 is of extraordinary relevance to designing NPs of particular targets in the nanomedicine field. Bare silica surfaces are characterized by two main functional groups, siloxane bridges (Si-O-Si) and silanols (Si-OH), that impart a hydrophobic/hydrophilic character to the silica surface based on their nature, density, and distribution on the surface [10]. Silica nanomaterials and especially the mesoporous ones, which can be easily high scale, prepared following a cheap method are characterized by a very high surface area and significant biocompatibility. This makes them excellent candidates for nanomedicine purposes such as bone tissue engineering, artificial implants, and especially drug-delivery systems [11]. This chapter discusses the nanomaterials in use in the biomedical field along with the relevant properties guiding toward their better performance. The chapter starts with the description of the engineered metallic nanomaterials and presenting nanoparticle-based imaging methods, then it elucidates the ceramic nanomaterials used in the implantation, further it gives insights into the properties of ceramic fluorescent materials, and, finally, the it discusses the use of silica nanoparticles in the drug delivery systems

178 Nanomaterials in Healthcare

10.2 STRUCTURAL ENGINEERING OF NANOPARTICLES Biomedical imaging techniques have been significantly improved over the past few decades. The structure and function of cancer tissues within soft and solid tumors can be revealed by various types of radiation techniques including radio waves and visible/near-infrared light that are employed in magnetic resonance imaging (MRI) and optical imaging techniques, respectively. Moreover, X-rays, gamma rays, and annihilation photons are competent to interrogate the cancerous tissue via X-ray computed tomography (CT), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In biomedical imaging techniques, sometimes sufficient contrast (based on electron/ proton density, tissue relaxation times, inherent optical and acoustic properties) can be provided by body tissues themselves. But in most cases, contrast agents, intended to provide or improve the imaging signal, are necessary to be introduced into a body. By taking advantage of nanotechnology and use of nano­ materials as a contrast agent, the traditional optical/magnetic-based imaging techniques have signifi­ cantly improved for abnormalities visualization and have also opened the way for the development of new methods for early-stage cancer detection [12]. Both magnetic and optical properties of nanoparticles are governed by their physiochemical features, which in turn determine sensitivity and resolution of different imaging methods. In this regard, the structure control (i.e., size and shape) and the surface chemical functionalization of NPs are the ways for enhancement of their performance. Efficient surface modification of NPs with organic and inorganic molecules provides improved physiochemical characters, leading to various clinical usages such as nano-based imaging [13], gene delivery, and drug loading [14]. Detailed structural features of nanoparticles will be discussed in the following. “Other surface properties and new studies on their improvements in clinical use can be briefly discussed with examples in this particular section”.

10.2.1 Size of Nanoparticles The nanoparticles represent distinct biological and physiochemical futures when compared to their counterpart particles at bigger sizes. In many applications of novel nanoparticles, the size, on one hand, appears as a pivotal factor due to relatively larger surface area and quantum effects arising from particle size reduction. On the other hand, by shrinking the size, nanoparticles become less obtrusive. This represents a determinative and fundamental factor in many favored biomedical purposes [15]. Effective tumor targeting and cellular uptake are largely influenced by nanoparticle size. Nanoparticles smaller than 100 nm are generally appropriate for anticancer drug delivery because of their selective and preferential accumulation at tumor sites deriving from enhanced permeability and retention effects. The nanoparticles can penetrate deeply and uniformly into the tumor by overcoming interstitial transport barriers that arise from some features of tumor microenvironments, including increased interstitial fluid pressure, aberrant vasculature, and dense extracellular matrices. Appropriate nanoparticle size is within the 10–100 nm range. Nanoparticles larger than 100 nm have been easily engulfed by the reticulo­ endothelial system; also, those having smaller sizes ( 8.0, acidic: IP < 5, and others in between these two groups) provides insight into the big picture of the binding the possible mechanisms of proteins interaction with HA surface suggesting (i) specific complexing of carboxyl groups and surface-exposed Ca and (ii) non-specific electrostatic interaction of protein positively charged protein domains with negatively charged HA surface sites. When considering the morphological features of HA and their effect on the adsorption of the proteins, it has been found by Kandori et.al. [62], that (0001) surfaces of nano-HA exhibit a better affinity to the basic proteins (e.g., lysozyme), while the (01-10) surfaces demonstrated the preference to the adsorption of acid proteins (e.g., Bovine Serum Albumin, BSA). Combining these findings with the previously revealed peculiarities of the structure of (01-10) surfaces, the idea of searching for the possible effects of the different surface terminations over the adsorption of the proteins arouses. Indeed, the studies of the adsorption of BSA on two types of biomimetic HA nanoparticles exhibiting different ratios of (010)_Carich/(010)_P-rich terminations (namely, 2:1 and 1:2.2) revealed that the BSA adsorbed at the nano-HA exhibiting more of (010)_Ca-rich terminations resulted to better maintain its native conformation, while the interaction of BSA with the sample exposing more (010)_P-rich type of structure led to the partial denaturation of the protein secondary structure [61]. Further, the work by Wallwork et al. [64], provides the evidence of the differences of the binding of BSA and recombinant mouse amelogenin with the HA crystals in terms of the proteins assembly and orientation on the surface; and, furthermore, highlighting the differences of the affinity of these proteins to the different crystal faces within the same solid.

10.3.4 Luminescent Lanthanide Hydroxyapatite-Based Nanomaterials A step further in the research of hydroxyapatite nanoparticles has consisted of the preparation of luminescent lanthanide-doped hydroxyapatite (lanthanide [65] = Eu3+, Tb3+, Er3+, La3+, etc.) for

188 Nanomaterials in Healthcare applications in medical and biological imaging. Lanthanide unique optical properties arise from elec­ tronic transitions within 4f shells or from 4f to 5d shells. Because of the incorporation of the lanthanide into the crystal structure, the luminescent nanoparticles exhibit a long fluorescence lifetime, a high quantum yield, sharp emission peaks, color tuning depending on the lanthanide, and good resistance to photo-bleaching from environmental conditions [66]. As a host material, hydroxyapatite provides a versatile crystal lattice to substitute Ca2+ with the dopant ion, whereas lanthanides offer the detectable and stable fluorescence signal, being possible to track its fate in cells by laser confocal fluorescence microscopy. In addition, hydroxyapatite nanoparticles exhibit notable properties such as bio­ compatibility, bioactivity, biodegradability, absence of toxicity or inflammatory and immune responses, high loading capacity of biomolecules and drugs, and ability to release them at acidic pHs, as those found at tumor sites or within lysosomes [55]. These properties are advantageous compared to those of tra­ ditional fluorescence labeling procedures based on organic dyes or fluorescent proteins, which are prone to problems such as short luminescence lifetime, broad-spectrum profiles, poor photochemical stability, and potential toxicity to cells or to quantum dots, which are somehow cytotoxic and show photoblinking [67]. All these characteristics make the doped lanthanide nanomaterials excellent nanoprobes for applications in medical diagnostics and targeted therapeutic applications [68] and thus have attracted the attention of different researchers for this type of application [6]. In the past decade, most studies with cells using Tb3+-, Er3+-, Eu3+-, Dy3+, or La3+-doped hydro­ xyapatites were devoted to studying their cytocompatibility in vitro (see the review from T. Tite et al.) [69]. Only a few of them were performed in vivo, like the one devoted to studying peculiar characteristics of synthetic apatite transformation when implanted in bone. Future theranostic applications in which the lanthanide-doped hydroxyapatites will be employed as luminescent probes and at the same time as drug delivery platforms must consider they are colloidal systems. The study of their particle size distribution and surface-related properties, i.e., the variation of ζ-potential of the suspensions vs. pH, are of great importance to predict the tendency of the colloid to disperse or aggregate in physiological conditions in the blood (pH around 7.4) or the tumor microenvironment (pH around 5–6). Furthermore, the surface features affect the formation of the protein corona around the nanoparticles when suspensions are injected into the biological media. In general, the aggregation must be minimized to avoid vein obstructions and facilitate cell uptake. In this regard, the importance of ζ-potential measurements is exemplified in the preparation of bonelike and cytocompatible luminescent Eu3+-doped citrate-coated apatite nanoparticles by the thermal decomplexing method [70]. The study reveals there is a synergic effect of Eu3+ doping and citrate coating on decreasing the ζ-potential to more negative values, thus decreasing the tendency of the colloid to aggregate.

10.3.5 Silica-Based Nanomaterials Among the biomaterials investigated in the nanomedicine and nanotechnology fields, silica nanoparticles are garnering interest, owing to their different surface features, diverse surface properties, and unique peculiarities, making them suitable for various biomedical applications such as promising drug nanocarriers. Silica (SiO2) is solid with a density between 2 and 3 g/cm3 and a high melting point (ca. 1700 °C). Its structure consists of a network of [SiO4] tetrahedral units arranged in different ways, resulting in various silica polymorphs. The high pliability of the Si-O-Si angle that connects two tetrahedral building blocks gives rise to different silica materials of mainly crystalline or amorphous allotropic forms [11]. First, the crystalline silica surfaces that are consisting of [SiO4] tetrahedral units arranged in an ordered and regular way are usually prepared by mixing or grinding. The most common high-density crystalline polymorphs are α-quartz, α-tridymite, and α-cristobalite [11]. It is noteworthy that crystalline silicas showed toxic effects when interacting with cellular systems in contrast to the interesting bio­ compatibility of amorphous silica nanoparticles. Therefore, continuous investigations in the biomedical and drug delivery fields have been devoted to studying the amorphous silica nanomaterials, featuring

10 • Relevant Properties of Metallic and Non-Metallic Nanomaterials 189

FIGURE 10.4 Schematic representation of the possible formation pathway of mesoporous silica using sur­ factants as templating agents. Inset shows the different types of functionalities exposed at the surface.

small particle sizes and high surface areas and including mainly fumed silica, colloidal silica, and mesoporous silica nanoparticles. Fumed silica (Cab-O-Sil in the USA or Aerosil in Europe) is synthe­ sized by hydrolyzing silicon tetrachloride vapor (SiCl4) at a high temperature (1100–1400 °C) in an O2/ H2 flame with subsequent fast quenching at room temperature [71]. As well established in the literature, this rapid quenching during the synthesis gives rise to metastable strained ring structures responsible for the high surface reactivity that characterizes the fumed silica nanoparticles [72]. Another interesting type of nonporous amorphous silica for biomedical uses is the colloidal silica nanoparticles prepared by hydrolysis of Si alkoxides in either homogenous systems following the conditions of the well-known “Stӧber method” or in heterogeneous systems of inverse micelles made in water-oil reverse micro­ emulsions with a subsequent condensation step at room temperature. This synthetic approach results in colloidal silica nanoparticles with a mass fractal structure presenting a huge and interesting amount of silanol groups located even below the external surface of the silica nanomaterials [9]. Moreover, it is worth mentioning that mesoporous silicas are also selected as excellent candidates in drug delivery systems due to their high biocompatibility with biological systems, as well as easy, cheap, and high-scale preparation routes. This type of ordered silica nanomaterials is synthesized by using organic surfactants as templates: the silica source and the surfactants are self-assembled into spherical micelles; this step is followed by the condensation of the silica source leading to the formation of rod-like micelles, finally, resulting in the formation of amorphous silica structures of periodic patterns upon the removal of the surfactant (as shown in Figure 10.4). Two well-known examples of mesoporous silicas are Mobil Crystalline Matter 41 (MCM-41) and Santa Barbara Amorphous (SBA), characterized by very high spe­ cific surface areas (690–920 m2/g) and tunable pore diameters of 2–4 and 5–30 nm, respectively.

10.3.6 Silica Surface Structure Since the silica surface represents the interface between the silica bulk and the external environment (biological system), the physicochemical features that characterize the interaction between the (bio)

190 Nanomaterials in Healthcare molecules and the surface of silica nanoparticles have been investigated at the atomic level. In this respect, the surface properties of bare silica should be first highlighted. Bare silica surfaces are characterized by the presence of two main functional groups: the siloxane (Si-O-Si) bridges and the silanol (Si-OH) groups. The distribution of these chemical functionalities, along with their nature and density, are directly responsible for the hydrophilic/hydrophobic character of the surface and the physical-chemical behavior towards (bio) molecules. Based on the synthetic approach, amorphous silica exhibits Si-OH groups (usually in mutual hydrogen bond interactions) are located at the surface as well as in bulk, as a result of incomplete con­ densation during the polymerization in the synthesis of the silica solid. Silanol groups are responsible for the hydrophilic behavior of the silica surface due to their ability to interact with polar groups or molecules via hydrogen bonds. Upon heating at a very high temperature, these H-bonded Si-OH groups are con­ densed, resulting in the formation of siloxane bonds (highly strained (Si-O)2 and (Si-O)3 rings) and water (which can be removed by a simultaneous pumping process). Through this dehydration process, the silica surface becomes richer in isolated Si-OH groups (very far apart) surrounded by siloxane rings (the different surface functionalities are demonstrated in the inset of Figure 10.4). These results in the hydrophobic character of the silica surface which starts to favor the adsorption of molecules through dispersive inter­ actions [11]. It is worth noting here that the investigation of the different types of Si-OH populations on the silica surface is crucial to understanding the noxious and beneficial effects of silica when dealing with the interaction with the human body. A recent work published by Pavan et al. [73] highlighted the presence at the surface of a special family of weakly interacting “nearly free silanols (NFS)”, which establish weak mutual interactions as they are distant by 4.0 to 6.0 Å, and which are supposed to be responsible for the silica particles toxicity since they are energetically favorable moieties to interact with cell membrane components, driving toxicity, lung inflammation, or pathological effects.

10.3.7 Silica in the Drug Delivery Field When an anticancer drug is administrated orally or via injection, it spreads within all parts of the body and only a small amount of the drug reaches the target site, resulting in a less efficient therapy accompanied by severe side effects since both healthy and diseased tissues are damaged. Continuous efforts have been devoted in the drug delivery field to design nanocarriers that transfer the anticancer drug exactly to the target tissue and release it in specific conditions for a long duration. Interestingly, silica nanoparticles and especially the mesoporous ones (SBA-15 and MCM-41) have been selected as excellent candidates for drug delivery systems due to their unique properties. They can be synthesized easily through high scale and cheap preparation routes: this makes them of high interest in different biological applications. Moreover, their unique surface properties make these silica nanoparticles suitable for the confinement, storage, and release of drugs within a living body. The hydrophilic and hydrophobic behaviors of the surface highlight the biocompatibility of the silica nanomaterials able to load different molecules such as peptides, drugs, and DNA to target different diseases [74]. Furthermore, the high surface areas of the mesoporous silicas result in the capability to incorporate high doses of drugs into the pores. In addition, the silanol groups on the walls of the silica nanomaterials provide great control for the drug release in the target site. The lack of toxicity, the furtive behavior of the immune system, the porosity, and the distinguishable surface properties of mesoporous silicas [11] are key parameters to deeply investigate and understand the detailed picture of the drug/silica system for various applications in the biomedical field.

10.4 OVERVIEW This chapter reviews some of the interconnections between the structural and surface features of several types of inorganic materials (metallic and ceramic ones), such as size, shape, surface structure with their

10 • Relevant Properties of Metallic and Non-Metallic Nanomaterials 191 functional feedback, and in detail discussed the surface features of some biomaterials providing them with good biocompatibility. With all the mentioned relevant features, the research efforts to drive a benefit of structural engineering in medical diagnosis techniques such as MR and SERS imaging were widely explored. Structural engineering of nanoparticles is a game changer for future clinical application and more controllable synthetic methods will direct to desire properties of nanomaterials. Given the fundamental knowledge of the key parameters of the biomimetic hydroxyapatite linking, the surface structure of the materials at the atomistic level with its effect on the state of the adsorbed molecules in the biological medium, the development of the new typologies of citrate-coated materials for biomedical applications is among the ongoing trends in the field. Also, the significant efforts are to be devoted to the further enhancing of biocompatibility of hydroxyapatites by doping it with various ions (i.e., those found in vivo) to improve their biomedical applicability. Furthermore, doping hydroxyapatite with lanthanides is also an ongoing field of research in which efforts are to be devoted to demonstrate its use in medical imaging and drug delivery (theranostics). In this respect, particle size, shape, and surface structure of the luminescent lanthanide-doped hydroxyapatite NPS need to be engineered to avoid particle aggregation and control the interactions within the biological medium. Understanding the silica surface features is of high interest for bio­ medical applications involving the adsorption processes on silica surfaces. As a recent discovery in this field, a specific family of silanols referred to as “nearly free silanols (NFS)” is a surface deter­ minant of the toxicity of silica samples irrespective of their crystalline or amorphous structures. More future studies should be devoted to the investigation of the molecular mechanisms that orchestrate human immune responses and silica biomaterials where the NFS might have a critical role. A further deep knowledge of the surface chemistry of silica materials could provide new insights for future innovative biomedical applications.

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Exosomes and Their Theragnostic Applications in Healthcare

11

Abhishekh Tiwari, Zainab Godhrawala, and Atul Chaskar National Centre for Nanoscience and Nanotechnology, University of Mumbai, Mumbai, Maharashtra, India

Contents 11.1 11.2 11.3 11.4 11.5

Introduction Sources for Exosome Isolation Mechanism of Exosome Biogenesis Structure, Composition, and Function of Exosomes Exosomes for Theragnostic Applications 11.5.1 Native Exosomes for Theragnostic Applications 11.5.2 Engineered Exosomes for Theragnostic Applications 11.6 Absorption and Distribution of Exosome-Based Theragnostic System 11.7 Challenges Related to Exosomes for Theragnostic Application 11.8 Conclusion and Future Prospective References

195 196 197 199 201 202 203 205 205 206 206

11.1 INTRODUCTION With the advent of nanotechnology, diagnostic and therapeutic medicine have gained prominence in the last few decades. Nano-based therapeutic systems like liposomes, hydrogels, dendrimers, solid lipid nanoparticles, etc; have been extensively utilized for therapeutic application. These systems in conju­ gation with various agents exhibit superior diagnostic and therapeutic thus enabling better medical results. However, the artificial nature of the nano-based systems exhibits certain disadvantages like immunogenicity, restricted tissue bioavailability, limited excretion rate, etc.; for clinical use [1].

DOI: 10.1201/9781003322368-11

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196 Nanomaterials in Healthcare Extracellular vesicles (EVs) are membranous structures which are derived from different types of cells. Based on the origin of these structures, size, composition, biomarkers and morphology, these structures are classified as exosomes, micro-vesicles, and apoptotic bodies. Different EVs play a wide range of functions in the cell, through a variety of physiological processes [2]. Exosomes are the most common type of EVs characterized by lipid bilayer membranous vesicles and their nano size. These exosomes are produced by all types of cells via the endosomal pathway [3]. Since the 1980s, exosomes were considered cellular waste found in damaged cells or homeostatic by-products formed in different processes of the cell. These exosomes were assumed to be irrelevant in the biological processes. Until recently, they were deemed important in cellular communications and reprogramming owing to their structural and functional composition [4]. Exosomes are bio-nano particles with sizes ranging from 30 nm to 100 nm that are secreted by the majority of living cells but do not have a strict definition [5]. Exosomes are membrane-bound vesicles with a diameter of a few nanometres that contain a variety of biomolecules, such as lipids, proteins, and nucleic acids [1]. Several techniques, including ultracentrifugation, immune isolation, and ultrafiltration, can be employed to separate and purify exosomes [6]. Different previously reported drug carriers exhibit limitations such as poor biocompatibility, inferior bio-distribution, unintended immunological response, etc. These limitations have instigated a search for better nanocarriers for drug delivery [7]. Liposomes have been successfully used as a delivery system in the treatment of various diseases, particularly cancer. Due to their structural alikeness with the cell membrane, liposomes offer superior therapeutic carrying capacity. However, liposomes exhibit certain constraints owing to their synthetic nature, which include poor targeting, the generation of a cytokine storm and immune response, morphological instability, and the incapacity to cross biological barriers, like the blood-brain barrier (BBB). It is well established that all eukaryotic cells release exosomes, their characteristics may differ on the basis of their source and their status during the release. Exosomes may thus provide a prognosis for a variety of diseases, including chronic inflammation, cardiovascular, neurodegenerative disease, diseases related to lipid metabolism, renal disease and cancer [8]. Exosomes, a natural alternative to liposomes, can therefore be advantageous for their theragnostic use [9]. Exosomes have recently gained popularity in clinical applications as a biomarker and a nanocarrier [8]. Thus, this chapter focuses on the theragnostic applications of native as well as engineered exosomes, their absorption, distribution, and associated challenges. In this context, it is critical to comprehend the reported sources for exosome isolation, the mechanism for exosome biogenesis, as well as their structure, composition, and function.

11.2 SOURCES FOR EXOSOME ISOLATION The behaviour of exosomes in the body determines its medical outcome. Properties like migration, distribution, pharmacokinetics, etc; are determined by the composition of the exosomes. The origin of the exosomes plays a crucial role in governing the composition of the exosomes [10]. Hence, the selection of the source of the exosomes is vital for its biological implications and subsequent use as a theragnostic agent [7]. The origin of the exosomes, i.e. the cells from which the exosomes are derived, determines the contents inside it. These exosomes carry out different functions like signalling and recognition of antigen-presenting molecules, cell-to-cell communication etc. [11,12]. The majority of the studies on exosomes utilize exosomes from mammalian cells. Exosomes are isolated from mammalian cells like dendritic cells, mesenchymal stem cells (MSCs), macrophages and tumour cells. These exosomes express proteins which are similar to the surface of proteins of the recipient cells thus imparting better absorption capabilities to the exosomes. Cells produce exosomes as

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signal-carrying molecules under various conditions. They are commonly found in bodily fluids like blood, cerebrospinal fluid (CSF), saliva, and breast milk [13]. However, exosomes of mammalian origin exhibit limitations like low yield, long and tedious processes, and irregularity in the quality of exosomes, thus restricting their clinical applications [14,15]. Plant-derived exosomes (PDEs) overcome a few of these limitations as the processes involved are easier to control, upscale, and cost-effective. PDEs are biocompatible and are biased towards tumours through specific endocytosis mechanisms. Exosomes have been isolated from a variety of fruits like mangoes, grapes, strawberries, etc; rhizomes like ginger; roots like carrots; and vegetables like tomatoes, etc. These exosomes also exhibit cross-species activity and minimize off-target effects, and thus can be utilized as potential theragnostic tools for biomedical applications [15].

11.3 MECHANISM OF EXOSOME BIOGENESIS The biogenesis of exosomes is an energy-dependent process carried out by a variety of membrane-bound proteins like the tetraspanins and the endosomal sorting complex required for transportation (ESCRT) [16]. Exosome development is instigated after the production of early cell membrane-derived endosomes via endocytosis. Subsequently, multivesicular bodies and late endosomes are formed (Figure 11.1). The infolding of the plasma membrane results in the formation of endosomes, which go through the endosomal system (or endocytic pathway), and have been divided into three compartments: 1) early endosomes; 2) late endosomes, and 3) recycling endosomes [17]. On the fusion of early endosomes with endocytic vesicles, they can undergo degradation, recycling, or secretion of biomolecules. Constituents or biomolecules needed to be recycled are sorted into recycling endosomes, rest early endosomes are transformed into late endosomes. Accumulation of intraluminal vesicles (ILVs) in the late endosomes takes place after the inward budding of the endosomal membrane. During this process, cytosolic ele­ ments (e.g. proteins, nucleic acids, and lipids) are packed into small vesicles inside the late endosomes. Late endosomes containing many small vesicles inside them are called multivesicular bodies (MVBs), which further form exosomes when the endosomal system produces intraluminal vesicles (ILVs) [2]. Figure 11.1 shows exosome biogenesis [18]. The formation of an intraluminal vesicle (ILV) requires two distinct processes. The first process is the reorganization of the endosomal membrane to become highly enriched in tetraspanins. Mainly the tetraspanins CD9 and CD63 are important for the development of exosomes. The second step involves the induction of the ESCRT at the ILV formation site [2]. Multivesicular bodies (MVBs) are formed during the endocytic pathway. They consist of intraluminal vesicles in their lumen [19]. Intraluminal vesicles (ILVs) degrade, recycle, or exocytose the proteins, lipids, and nucleic acids in the late endosomes. Intraluminal vesicles (ILVs), with specific endosomal compartments rich in proteins, lipids, and cytosolic components, are formed by the inward budding of the exosomal membrane. These MVBs are then carried to the plasma membrane through the structural network of the cytoskeleton and microtubules. If the content of the MVBs is designated for degradation, they fuse with the lysosome, or they are sent to the cellular membrane to release ILVs. When this fusion occurs between the cellular membrane and multivesicular bodies to secret all ILVs; the ILVs are called exosomes [16,20]. The physiological conditions of the parental cell determine exosome biosynthesis and composition [7]. The ESCRT proteins TSG101, STAM1, Alix, and CD9 regulate pathways involved in the packaging and biosynthesis of exosomes. The biogenesis of exosomes can undergo two pathways: 1) ESCRTdependent pathway and 2) ESCRT-independent pathway. The ESCRT-dependent machinery mainly comprises four complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) and associated proteins. The role of the ESCRT mechanism is to sequester ubiquitinated proteins to specific regions of the

198 Nanomaterials in Healthcare

FIGURE 11.1 Exosome biogenesis [ 1].

endosomal membrane through the ubiquitin-binding subunits of ESCRT-0. This is known as ubiquitindependent cargo clustering. Once the ESCRT-0 complex binds with ESCRT-I and ESCRT-II complexes, subsequently this complex will attach to ESCRT-III. ESCRT-III is a protein complex that is responsible for budding. As the budding process is complete forming the ILVs, the ESCRT-III complex is separated from the membrane of the MVBs by VPS4 sorting protein via an energy-dependent reaction. In the ESCRT-dependent mechanism, the accessory proteins (e.g. VPS4 ATPase) aid in dissociation and recycling [8,21]. The ESCRT-independent pathway is another route for sorting exosomal cargo into MVBs; this mechanism relies on raft-based microdomains for lateral cargo segregation within the endosomal membrane. Sphingomyelinases, which hydrolyze phosphocholine to form ceramides, are abundant in these microdomains. In the presence of ceramides, the membranes demonstrated lateral phase separation and microdomain coalescence. Moreover, there are possibilities of exhibiting spontaneous negative curvature which might result due to the presence of a cone-shaped ceramide structure in the endosomal membrane. As a result, this mechanism highlights the significance of lipids in exosome biogenesis. [8]. Alternatively, the syndecan-syntenin-ALIX pathway is an ESCRT-independent pathway for exosomes biogenesis that involves heparanase, syndecan heparan sulfate proteoglycans, ADP ribosylation factor 6 (ARF6), phospholipase D2 (PLD2), and syntenin, including vesicle formation and protein loading. Syntenin-ALIX interaction facilitates ILV formation, and it is dependent on the availability of heparan sulfate, syndecans, and ALIX [22]. Heparanase releases syntenin-1, syndecan, and CD63 contained in exosomes, through the enzymatic breakdown of heparin sulphate chains on syndecans, in which ARF6 and PLD2 control ILVs [2,23].

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11.4 STRUCTURE, COMPOSITION, AND FUNCTION OF EXOSOMES Exosomes that are produced through biogenically derived processes have a structure, components, and molecular processing that mirror the activities in their parental cells; as a result, the exosomes’ com­ ponents might contain useful agents [15,24]. These are uniformly formed nanovesicles with sizes between 40 and 150 nm. A lipid bilayer made up of several phospholipids, including phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, ceramide, and cholesterol makes up the structure of these nano-vesicles. Different biomolecules including RNAs, DNAs, and proteins like heat shock pro­ teins (HSPs), BAR, and Rab are among the cytosolic components. These cytosolic components interact with the target cell, causing upregulation or downregulation of the genes in the target cell, as well as changes in the shape of the exosomes [25,26]. The morphology and size of exosomes play an important role in their absorption in tissues. Cells derived from Human Mast Cell 1 (HMC-1) have been reported to produce different types of exosomes [26]. These exosomes of different morphologies can be classified as follows: 1. Single Vesicular Exosomes: The most common forms of exosomes comprising of bilipid mem­ branous structure enclosing the cytosolic apparatuses, which have an average size of 71 ± 58 nm. 2. Double Vesicular Exosomes: These exosomes are composed of smaller vesicles enclosed within larger exosomes. The shape of the larger vesicle can vary from spherical to slightly elongated with an average size range of 171±101 nm. 3. Triple Vesicular Exosomes: They contain multiple small vesicles in one larger vesicle ranging from 65–380 nm in size. 4. Small Double Vesicular Exosome: This category of exosomes is similar to double vesicular exosomes. The discontinuity of exosome inner membranes distinguishes this exosome type from double vesicular exosomes. Their average size is in the range of 54±10 nm. 5. Oval Vesicular Exosomes: These are elongated, single bilipid membrane with two axes of symmetry perpendicular to each other, and the average size of these exosomes is 114±86 nm. 6. Small Tubular Exosomes: With an average size of 145±87 nm, it consists of a bilayer membrane tubular vesicle. 7. Large Tubular Exosomes: It is analogous to small tubular exosomes, but they are bigger in size greater than 700 nm. The investigators also observed exosomes that could not be divided into these categories. It is, however, important to note that the experimental procedures used for the study resulted in the disintegration of the structure of those exosomes. These exosomes had some distinctive features, which are listed below (Figure 11.2): 1. Coated Exosomes: In these exosomes, electron-dense spikes protruded from/are partially attached to the membrane structure. As a result of these protrusions, membrane fusion may enable attachment to the target cells during theragnostic cargo delivery. 2. Filamentous Exosomes: These exosomes consist of a filamentous structure that fills the lumen of these small and tubular exosomes. 3. Electron-dense Exosomes: In these exosomes, electron-dense materials were present, which might be granules of the mast cells that produced the exosomes. There are numerous surface proteins, including CD9, CD63, CD55, CD59, and CD47 that carry out a variety of physiological roles such as exosome aggregation via the receptors on the recipient cell’s

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FIGURE 11.2 Different morphologies of exosomes derived from HMC-1 cells. A – single vesicular exosomes. B – double vesicular exosomes, C – triple vesicular exosomes, D – small double vesicular exosomes. E – oval vesicular exosomes, F – small tubular exosomes, G – large tubular exosomes, H – coated exosomes, I – Filamentous exosomes, J – electron dense exosomes.

surface and facilitate avoiding immune cells to prolong their circulation in the body [27,28]. Exosomes comprise different enzymes, including calcium-dependent enzymes that transport multivesicular struc­ tures to the plasma membrane before releasing them into the extracellular environment. Additionally, it also contains proteins involved in cell adhesion, membrane fusion, transport, and antigen presentation [29]. The typical makeup of exosomes is displayed in Table 11.1. It describes the proteins, lipids, and RNA types found in exosomes as well as their functions and their examples. TABLE 11.1 SR NO. I) 1.

2. 3. 4. 5.

Exosome composition and roles of main components [ 16, 30] CATEGORY

Protein Tetraspanins

ESCRT machinery/MVB biogenesis Heat Shock Proteins (Hsp) Membrane transport and fusion Major Histocompatibility Complex (MHC) molecules

6.

Cytoskeletal proteins

7. 8.

Adhesion Glycoproteins

9.

Growth factors and cytokine

ROLE

EXAMPLES

Exosome biogenesis, exosomes CD9, CD63, CD37, CD81, CD82, cargo selection, targeting, and CD53 uptake Exosome biogenesis Alix, TSG-101 Exosomes released, signalling Exosome secretion and uptake

Hsp90, Hsc70, Hsp60, Hsp20, Hsp27 GTPases, Annexins, Flotillin, Rab GTPases, dynamin, syntaxin MHC Class I, MHC Class II

Antigen presentation to generate an immunological response Exosome biogenesis and Actin, Cofilin, Tubulin secretion Exosome targeting and uptake Integrin-α,-β, P-selectin Exosomes targeting and uptake β-galactosidase, O-linked glycans, N-linked glycans. Exosome targeting and uptake, TNF-α, TGF-β, TNF-related signalling apoptosis-inducing ligand (TRAIL)

11 • Exosomes and Their Theranostic Applications in Healthcare TABLE 11.1 (Continued) SR NO.

201

Exosome composition and roles of main components [16,30]

CATEGORY

10.

Other signalling receptors

II) 1. 2.

Lipid Sterols Sphingolipids

3. 4.

Sphingolipids Phospholipids

5.

Phospholipids

6.

Phospholipids

7.

Phospholipids

8. III) 1. 2.

Glycosphingolipids Metabolic enzymes Lyases Peroxidase

IV) 1.

Nucleic acid RNA

ROLE

EXAMPLES

Exosome targeting and signalling including apoptosisinduction and iron transport

Fas ligand (FasL), TNF receptor, transferrin receptor (TfR)

Exosome secretion Cargo sorting and exosome secretion Exosome rigidity and signalling Exosome formation, signalling, and uptake Exosome formation, structural characteristics Exosome formation, structural characteristics Exosome formation, structural characteristics Exosome rigidity

Cholesterol Ceramides Sphingomyelin Phosphatidylserine Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Gangliosides

Gluconeogenesis Enolase-1 Protection from reactive oxygen Thioredoxine peroxidase species Cell-to-cell communication

mRNA

The fundamental role of exosomes is to maintain communications between cells. For this, the cells produce exosomes through the process called exocytosis. These exosomes are then endocytosed by the target cell via a process called endocytosis. This allows cells to transmit signals for communication between nearby and distant cells [1]. Exosomes can have positive as well as negative physiological effects. Exosomes play beneficial role in many fundamental physiological processes like inflammation [31,32], cardiovascular disease [33,34], organ development [35], immune system response [36], antigen representation [37], neuronal communication [38], reproductive performance [39], and many more. They also have a role in a variety of diseases, such as aiding in viral infections and prion propagation [40] and cancer progression [41]. They might transfer and accumulate some cytotoxic, defective proteins, thus playing a role in the development of neurodegenerative disease [42,43].

11.5 EXOSOMES FOR THERAGNOSTIC APPLICATIONS Exosomes are naturally formed as a carrier for transporting a range of biomolecules as messages between two cells. Since the proteins expressed on the surface of the exosome serve as a diagnostic tool, these exosomes can be used as theragnostic agents. On the other hand, intracellular elements such as mRNAs, HSPs, and others function as therapeutic tools [44]. Exosomes are used as theragnostic agents since other traditional nano-based theragnostic agents possess limitations. Poor biocompatibility, ineffective biodis­ tribution, unintended immunogenic reaction, etc. are a few of these limitations. Exosomes include

202 Nanomaterials in Healthcare particular cell surface proteins, which provide them with targeting abilities and make it easier for them to interact with the recipient cell’s surface, thus avoiding the possibility of delivery at a non-targeted site [7]. Exosomes do not cause resistance, unlike certain conventional and some nano-based therapeutic methods, since they alter the machinery that causes resistance in the recipient cell. Other benefits of exosomes include longer transit times, less inflammatory response, defence against phagocytic cells, and improved enhanced permeability and retention (EPR) effects. These characteristics make it possible to effectively use exosomes as theragnostic agents in medicine [45]. However, a highly effective dose of exosomes is required for clinical applications due to their unintended internalisation by immune cells [46]. Exosomes have therefore been combined with other molecules, such as aptamers, dyes, nano­ particles, etc. to circumvent these restrictions and give exosomes the ability to serve as a diagnostic and therapeutic tool. Exosomes can be functionalized with nanoparticles or loaded with these modalities to be employed as theragnostic agents in medicine. Furthermore, these agents can be included in the exosome biogenesis pathway by being incubated with the cells of origin or loaded into the exosomes using methods like electroporation, ultrasonication, saponin modification, etc. [45]. Exosomes’ theragnostic capabilities have been investigated in both their native (using exosomes as a theragnostic application directly after an extraction) or engineered (using exosomes as theragnostic application after modification) forms, using methods such as recombinant DNA technology, conjugation or encapsulation of dyes or nanoparticles with enhanced optical functionalities. This aspect of exosomes will be covered in greater detail in subsequent sections.

11.5.1 Native Exosomes for Theragnostic Applications Exosomes are naturally composed of a plethora of biomolecules such as proteins, siRNAs, mRNAs, and so on, which have been shown to alleviate a variety of diseases in the recipient cell. These exosomes can thus act as carriers or theragnostic agents, facilitating the delivery of various biomolecules and eliciting a therapeutic response. Exosomes bind to cell surface proteins on the recipient cell and are then inter­ nalized by the target cell via endocytosis. When these exosomes are endocytosed, they cause the upregulation or downregulation of genes and proteins in the recipient cells, resulting in a therapeutic response. Exosomes have also been used as a theragnostic agent, and have thus been delivered through carriers such as hydrogels. Photoacoustic imaging-guided therapeutics for erectile dysfunction have been used with hydrogel loaded with stem cell-derived exosomes extracted from adipose-derived mesen­ chymal stem cells. Theragnostic system accumulation was improved by in-situ gelation of these hydrogels, allowing for better imaging and therapeutic effect. Polydopamine nanoparticles (PDNPs) incorporated a temperature-sensitive poly (ethylene glycol)-poly(-caprolactone-co-lactide) (PELA) hydrogel which was used to encapsulate stem cell-derived exosomes. As biocompatible photoacoustic contrast agents, PDNPs were chosen, while PELA hydrogels were used to control exosome release [47]. The accumulation of exosomes in soft tissues was made possible by another technique known as ultrasound-targeted microbubble disruption (UTMD). Ultrasound-targeted microbubble destruction (UTMD) has recently emerged as a novel technique for delivering tissue-specific genes. The cavitation effect within the microvasculature of target tissues is used to deliver nucleic acid drugs, which is especially useful in local delivery and tissues with biological barriers. However, before UTMD, microbubbles should be loaded with genes or drugs. Ultrasound microbubbles are currently ineffective for gene loading and have poor long-term stability, limiting their potential. Upon release of nucleic acid from the damaged micro­ bubble, the endocytosis efficiency is very low [48]. Exosomes can be delivered specifically to the refractory tissues using UTMD to enhance localized vessel permeability. Exosomes’ potent and precise delivery into the muscle, adipose tissue, and the heart opens up new possibilities for organ-specific gene therapy. This method showed a higher exosome accumulation in the targeted tissues, which was confirmed by the UTMD exosome’s fluorescence intensity being higher than in the control group [49]. Plant-derived exosomes, in addition to animal-derived exosomes, have been used as theragnostic systems. Plant-derived exosomes are superior to mammalian exosomes in terms of cost, availability,

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sustainability, and so on. The phytochemicals and RNAs found in exosomes also contribute to their therapeutic properties. Ginger-derived exosome-like nanoparticles (GELNs) containing RNAs have been demonstrated to play an important role in gut microbiome maintenance. Interaction of these RNAs with the genes of microorganisms like Lactobacillus rhamnose is essential for initiating the aryl hydrocarbon pathway. However, it has been reported that the target capabilities of these exosomes are limited by their membrane lipid composition [50]. Thus, there is a need for modifications of exosomes to overcome this constraint.

11.5.2 Engineered Exosomes for Theragnostic Applications Exosomes are engineered to enhance their targeting capabilities for subsequent delivery of desired cargo to the target site. The bioavailability of exosomes is restricted because of the rapid endocytosis by the immune cells resulting in a high therapeutic index. Different approaches have been explored by researchers to engineer exosomes to increase their bioavailability. These strategies also facilitated enhanced targeting capabilities, better stability in serum, avoiding endocytosis by immune cells, and imparting diagnostic capabilities to the exosomes. These approaches are used separately or in arrangement to enhance the efficiency of the theragnostic system. Figure 11.3 shows different modifi­ cations for the exosome-based theragnostic system.

FIGURE 11.3 Different modifications for exosome-based theragnostic system.

204 Nanomaterials in Healthcare Exosomes have been synthesized via a range of methods, such as molecular biology techniques, in which the biogenesis of exosomes is triggered by the introduction of a particular protein into the cell, resulting in the expression of that protein on the surface of the produced exosome. BACE1 is a target in Alzheimer’s disease, and it has been revealed that siRNA delivery utilising modified exosomes can knock down this protein. The exosomes were produced by transfecting dendritic cells with Lamp2b protein fused with RGV peptide, which caused the dendritic cells to make exosomes expressing Lamp2b protein coupled with RGV peptide. These exosomes were used to transport GAPDH siRNA to neurons, oligodendrocytes, and microglia in the brain [51]. This suggests that exosomes can circumvent the challenges of conventional drug delivery systems in crossing the blood-brain barrier (BBB). Exosome functionalization or loading with dyes, nucleic acids, peptides, or nanoparticles to give them theragnostic capabilities is another technique used for the design of the theragnostic system. For example, exosomes loaded with vanadium carbide and functionalized with the arg-gly-asp peptide for NIR-sensitive photothermal therapy The nuclear uptake of the theragnostic system by the target MCF-7 breast cancer cells was improved by the peptide’s functionalization [52]. Exosomes from mouse’s Raw 264.7 cells were coupled with the arg-gly-glu peptide for combinatorial treatment against cancer cells in a similar manner. Tumour cells were killed by a combination of NIR-sensitive indocyanine dye and paclitaxel loaded in these exosomes [53]. Different techniques can be used to functionalize nanoparticles, dyes, peptides, etc. According to a study, EDC-NHS carbodiimide chemistry was used to functionalize superparamagnetic iron oxide nanoparticles (SPIONS) with transferrin. This led to the formation of SPIONS functionalized with transferrin, which connected with transferrin receptors on exosomes generated from blood plasma and carrying the peptide BAY55–9837 for the treatment of type 2 diabetes mellitus. The SPIONS served as the diagnostic tool and helped monitor the response of the system [54]. This functionalization approach can also be used to functionalize peptides onto exosomes to deliver oligonucleotides [55]. Exosome loading with various targeting as well as theragnostic agents has been shown to improve the theragnostic system’s accumulation and retention capabilities. Passive loading, or loading the ther­ agnostic agent during or after exosome isolation, has been employed in the development of these theragnostic systems. The pre-isolation approach involves the incubation of a theragnostic agent with the source cells, which results in the exosomes encapsulated with these agents. Exosomes containing silver nanoclusters and doxorubicin were isolated by incubating the precursor i.e. the silver salt and doxoru­ bicin with various normal and cancerous cells. Theragnostic system accumulation was found to be greater in cancerous cells compared to normal cells by using in-situ synthesised silver nanoclusters as a targeting agent and diagnostic tool [45]. This technique encapsulates these drugs through the exosomal biogenesis pathway [25]. The alternative method involves adding these substances exogenously into the exosomes after they have been isolated and purified. For instance, utilization of the focused electroporation technique to create RNA-encapsulated exosomes for delivery is a simpler and more effective way. This method is simpler than previously described ones since it doesn’t require extensive and complicated molecular alterations to the cytosolic components, or source cells for producing RNA-encased exosomes [56]. This technology enables therapeutic agents such as DNA and miRNA to be loaded into the exosome while being tagged with fluorescent molecules (diagnostic modality) such as supercharged proteins generated by altering the lysine to arginine ratio and NIR-sensitive dyes for theragnostic applications [57,58]. Manganese carbonyl (MnCO) has recently been demonstrated to have anticancer action due to its capacity to react with H2O2 to form carbon monoxide (CO), which depletes oxygen from haemoglobin and damages mitochondria. However, CO is highly toxic, making its use in clinical settings challenging. Exosomes have also been investigated as carriers that, upon reaching the target region, release gases like carbon monoxide (CO). A tumour-derived exosome laden with manganese carbonyl (MnCO) demon­ strated persistent CO gas release in a high H2O2 environment. These mitochondria-toxic substances destroy only hypoxic tumour cells, protecting healthy cells from damage [59].

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Animal-derived exosomes have limitations that include high isolation costs, complex infrastructure, scale-up constraints, etc. Exosomes from various species, such as plants, can be used to get around these restrictions. As they exhibit cross-reactivity across different species, plant-derived exosomes can also be utilized for biomedical applications. Indocyanine green-loaded exosomes from aloe plants have been investigated for theragnostic use in tumour cells, including B16F10, 4T1, and MCF-10A [60]. These plant-derived exosomes, however, show constraints, including a restricted number of target-binding surface proteins that have an affinity for a particular cell, which limits their utility in their unaltered state [60].

11.6 ABSORPTION AND DISTRIBUTION OF EXOSOMEBASED THERAGNOSTIC SYSTEM The pharmacokinetic behaviour of the exosomes plays a vital role in determining their efficiency as a theragnostic system. Hence, understanding their absorption and distribution in-vitro and in-vivo is essential. Theragnostic systems based on exosomes are absorbed by target cells via a process known as endocytosis. This is an energy-dependent process. Different endocytic pathways can be used by exosome-based theragnostic systems to ensure their effective absorption by the target cells. The primary pathway used by aptamers conjugated doxorubicin-loaded exosomes is clathrin-mediated endocytosis. Endocytosis inhibitors (eg. dynasone) decreased the absorption of the engineered exosomes by the target CEM cells via inhibition of membrane receptors, showing the significance of these receptors in the endocytosis mechanism. Other endocytic mechanisms, such as the caveolae-mediated pathway and micropinocytosis, are also implicated in theragnostic system absorption, as evidenced by the decreased absorption of these engineered exosomes by target cells after being treated with their inhibitors [61]. The in vivo absorption of the theragnostic system is susceptible to a variety of variables. Reduced absorption of the theragnostic system can be caused by features like the functional element attached to the exosome’s surface and the process of functionalization [62]. Understanding the theragnostic system’s distribution in vivo is crucial in comprehending how these systems behave. In a study, Rashid and colleagues found that tumour-derived exosomes were distributed in the lungs and targeted tumour cells in a time-dependent manner. The theragnostic system’s dispersion is significantly influenced by the functional moiety attached to the exosomes [63].

11.7 CHALLENGES RELATED TO EXOSOMES FOR THERAGNOSTIC APPLICATION The lack of appropriate techniques to assure sufficient exosome yields is a major obstacle in the ther­ agnostic application of exosomes. Liposome extruders are used to create synthetic vesicles in order to get around this restriction. However, clinical contexts have not yet been adequately examined for this approach [64]. Although plant-derived exosomes produce excellent yields of exosomes in comparison to mammalian exosomes, only a limited number of these exosomes exhibit specificity for the target site. Therefore, exosomes necessitate further modifications via different methods. Modifications of exosomes often result in morphological alterations in exosomes. These alterations include changes in the composition of phospholipids and changes in the surface proteins responsible for targeted interactions. Modifications also result in changes in the cytosolic composition and loss of asymmetry causing decreased absorption of these exosomes by the recipient cells. Most of these changes

206 Nanomaterials in Healthcare occur as a result of modifications carried out before the isolation of exosomes. These pre-isolation modification procedures often are laborious and require expensive infrastructure [29]. The lack of appropriate methodologies to isolate monodisperse exosomes also acts as a barrier to its applicability as a theragnostic system. Currently available methods produce polydispersed exosomes which reduce the absorption of these theragnostic systems at the target site [60]. Exosomes’ clinical use is further constrained by a limited understanding of the type of their contents and in vivo behaviour.

11.8 CONCLUSION AND FUTURE PROSPECTIVE Exosomes-based theragnostic systems are intrinsically biocompatible, exhibit superior EPR effect, and can bypass lysosomes, thus proving to be an alternative to synthetic nanoparticles. A poor understanding of their heterogeneity and complexity is a limiting factor for their utilization as a theragnostic system. Exosomes enable cellular communication and thus are pathologically relevant. However, their utilization as the theragnostic system is restricted by the difficulties in their isolation, purification, and subsequent modifications. Mammalian exosomes require a sophisticated infrastructure, resulting in a high cost for the system developed. Plant-based exosomes can overcome this limitation and thus is an interesting area for future exploration. However, more information on the composition of these exosomes, strategies to isolate, purify and modify them, and their potential application as a theragnostic agent needs to be explored for future application.

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Nanogels for Theranostic Applications in Healthcare

12

Vaishali Pawar1, Amreen Khan1,2, Shruti Pendse1, Rupali Bagale1, Akshara Adapa1, and Padmini Chandra1 1

NanoBios Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 2 Center for Research in Nanotechnology and Science, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 12.1 12.2

Introduction Applications of Nanogels 12.2.1 Nanogels for Targeted Drug Delivery 12.2.1.1 Active targeting 12.2.1.2 Passive targeting 12.2.2 Nanogels for Stimuli-Responsive Drug Delivery 12.2.2.1 Temperature-responsive nanogels 12.2.2.2 pH-responsive nanogels 12.2.2.3 Light-responsive nanogels 12.2.2.4 Magnetic-responsive nanogels 12.2.3 Nanogels for Poorly Water-Soluble Drugs 12.2.4 Nanogel for Gene Delivery 12.2.5 Nanogels for Brain Drug Delivery 12.2.6 Nanogels in Diagnosis and Imaging 12.3 Challenges and Future Perspective 12.4 Summary and Conclusion References

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DOI: 10.1201/9781003322368-12

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212 Nanomaterials in Healthcare

12.1 INTRODUCTION Hydrogels are cross-linked hydrophilic polymers with the ability to retain a large amount of water [1]. Cross-linking can be either via chemical or physical methods [2]. Nano-sized systems have shown lower degradation, controlled release of the drug, and enhanced penetration effect. Following the benefits, the nanosystem design of nanogels as nano-sized hydrogels started to develop [3]. The incorporation of moieties can be done by taking advantage of material properties of nanogels like charge and immobilization for electrostatic interactions. In any of the cases, the release of molecules is assisted moistly by hydrolytic degradation [4]. The densely and highly branched cross-linking is owed to the molecules’ sustained release for a prolonged time provided the mechanical properties of nanogel remains within the desired limit. The main component of nanogel which also defines its properties is a polymer as the basic ingre­ dient. Different types of natural and synthetic polymers have been utilized in nanogel formation [5]. Characteristics like charge, porosity, stiffness, and degradation can be varied by the chemical compo­ sitions of the polymers. The selection of polymer is also done based on its interaction with the active molecule specifically in case of entrapment or encapsulation [6]. The properties of another component, the cross-linker, demonstrate the potential of altering the mechanical properties of nanogels. Thus, each component and its combination gave importance in nanogel formation and its application. Nanogels can also be designed in response to different stimuli including light, pH, and temperature through functionalization with different groups [7]. Surface modifications of nanogels also provide the advantage of specifically targeting the active moiety to a particular site. In the case of targeted delivery, properties like swelling, softness, and site-specificity become important considerations [6,8]. The threedimensional network formed by hydrogel further creates a way to hold the water molecules through absorbing and swelling. The extent of swelling and deswelling of nanogels have a characteristic approach like composition, cross-linker hydrophilicity, and degree of the cross-linked network [8]. All of this restricts the mobility of the polymer chain and further freedom of conformation. The water-holding capacity and maintaining intactness of nanogel structure correlate well with biologically active mole­ cules when carried by gel pores. Nanogel utilization in biosensing, biochemical separations, cell culture systems, and anticancer therapy are other advantages. Moreover, the long-term and leading trend can be seen in the delivery of diagnosis, imaging, and contrast agents. Such a combination of organic nanogels and incorporation of inorganic agents are classified under nanohybrids, which have wide applications in multipurpose medical conditions [9]. The core-shell structure of nanogels comprises two layers that contribute to the efficiency of drug delivery. The inner layer, also known as the core, functions in carrying the delivering agent and the outer layer, the shell, plays a role in protecting the core. Since a majority of the drugs are unstable and express low solubility, the core-shell structure of the nanogel can be helpful in loading and protecting the drug from the incompatible surrounding environment. The core and shell structure of nanogels can be further modified for distinct features such as targetability, retention time, controlled release, stimuli-responsiveness, and stability [10]. Several distinct characteristics of nanogels make them ideal for their application in biomedical as drug delivery agents, diagnostic, imaging, and theranostic systems. Characteristics such as high bio­ compatibility and high biodegradability of nanogels ensure no adverse effects on bodily organs upon administration. The small size of nanogels enables enhanced permeation capability, penetration into target sites including solid tumors, tissue, and infracted area, and intracellular drug delivery. Another important advantage of nanogels is their ability to carry both hydrophilic and hydrophobic drugs by functional group modification. Finally, the stimuli-responsive behavior of nanogels allows the triggered and controlled delivery of drugs at the target site with high selectivity, thus increasing the therapeutic efficacy [11]. With diverse applications of nanogel, in this chapter, we have compiled the basics and

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recent developments in nanogels. Covering the synthesis of nanogels in different drug delivery areas, the sections also focus on the properties and suitability of nanogels as effective carriers.

12.2 APPLICATIONS OF NANOGELS 12.2.1 Nanogels for Targeted Drug Delivery The versatility of nanogel-based drug delivery platforms provides a great opportunity to use them as a targeted drug delivery vehicle. These nanogels can be used for delivery of the drug at the desired location such as an organ, tissue, or cell. However, the non-specific nanogels can be easily cleared from circu­ lation or can be easily taken by non-targeted cells. These nanogels, therefore, need to be modified through active and passive targeting strategies to reduce imprecise targeting and increase circulation time, permeation, and stability of the nanogel. Both the passive and active targeting are shown in Figure 12.1. In active targeting, the nanogel surface platform is combined with the ligand, which interacts with the antigens or receptors present on the target cells specifically. Passive targeting involves modification of nanogels to increase the passive accumulation of the drug through the effect of the enhanced permeability and retention (EPR). To increase the EPR effect, which is equivalent to circu­ lation time in blood, the nanogels need to be surface modified to reduce opsonization and recognition that leads to its removal from the body system [12].

12.2.1.1 Active targeting Monoclonal antibodies are such protein moieties that are of particular interest due to their specific interaction with tumor cell membrane proteins. Combining the polymer from the gel to the monoclonal antibodies (mAb) can be done through Schiff base formation. Aldehyde-functionalized cl-PEG-b-PMA nanogels were connected to mAb CC49 produced against a tumor-associated glycoprotein 72 (TAG-72). As conjugating mAb with nanogels did not affect its specificity, decorating the surface of nanogel with

FIGURE 12.1 Types of targeted drug delivery in nanogels.

214 Nanomaterials in Healthcare mAb CC49 retains its strong interaction with the surface-immobilized antigen [13]. To target CD4+ T cells or CD4 lymphoma cells, a random copolymeric nanogel was created using poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide ethyl methacrylate (PDSMA) that was con­ jugated with anti-CD4 antibodies. This leads to selective targeting and decreases the non-specific uptake of the drug mertansine present in nanogel [14]. In recent times, a dual-targeting drug delivery system has been worked upon, where two different biomarkers are taken into consideration for active targeting of tumor cells. Hyaluronic acid nanogel combined with epidermal growth factor receptor (EGFR) and CD44 boost therapeutic protein delivery in SKOV-3 human ovarian cancer and MDA-MB-231 human breast cancer. The over-expression of EGFR and CD44 on the cancerous cell leads to increase uptake of the Granzyme B therapeutic protein present in the nanogel through receptor-mediated internalization. When compared with single targeting, this dual-targeting approach gave much more enhanced permeation of therapeutic protein in the cancerous cells [15]. Mesenchymal stem cell membranes are comparatively easy to isolate and contain the molecular recognition moiety that interacts with the tumor cells with high affinity. This quality of the stem cell membrane has been exploited to develop the targeted active drug delivery system for tumor inhibition. Stem cell membrane coated gelatin nanogel (SCMG) was prepared where doxorubicin hydrochloride loaded gelatin hydrogel are surface fused with stem cell membrane vesicle for tumor recognition purpose [16]. One of the signs used in targeting ligands for anti-cancer drug delivery is RGD (Arg-Gly-Asp) motif as they are recognized by the integrins that are over-expressed on cancerous cells. Cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2) nanogel was linked with the RGD peptide and those nanogels were incubated with HeLa cells. It was observed that the RGD-CHP nanogels were uptaken by the cancerous cells through integrin receptor-mediated endocytosis, particularly through clathrin-mediated endocytosis and micropinocytosis [17].

12.2.1.2 Passive targeting The Janus nanogel prepared from PLGA–PEG–PLGA with a pro-drug of Taxol, PEGylated Taxol was prepared that become one of the prospective polymeric carriers for the improved delivery of Taxol prodrug which is a hydrophobic drug molecule towards the target tumor tissue through the EPR effect. The pre­ pared Janus nanogel effectively inhibited the growth of the tumor when compared with the clinically used Taxol [18]. Carboxy methyl cellulose (CMC) nanogel containing disulfide linkages are very sensitive to the reductive environment. Tumor tissue region contains four times higher concentration of Glutathione generating reductive environment over there. These CMC nanogels get de-cross-link very quickly leading to the release of the drug entrapped inside the nanogel. These gels were also having high drug loading capacity and show a higher concentration in plasma with much more retention time. Doxycycline-CMC nanogel had prolonged circulation in the blood and significant drug accumulation inside the tumor tissue site [19]. As Glutathione maintains redox homeostasis in the tumor region, magnetic composite nanogel was developed containing redox-active polymer methacryloyloxyethylphosphorylcholine (MPC) and N, N′-bis(acryloyl)cystamine (BACy) as the outer covering and acid-degradable Fe3O4 nanoclusters as the core that reduces Glutathione concentration through thiol-disulfide exchange reaction. The more promising thing regarding this nanogel was it showed increased EPR effect, and superior cellular uptake efficiency providing a better platform for passive targeting and photodynamic therapy [20]. A zwitterionic nanogel was prepared using sulfobetaine methacrylate (SBMA) and N, N′-bis-(acryloyl) cystamine (BAC) loaded with doxycycline. These nanogels are resistant to surface adsorption of proteins, thus preventing protein corona formation, which eventually leads to the prolonged circulation of these nanogels into the blood. They also possess excellent stability, a lower degradation rate, and increased drug release [21]. Another strategy for drug targeting is through nanomotors, which are synthetic motors converting chemical energy into the driving force for drug delivery. A biodegradable (Pt/ CaCO3@HA-CB) system was created where Pt/ CaCO3 acts as a nanomotor and Cuccurbituril-conjugated hyaluronate (HA-CB) is the nanogel com­ ponent. The presence of H2O2 and acidic conditions in the tumor site degrade the nano-motor part leading to the release of encapsulated HA-CB nanogel that are eventually uptaken by cancerous cells through receptor-mediated endocytosis [22].

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FIGURE 12.2 Types of stimuli-responsive nanogels used in drug delivery.

12.2.2 Nanogels for Stimuli-Responsive Drug Delivery The demand for nanogels is increasing in the field of drug delivery. It is gaining popularity due to its high water absorption property, targeted drug delivery, adhesion to biologically active molecules, etc. Dual stimuli-responsive nanogels are made using organic and inorganic polymers. These nanogels are easily assembled by using different polymers having functional groups; e.g. pH- and temperature-responsive nanogels are used extensively for controlled release [23]. Nanogels can contain both hydrophobic and hydrophilic drugs loaded with quantum dot-polypeptide. The release rate of these drugs can be controlled by varying pH and temperature [24]. Figure 12.2 shows various stimuli-responsive nanogels.

12.2.2.1 Temperature-responsive nanogels There are temperature-responsive nanogels that respond to temperature as a stimulus for drug delivery. At a certain temperature, these nanogels have volume phase transition (VPTT). Poly(N-isopropyl acrylamide) (PNIPAM) is a polymer that has a lower critical solution temperature in an aqueous solution and the particle size of this polymer decreases drastically when the temperature increases above its set VPTT [25]. In drug delivery, positively temperature-responsive nanogels are preferred because the drugs, in this case, are entrapped within the nanogels before the temperature-triggered release which prevents the early release of the drug from the network of nanogel [26]. For producing temperatureresponsive nanogels, VPTT and phase transition temperature are the most important parameters. At inflammation sites, the body temperature is higher than the normal body sites. Therefore, VPTT slightly higher than the biological temperature is favored [27]. Incorporation of hydrophilic monomers into the PNIPAM chain has increased the VPTT, but in recent research, non-PNIPAM nanogels having higher VPTT are used in drug delivery. These nanogels have high sensitivity to changes in temperature by having narrow VPTT. These nanogels are made using inorganic clay as a cross-linker [28]. The drug loading capacity can be increased by producing hollow nanogels by colloid templated polymerization and then removal of the core that is used as a template [29].

12.2.2.2 pH-responsive nanogels The pH value differs in different body tissues within the human body. The pH of the normal tissue is 7.4. Likewise, most of the tumor cells/tissues exhibit lower pH. These cancerous cells are very fast growing and therefore they uptake nutrients at a faster rate leading to insufficient oxygen and energy supply and production of lactic acid making the conditions acidic [30]. The extracellular pH is 6.5–6.8 and the

216 Nanomaterials in Healthcare intracellular endo/lysosome has an even lower pH of 5.0–5.5. Therefore, the pH-sensitive nanogel for drug delivery will be very useful [31,32]. Nanogels composed of cross-linked polyelectrolytes containing both weakly acidic or basic groups are being developed for pH-targeted tumor theranostics. There is a pH-dependent charge conversion property that switches from an acidic environment from positive to negative to enhance the cellular uptake of drug-loaded nanogels and kill cancer cells. There are many such organic-inorganic nanogels developed for pH-targeted drug delivery [33], pH-dependent swelling, and shrinking properties that show a positive surface charge in the acidic environment are being developed via cationic polymer-mediated AuNC self-assembled nanogels. The fluorescence property was enhanced by cross-linking of AuNCs by the phenomenon of aggression-induced emission. Also, peptides and antibodies are loaded, which enhances cellular uptake [34].

12.2.2.3 Light-responsive nanogels The light-responsive nanogels consist of two categories. The first category consists of metal nano­ particles like Au, Ag, Pt, and Ge, which have a photothermal capability. With minimal irradiation energy, these metal nanoparticles lead to high absorption leading to efficient light-heat conversion due to surface plasmon resonance (SPR) [35]. Carbonaceous nanomaterials (carbon nanotubes, graphene, etc.), semiconductor nanomaterials (CuxS, CuxSe, CuxTe, etc.), and magnetic nanoparticles (FexOy, etc.) have also shown efficacy in converting light into heat [36,37]. These nanoparticles can be successfully incorporated into polymeric nanogels, which offer photothermally induced drug release for anticancer applications [38]. The negative thermoresponsive polymer poly (NIPAAm) is conjugated with Au nanoparticles, which upon NIR irradiation, produce heat at a certain wavelength of light for cancer photothermal treatment [39]. The second category involves photoactive groups like azobenzene, spir­ obenzopyran, triphenylmethane, or cinnamonyl. These functional moieties change size and shape upon irradiation. The trans and cis conformation of photoisomerized azobenzene have two phenyl rings linked by an N=N double bond where the isomers can be switched using ultraviolet (UV) light [40].

12.2.2.4 Magnetic-responsive nanogels Magnetic-responsive nanogels consist of magnetic materials like iron oxide nanoparticles. As iron is the most abundant metal found inside the human body, these iron oxide nanoparticles are non-toxic and biocompatible and widely used in theragnostics [41]. Based on the external magnetic field, these nanoparticles can be transferred and directed toward the targeted site for drug delivery. Magnetic poly (vinyl pyrrolidone) nanogels are synthesized with the chemotherapeutic drug Bleomycin A5 Hydrochloride (BLM). When these nanogels were injected into rabbits with auricular VX2 tumors, and a permanent magnet over the surface of the tumor, there was a significant tumor size reduction seen within 24 hours. Similarly, magnetic polyacrylamide nanogels were also injected into rabbits depicting effective results [42].

12.2.3 Nanogels for Poorly Water-Soluble Drugs Nanogels are commonly used for the delivery of hydrophilic drugs having several properties like deformability, tenability, colloidal stability, functionality, etc., which solves problems of the barriers seen in delivering especially hydrophobic drugs. These hydrophobic drugs can be efficiently delivered without compromising their therapeutic effects to the targeted site via nanogels. Microgels are introduced into nanogels, which are core-shell structure nanogels that have hydrophobic domains for delivering hydrophobic drugs [43]. The degree of loading the hydrophobic drug into microgels can be increased via electrostatic, van der Waals interaction, etc. There are various strategies used to enhance the loading of hydrophobic drugs into nanogels. Compartmentalization of hydrophobic nanoparticles into microgels is in such a way wherein the drug is entirely encapsulated inside the microgel matrix. Multiple hydrophobic

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drug nanoparticles can also be loaded [44]. Nexinhib20 is a drug used for the treatment of pulmonary inflammation. This drug is loaded into the core of poly(lactic-co-glycolic acid) nanoparticles that are then encapsulated inside PEG microgels via the water-in-oil emulsification method [45]. Thermosensitive nanogel can be used for microgel which maintains swollen state below its VPTT to enable higher hydrophobic drug loading. Oligo (ethylene glycol)-based thermosensitive microgels were used for the chemotherapeutic agent dipyridamole. The other strategy is to involve hydrophobic moieties in the cross-linked polymer of microgel and then increase the drug loading capacity of hydrophobic drugs. Hyaluronic acid has a cholesteryl group (CHHA) and CHHA modified with amino acids was prepared for drug delivery, which was successfully incorporated into nanogels to enclose poorly watersoluble drugs and also increase the solubility [46].

12.2.4 Nanogel for Gene Delivery Delivery of genes is very crucial and the suitability of a good carrier system is very demanding [47,48]. Possible limitations of gene-carrying delivery systems are prone to be cleared faster as chances of intracellular chemical and enzymatic degradation are very high [49]. Polymers can turn out to be a good choice as they are available in different molecular weights, charges, and easy modifications for linking targeted moieties. Moreover, the reactivity in physiological conditions can also be controlled and easy to scale up [50]. Further, softness, swelling, and the ability to maintain their activity in biological conditions under correct conformation can be an added advantage. The viscous depot and gel-like behavior feature various physicochemical properties like the high degree of cross-linking and porosity [51]. Various approaches have been implied for gene delivery by nanogels, especially through a stimuliresponsive release, as shown in Figure 12.3. The ability to accumulate and release the payload to the desired site and structural changes can be facilitated by a change in temperature [52]. Poly(N-isopropyl acrylamide) (PNIPAM) is one such polymer that is thermosensitive and has been used to provide thermal responsive behavior. When combined with polyethyleneimine through radical graft copolymerization, PNIPAM can encapsulate and carry the tumor suppressor gene TRP53. This cationic thermo-responsive nanogel is considered a system with higher transfection efficiency [48]. Likewise, other stimuliresponsive nanogel-based delivery systems have been studied for carrying genes. DNA-nanogel complex formed by interaction has been seen by linear stabilization of cationic glycopolymer that further facilitated cellular and gene uptake. For the co-delivery of plasmid DNA and protein, a delivery system containing methyl ethyl methacrylate and 2-lactobionamidoethyl methacrylamide by reversible

FIGURE 12.3 Types of stimuli-responsive nanogels used in gene delivery.

218 Nanomaterials in Healthcare addition-fragmentation chain transfer polymerization was introduced. The release profile of the nanogel encapsulating gene showed burst release [53]. Genes along with growth factors have also been studied in different carrier systems including nanogels [54]. Genes, specifically siRNA, have been effectively co-delivered for various applications to the desired site by polymeric nanogels. A few challenges like endosomal entrapment of nanogel com­ plexes can also be dealt with by effectively utilizing the co-delivery approach [55]. Protamine-based nanogel has been used for the co-delivery of epigallocatechin-3-O-gallate and competent siRNA with a promising strategy. This single nanogel system with diverse components has been effective in increasing the cytotoxicity of drug-resistant cell lines by many folds as compared to only drugs. Selectivity and tumor growth inhibition activities were demonstrated efficiently by the multi-component carrier system [56].

12.2.5 Nanogels for Brain Drug Delivery The delivery of drugs to the brain is hampered by the blood-brain barrier (BBB), which results in a decrease of therapeutic effect. Studies have shown that a few factors of compounds of nanoparticles can penetrate the BBB and reach the brain [57]. Enhanced permeability and retention (EPR) effect, endocytosis, and receptor-mediated transcytosis are some of the ways through which drugs can be delivered to brain [58]. Therapeutic agents are delivered intravenously or intranasally before or after surgery to target the brain tumor. MRI traceable instant gelated nanogel is prepared using negatively charged carboxymethyl cellulose-grafted poly(N-isopropylacrylamide-co-methacrylic acid) and posi­ tively charged gadopentetic acid/branched polyethylenimine [59]. Two drugs, olaparib and etoposide, were delivered using spray delivery system of bioadhesive hydrogel consisting of pectin and poly (ethylene glycol)-block-polylactic acid (PEG-b-PLA). Burst release of 5% was observed with 100% drug release in 48 hours. Due to the bioadhesive nature of hydrogel, it adheres to the brain tissue and was not washed away by interstitial fluids [60]. Likewise, the various drugs were delivered to the brain by different methods.

12.2.6 Nanogels in Diagnosis and Imaging To improve current therapies for lethal diseases, nanogels were developed with the theragnostic approach. Nanogels are fabricated in such a way to diagnose and deliver the drug at the same time, thus offering a better theragnostic platform. These nanogels are of prime interest for cell imaging purposes when compared with a conventional imaging agent as it is biocompatible, less toxic, and possess betterenhanced permeation and retention effect. Different forms of diagnosis and imaging agents containing nanogels are shown in Figure 12.4. Nanogels satisfying the dual need of being onco-theragnostic were developed using alginate and superparamagnetic iron oxide nanoparticles (SPONs) along with the incorporation of doxycycline in the alginate nanogel. Due to inherent magnetic properties, SPONs can be used for MRI imaging. These nanogels not only act as a good MRI contrasting agent but also can be used for magnetically targeted drug delivery purpose [60]. To get fluorescence imaging, a hybrid nanogel was developed containing Ag-Au bimetallic nano­ particle core, thermo-responsive polyethylene glycol as the shell, and hyaluronic acid present on the surface that specifically targets the tumor cells. The visible fluorescence for imaging the melanoma cells was generated by the bimetallic Ag-Au core. The bimetallic core can change the intensity of the fluo­ rescence by sensing the temperature difference through thermo-responsive PEG [61]. To further have dual-color fluorescent imaging, a cyclodextrin nanogel was fabricated containing spiropyran and 4-amino-7-nitro-1,2,3-benzoxadiazole (NBDNH2) encapsulated in it. The spiropyran molecule acts as an acceptor under ultraviolet or visible light and gives out fluorescence used for imaging purposes.it also quenches or recovers the fluorescence generated by NBDH2 thus helping to achieve

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Nanogels

Supermagnetic Iron Oxide Nanoparticlesnanogel MR Imaging

Au Nanoparticles-nanogel Anti-oxidant enzyme-nanogel

Dark field microscopy CT Imaging

QDs/Fluorophore-nanogel

Ultrasound Imaging

Fluorescence Imaging

FIGURE 12.4 Different forms of diagnosis and imaging agents containing nanogels.

dual-color fluorescent imaging. This covalent interaction between the two fluorophores provides photoswitchable property along with less dye leakage and long-term stability. The photo responsiveness of this nanogel was within 60seconds and switching behavior was observed every time of its use [62]. Using the aggregation phenomenon, it is easy to control the particle size of the nanogel and therefore can passively target the cells for better uptake. The stabilized aggregated nanogel particles were labeled with fluor™ 750C2maleimide dye and were effectively up taken by the lungs. This dye-labeled nanogel persisted at the target site for around 48 hours and then was enzymatically degraded and cleared out without any traces of toxicity [63]. The unique properties of quantum dots such as photostability, wideband excitation, and high quantum yield make them one of the best candidates for cell imaging purposes. For cancer cell imaging, the incorporation of cadmium-telluride quantum dots in chitosan nanogel was carried out. It was observed that there was no complete quenching of fluorescence even in the cellular localization on quantum dots under the experimental pH conditions. Owing to the small size of QD-CNGs, they were easily uptaken by the cells through phagocytosis [64]. Lysozyme-dextran nanogel containing gold nanoparticles and doxycycline was also fabricated for theragnostic purposes. The dextran shell endows the prepared nanogel with stealth property through which it can easily bypass the immune system detection and therefore have prolonged circulation time. The hydrophilic dextran makes the nanogel much more stable under physiological conditions. Au-Lys-DexDox nanogel act as a contrasting agent for optical imaging of cell along with being a drug carrier [65]. Incorporation of fluorescein isothiocyanate (FITC)-labeled peptides and Au nanoparticles in cross-linked polyamine core PEG nanogel was used to have a real-time fluorescence imaging of hepatic tumor cells to an apoptosis-inducing agent, staurosporine. In the case of normal cells, caspase 3 is in an inactivated form that causes quenching of fluorescence signal due to fluorescence resonance energy transfer between gold nanoparticles and FITC. In apoptotic cells, the peptide is cleaved by caspase 3 activation leading to a strong fluorescence signal that appears [66]. Cancer cell imaging along with photothermal therapy was obtained through FITC labeled Au@IPNpNIPAAmnano-gel. The uptake of this nanogel by HeLa cells was dependent on dose concentration and time. The majority of Au@IPN-pNIPAAmnanogels were observed inside the lysosome. Due to the scattering properties of gold nanoparticles, Au@IPN-pNIPAAmnanogels acted as the contrasting agent to form cellular imaging through simple dark-field microscopy [67].

220 Nanomaterials in Healthcare Nanogels prepared from cholesterol-bearing pullulan were mixed with protein-coated quantum dots and were highly stable due to the electrostatic interaction between the QDs and amino group present in the polymeric pullulan matrix. Due to their stability, they were internalized by the HeLa cancerous cell line without any aggregation. The quantum dots presented uniform fluorescence distribution inside the cytoplasm of HeLa cells. The uptake of these nanogels bearing QDs was much more than the con­ ventional way of using QDs through conjugation with liposomes [68].

12.3 CHALLENGES AND FUTURE PERSPECTIVE The nanogel-based platform has been one of the prime interests in nanotechnology-based pharmacology due to its diverse properties. Through varied modifications, nanogels possessing several properties and serving multiple functions can be achieved. This characteristic of nanogels helps tremendously to attain targeted and enhanced drug delivery. Camouflaging the nanogel through a specific biological membrane can be explored thus creating an intelligent actively targeting drug delivery system. Instead of using acrylate-based polymers, nanogels can be fabricated using polysaccharides and stable proteins that have no or very less toxicity. Much more emphasis should be put on developing mechanisms to generate stimulus-responsive nanogels that will transport a significant amount of drug efficiently in the presence of different barriers. Instead of having a rigid nanogel, its viscoelasticity should be taken into consid­ eration as it enhances its accessibility to the target site. Nevertheless, there are several problems asso­ ciated with the clinical use of nanogels. The hybrid nanogels that are widely developed for theragnostic purposes face problems related to uncontrollable size, the biocompatibility of the components used, and the lack of proper clinical data showing the safety of the prepared nanogels. Surface functionalization or surface modification that assists in targeted delivery can negatively affect the biocompatibility, stability, circulation and retention in blood, and permeation as well. Quantum dots that are incorporated in nanogels for imaging purposes can show cytotoxicity if prepared through heavy metals. Engineering of nanogels should be done in such a way that there is complete clearance of nanogels before the release of the quantum dots in the body system. Moreover, the unreacted monomers or any residual surfactant that are present in the nanogel can cause lethal effects. The presence of non-covalent bonds in the nanogel needs to be considered as it can cause leaking of the drug or targeting moiety eventually affecting its release. Fine-tuning of many components of nanogels and thorough analysis of the pharmacokinetics and pharmacodynamics needs to be done before their clinical application. A large amount of in vivo and in vitro studies need to be performed before their clinical trial. To date, the nanogel preparation is restricted to the experimental level, but to bring this to people, the developmental procedure for large-scale pro­ duction and its cost-effectiveness need to be understood and studied. Even though nanogels have evolved a lot encasing several moieties and becoming a better system for targeted drug delivery, much remains to be done that will help to discover the multifunctionality of those gels. If all the negative issues related to nanogels are properly addressed, this drug delivery and the theragnostic system can be converted into a better category of pharmaceuticals, thus improving clinical care.

12.4 SUMMARY AND CONCLUSION Applications of nanogels in the field of biomedical have made tremendous progress in the last years. Due to the hydrophilic nature of nanogels, delivery of hydrophilic drugs is easily achieved; however, recent developments have allowed the fabrication of nanogels for the successful delivery of hydrophobic

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drugs too. Overtime nanogels have been adapted to carry several drugs, peptides, proteins, and genes. A single nanogel has evolved to carry multiple drugs and respond to multiple stimuli, thus making them highly efficient in delivering drugs at a target site, increasing therapeutic efficacy, and also making them an ideal theranostic system. This is achieved due to continuous advancement in understanding the properties of nanogels and using this knowledge in fine-tuning the characteristics to our advantage. Nanogels are engineered to make responsive to pH, light, and temperature by functionalized polymers or ligands that help in their action at the target site with controlled release of the drug. The biocompatibility and biodegradability properties of nanogels make them highly safe for use in vivo with minimum toxicity or adverse effects. The development of hybrid nanogels providing multipurpose action is prime in biomedical applications. These hybrid nanogels not only penetrate the target site but also provide highresolution contrast imaging, helping in diagnosis and then delivering the drug in response to environ­ mental stimuli. Several research studies have exploited these properties of nanogels and have been successful in tailoring the nanogels against cancerous tumors. Nanogels can be used to impact diseases such as cancer; additionally, the varied range of possible alterations will provide application in bio­ sensing, diagnosis, imaging, theranostic, and gene delivery. Although great progress has been made in the field of nanogel design, the number of nanogels explored in clinical studies remains low. The complicated system and minute structural characteristics require careful monitoring of the nanogel to obtain the desired outcome. In conclusion, the biomedical application of nanogels requires biocompatibility, advancement in the design, and a detailed study of the in vivo behavior of nanogels to reduce the side effects.

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Theranostic Application of Nanofibers in Tissue Engineering

13

Atul Chaskar1, Namrah Azmi2, and Dhriti Shenoy1 1

National Centre for Nanoscience and Nanotechnology, University of Mumbai, Mumbai, Maharashtra, India 2 Dept. of Physics, Faculty of Natural Sciences, Norwegian University of Science and Technology, Trondheim, Norway

Contents 13.1 Introduction 13.2 Nanofiber-Based Scaffold for Drug Delivery 13.3 Nanofiber-Based Stem Cell Therapy and Labeling 13.4 Nanofiber-Based Scaffold Construction and Modification 13.5 Multifunctional and Smart Nanofiber-Based Scaffolds 13.6 Conclusion and Future Prospects References

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13.1 INTRODUCTION In the past few decades, enormous research has been done to explore tissue engineering and regenerative medicine. It is a highly multidisciplinary domain that uses the traditional knowledge of biology material science along with engineering sciences to develop a tissue or organ that not just resembles the original tissue or organ but also is able to carry out its functions [1]. Tissue engineering is a branch of regen­ erative medicine that attempts to repair and rehabilitate the damaged organs and tissues that cannot be repaired by conventional medication and therapies [2]. The material to be used to repair and rehabilitate the tissue for regeneration must possess the properties similar to the tissue. If not, there is a risk of rejection of the said tissue by the host body. So, to avoid severe immune rejection, low biocompatibility, and bi-functionality, tissue engineering uses the cells from the patients themselves. Apart from cells, scaffold i.e., the artificially or naturally engineered material which has the capability to cause desired cellular interactions and could help in formation of new functional tissues, along with it growth factors can also be used [3]. Nanomaterials have proven to be very good scaffold materials for tissue repair and DOI: 10.1201/9781003322368-13

225

226 Nanomaterials in Healthcare regeneration. This can be attributed to the ease with which we can control and fine-tune the properties of the nanomaterials just as our need. The feasibility to combine the various nanomaterials in order to enhance the desired properties and overcome their previous limitations have seemed to be the key for the upcoming innumerably possible nanomaterials [4,5]. It has been observed that the construction of the scaffold greatly affects the cellular microenvironment and cell functions [6]. The extracellular matrix, a naturally occurring framework that supports the tissues system in the body and aids in cell proliferation, migration, and differentiation, retains the cells and supports their movement within the tissues system. The nanofiber-based scaffolds not only provide a large surface area for cellular interactions and regeneration but also show ability to mimic the ECM [7]. This makes the nanofiber-based scaffolds better and more cyto-compatible scaffolds than any other nanomaterial-based scaffold [8]. Another benefit of nanofiber-based scaffolds is that they can be loaded with drugs or biological molecules e.g., growth factors [9] that are required for wound healing and tissue repair [10–12] so as to form a robust functional tissue [13]. The nanofiber-based scaffolds provide several advantages over the natural scaffold viz. high thermal stability, high encapsulation efficiency, controlled or sustained release of encapsulated compounds, etc. The rate of release of these encapsulated components depends on the thickness, porosity, structural defects, morphology, permeability, and chemical composition of the fiber as well as the amount of the encapsulated material [14,15]. The nanofibers of different types can be synthesised so as to suit our needs and applications. In recent years, core-shell nanofibers have emerged as efficient and innovative materials, especially for drug delivery applications. This is because they help to achieve dual drug delivery by delivering organic solvent-soluble as well as water-soluble drugs simultaneously to targets that require multiple medications at a time [16]. Different ways of implementing nanofibers in next-generation theragnostics have been demonstrated through Figure 13.1.

FIGURE 13.1 Different ways of implementing nanofibers in next-generation theragnostics.

13 • Theranostic Application of Nanofibers in Tissue Engineering 227

13.2 NANOFIBER-BASED SCAFFOLD FOR DRUG DELIVERY Extracellular matrix or EMC is the naturally occurring physical scaffold that not just provides support for the cellular structures in a tissue but also helps in cell-to-cell communication, cell adhesion, growth, differentiation, etc. The proteins, glycosaminoglycan, and glycoconjugate make up the EMC and are capable of initiating some of the essential biochemical reactions required for the homeostasis and tissue morphogenesis [17]. Nanofiber-based scaffolds are able to mimic the structure of the collagen network present in the EMC. In addition to mimicking the structure of the EMC, they are able to simulate the topological cues of the collagen fibers, and also provide good mechanical strength and suitable microenvironment to the cells for adhesion and proliferation [1]. For developing these nanofiber-based scaffolds, natural polymers are more favoured as their low cytotoxicity, excellent biocompatibility, and their more similar chemical structure to that of the native EMC which helps in cell proliferation and attachment [18]. However, they exhibit inferior mechanical strength and stability. These limitations of the natural polymers are not seen in the synthetic polymers. However, the synthetic polymers, even though can be made biocompatible and non- toxic often fail to provide the required microenvironment for the cells to grow. So, by combining two or more polymers or adding nanoparticles to the polymer, multifunctional scaffolds are created to overcome the limits of the single polymer. Nevertheless, the structure, composition, and role of EMC is not as simple. As it is not just an interconnected network of collagen, the most efficient biomimetic scaffold should not just have a similar architecture as that of the EMC, but it should also be able to initiate biochemical reactions like the natural scaffold. To achieve this, the scaffold needs to have the drug molecules or other biomolecules like growth factors or antibacterial/antifungal nanomaterials that will help in rapid healing of the wound by accelerating the growth of new cells or prevent infection to the healing tissue. Different types of electrospinning techniques are used to load these drugs or biomolecules or nanoparticles on the nanofiber-based scaffold and the techniques are selected according to the nature of drug/biomolecule/nanoparticle and their release kinetics. 1. Uniaxial Electrospinning: In this technique, there is a single nozzle for the polymeric solution to get drawn into nanofibers. It can further be divided into two types: a. Simple Electrospinning: The polymer is electrospun into a nanofiber, and then the drug/ biological molecule is loaded on its surface by electrostatic interaction, hydrophobic interaction, hydrogen bond contact, and/or van der Waals interaction of the drug/bio­ molecule with the polymer’s surface functional groups. The nanomaterials or metal ions can be positioned on the surface of the nanofiber without affecting the structure of the fibers subsurface layer. b. Simple Blending Electrospinning: In this electrospinning technique, drug/biomolecule/ nanoparticle is mixed with the polymeric solution prior to electrospinning. This mixing thus gives uniform distribution of the drug/biomolecule/nanoparticle throughout the nanofiber. Here, the drug molecules/biomolecules/nanoparticles are released via simple diffusion and can be used for long-term delivery of drug. However, there is a chance of having burst release of drugs, as the distribution of drug/biomolecule/nanoparticle is random and uneven, and so this method is not suitable for sensitive drug molecules, proteins, and DNA may denature as organic solvent used can modify their biochemistry [19,20]. 2. Multiaxial Electrospinning: In this type of electrospinning, more than one nozzle; like two in coaxial, three in triaxial, and four in quad-axial are used. The drug can be mixed with polymer

228 Nanomaterials in Healthcare

FIGURE 13.2 Different types of electrospinning and its applications for various tissue engineering.

in the core or inner layers or shell layer depending on its release kinetics. It can also be used to deliver two different types of drugs at same site of injury. The rate of release of drugs depends on the rate of degradation of the shell layer. With multiaxial electrospinning, we can achieve sustained release of drug molecules for a long period of time as the burst release of the drug can be avoided [9,21,22]. 3. Emulsion Electrospinning: Here the drug is first encapsulated in immiscible droplets and it is then dispersed in the polymer solution. This is utilized for the sustained delivery of proteins, DNA, and sensitive therapeutic molecules whose properties are affected by the organic sol­ vents used to dissolve them. It is less complicated technique and shows better reproducibility [19]. Figure 13.2 explains the various electrospinning techniques along with application of electrospun fibers for various tissue engineering. One of the most common natural polymers used in tissue engineering, gelatine, is produced from col­ lagen and has great biocompatibility and bio-absorptivity. However, it has low mechanical stability and is susceptible to enzymatic breakdown. Poly lactic-co-glycolic acid (PLGA), an FDA-approved synthetic polymer has good biocompatibility, controlled biodegradation, and suitable mechanical strength but fails to provide suitable microenvironment for cell growth and differentiation. By combining both the polymers, the limitations of both the polymers can be overcome. For simultaneous delivery of multiple drugs, these polymeric nanofibers can be embedded with nanoparticles. Like PLGA/gelatine, nanofiber is embedded with mesoporous ZnO for simultaneous delivery of hydrophilic and hydrophobic drugs[23]. Loading biphasic calcium phosphate (BCP) nanoparticles in a polyvinyl alcohol (PVA)-gelatine nano­ fiber mat has shown to improve the mechanical strength of the nanofiber along with accelerating the osteogenic differentiation, thereby increasing the rate of healing of the bone. This can be attributed to the chemical composition of BCP. BCP is a ceramic made up of β-tricalcium phosphate and hydroxyapatite. Both of them contain the naturally occurring ions that make up the bone matrix. During bone healing, the β-tricalcium phosphate gets dissolved and hydroxyapatite is used for bio-resorption for bone healing [24]. Gelatine in combination with a silica matrix has shown an uncanny similarity to that of the bone matrix. This gelatine-silica matrix together with polycaprolactone (PCL) microfilaments not only pro­ vides a suitable microenvironment for tissue regeneration but also acts as a dual drug delivery scaffold for sequential release of angiogenic and osteogenic factors [25]. Co-electrospinning of gelatine and PCL

13 • Theranostic Application of Nanofibers in Tissue Engineering 229 nanofiber has proven to be an effective controllable drug delivery system for bone regeneration. With an outer shell of gelatine, there is enhancement in cell adhesion while the PCL layers provide mechanical strength and help to control the rate of drug release. It is realized that the rate of drug release is inversely proportional to the PCL content [22]. Another biomimetic scaffold of PCL/gelatine nanofiber was developed in which alginate hydrogel was used to mimic the gel-like matrix in EMC. Here the nanofibers were loaded with the PLGA nanoparticles which contained kartogenin, a bioactive molecule that could help to promote chondrogenic differentiation [26].

13.3 NANOFIBER-BASED STEM CELL THERAPY AND LABELING Stem cells are the naturally occurring pluripotent cells in the body that help in wound healing and tissue regeneration by replacing the affected cells. They release growth factors and induce/ cause cell signaling to modulate immune response, thereby promoting angiogenesis [27]. Cell-free therapies are the methods where a nanomaterial-based scaffold is loaded with growth factors or potent signaling molecules for guided growth of stem cells. These molecules may be specific for a given tissue and are associated with cell migration, adhesion, proliferation, and differentiation [28]. Apart from this, the nanoparticles can be used for a non-invasive tracking of these stem cells to monitor the tissue growth. Super paramagnetic iron oxide nanoparticles (SPIONs) are the most widely used nanoparticles for monitoring the stem cells by MRI. Apart from these, polymeric nanofiber-based scaffolds have also been used for tagging and monitoring various types of stem cells [29]. Electrospun nanofibrous scaffolds have been thriving in assisting and preservation of chondro­ genic incorporation of stem cells. These constructs have set the expectation for cartilage tissue en­ gineering implementations. Activity amongst the stem cells and nanofibers are important in a cellscaffold model while utilizing them for varied tissue engineering uses [30]. The mesenchymal stem cells (MSCs) in coexistence with aligned nanofibrous scaffolds carry favour for engineering aniso­ tropic fibrocartilage. Cyclic rigidity of fibrin gel positively disposes fibrous gene indication and cyclic contraction of hydrogel scaffold fortifies MSC chondrogenesis. Dynamic tensile loading on MSCplanted aligned nanofibrous constructs disposes fibrous marker typical of fibrocartilage, and enhances the properties during manufacturing of collagenous ECM and allows the increment of practical characteristics of scaffold [31]. Curcumin-integrated chitosan/polyvinyl alcohol/carbopol/poly­ caprolactone have been availed to investigate the biological correspondence using the mesenchymal stem cells acquired from buccal fat pad. It proves to assist mesenchymal stromal cell regeneration on curcumin-loaded nanofibers in-vitro [32]. Human mesenchymal stem cells (MSCs) lately occur to be implemented as a remedy of osteo­ arthritis in scientific experiments as they are favourable substitute to cell origin for cartilage regenera­ tion. Their differentiation can be analyzed, employing nanofiber-based poly-ethersulfone (PES) scaffold in order to achieve chondrocytes [33]. The development and differentiation characteristics of human MSCs along with their osteogenic and chondrogenic by-products planted in electrospun poly lactic-coglycolic acid (PLGA) nanofiber have been examined. PLGA nanofibers are compatible with hMSCs along with osteoblasts and chondrocytes as well that are acquired from MSCs. Thus, hMSCs carry on reproducing upon planting in PLGA nanofiber [34]. On other hand, bone marrow mesenchymal stem cells (BMSCs) applied in stem cell therapy had been displayed as a favourable perspective for the cure of injury through triggering the growth of epi­ thelial cytoplasm and angiogenesis in injuries. Feasibility of implanted BMSCs and increase amelio­ ration of injuries can be escalated by a connective treatment utilizing chitin nanofiber hydrogel as a biomaterial accompanying BMSCs [35]. Electrospun polycaprolactone (PCL) scaffold, which exhibited

230 Nanomaterials in Healthcare strong strength for the differentiation of rat bone marrow stromal cells (BMSCs) and in vitro calcium phosphate mineralization and collagen accumulation, was initially implemented as a nanofibrous model for bone regeneration. PCL nanofibrous scaffold implanted with mesenchymal stem cells (MSCs)planted scaffolds in rat omenta demonstrated required osteogenic differentiation capabilities and in vivo ECM production [36]. Electrospun blend of polyvinyl alcohol/polycaprolactone (PVA/PCL) scaffolds with autologous bone marrow MSC (BM-MSC) have been developed for in-vivo cartilage tissue engineering. These PVA/PCL nanofiber scaffolds assisted proliferation and chondrogenic differentiation of MSC and showed an increase in capacity to assist primary cell adjunct and its development [37]. Mouse fibroblast cells and bone marrow-derived MSCs were utilized to report electrospun PLGA construct that were believed to deliver the mechanical characteristics required for tissue engineering similar to that of the cartilage [38]. Human tendon stem/progenitor cells (hTSPCs) have high potential for osteogenesis, adipogenesis, and chondrogenesis, which favours cell differentiation to the osteo-lineage in conjunction with a poly (L-lactic acid) nanofibrous scaffold [39]. Also, a nanofiber-based ex vivo stem-cell growth technology and pro-angiogenic growth factors upregulation of human umbilical cord blood (UCB) – derived progenitor cells accompanied by the objective of alleviating neo-vascularization as a therapeutic approach for myocardial ischemia and peripheral vascular illness through improvement of angiogenic potential of curative stem cells were utilized. Pro-angiogenic growth factors overexpression in progenitor cells improves autologous or allogenic stem cell therapy for ischemic illnesses [40]. Furthermore, embryonic stem cells (ESCs) have been investigated for their capacity of differenti­ ation into neural lineages on being planted into nanofibers. Alignment of nanofibers orientation was used to administer coordination/acclimatization of neural lineage accompanying neutrite protrusion [41]. Additionally, human induced pluripotent stem cells (iPSCs) were evidenced to have favourable prospective for regenerative drug and tissue engineering usage. Increased capability of iPSCs to dif­ ferentiate into osteoblast-like cells was observed through proceeding simultaneously with nanofiberbased PES scaffold planted iPSCs. Flattened and extended architecture, compatibility of iPSCs on PES nanofiber establishes biological correspondence of PES nanofibrous scaffold [42]. Furthermore, catechin-coated polymer nanofiber constructions significantly improve in-vivo bone formation of human adipose tissue derived stem cells (hADSCs) implantation in mice. The innate biochemical performance of catechin provided the constructs with antioxidant and calcium constructing features, leading to enhanced attachment, production, mineralization, and osteogenic differentiation of hADSCs [43].

13.4 NANOFIBER-BASED SCAFFOLD CONSTRUCTION AND MODIFICATION In tissue engineering, scaffold is pivotal due to its anchorage formation properties that are necessary for cellular inter-linkages. Therefore, the evolution of an absolutely perfect scaffold is profoundly necessary to supply appropriate cell material and cell-cell junction exposure. Scaffold made up of electrospun nanofi­ bers take over some distinctive properties like an equivalent diameter to the surrounding extracellular matrix (ECM) along with sufficient surface area to provide cell bonding and deposition of bioactive components [44]. Porous nanofibrous scaffolds of higher surface-to-volume ratio are noticed to be com­ parable to cross-linked porous collagen fiber (50–500 nm) observed in the surrounding ECM. Morphology of nanofiber mimicking the surrounding ECM allows productive duct for oxygen and nutrient passage [45]. Nanofibers impersonate the collagen fibers of ECM and due to this versatile nature and mobility elec­ trospinning is broadly accepted for their development. Electrospinning process integrating water whirlpool

13 • Theranostic Application of Nanofibers in Tissue Engineering 231 to gather nanofibers leads to an extremely porous group of filaments, which therefore, are subsequently used as tissue engineering scaffolds. This electrospinning when merged with freeze-drying technique produces a blend of matter where a nanofiber is installed in a 3D porous aerogel [46]. Incorporation of hydrogel into the electrospun nanofibers allows imparting harmonious properties to scaffolds. Currently, nanofibers (in micron size) integrated into hydrogel scaffold have arbitrary distri­ bution throughout the scaffold with dispersed macro porous architecture [47]. In some cases, assimilation of regenerated cellulose (rCL) nanofiber in chitosan (CS) hydrogel could be executed because a natural hydrogel scaffold often displays deficient mechanical power, crucial for bone tissue regeneration. This rCL/CS blend scaffold illustrates distinctive porous architecture, increased contractile power, improved ore-osteoblast cell (MS3T3-E1) applicability, adhesion, and perforation [48], whereas mixed printable biomaterials consisting of alginate and gelatine hydrogel structures loaded with carbon nanofibers can be contrived for generation of electro-conductive printable 3D scaffold. This protocol/fabrication method permits formation/production of a strengthened blend with enhanced mechanical behaviour [49]. Development of a 3D vascularized tissue allows scientific as well as technological improvements in tissue engineering, organ reconstruction, and drug screening. Mainly in osteoblast behaviour, rigidness of the scaffold has been recognized as a key aspect. (The rigidness of the scaffold plays a crucial/vital role in osteoblast behaviour.) This scaffold should be powerful for effortless control during resection and should have equivalent assimilation amount to that of novel tissue reinvention [47]. Furthermore, developments of nanofibers from natural polymers are multifaceted owing to their advantageous utilization as scaffolds for tissue engineering. Gelatine in nanofibrous forms has been absolutely/intensively investigated for biomedical implementation due to its innate biocompatibility, biodegradability, and non-toxicity. These nanofibers are used as scaffolds for tissue engineering due to their expanded surface area, multifaceted nature, and adaptable porosity [50]. Reconstruction of cellulose nanofibers in the form of acetate-free nanofibers through alkaline de-acetylation of gyrated nanofiber and inactivation with silver can be done to obtain a scaffold with anti-bacterial properties for implementation in osteo-integration [51]. Chitosan nanofiber scaffolds utilized as 3D cardiac culture representative structures were screened for their suitability for cardiac tissue engineering. The fibronectin-coated chitosan fibers produced by elec­ trospinning improves attachment of the cell to the fibers and emigration into the inter-fibrous network. This results in chitosan nanofibers that probably hold on to their cylindrical architecture in long-term cell cultures and neonatal rat cardiomyocytes on the fibers displaying better cell adhesion and expansion. This property is owing to the emergence of enormous tissue-like cell milieu through co-cultures with fibroblasts, specifying utilization of 3D chitosan nanofibers as a capable construct to grow heart tissues [52]. Owing to the well-recognized/validated biological correspondence to H9C2 cells, a polyurethane (PU)/chitosan (CS)/ carbon nanotubes (CNT) blend nanofibrous scaffold can be electrospun for its application in cardiac tissue engineering. This electro-conductive nanofiber scaffold might be taken into account/consideration as a favourable scaffold for infarcted myocardium regeneration for its precise biological compatibility to assist cell adhesion and perforation [53]. An electrospun nanofiber scaffold made up of soya bean protein nanoparticle (SPN) and then altered with poly hydroxybutyrate (PHB) is developed. This nanofiber scaffold shows an increase in cell compatibility, thus proving itself appropriate for implementation in skin tissue engineering. This nanofiber scaffold can provide excessive hydrophilicity and can resemble phys­ icochemical behaviour of natural ECM, which are preferable properties for any scaffold for its biomedical implementation [54]. MoS2 and rGO nanosheets manufactured and integrated into silk fibroin (SF) nanofibers act as mechanical promoters. Subsequently, either of MoS2 nanosheets and rGO nanocompo­ sites can be utilized as an electrically conductive agent for cardiac tissue engineering [55]. Also, as proven, resorbable polymer electrospun nanofiber (RPEN)–based materials with high surfaceto-volume ratios along with porous forms have magnificent pore inter-relations that are appropriate for development and evolution of distinct sorts of cells. RPENs implied for bone tissue engineering can be categorized as bioactive materials that are deposited into the nanofibers where nanoparticle encapsulation prior to electrospinning is considered as a systematic method to increase firmness of nanofibers. RPENs can also be bioactive matters immobilized onto nanofibers or the ones encapsulated into nanomaterials

232 Nanomaterials in Healthcare before scaffold development. Utilization of these stacked bioactive matters into electrospun nanofibrous scaffold or implementation of bioactive matters on aligned nanofibrous scaffold proves to be advanta­ geous for nerve tissue regeneration. This loading of bioactive substances is also a captivating way for vascular tissue and skin tissue engineering [56]. On the other hand, insertion of polyphenols can be done into the polymers during electrospinning to produce polyphenol comprising nanofibrous scaffolds with enhanced properties. Additionally, enrich­ ment of nanofibrous scaffold with powerful mechanical properties can be carried out by sufficient covalent and non-covalent interconnections to catechol/pyrogallol groups with polymers supplying physically and chemically cross-linked networks. Similarly, polyphenols can improve adherent beha­ viour of electrospun nanofibers, enabling the infiltration of media culture into nanofiber pores through in vitro, which might profoundly encourage cell connection, perforation, and proliferation [43]. Poly(3-hydroxibutyrate-co-3-hydroxivalerate) (PHBV) nanofibers are multifaceted nanofibers and considered a favourable perspective for axonal guidance in peripheral nerve repair. PHBV fiber as a strengthened scaffold inside hydrogen is noteworthy of enhancing mechanical behaviour of the assembly due to strain transmission connecting the matrix and support. Formational comparison of PHBV nanofibers and ECM in skill leads to a good performance of fibroblast cell attachment and perforation [57]. A blend of polylactic acid/polycaprolactone (PLA/PCL) electrospun nanofiber accommodating nano­ hydroxyapatite (nHA) and zeolite for encouraging the perforation of human dental pulp derived stem cells (hDPSCs) with probable implementation in dental tissue engineering is developed as nHA and zeolite subsistence in scaffold composition could have a beneficial consequence on osteo-conductivity and osteo-inductivity of scaffold [58]. A versatile bio-intersecting tissue constructed autologous scaffold is made of modifying chunk polyurethane and is tubular in shape. It has ordered nanofiber morphology, changeable mechanical behaviour, and a hydrophilic PEGylation junction. It is capable of promoting simultaneous attachment, directional expansion, and perforation of a New Zealand rabbit autologous urethral epithelial cells (ECs) and smooth muscle cells (SMCs). It is significant that amphiphilic PU altered with a suitable hydrophilic PEGylation nanofiber juncture causes/triggers/initiates host immune cell death to obstruct inflammatory retaliation [59]. Conductive electrospun nanofiber scaffold consisting of conductive polypyrrole (PPy) polymer was developed to quicken amelioration of injured tissues. These conductive nanofiber scaffolds are specifically suitable for their utilization in anatomy to produce electrical signals in cardiovascular muscles, including heart muscles/nerves. As per reports, nanofibers consisting of polypyrrole improve growth and differentiation of nerve cells. Polylactic acid/polypyrrole nanofiber scaffold activates growth of nerve cells, attachment, proliferation and differentiation of nerve cells. Polypyrrole/polycaprolactone/ gelatine nanofiber scaffold proves to be worthy factor for cardiac muscle tissue regeneration [60]. Also, several attempts are made to formulate and design an electroactive electrospun nanofiber scaffold (EENFS), articulating with electroactive tissues to enhance the accessibility of implementation of electroactive tissue in tissue engineering. These genius scaffolds transfer electrical signals or transform a stimulus to electricity and then transport these electrical stimulations effortlessly and precisely to the appropriate cell or tissue. Stimulation of externally electrical simulations can also provoke a bioactive component release from a well-designed EENFSs [61]. Figure 13.3 summarizes the use of different polymer materials into fabrication of nanofiber scaffold materials for construction and modification.

13.5 MULTIFUNCTIONAL AND SMART NANOFIBERBASED SCAFFOLDS Multifunctional scaffolds made up of smart materials can be applied in various tissue engineering domains because they can robustly take part in the procedure of supplying the biological indication that

13 • Theranostic Application of Nanofibers in Tissue Engineering 233

FIGURE 13.3 Different types of polymers used to construct nanofiber-based scaffold.

conduct and administer cell functions. These are acquired from unique functional and smart materials; those permit modulation of the behaviour and characteristics of the constructs and can also conduct various important chores at the same time. These scaffolds should accommodate varied necessities including bioactivity, biocompatibility, administrable biodegradability, proper mechanical toughness, morphology, and porosity, along with assisted delivery of chemical and biological signal to eradicate infection from pathogens as well as decrease immune response, while promoting cell adhesion, growth, and trigger osteo-differentiation and also angiogenesis. These designs’ capacity to promote tissue development and regeneration has been demonstrated in vitro and in vivo utilizing diverse animal models. A smart electroactive polymer employed in tissue engineering permits the triggering of cell attachment, perforation, and differentiation through electrical stimulus (ES). The indulgence of aniline particles in polymeric materials can supply constructs with electroactive properties. Shape memory substances have also been implemented as a unique category of stimulus reactive substances with enhanced ability for the achievement of smart tissue engineering scaffolds utilizing the slightest intrusive transplantation owing to their inherent shape retrieval characteristics permitting the delivery of heavy models in a compact-sized shape via a tapered duct in the body, and the recuperation of its native shape on activation through an exterior impulse like temperature, ultrasound, etc. [62]. “Smart” materials can undergo modifications in their physiochemical characteristics as an effect of exterior impulses like heat, light, and electric field. These varieties of moieties are comparatively favourable for wound healing usage, as they can dispense various superiority juxtaposed to natural substances such as managed and requested delivery of therapeutic agents. Contradictory to other biomaterials like hydrogels, electrospun constructs have lately been fabricated, utilizing ways that include smart materials [63]. The “smart” nanofibrous scaffolds should be capable of protecting the delivered materials from denaturation and support their biological activities, providing various traditional nanofiber development strategies. A “smart” nanofibrous construct that is able to promote cell-matrix association via bioinspired aspects, and incorporating suitable biological action through managed liberation of integrated biological molecules can be essential for scaffold-based tissue engineering [64]. Further, cell alignment is majorly important for various tissues to obtain their in vivo biological functions, incorporating vascular tissue, musculoskeletal system, myocardial tissue, corneal stromal, tendons, and nerves. Henceforth, fabrication of smart materials that imitate the native ECM to impart the cell orientation is important for tissue engineering [65]. Identifying ways to develop cell-responsive,

234 Nanomaterials in Healthcare smart and advance hydrogel constructs which could be altered to imitate the bi-directional molecular function happening/occurring among the cells and the native ECM could be developed [66]. On the other hand, smart nanofiber scaffolds including various gradient signals can be developed for imitating the cellular and architectural properties of in vivo cellular surroundings [67]. Smart scaffolds have lately come up to attain this prerequisite by imparting bio-responsive and morphologically modified scaffolds. These constructs can transport and deliver the biomolecules in a controlled and configurable mode. Fascinatingly, few of the smart scaffolds can tune the host tissue reaction and additionally enhance the therapeutic potential of scaffolds in vivo. Primary cells in a 2D sheet organization undergo morphogenetic modifications to configure 3D tissues in the body. Origami-based smart scaffolds motivated by this concept help to develop complex tissue scaffolds. Hydrogels are perfect substances to fabricate origami-based smart constructs as their amount of swelling can lead to interior strains in the material. Henceforth, smart scaffolds could be fabricated to impart these biophysical and biochemical signals and provide distinctive immuno-informed biomaterials. Smart scaffolds applied as delivery materials can particularly merge with target tissues or organs in the body and deliver their matter with specific release kinetics as a result of signals from target cells or native ECM. Owing to the emergence of novel therapeutic agents, it is important to compose compar­ atively smart constructs to release such agents in a potentially monitored way [68]. One such fabricated blend of nanofibers functionalized with plasmonic titanium nitride (TiN) nanoparticles is favourable for the manufacturing of smart scaffold for tissue engineering and allows novel approaches for theranostic applications. In a study, an electrospinning of polycaprolactone (PCL) done along with laser synthesized TiN nanoparticles at different controlled ratios in to develop a new hierarchical blend nanofiber for tissue engineering have been screened [69]. Another type of smart materials is piezoelectric scaffolds, which could develop electrical signals in acknowledgment/response to the implied stress. Therefore, they can trigger the indications and improve the tissue regeneration at the damaged area. This implements more often to neural tissue because the electrical charges are important for cell function. The significant benefit of these piezoelectric scaffolds is that electrical potential can be developed controllably under the influence of mechanical forces, without the necessity to utilize uncontrollable electrodes [70]. Also, electroactive biomaterials are a part of a new generation of “smart” biomaterials that allow the direct delivery of electrical, electrochemical, and electromechanical stimulations to cells. The family of electroactive biomaterials includes conductive polymers, electrets, piezoelectric, and photovoltaic materials. Conductive polymers feature great elec­ trical stimulus control; outstanding electrical and optical characteristics; a high conductivity/weight ratio; and may be made biocompatible, biodegradable, and porous [71]. A type of self-assembling “smart” peptide nanofiber-based hydrogels (NFHGs) resulted in a good execution in enhancing wound healing due to the biomimetic structure, non-immunogenicity, and noncytotoxicity characteristics of the peptide nanofiber matrix. Various other types of functional NFHGs utilized as skin engineering scaffolds have also been reported, like chitosan/vinol/hydroxyapatite/ce­ fixime blend NFHGs with antibacterial ability, self-assembled peptide nanofibers, and epidermal growth factor composite hydrogel [72]. Smart dressings with the capability of healing and monitoring wounds through the aid of sensoracquainted dressings have been introduced. A smart europium (III) coordination polymer functionalized polyacrylonitrile nanofiber was developed as a detector to investigate healing condition. Recently, polyvinyl alcohol/sodium alginate (PVA/SA) nanofibers were noticed to potentially operate as a smart dressing for wounds due to their blending with pH and the thermo-responsive sodium alginate-g-N-isopropylacrylamide for managed delivery of diclofenac sodium as an anti-inflammatory drug to infected wounds [73]. A supplementary element of “smart” drug delivery system is the nanofibrous nonwoven fabric. Lately, electrospun polymeric nanofibers were found to be effective contenders for tissue engineering and drug delivery system uses. The effective loading ability, side-by-side delivery of various molecules, and eco­ nomical availability are the largely captivating characteristics of electrospun models for their application in drug delivery. Relying on various criteria influencing the drug−polymer intermolecular forces, it is ben­ eficial to control the drug release in a way that the release procedure can take up to many weeks [74].

13 • Theranostic Application of Nanofibers in Tissue Engineering 235

13.6 CONCLUSION AND FUTURE PROSPECTS Electrospun nanofibers owing to their resemblance with the morphology of surrounding ECM and its architecture offer a suitable environment for tissue regeneration. Certain cell-seeded nanofibers dem­ onstrate the capacity to support chondrogenic differentiation of various stem cells. These nanofibers on incorporation into 3D scaffolds through integration of certain polymers and other advance materials, generate biomimetic structures exhibiting great potential in tissue reinnervation. Though significant progress has been achieved in nanofiber production technologies and its use in tissue regeneration, there is insufficient mechanistic understanding, which limits the field’s rate of evolution. More structured and numerical investigations are required to demonstrate the association among architectural properties, drug delivery, stem cell function, and tissue regeneration. Integrative perspective and participation are required to productively address the problems and promote the discipline.

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238 Nanomaterials in Healthcare [58] S. Mohandesnezhad, Y. Pilehvar-soltanahmadi, and E. Alizadeh, “In vitro evaluation of Zeolite-nHA blended PCL / PLA nanofibers for dental tissue engineering,” Mater. Chem. Phys., vol. 252, no. May, p. 123152, 2020, 10.1016/j.matchemphys.2020.123152. [59] Y. Niu et al., “Designing a multifaceted bio-interface nanofiber tissue-engineered tubular scaffold graft to promote neo-vascularization for urethral regeneration” 2020, 10.1039/C9TB01915D. [60] M. Zarei, A. Samimi, M. Khorram, M. M. Abdi, and S. Iman, “Fabrication and characterization of con­ ductive polypyrrole/chitosan/collagen electrospun nano fi ber scaffold for tissue engineering application,” Int. J. Biol. Macromol., vol. 168, pp. 175–186, 2021, 10.1016/j.ijbiomac.2020.12.031. [61] X. Zhang et al., “Electroactive electrospun nanofibers for tissue engineering,” Nano Today, vol. 39, p. 101196, 2021, 10.1016/j.nantod.2021.101196. [62] M. Kaliva, M. Chatzinikolaidou, and M. Vamvakaki, Multifunctional Tissue Engineering Scaffolds, no. 25. The Royal Society of Chemistry, 2017. [63] A. Memic, T. Abudula, H. S. Mohammed, K. J. Navare, T. Colombani, and S. A. Bencherif, “Latest progress in electrospun nano fibers for wound healing applications,” ACS Appl. Bio Mater., vol. 2, pp. 952–969, 2019, 10.1021/acsabm.8b00637. [64] W. Li and J. A. CooperJr., “Fibrous scaffolds for tissue engineering”, Biomaterials for Tissue Engineering Applications, pp. 47–73, 10.1007/978-3-7091-0385-2_3. [65] X. Zhao, Y. Lin, and Q. Wang, “Virus-based scaffolds for tissue engineering applications,” WIREs Nanomed. Nanobiotechnol., vol. 7, no. 4, 10.1002/wnan.1327. [66] T. G. Kim, H. Shin, and D. W. Lim, “Biomimetic scaffolds for tissue engineering”, Adv. Funct. Mater., pp. 1–23, 2012, 10.1002/adfm.201103083 [67] A. Seidi, K. Sampathkumar, A. Srivastava, S. Ramakrishna, and M. Ramalingam, “Gradient nanofiber scaffolds for tissue engineering”, J. Nanosci. Nanotechnol., vol. 13, no. 7, pp. 4647–4655, 2013, 10.1166/ jnn.2013.7187. [68] S. Ahadian and A. Khademhosseini, “Smart scaffolds in tissue regeneration,” Regen. Biomater., vol. 5, no. 3, pp. 1–4, 2018, 10.1093/rb/rby007. [69] V. P. Nirwan, E. Filova, A. Al-kattan, and A. V Kabashin, “Smart electrospun hybrid nanofibers func­ tionalized with ligand-free titanium nitride (TiN) nanoparticles for tissue engineering”, 2021. [70] A. Zaszczynska and A. Gradys, “Piezoelectric scaffolds as smart materials for neural tissue engineering”, 2020. [71] R. Balint, N. J. Cassidy, and S. H. Cartmell, “Conductive polymers: Towards a smart biomaterial for tissue engineering,” Acta Biomater., vol. 10, no. 6, pp. 2341–2353, 2014, 10.1016/j.actbio.2014.02.015. [72] Q. Fu et al., “Nanofiber-based hydrogels: Controllable synthesis and multifunctional applications,” vol. 1800058, pp. 1–19, 2018, 10.1002/marc.201800058. [73] E. A. Kamoun, S. A. Loutfy, Y. Hussein, and E. S. Kenawy, “Recent advances in PVA-polysaccharide based hydrogels and electrospun nanofibers in biomedical applications: A review,” Int. J. Biol. Macromol., vol. 187, no. April, pp. 755–768, 2021, 10.1016/j.ijbiomac.2021.08.002. [74] P. Nakielski et al., “Multifunctional platform based on electrospun nanofibers and plasmonic hydrogel: a smart nanostructured pillow for near-infrared light-driven biomedical applications”, 2020, 10.1021/ acsami.0c13266.

Role of Nanomaterials in Biosensing Applications

14

Jasmeen Kaur and Menam Pokhrel NanoBios Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 14.1 14.2 14.3 14.4

Introduction Biosensors: An Overview Nanomaterials – Characteristic Features for Biosensing Applications Nanomaterials-Based Biosensing for In-Vitro Diagnostics 14.4.1 Metal Nanoparticles 14.4.2 Metal Oxide-Based Nanomaterials 14.4.3 Carbon-Based Nanomaterials 14.4.4 Nanocomposites 14.5 Challenges and Future Prospects 14.6 Conclusion References

239 240 242 245 245 249 250 252 253 254 255

14.1 INTRODUCTION The imperative use of biosensors for tracking several biological entities and monitoring various bio­ chemical reactions has lately acquired considerable attention for numerous biomedical applications, especially disease diagnosis and health monitoring. Biosensors are analytical devices that utilize bio­ logical elements (such as antibodies, nucleic acids, etc.) as bioreceptors to detect different disease biomarkers that are further quantified with the help of a transducer (optical, electrochemical, etc.) [1]. A quick and reliable in-vitro analysis of various biological samples, including blood, tissue, urine, saliva, sweat, etc., has opened paths for early diagnosis and better disease management [2,3]. The growing utilization of biosensors has outmoded the conventional laboratory methods of disease diagnosis, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), polymerase chain reaction (PCR), etc., with respect to better detection accuracy and rapid processing time with no bulky DOI: 10.1201/9781003322368-14

239

240 Nanomaterials in Healthcare instrumentation or requirement of skilled personnel. Biosensors, ideally, should be user-friendly, costeffective, highly stable, and able to rapidly and accurately detect low abundant target analytes from the original matrix (biological sample) with high precision, using a small sample volume. However, there are several challenges faced by the current biosensing devices, such as efficient signal collection and lower limit of detection that need to be addressed to achieve the ideal characteristics. Recent developments in the area of nanoengineering and interdisciplinary research have allowed for the development of nanosized materials, that, when incorporated into biosensors, assist in the amelioration and sometimes solving the challenges with improved analytical performance (i.e., high sensitivity and better specificity). Nanomaterials, as the word suggests, are nano-sized materials with at least one dimension measuring between 1 to 100 nm [4]. Their nanoscale size and correspondingly high surface area-to-volume ratio contribute to a number of their unique physical and chemical properties that have been exploited to develop extremely sensitive and robust biosensors. They have been integrated into biosensors for multiple purposes such as bioreceptor immobilization, signal enhancers, electrocatalysts, transducers, and even sample preparation. Different types of materials such as organic (carbon-based), inorganic (metal and metal oxides), or polymeric have been used to synthesize nano-dimensional structures (0D, 1D, 2D, or 3D) that have been used in bioanalytical devices, mainly to improve their precision, speed, and their ability to perform multiple detections in miniaturized formats [5]. Nanomaterial-based biosensing has been applied to the in-vitro diagnosis of several metabolic analytes, along with various infectious and non-infectious diseases. Since, the development of the first glucose biosensor by Clark and Lyons, biosensors with improved analytical performance have been developed for multiple disease biomarkers. This chapter first briefly describes the components and types of biosensing systems, followed by an in-depth discussion of the role of nanomaterials as biosensing moieties. The different types of nanomaterials commonly employed in biosensing systems for disease detection are further discussed with examples. Some of the recently developed nanomaterial-based biosensors for disease diagnosis have also been summarized and tabulated. Finally, the difficulties encountered in translating these biosensors for clinical applications are discussed along with future outlooks.

14.2 BIOSENSORS: AN OVERVIEW Biosensors are analytical systems that utilize biological elements as bioreceptors along with a physicochemical component to generate measurable signals with the aim to detect different molecular and biomolecular analytes [1]. The biorecognition elements such as enzymes, antibodies, nucleic acids, etc. are responsible for recognizing and interacting with the specific analyte of interest. The analyte-receptor interaction is then converted into a measurable signal, such as optical, electrochemical, etc. by the transducer, which is finally detected and interpreted by a detector. The detected signal can be directly or inversely proportional to the concentration of the target analyte. A schematic figure representing the components of a typical biosensor is shown in Figure 14.1(a). Biosensors have been employed in a wide range of applications including disease detection, health monitoring, drug discovery, food safety, environmental monitoring, etc. The scope of this chapter is limited to the application of biosensors in the detection and monitoring of numerous diseases such as metabolic diseases, cardiovascular diseases, infectious diseases, neurological diseases, various types of cancers, etc. Typically, the performance of different biosensing systems is assessed by comparing their char­ acteristics, for instance, selectivity, sensitivity, stability, and reproducibility. The selectivity, sensitivity, and stability of the biosensors generally depend on the type of biorecognition element employed. Ideally, the biorecognition elements should not degrade over a period of time (stability) and have a high binding affinity for the target analyte such that they should be able to detect the target analyte in a sample

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FIGURE 14.1 (a) Schematic representation of the components of a biosensor; (b) schematic representation of different types of nanomaterials and their role in biosensing applications.

containing other contaminants (selectivity), even if present in trace amounts (sensitivity). The different biorecognition elements used in biosensing systems can be the conventional ones such as enzymes, antibodies, nucleic acids, whole cells, etc., or the recently emerged ones such as aptamers, molecularly imprinted polymers, phages, affibodies, etc. [6]. Based on the nature of the biorecognition element used, biosensors are broadly classified as catalytic-based or affinity-based biosensors. While catalytic bio­ sensors use enzymes or microorganisms as bioreceptors and use the catalytic reaction on their surface to

242 Nanomaterials in Healthcare generate signals, affinity biosensors use antibodies, nucleic acids, aptamers, MIPs, etc. as bioreceptors and use their specific interaction with the analyte to generate signals [7]. Along with the biorecognition elements, the different transduction methods employed in the bio­ sensing systems also define their stability, sensitivity, and reproducibility. The transducer should be temperature-insensitive (stability), able to detect weak signals (sensitivity), and accurately provide alike results every time a sample is measured (reproducibility) [8]. The biosensing systems may operate through various transduction methods such as optical, calorimetric, electrochemical, piezoelectric, etc., to produce detectable signals. Of these, optical and electrochemical transducers are the most common among different biosensors for disease detection. Optical biosensors detect and measure changes in different optical phenomena, such as absorption, reflection, transmission, refraction, fluorescence, luminescence, surface plasmon resonance, etc., induced by optical transducers in response to the analytebioreceptor interaction [9]. The optical signals generated have several advantages over other physical signals, including high sensitivity, stability, immunity to external disturbance, and low noise [10]. Electrochemical biosensors, on the other hand, detect and measure the electric signals generated by the electrochemical transducer on its surface in response to the interaction of the surface-immobilized biorecognition element with the target analyte. The electrochemical transducer is usually a working elec­ trode (WE) that generates signals such as changes in current (amperometric/voltammetric), voltage (potentiometric), or resistance (conductimetric/impedimetric) [11]. Biosensors have arisen as an alternative to routinely applied diagnostic techniques and must provide rapid, easy-to-use, and cost-effective means for highly specific and sensitive detection of analytes [12]. The classical biosensing systems, although provided the advantage of simplicity and rapidity, had limitations in terms of signal strength, stability and sensitivity. Incorporation of nanomaterials helped improve sensitivity by signal enhancement due to greater bioreceptor immobilization. Additionally, other characteristics of nanomaterials such as high electrocatalytic activity and better transduction helped improve performance by overcoming the limitation of conventional biosensing systems. Hence, to fulfill the growing demand for analytically sound in-vitro molecular biosensors for biomedical diagnosis, nanomaterials have been extensively studied for their added advantage to the analytical performance of these biosensors [13,14].

14.3 NANOMATERIALS – CHARACTERISTIC FEATURES FOR BIOSENSING APPLICATIONS The term ‘nanomaterials’ refers to materials with any internal or external structure in the nanoscale dimension (1–100 nm) [15]. Based on the structural dimensionality, nanomaterials can be categorized as zero-dimensional (0D), one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) nanomaterials. 0D nanomaterials have all dimensions within the nanoscale, i.e., < 100 nm (for example, nanoparticles and nanoclusters) while the 3D nanomaterials have all three dimensions outside the nanoscale, i.e., > 100 nm (for example, nanoflowers, nanopillars). In the same way, 1D (for example, nanotubes, nanorods, nanofibres, and nanowires) and 2D (for example, nanofilms, nanosheets) nano­ materials have one and two dimensions greater than 100 nm, respectively. The different dimensionality of the nanomaterials is possible due to the ability of certain materials to exist in certain forms. Different inorganic, organic, or polymeric materials have been used in the fabrication of nanostructures. Inorganic material-based nanostructures include metal nanoparticles (for example, gold, silver, platinum, palla­ dium, etc.) or metal oxide nanoparticles (for example, zinc oxide (ZnO), copper oxide (CuO), iron oxide (Fe2O3), aluminium oxide (Al2O3), etc.). Organic material-based nanostructures are made up of carbon or graphene derivatives, including carbon nanotubes, graphene oxide, etc. The properties of these materials at nano-size vary greatly from their bulk counterparts. Nanomaterials have high surface energy,

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high surface-to-volume ratio, enhanced electrical conductivity, excellent catalytic activity, great mag­ netic properties, and so on [16]. These specific properties of nanomaterials deem them useful in multiple applications such as drug delivery, biosensing, bioimaging, biotherapeutics, etc. [17]. In the area of biosensing, the specific properties of nanomaterials integrated into the biorecognition or transducing elements are directly correlated to the sensing abilities of the biosensor [18]. Nanomaterials can be employed in a biosensor for multiple purposes such as enhanced immobilization of bioreceptor molecules, non-enzymatic biosensing, sensitive transduction, and analyte separation from testing samples [Figure 14.1(b)] [19]. Some of the salient characteristics of the nanomaterials useful as biosensing moieties are described herein. Nanomaterials have a characteristic high surface area that allows ample functional groups for efficient adsorption/immobilization of biorecognition molecules [5]. The higher the immobilized moieties, the better will be the range of detection and the sensitivity of the biosensor as a result of pre-concentration and affinity-based segregation of the analyte to the specific biorecognition moiety. An electrochemical impedance spectroscopy (EIS)–based biosensor developed for the detection of epidermal growth factor receptor 2 (ErbB2), a breast cancer biomarker, used a nanocomposite immunoelectrode consisting of a 3D, porous graphene foam (GF) modified with elec­ trospun carbon-doped titanium dioxide nanofibers (nTiO2) [20]. The large specific surface area of the GF–nTiO2 composite permitted highly stable and enhanced immobilization of the anti-ErbB2 antibodies using EDC−NHS coupling and better impedance response towards analyte ErbB2 [Figure 14.2(a)]. Also, their high porosity allowed access to the sensing surface for the target analyte. Further, the integration of the nanocomposite immunoelectrode into a microfluidic device allowed miniaturization, enabling a detection limit of 1fM. Nanomaterials also possess enzyme-like or catalytic properties that allow their use as nanocatalysts in electrochemical biosensors for signal enhancement. These nanomaterials exhibit a higher electroactive area and have the ability to transfer electrons to the electrodes in the absence of enzymatic redox moieties. For instance, Ahmad et al. synthesized hierarchical copper oxide (CuO) nanoleaves and used them to develop an electrochemical, non-enzymatic glucose biosensor [22]. The CuO-based glucose sensors can oxidize glucose directly on the electrode surface and hence are the best candidate for nonenzymatic biosensors. A low-temperature hydrothermal method was used to synthesize hierarchical CuO nanoleaves, which were then cast on a glassy carbon electrode (GCE). The CuO nanoleaves modified GCE was used as a working electrode in a three-electrode electrochemical cell and showed enhanced electro-catalytic activity for the direct electro-oxidation of glucose in 100 mM sodium hydroxide (NaOH) electrolyte. The developed biosensor resulted in enhanced sensitivity (1,467.32 μA/(mM cm2)) with a LOD of 12 nM. The optical and conductive properties of nanomaterials allow their use as transducers in biosensing systems for signal enhancement. Specific nanoscale optical properties such as surface plasmon resonance (SPR) or surface-enhanced Raman spectroscopy (SERS) are exploited for optical transducers to allow better optical signaling. The group of Huang reported a silver@graphene oxide (Ag@GO) nanocomposite-based optical sensor that uses the SPR property of silver nanoparticles to detect several biomolecules, i.e., dopamine, ascorbic acid, and uric acid [23]. The addition of the analytes to the nanocomposite solution led to the aggregation of nanoparticles that, in turn, influenced the SPR absorption band. The difference in the absorption spectrum was monitored to identify the presence and concentration of the selected biomolecules. Likewise, the conductive properties of nanomaterials are exploited for electrochemical transducers to transmit, enhance, and modulate electrical signals for better electrical signalling. Nanostructured electrochemical transducers allow effective mass transport towards the electrode (owing to their high ratio of surface area to volume ratio), nonplanar diffusion, and reduced capacitive current owing to their nano size [19]. Nanomaterials have also been incorporated into biosensing systems for their use in sample prepa­ ration and segregation, i.e., to pre-concentrate the sample analytes and avoid biofouling. Biofouling occurs due to the presence of interfering particles in the sample other than the target analyte, and can significantly affect the specificity of the biosensor. Therefore, the analyte of interest needs to be seg­ regated in such a way that the interfering moieties are unable to interact with the detection base.

244 Nanomaterials in Healthcare

FIGURE 14.2 (a) Illustration depicting the conjugation of anti-ErbB2 on the surface of GF and GF-nTiO2 electrodes using EDC-NHS chemistry, followed by oxygen plasma treatment. Reprinted with permission from [ 20] Copyright (2016) American Chemical Society. (b) Schematic of iMEX assay. Antibody (against CD63) coated magnetic beads capture the exosomes directly in plasma, followed by binding of HRP-labeled targeting antibodies. Chromogenic electron mediator, TMB is finally mixed with magnetic beads to generate current signals in the presence of HRP. HRP-horseradish peroxidase; TMB-3,3′,5,5′-tetramethylbenzidine. Adapted with permission from [ 21] Copyright (2016) American Chemical Society.

Nanomaterials can prove valuable in analyte segregation and sample preparation. Separation of the analyte of interest from the sample can be done either through size-based separation or affinity-assisted separation. Sun and co-workers synthesized an isoporous silica-micelle membranes (iSMM) on the electrode surface to act as an anti-biofouling layer to electrochemically detect chloramphenicol (CAP) in whole blood samples with no pretreatment [24]. The iSMM used size-based (molecular sieving) and charge-based separation to pre-concentrate the drug molecules. This way the iSMM allowed only small and neutral/lipophilic analytes to permeate through it and be detected by the electrode beneath through impedance spectroscopy, thereby avoiding biofouling. The impedance spectroscopy allowed easy detection of change in resistance for charge transfers between the interface of iSMM and electrode. The resistance value corresponds to iSMM permeability and exchange of electrons at the electrode surface. Likewise, 3D nanoarrays such as nanopillars [25] and nanowires [26] offer nanoporous sieving matrices and have been fabricated on chips for the separation of DNA molecules under DC electric fields based on

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their sizes. Affinity-assisted separation, on the other hand, uses magnetic nanoparticles to pre-concentrate the analyte of interest. In the presence of an external magnetic field, magnetic nanoparticles modified and functionalized with biorecognition capture moieties (such as antibodies, aptamers, etc.) not only preconcentrate the target analyte and prevent biofouling but also attract the analyte-bioreceptor complex towards the electrode surface and enhance the sensitivity. Jeong and co-workers reported an iMEX system that is an integrated magnetic−electrochemical test system for exosomes. [21]. The magnetic bead-based enrichment of exosomes allowed preconcentration of exosomes from blood samples owing to the fast-binding kinetics between the exosomes and the antibodies [Figure 14.2(b)]. This additionally allowed the concentration of the signal sources on the electrode surface, thereby improving sensitivity. To sum up, the nanomaterial’s ability to immobilize a high number of bioreceptors, support faster electron transfer, tune SPR, and good catalytic properties are some of the characteristics which help nanomaterials enhance the sensitivity towards analyte detection, specificity to the target analyte, and overall signal output of the biosensing systems.

14.4 NANOMATERIALS-BASED BIOSENSING FOR IN-VITRO DIAGNOSTICS The advancements in the area of genomics and proteomics have led to the identification of certain disease-specific biomarkers, detection of which has become one of the primary practices in routine clinical diagnosis [27]. Conventional laboratory-based diagnostic tools usually employed to detect these biomarkers are quite effective. However, they are expensive, time-consuming, involve bulky instru­ mentation, require trained personnel, and are not user-friendly [12]. Biosensing has emerged as a rapid, cost-effective, and portable alternative to laboratory-based, benchtop diagnostic methods. The oppor­ tunity to rapidly detect specific disease biomarkers that either indicate pathogenesis or are physiologi­ cally relevant has allowed early-stage diagnosis of diseases and has created room for better and personalized treatments. This has further helped in the reduction of the cost of patient care, particularly associated with the advanced stages of diseases such as cancer, respiratory illness, cardiovascular dis­ eases, etc., the incidence and progression of which are at their maximum high. Further, the incorporation of nanostructured materials in biosensing devices for in-vitro diagnostics has allowed better capture of rare biological target analytes and thereby led to the enhancement of the resulting signal transduction. Nanomaterials with their advanced nano-properties are the perfect entities to augment the biosensor’s ability to detect the biomarkers in the micro and nano range with high sensitivity and specificity for in-vitro diagnosis. The function of the biosensing component (such as bioreceptor or electrode com­ ponent) dictates the choice of nanomaterial to be used that can be fabricated from inorganic, organic, or composite materials [28]. Different nanomaterials such as gold nanoparticles, silver nanoparticles, magnetic nanoparticles, graphene, carbon nanotubes, polymeric nanocomposites, nanoarrays, etc. are being extensively integrated into biosensing systems owing to their unique characteristics in order to develop biosensors with enhanced detection limits down to single-molecule detection. Some of these nanomaterials have been discussed in detail in the following sections describing their potential for enhanced biosensor performance along with examples. Table 14.1 summarizes some of the recent biosensors that use nanomaterials for sensitive detection of disease biomarkers.

14.4.1 Metal Nanoparticles Metal nanoparticles exhibit unique features including their shape and size, ease of synthesis, purity, core composition, surface properties and stability. These exceptional characteristics are exploited in biosensors

1D

0D

Chronoamperometry Surface Plasmon Resonance Electrochemical Impedance Spectroscopy Cyclic voltammetry Electrochemical Impedance Spectroscopy Fluorescence Amperometry Cyclic voltammetry Electrochemiluminescence

AgNCs

Magnetic beads

Magnetic nanoparticles

ZnO nanotubes CuO Nanowires CuO nanorods

SWCNT

ZnO nanorods

– 1.2 pM, 22 pM and 13 fM

70 µM 2 µM

0.22 µM 1 pg/mL

[ 21]

3 × 104 exosomes 41 IU/mL 10 CFU/mL

[ 47]

[ 44] [ 45] [ 46]

[ 42] [ 43]

[ 40] [ 41]

[ 37] [ 38] [ 39]

6 pM 0.001 ng/mL 10 nM

0.5 nM 0.37 fg/mL 12 pg/mL

[ 34] [ 35] [ 36]

[ 33]

0.525 µmol/L

Neurodegenerative syndromes SARS-CoV-2 virus Cancer biomarker Hepatocellular carcinoma Alzheimer’s E. coli Neurodegenerative syndromes Ovarian cancer

[ 29] [ 30]

REFERENCES

[ 31] [ 32]

7.8 HAU 0.01 μg/mL

LIMIT OF DETECTION

Cardiovascular diseases 20 fg/mL Tumour marker 2.2 pg/mL

Influenza A virus Ovarian cancer

APPLICATION

Cancer, COVID 19 Multidrug resistance bacteria Glucose Diabetes Cardiac Troponin-T Acute myocardial infarction Glucose Diabetes Glucose Diabetes Glucose and H2O2 Diabetes and Oxidative stress biomarkers Nucleic acids, Hg2+, and Biomolecules thrombin

CD63-positive Exosomes Interferon-α Gram-negative bacteria

Beta-amyloid oligomer Lipopolysaccharide Dopamine

AgNPs

Linear Sweep Voltammetry Cyclic voltammetry Fluorescence

Hemagglutinin Platelet-derived growth factor Creatine kinase-MB Carcinoembryonic antigen Dopamine

ANALYTE DETECTED

Main protease (Mpro) Mucin 1 Alpha-fetoprotein-L3

Colorimetric Colorimetric

TYPE OF BIOSENSOR

Chronocoulometry Differential Pulse Voltammetry Differential Pulse Voltammetry Colorimetric Electrochemiluminescence Square wave voltammetry

AuNPs

NANOMATERIAL USED

Nanomaterial-based biosensors for the detection of disease biomarkers

NANOMATERIAL DIMENSIONALITY

TABLE 14.1

246 Nanomaterials in Healthcare

Breast cancer

Diabetes Phenylketonuria Metabolites

Uric acid, Cholesterol, Triglycerides

Diabetes

Glucose Phenylalanine

Glycated hemoglobin (HbA1c)

Dopamine, Ascorbic Biomolecules acid, and Uric acid Dopamine and Uric acid Neurological illness

ErbB2

Cancer cells Bacterial infection

Alzheimer’s

Diabetes Metabolic biomarker Diabetes Metabolic biomarker Non-small cell lung carcinoma Epithelial derived tumours Biomolecules

[ 54]

[ 53]

[ 52]

[ 48] [ 49] [ 22] [ 50] [ 51]

1 μM, 0.3 mM, 0.2 mM

3.90 µM 3.0 nM

[ 61]

[ 59] [ 60]

49 nM, 634 nM, [ 23] and 927 nM 0.37 μM and [ 57] 0.61 μM 0.072 % [ 58]

1300 cells/mm2 [ 55] 0.9*102 [ 56] CFU/mL 1 fM [ 20]

1.58 μM, 0.06 μM, 0.09 μM, 0.10 μM And 6.45 μM 0.16 ng/mL

1 pM

3 x 10−3 M 16 pM 12 nM 0.075 µg/mL 1 fg/mL

Abbreviations: AuNPs – gold nanoparticles, AgNPs – silver nanoparticles, AgNCs – silver nanoclusters, AuNCs – gold nanoclusters, ZnO – zinc oxide, CuO – copper oxide, SWCNT – single-walled carbon nanotubes, MWCNT – multi-walled carbon nanotubes, GO – graphene oxide, rGO – reduced graphene oxide, GF-nTiO2 – graphene foam modified with titanium dioxide nanofibers, Zr-MOF – zirconium metal-organic frameworks, Fe3O4(TMC) – trimethyl chitosan functionalised magnetic nanoparticles, PAni – polyaniline, Pt NPs – platinum nanoparticles, ErbB2 – epidermal growth factor receptor 2, HAU – hemagglutination unit.

Cyclic voltammetry Differential Pulse Voltammetry Cyclic voltammetry

Differential Pulse Voltammetry Electrochemiluminescence

SWCNT and MWCNT Zr-MOF/ Fe3O4(TMC)/ AuNCs CNT-CuO ZnO nanorods/ Au NPs PAni hydrogel /PtNPs

Ag@GO

Electrochemical Impedance Spectroscopy Surface Plasmon Resonance

GF–nTiO2

Filopodia E. coli

Surface Plasmon Resonance Fluorescence

Nanocomposites

β-amyloid

Ascorbic acid, Dopamine, Uric acid, Tryptophan, and Nitrite

Folic acid protein

Glucose Vitamin D3 Glucose 25-hydroxyvitamin D3 cytokeratin 19

Square wave voltammetry

Differential Pulse Voltammetry Differential Pulse Voltammetry

Polyurethane nanopillar array SU-8 nanopillars ZnO Nanoarrays

Graphene nanosheets

rGO

Fluorescence Cyclic voltammetry Amperometry Fluorescence Surface Plasmon Resonance

3D

2D

MWCNT CuO nanoleaves GO

14 • Role of Nanomaterials in Biosensing Applications 247

248 Nanomaterials in Healthcare to achieve high detection sensitivity. Noble metal nanoparticles, such as gold, silver and platinum nano­ particles have long been used in bioanalytical devices owing to their simple production, easy surface modification, and good biocompatibility with excellent optical and electrical properties. Their optical properties, in particular, the SPR is quite interesting and can be tuned with the change in size, shape, or dielectric constant of the environment and also, in the presence of other nanoparticles (aggregation) [5,62]. The biorecognition event in a gold or silver nanoparticle-based biosensor can cause a shift in the resonant frequency (localized surface plasmon resonance (LSPR)). When this shift is particularly in the visible region, we can detect changes in the color of the nanoparticles through the naked eye. A wide range of colorimetric biosensors have been developed using this phenomenon for disease-specific biomarkers. Liu et al developed a colorimetric immunosensor to detect the Influenza A virus (IAV) using antihemagglutinin antibody-modified gold nanoparticles [29]. Among various subtypes of influenza virus, H3N2IAV is an emerging virus with high infection ability. The emergence of new subtypes can show problems regarding economic aspects, control measures, and surveillance. Therefore, early detection is required to avoid these circumstances and control the spread of the virus. This virus has numerous recognition sites on its surface, so the influenza-specific antibody conjugated gold nanoparticles align on these recognition sites, thereby causing aggregate formation. These aggregates cause a red shift in the absorption spectrum as a result of plasmon coupling between the gold nanoparticles and can be visually detected as a color change from red to purple. The absorption measurements using UV-vis spectro­ photometer can sensitively quantify the H3N2 IAV with a detection limit of 7.8 hemagglutination units (HAU) [29]. Hasan and co-workers also used the phenomenon of gold aggregation to colorimetrically detect cystic ovarian cancer biomarker, platelet-derived growth factor (PDGF) [30]. He synthesized aptamer-modified gold nanoparticles that aggregated in the presence of PGDF, thereby causing a color change from pink to purple due to plasmon shift. The biosensor was specific and sensitive enough to detect PGDF within a linear range of 0.01–10 μg/mL and a LOD of 0.01 μg/mL. Recently, Retout et al. used bridging peptides with specific sequences to induce the aggregation of silver nanoparticles into hyperbranched nanostructures by means of diffusion-limited aggregation (DLA) [Figure 14.3(a)] [34].

FIGURE 14.3 (a)i. Schematic of the peptide-induced aggregation of the AgNPs-BSPP into hyperbranched nanostructures. ii. Mechanism of amino acids to bridge AgNPs-BSPP via electrostatic or hydrophobic inter­ actions (pi–pi) with BSPP. iii. Figure illustrating the sensing mechanism of Mpro. 1) Mpro (present in the sample) incubated with M1 for 4 hours to allow the cleaving of M1, thereby releasing the bridging motif. 2) Bridging motif induced aggregation of the AgNPs-BSPP, measured in the form of optical changes proportional to the concentration of Mpro. Adapted with permission from [ 34] Copyright 2022 American Chemical Society (b) Schematic showing the sensing mechanism of His-Ag nanoclusters and GSH-DHLA-Ag nanoclusters for the ratiometric fluorescence detection of dopamine. Reprinted with permission from [ 39] Copyright 2020 Elsevier.

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They exploited this phenomenon to develop a colorimetric biosensor for the detection of the SARS-CoV2 virus by detecting the enzymatic activity of the enzyme, the main protease (Mpro). They designed a recognition sequence for the enzyme Mpro, i.e., the peptide M1 which was conjugated with a bridging motif and a negatively charged quenching site [Figure 14.3(a)]. Mpro (present in the sample) was incubated with M1 for 4 hours to allow the cleaving of M1, thereby releasing the bridging motif. The sample was then incubated with bis(p-sulfonatophenyl) phenyl phosphine (BSPP) coated AgNPs during which the bridging motif induces aggregation of the AgNPs-BSPP, measured in the form of optical changes that are proportional to the concentration of Mpro [34]. Gold and silver nanoparticles have also been employed extensively in electrochemical biosensing owing to their high electrical conductivity due to the presence of free electrons, stable immobilization of biomolecules retaining their biological activity and electron transfer ability between immobilized bio­ molecules and electrode surfaces [63]. They have been employed as labeling carriers, catalysts, and electrodes in different electrochemical analytic biosystems. The group of Lin developed a ratiometric electrochemical immunoassay for tumour marker, carcinoembryonic antigen (CEA) using polythionine–Au composites (PTh–Au) modified electrode in potassium ferricyanide (K3[Fe(CN)6]) containing solution as the electrolyte [32]. The PTh–Au was utilized as an internal reference signal and the K3[Fe(CN)6] as an indicator signal, the ratio of which was used to detect the binding and concentration of CEA. Fang et al. used Au-nanocatalyst (AuNC) label mediated redox species [outer-sphere-reaction-philic (OSR-philic) and inner-sphere-reaction-philic (ISR-philic)] and reported an electrochemical biosensor for the ultrasensitive and incubation-free detection of creatine kinase-MB (CK-MB), a cardiac biomarker [31]. An electro­ chemical biosensor that used gold both as an electrode, as well as a labeling carrier, was also developed to detect dopamine [33]. The sensor employed gold substrate as the working electrode modified by a selfassembled monolayer of 11 mercaptoundecanoic acid (11-Mua), followed by layer-by-layer (LbL) assembly of poly(ethyleneimine) (PEI) and finally a layer of flavin adenine dinucleotide (FAD) conjugated glutathione stabilized AuNPs [33]. The gold nanoparticles increase the sensitivity of the sensor due to a higher electroactive area compared to the bare substrate; and the use of FAD, i.e., a cofactor that is cheaper than enzymes and does not require membranes for immobilization purposes, made the sensor more spe­ cific. Silver nanoparticles have also been employed in the electrochemical analysis, for instance, as signal enhancers in an ultrasensitive electrochemical biosensor developed to detect the negatively charged lipopolysaccharide (LPS) and hence also E. coli as a model for gram-negative bacteria [38]. The further size reduction of metal nanoparticles gives rise to metal nanoclusters, that no longer have SPR or conducting properties of nanoparticles but show bright luminescence [64]. The wide use of metal nanoclusters as fluorophores in fluorescent biosensors is based on their size-dependent tunable fluorescence, good photostability with high emission rates, and biocompatibility. The group of Luo developed a ratiometric fluorescence biosensor for the detection of dopamine using different ligand stabilized silver nanoclusters (AgNCs) [39]. Based on the different electron-donating abilities of the different ligands, histidine (His) templated AgNCs and glutathione (GSH) and dihydrolipoic acid (DHLA) protected AgNCs emitted fluorescence at different wavelengths. In the presence of dopamine, the fluorescence of His-AgNCs quenched while the fluorescence intensity of GSH-DHLA-AgNCs enhanced [Figure 14.3(b)] [39].

14.4.2 Metal Oxide-Based Nanomaterials Similar to metals, nanostructured metal oxides (NMOs) also exhibit phenomenal morphological, optical, and electrical properties as a result of which they are being used extensively in biosensing systems for their role in improved bioanalytical performance. Nanostructured oxides of metals such as zinc oxide (ZnO), copper oxide (CuO/Cu2O), cerium oxide (CeO2), silicon dioxide (SiO2), titanium dioxide (TiO2), iron oxide (Fe3O4), etc. offer enhanced matrices for the immobilization of biorecognition molecules because of their remarkable electron-transfer kinetics and good adsorption capability. The NMOs ensure better attachment of bioreceptors, such as enzymes, antibodies, nucleic acids, etc. per unit mass of

250 Nanomaterials in Healthcare constituents resulting in overall signal amplification and improved biosensing characteristics [65]. Different morphologies of NMOs such as nanorods, nanowires, nanofibers, etc. have been employed in multiple biosensors to develop innovative nanodevices with elevated performance. ZnO nanostructures have been extensively investigated as a suitable material for protein (enzyme or antibody) immobilization because of their biocompatibility, stability, an exclusive band gap of 3.37 eV and high isoelectric point (IEP = 9.5) [66]. For instance, ZnO nanorods were used for the sensitive electrochemical detection of Glucose. The ZnO nanorods grown on ITO-coated glass substrates had a uniform diametric distribution and high surface-to-bulk ratio that gave the uniformly distributed enzyme, glucose oxidase (GOx), a better electrical contact with the electrode [42]. The group of Prasad demonstrated an ultra­ sensitive, electrochemical detection of cardiac Troponin-T (cTnT) using functionalized nanostructured ZnO sensing electrodes on flexible porous polyimide substrates. Electrochemical Impedance Spectroscopy (EIS) was used to measure the adsorption of the analyte (cTnT) present in an ionic buffer to the functionalized sensing electrode (Anti-cTnT/Nanostructured ZnO) [43]. The antibody functio­ nalized nanostructured surfaces allowed enhanced charge transfer and therefore could sensitively detect cTnT with a detection limit of 1 pg/mL. CuO is another widely used NMO in electrochemical sensing of disease biomarkers because of its inherent catalytic properties, cost-effectiveness, and easy synthesis. Zhang et al. synthesized CuO nanowires and used their electrocatalytic behaviour to fabricate a nonenzymatic glucose biosensor. The prepared CuO nanowires modified working electrode displayed excellent sensitivity (648.2 μA cm−2 mM−1) towards glucose detection owing to their large electroactive surface and enhanced electron transfer for the electrooxidation of glucose in the presence of an alkaline electrolyte (50 mM NaOH) [45] [21]. Nanostructured iron oxides, specifically magnetic nanoparticles have been significantly used in bioanalytical applications owing to their biocompatibility, high magnetic susceptibility and less back­ ground interference with bio-samples [67]. Magnetite (Fe3O4) nanoparticles are the most commonly employed iron oxide nanoparticles in biosensing systems due to their distinctive electric and magnetic (superparamagnetism) properties. Mainly, they have been used as a pre-concentrator, for efficient sep­ aration of analytes under the influence of a magnetic field. They have also been used as nanocarriers for electroactive moieties, electrode modifiers, and signaling agents. Andrade and the group used chitosancapped magnetic nanoparticles (Fe3O4-Chit) modified with antimicrobial peptide (Synoeca-MP) for impedance-based detection of gram-negative bacteria. The Fe3O4 nanoparticles act as signal amplifiers in the biosensor enabling a limit of detection of 10 CFU/mL [41]. Saylan et al. employed magnetic nanoparticles to fabricate plasmonic biosensors using metal affinity interaction for the detection of interferon-α. The affinity of the nanoparticles towards the interferon analyte and the corresponding change in reflectivity on the plasmonic surface was analyzed to detect and quantify interferon-α [40]. The magnetic nanoparticle-assisted plasmonic sensor allowed real-time detection with a LOD of 41 IU/mL. Lately, many reported biosensing systems have incorporated magnetic nanoparticles as core-shell bimetallic structures in conjunction with other metal nanoparticles for enhanced efficiency as a result of their synergistic effect [68]. Other than optical and electrochemical biosensing, magnetic nanoparticles have also been employed in other transduction techniques such as piezoelectric [69] and magnetoresistive [70] biosensors.

14.4.3 Carbon-Based Nanomaterials Carbon-based nanomaterials have received enormous research importance due to their potential sensor applications, specifically in the area of electrochemical sensing of different biomolecules. They display several unique properties including a large surface-to-volume ratio, high electrical conductivity, electron mobility at room temperature, biocompatibility, long-term chemical stability, and robust mechanical strength [71]. These exceptional characteristics have enabled carbon-based nanomaterials to deliver high-performance biosensors with high sensitivities and low detection limits. The ability of carbon atoms to undergo hybridization thereby forming different structures with various physical properties and

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geometrical shapes such as tubes, sheets, etc. has allowed it to exist in polymorphic forms such as carbon nanotubes, graphene, diamond, fullerenes, etc. Carbon nanotubes (CNTs) and graphene are the most common carbon-based nanomaterials used as electrode materials in the recently developed electro­ chemical biosensors. Carbon nanotubes (CNTs) are cylindrical nanostructures made up of rolled-up graphite sheets. They can be composed of either a single graphitic layer, i.e., single-walled carbon nanotubes (SWCNTs) or multiple coaxial graphitic layers, i.e., multi-walled carbon nanotubes (MWCNTs). Both have been used extensively in electrochemical biosensing systems because of their excellent electrical and thermal conductivity and highly tunable physical and chemical properties [72]. Guan et al. fabricated a hybrid CNT composite (MWCNT-COOH and SWCNT-OH) modified GCE as the working electrode for the simultaneous electrochemical detection of dopamine and uric acid. The net-like composite structure extensively enhanced the electrochemical activity of the sensor due to accelerated electron transfer reactions and by providing multiple active sites for electrocatalytic oxidation of the analytes [57]. Recently, Bora and colleagues used nitrogen-doped MWCNT for electrochemical detection and mon­ itoring of vitamin D3. The nitrogen-doping introduced additional chemically active sites to the carbon structure, thereby allowing a greater number of places for the analyte binding and increased electron transfer between the electrode and the electrolyte, increasing the overall conductivity compared to bare graphite or pristine CNT [49]. Apart from promoting electron transfer and increasing conductivity, CNTs have also been used as substrates for further functionalization and to facilitate the adsorption of bio­ molecules. For instance, the facile binding of single-stranded DNA (ssDNA) to SWCNT in the form of helical wraps around the surface of nanotubes has been extensively studied [73]. Huang et al. exploited this phenomenon to detect DNA using the electrochemiluminescence (ECL) method. The selective assembly of SWCNTs to ssDNA bound ITO electrode (and not the hybridized dsDNA) helps in cata­ lyzing the electrochemical reaction of an ECL co-reactant, tri-n-propylamine (TPrA), thereby amplifying the ECL signals [Figure 14.4(a)] [47]. Graphene is a two-dimensional, sp2 hybridized carbon layer arranged in a hexagonal lattice. It displays unique structural (mechanical strength, flexibility) and electrical (efficient electron transfer) features that allow their use in highly sensitive biosensing platforms. Graphene and its derivatives including graphene oxide (GO) and reduced graphene oxide (rGO) have been used in electrochemical sensors as signaling carriers and electrocatalysts to selectively and accurately detect biomolecules. He et al. fabricated an rGO deposited gold electrode modified with folic acid, for the electrochemical detection of folic acid proteins (also known as the folate receptor). The electrophoretically deposited rGO results in increased currents that are significantly decreased upon target binding [52]. This decrease in current is measured, to sensitively detect folic acid protein up to a detection limit of 1 pM. Wang and co-workers reported a biosensing electrode with free-standing graphene nanosheets as a sensing layer over Ta wire as the base electrode for the simultaneous detection of multiple biomo­ lecules including ascorbic acid, dopamine, uric acid, tryptophan, and nitrite in human serum samples. The graphene nanosheets fabricated using the chemical vapour deposition (CVD) method are exposed to many edge defects (that means different band-gap energy levels) and hence, to the active sites affecting the oxidation potential of analytes and hence ensuring higher analyte selectivity [53]. The nanosheets also displayed high adsorption of the analyte due to the high surface area, and high con­ ductivity due to higher electron transfer. Apart from good electrical properties, graphene oxide also possesses a number of remarkable optical properties. The fluorescence quenching ability of graphene oxide was exploited by Gupta and colleagues to develop a fluorescent biosensor for 25-hydroxyvitamin D3 (25(OH)D3). In the presence of the analyte 25(OH)D3, the fluorescently labelled aptamer binds to the analyte and hence its fluorescence remains unchanged [50]. However, in the absence of the analyte, the fluorescently labeled aptamer is adsorbed onto the graphene sheets by π–π interaction that leads to quenching of the fluorescence owing to Förster resonance energy transfer (FRET). Thus, changes in the fluorescence intensity are measured to detect 25(OH)D3 with a LOD of 0.15 µg/mL. Chiu et al. used COOH modified GO sheets on Au film to fabricate a SPR immunosensor to detect lung cancer biomarker, cytokeratin (CK) 19 [Figure 14.4(b)]. The enhanced field energy propagation intensity of

252 Nanomaterials in Healthcare

FIGURE 14.4 Schematic representation of the self-assembly of SWNTs with (capture) ssDNA for ECL emission [top] and its regulation when the capture DNA hybridizes with the perfect complimentary ss DNA (pc-DNA) to form a dsDNA [bottom]. Reprinted with permission from [ 47] Copyright 2018 Elsevier. (b) Illustration of an immunoassay-based detection of lung cancer antigen, CK 19 on binding with the anti CK 19 antibody immobilized on the GO-COOH based SPR chip. Adapted with permission from [ 51] Copyright 2018 Elsevier. (c) Schematics of the sensing mechanism of the PAni hydrogel/PtNPs hybrid electrodes loaded with PtNPs and enzymes. Adapted with permission from [ 61] Copyright 2015 American Chemical Society.

the SPR sensor owing to the use of COOH-modified GO sheets resulted in the highly sensitive detection (LOD – 1 fg/mL) of human plasma CK19 [51].

14.4.4 Nanocomposites Nanocomposites are multi-phasic materials; where at least one composite phase is of nanoscale, and so they take advantage of the synergistic effect of physical, chemical, and optical properties of the various materials together [74]. With advancements in nano-science and technology, nanocomposite structures are extensively being designed and utilized for biosensing purposes as they present with better elec­ trochemical and optical properties that result in improved sensing performance. Based on the different matrices that integrate various nanomaterials, nanocomposites can be classified into metal-based, metalcarbon-based, metal-organic frameworks (MOF) based or polymeric-based nanocomposites. The group of Ojani reported a metal/metal oxide nanocomposite to electrochemically detect phenylalanine in human serum samples (Rahimi-Mohseni et al. 2021b). A filter paper disc was modified with ZnO@Au hybrid nanoarrays along with phenylalanine hydroxylase (PHA) from the leaf-like extract of mosses that was further placed on the graphite screen-printed electrode for electrochemical detection. The ZnO

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nanorods were used for their higher immobilization of the enzyme PHA, while the Au nanoparticles were used for their good electrocatalytic activity. The reported paper-based nanocomposite structure was considered a compact and easy platform for phenylketonuria detection in real samples with a LOD of 3nM (Rahimi-Mohseni et al. 2021b). Metal-carbon nanocomposites exploit the large surface area provided by carbon nanomaterials to host different nanoparticles and increase the number of reactive sites. A carbon nanotube (CNT)-copper oxide (CuO) nanocomposite-based non-enzymatic glucose sensor was developed by Geetha and co-workers. The nanocomposite was employed as an electrocatalyst that improved the direct electron transport to the surface of the electrode and the metallic centres executed the catalysis [59]. The CNTCuO nanocomposite showed high specificity, sensitivity (LOD 3.90 µM), and quick response time (2 s) for glucose detection in artificial sweat solution. The MOF-based nanocomposites have been employed in biosensing applications as MOFs have more catalytic active sites that ensure fast electron transfer and better electrocatalytic activity [75]. An electrochemiluminescence sensor that used a nanocomposite of zirconium (Zr) MOF, trimethyl chitosan (TMC) functionalized Fe3O4 nanoparticles, and BSA capped Au nanoclusters as an ECL label for the anti-HbA1c monoclonal antibody, and rGO as the immobilization platform was designed and developed for detection of glycated haemoglobin (HbA1c) [58]. BSA-AuNCs were employed as luminophores because of their good biocompatibility and direct electron transition characteristics, while Fe3O4(TMC) nanoparticles were used as a co-reaction accelerator to promote the reaction rate between the lumino­ phore and co-reactant and hence produce an amplified ECL signal. The integration of the luminophore, i.e., BSA-AuNCs and the co-reaction accelerator, i.e., Fe3O4(TMC) NPs, within the Zr-MOF for their close proximity improved the sensitivity of the assay. The mutual synergistic effect of the different nanostructured materials making up the composite allowed signal amplification and improved sensitivity of the ECL system [58]. Polymer-based nanocomposites comprise inorganic/organic nanomaterials in a polymeric matrix. Conductive polymers have gained a lot of attention in the last decade because of their unique electro­ active properties as well as biocompatibility. Li et al. fabricated a 3D-porous polyaniline (PAni) hydrogel-based matrix with homogenously dispersed platinum nanoparticles (Pt NPs) and enzymes [Figure 14.4(c)], onto a GCE for the electrochemical detection of metabolites such as uric acid, cho­ lesterol, and triglycerides [61]. PAni is a hydrophilic, conductive polymeric hydrogel with a porous nanostructure that enhanced the transfer of electrons between the enzyme and the electrode, while the Pt NPs act as electrocatalysts for the electrochemical oxidation of hydrogen peroxide produced due to enzyme-substrate reaction. Due to the synergistic effect of conductive hydrogel, Pt NPs, and enzymes, the sensor showed high sensitivity toward the detection of several metabolites within three seconds [61].

14.5 CHALLENGES AND FUTURE PROSPECTS The use of nanomaterials as biosensing moieties has advanced the field of bioanalytics. However, there are still several gaps and restrictions associated with its use. Despite the extensive use of nanostructured materials for enhanced biosensing performances, their application is restricted to academic research laboratories only. Not many of these biosensors except the optical lateral flow pregnancy tests, the electrochemical glucose monitoring systems, and the recently developed home-based COVID-19 test have been made commercially available for clinical applications. The translation of the academic laboratory-developed biosensors into commercially viable industrial prototypes faces several difficulties. The limited robustness and reproducibility of nanomaterial-based biosensing systems are among the major challenges faced. The different nanomaterials employed for enhanced biosensor performance limit their long time use because of the complexity involved. The more complex the nanostructure is, the more

254 Nanomaterials in Healthcare difficult its reproducibility is. The uniformity in batch-to-batch morphology, size, shape, and their combinations during synthesis is extremely challenging. Future work should focus on optimizing easy synthesis with less batch variation and better control over the robustness and reproducibility of the nano-biosensors. Another significant hindrance to the commercialization of nanomaterials-based biosensors is the non-specific, off-target interaction of nanomaterials with sample biomolecules present in addition to the target analyte. The unintended adsorption of these biomolecules onto the sensor’s surface, i.e., surface fouling, hampers the detection sensitivity of the biosensor. For reliable biosensing applications for disease detection, the undesired interactions and aggregation of nanomaterials must be kept to a minimum. Future work should focus on a better understanding of the nonspecific interactions of nanomaterials and antifouling properties. Efforts must be made to prevent fouling for biosensing to be effective. Testing of the developed biosensors should be done in real-world matrices instead of distilled water or buffers. For consistent and precise results, the characterization process, production, and alterations of nanomaterials must be investigated and managed. Stability and functionality of the nanomaterials under different physiological conditions along with a good performance at varying tem­ peratures, and pH conditions are some of the other challenges that require consideration. Further, the quality, simplicity, and accessibility of biosensors can be enhanced through improved integration of electronics, microfluidics, and digitalization. These integrations will also improve bio­ sensors’ capacity for multiplexed biomarker detection with high patient compliance. Developing novel biosensing systems exploiting the rapidly developing field of nanotechnology and providing molecular analysis in the form of sophisticated tests at the patient’s bedside or point of care is the ultimate goal of the bioanalysis community. With continuous future advancements in biosensing technology, early disease detection will be accessible to the end users through clinics or home-based health monitoring. Monitoring disease conditions and keeping a track of their health will be much more convenient in the future.

14.6 CONCLUSION The use of nanomaterials in biosensing systems has allowed for improved analytical performance with better sensitivity and detection limits. The distinct characteristics of nanomaterials such as large surfaces, electrocatalytic power, transduction capacity, and inherent material qualities have boosted their use in biosensors, as discussed in great detail in this chapter. The application of nanomaterialsbased biosensing for in-vitro diagnosis raises new possibilities for early disease detection and better therapeutic outcomes. This chapter cites and discusses some of the recent nanomaterial-based bio­ sensors developed to detect several disease biomarkers. The use of nanomaterials in biosensing sys­ tems has overcome its traditional drawbacks, such as low capture efficiency, selectivity, and sensitivity. However, their numerous shortcomings such as fouling, stability and reproducibility limit their use in clinical settings. The current research focuses on more robust, rapid, and inexpensive biosensors along with enhanced analytical performance using the recent and fast-growing advance­ ments in the field of nanotechnology. The development of affordable, easy-to-use, and portable bioanalytical devices with embedded nanomaterials is inevitable in the near future, owing to the continuous research efforts being made in this domain. Simultaneously, there is a rising interest in real-time analysis of living cells in intact organisms. This not only allows rapid, accurate, and early detection but also provides room for a better prognosis. With the increasing interest in personalized medicine, different therapeutic strategies can further be applied to improve the patient’s outcome. Conflict of Interest: The authors report no conflict of interest.

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Application of TwoDimensional Materials for Cancer Theranostic

15

Barkha Singh1,2 and Ritika Uday Gaitonde3 1

Department of Biosciences and Bioengineering Centre for Research in Nano Technology & Science (CRNTS), Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 3 Department of Life Sciences, Ramnarain Ruia Autonomous College, Mumbai, Maharashtra, India 2

Contents 15.1 15.2 15.3 15.4

Introduction Properties of 2D Nanomaterials Synthesis of 2D Nanomaterials Application of 2D Nanomaterials in Cancer Therapy 15.4.1 Graphene and Its Derivative 15.4.2 Two-Dimensional Transition Metal Dichalcogenides (TMDCs) 15.4.2.1 Molybdenum disulfide (MoS2) 15.4.2.2 Tungsten disulfide (WS2) 15.4.2.3 MXenes 15.4.2.4 Xenes 15.4.3 Black Phosphorus (BP) 15.4.4 Boron Nitride (BN) 15.4.5 Metal Oxide Nanosheets 15.4.5.1 Manganese dioxide (MnO2) 15.4.5.2 Molybdenum oxide (MoOx) 15.4.5.3 Zinc oxide (ZnO) 15.4.5.4 Iron oxide (IO) 15.4.6 Layered Hydroxides (LDH) 15.4.7 Metal Organic Framework (MOF) 15.5 Conclusion References

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DOI: 10.1201/9781003322368-15

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260 Nanomaterials in Healthcare

15.1 INTRODUCTION Two-dimensional (2D) nanomaterials are novel nanomaterials comprising of tiny layers with a thickness of at least one atomic layer. 2D nanomaterials generally have lateral size in the range of 100 nm to a few micrometers, however, possess the thickness of only a few atomic layer, usually < 5 nm [1]. These include graphene, black phosphorus, MXenes (M is an early transition metal, A represents group 13 and 14 elements and X is carbon or nitrogen), transition metal dichalcogenides (TMDCs), transition metal oxides (TMOs), layered double hydroxides (LDHs), etc. Because of their enormous surface area, unique surface chemistry, as well as quantum size effect, 2D nanomaterials often have extraordinary physicochemical features that differ from their nanoparticle and bulk counterparts [2]. The multilayer structure (Figure 15.1) of these nanomaterials has strong in-plane bonding and weak van der Waals between the individual layers [3]. The advantages of such 2D nanomaterials that make them useful in biomedical applications are: 1. Have very high surface areas in comparison to other nanomaterials that enable adsorption of guest molecules. 2. Possess the ability to form biomedical nanocomposites with high mechanical and physico­ chemical properties. 3. They have an ultrathin structure that makes them light sensitive.

15.2 PROPERTIES OF 2D NANOMATERIALS 1. Light to heat conversion - 2D nanomaterials have excellent optical properties as they have spectral absorption peaks in the near infrared (NIR) region. Consequently, the probability of exciting free electrons by irradiation with light energy leads to high light absorption efficiency. Upon light irradiation, most 2D nanomaterials shift from the ground energy state to the singlet excited energy state, quickly proceeding to a triplet energy state, followed by decay back to ground state, in turn releasing the absorbed light energy to the surroundings as singlet oxygen. This phenomenon is very important in development of nanomaterials for photothermal therapy (PTT) and photodynamic therapy (PDT) as when heat is generated by NIR irradiation in the tumor, the temperature of the tumor rises rapidly resulting in regression of the tumor. Li et al., designed a thin MXene membrane with a photothermal conversion efficiency of 100% [4]. 2. Stability - 2D nanomaterials have closely packed crystal structures much like their bulk form as they have strong isotropic metallic bonds. The ultra-thinness and planar structure of these nanomaterial makes them very flexible, enabling their usage in drug delivery, tissue engineering, etc. They also demonstrate excellent lubricant properties due to the interlayer sliding and are superconducting at lower temperature, due to their metallic character. 3. Electronic properties - 2D nanomaterials have equivalent electronic properties as their bulk counterparts, contrary to the belief that their small size should hamper their performance. The defects caused by low or nil interlayer interactions in the 2D nanomaterials, which causes lower conductivity, can be exploited to change the conductivity of the materials. External stimulation causes electron movements in conducting and valence bands to change, which can lead to a reaction of the active electrons and cause reactive oxygen species (ROS) generation, which can be applied in PDT. The production of these radicals in the cancer cells leads to oxidative stress in the cells. This leads to cells death through multiple pathways like apoptosis, necrosis, disruption of tumor vasculature, etc. [5].

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FIGURE 15.1 Schematic to represent layered structure of 2D materials: (a) transition metal dichalcogenides, (b) MXene, (c) graphene.

4. Magnetic properties - Magnetism can be induced at room temperature in 2D nanomaterials like graphene, molybdenum disulphide (MoS2), etc. by doping of these materials. The inherent or induced magnetism can lead to application of these materials to various imaging and therapy techniques like magnetic resonance imaging (MRI). 5. Biocompatibility - 2D nanomaterials show a good promise in the biomedical field as they have low cytotoxicity towards non-tumorous cells as well as stability in biological fluids but still many nanomaterials suffer from poor biodegradability and toxicity, which hinders their clinical translation [6]. 6. Surface chemistry and functionalization - The surface of 2D nanomaterials is decorated with certain molecules or polymers like polyethylene glycol (PEG) to improve their physiological stability and biocompatibility. The large surface area of 2D nanomaterials provides a good platform for loading of therapeutic agents [7]. PEGylation improves the efficiency of drug delivery to target cells and also imparts “stealth” properties to the NS as it imparts it with longer circulating time that allows it to escape the reticuloendothelial system. PEGylation also reduces opsonization and prevents clearance of PEGylated NPs by the mononuclear phagocyte system and also decreases hemolysis and RBC aggregation. This provides the nanomaterials with a greater chance of survival in the body to kill cancer cells. 7. Fluorescence - 2D nanomaterials have sizable bandgaps that results in a good fluorescence on chemical treatments. The fluorescence can be utilized for sensing or bio-imaging application like in imaging of the tumor cells.

15.3 SYNTHESIS OF 2D NANOMATERIALS 2D nanomaterials are synthesized by two basic approaches: top down and bottom up. • The top-down method involves the splitting of the bulk material into nano scale materials. The techniques used in this method are:

262 Nanomaterials in Healthcare 1. Mechanical compression - It simply involves reducing the dimensions of bulk metals by applying mechanical force or repeated folding like separating graphene using scotch tape. However, due to low yields and uncontrollable thickness, this method is not feasible for biomedical applications. 2. Liquid exfoliation - It utilizes ultrasound, microwave, shear stress, thermal stress, or electrochemistry in a liquid medium to reduce the van der Waals forces between layers of bulk material to form nanoscale particles. Although water dispersible nanomaterials can be produced by this method, this method suffers from low material yields, a property that hinders its path to scalability. 3. Nanolithography - Nanomaterials are fabricated in a controlled manner by patterning or etching on a well-defined 2D substrate. This method is especially suitable for materials like MXenes that have strong interlayer bonds in the bulk materials. • Bottom-up method involves the production of 2D nanomaterials through chemical reactions between various molecules. This method encompasses: 1. Chemical Vapor Deposition (CVD)- In this method, a substrate is exposed to volatile compounds to react and produce a thin film on the surface of the substrate under high temperatures and in vacuum. This method can be used for the large-scale production of ultrathin nanomaterials. However, the primary disadvantages are its high cost, which is due to the extreme heating and cooling required, size limits, and constrained applications. 2. Hydro/solvothermal method- It involves controlled, high-temperature synthesis of nano­ materials in vacuum conditions by dissolving the bulk metals in water or other solvents. Even though this method is low cost and simple, the real-time nanomaterial formation cannot be observed, and the synthesis process is easily altered by the reaction parameters. This makes it difficult to control the particle size of the nanomaterials.

15.4 APPLICATION OF 2D NANOMATERIALS IN CANCER THERAPY Cancer is a serious and devastating disease claiming millions lives annually worldwide. According to GLOBOCAN 2020, cancer caused approximately 10 million deaths in 2020 alone [8]. Surgical, radiation, and chemotherapy treatments for cancer involve a variety of side effects. In the mid-1990s, a novel technique was developed called photothermal therapy (PTT), which proved to be a major milestone in cancer therapy. It is a non-invasive therapeutic technique involving targeted delivery of biocompatible nanoparticles, which when irradiated with NIR light leads to the thermal ablation of tumor cells, causing minimal damage to the surrounding healthy tissues and cells [9]. PTT is advantageous as it is non-invasive, high controllability, highly specific and has accurate spatial-temporal selectivity compared to traditional therapy techniques. NIR laser is used in PTT as NIR light has less tissue absorption that leads to deeper tissue penetration [10]. Many 2D nanomaterials have been used extensively in PTT because their good biocompatibility, good cytotoxicity, the spectral absorption peaks in the NIR range that greatly increases the depth of light transmission through the skin, amenable to surface modification and the ability of the absorption peak to be fine-tuned to a very narrow wavelength range which increases PTT specificity. 2D nanoparticles-based PTT can be used as it is or even in combination with other therapeutic and imaging modalities (Figure 15.2). For example, manganese dioxide loaded 2D black phosphorus nanosheets have been utilized for MR image guided synergistic PTT, PDT, and chemotherapy to treat cancer [11]. Hybrid nanosheets are an amalgamation of different NSs with varied properties such that the functions of these individual NSs can be combined to produce NSs having properties advantageous for

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FIGURE 15.2 Schematic representing application of 2D materials in biomedical application. Reprint with permission [ 2].

combined applications. Even though inorganic NSs have excellent optical, thermal, and mechanical properties, they need to be dispersed in an aqueous solution for chemical stability, that is provided when they are combined with organic NSs [12]. Attaching synthetic polymers at an atomic level, them enables the combination of advantageous properties of both NSs without phase separation inconveniences, while also drastically increasing solubility, stability, and also decreasing the toxicity [13].

15.4.1 Graphene and Its Derivative Single-layer graphene was first isolated from graphite by Geim and co-workers in 2004 at Manchester University [14]. Because of its 2D structure, graphene demonstrated outstanding mechanical strength,

264 Nanomaterials in Healthcare excellent thermal conductivity, and unique electrical characteristics, including high conductivity and charge carrier mobility. The properties of the material at two dimensions show significant dif­ ference as the dimensionality of the carbon form changes to zero dimension, one dimension, and three dimensions [15]. In terms of cancer theranostic applications, graphene and its derivatives possess high near-infrared (NIR) absorbance, which may be used as photothermal agents for effective cancer photothermal treatment (PTT). Graphene may be loaded with numerous types of biomolecules with high efficiency for applications in gene transfection and drug delivery because of its extraordinarily high specific surface area [16]. PEGylated graphene oxide nanoribbon was synthesized and loaded with chemotherapy drug doxo­ rubicin (DOX) for application in chemo-PTT. The material (PL-PEG-GONRs/DOX) showed 6.7-fold lower IC50 value against U87 cells than free DOX, indicating synergistic cell death caused by chemo­ therapy and PTT. PL-PEG-GONRs caused no cytotoxicity in U87 cells in vitro and no hematological toxicity in mice, demonstrating its biosafety [17]. Yin et al. fabricated a system using PEGylated GO nanosheets to co-deliver small interfering RNAs targeting the HDAC1 gene and G12C mutant K-Ras gene siRNAs (Figure 15.3a). The tumor targeting was enhanced by folic acid. The material showed synergistic effects combining gene therapy and photothermal activity against pancreatic cancer cells MIA PaCa-2 [18].

FIGURE 15.3 (a) The diagrammatic representation of the design and use of GO/PEG/FA/PAH/siRNA nanosheets a for Combined photothermal and gene therapy for treatment of Pancreatic cancer. Reprint with permission [ 18] (b) Diagrammatic representation of MoS2-PEI-HA nanosheets as a multifaceted platform for PET image guided targeting and multiple stimuli-responsive therapy of MCF-7-ADR cells. MoS2 nanosheets are synthesized by a liquid assisted exfoliation process. MoS2-PEI-HA nanosheets application for active CD44targeting delivery of DOX as well of the reversal of MDR by the way of P-gp protein inactivation.NOTA-64Cu functionalized MoS2-PEI-HA nanosheets for PET imaging. Reprint with permission [ 22]. (c) 2D Ultrathin MXene-Based Drug-Delivery Nanoplatform for Synergistic Photothermal Ablation and Chemotherapy of Cancer. Reprint with permission [ 23].

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In addition, different inorganic nanoparticles might be conjugated on the surface of nano-graphene to give additional optical and magnetic characteristics that could be advantageous for in vivo tumor multimodal imaging contrast [19]. A nanocomposite containing graphene oxide and silver nanoparticles was constructed for X-ray imaging and chemo-PTT (GO@Ag-DOX-NGR) [20]. DOX was used for chemotherapy and the nanocomposite showed drug loading efficiency of 82%. GO@Ag-DOX-NGR had significantly (P-values < 0.05 were considered statistically significant) greater anticancer efficacy in an in vivo mouse tumor model, with no obvious side effects on normal organs. DOX showed 8.4-times higher absorption in tumor than normal tissues. The drug release kinetics also showed 1.7-times higher DOX released in the tumor site than normal tissues using NIR laser as an external stimulus. GO@Ag-DOXNGR showed very good X-ray imaging ability, indicating its potential for combined diagnostics and treatment. Graphene oxide can also be used as a sensor for cancer environments, like anon-invasive, pH-based optical sensor of cancerous environments using GO flakes [21]. The material possessed emission in red/ near-IR which was used to detect the acidic environment of the cervical carcinoma HeLa cells.

15.4.2 Two-Dimensional Transition Metal Dichalcogenides (TMDCs) Graphene is extremely popular, owing to its many exceptional features but the lack of an electronic bandgap in it has inspired a search for 2D materials with semiconducting characteristics. Transition metal dichalcogenides (TMDCs), which are semiconductors of the type MX2, where M represents a transition metal atom (such as Mo or W) and X represents a chalcogen atom(such as S, Se, or Te), are a possible alternate choice [24]. The atoms in the TMDs are hexagonally packed. The monolayer structure possesses X-M-X configuration where metal atoms occupy space between two planes of chalcogen atoms. Many of the electrical, optical, mechanical, and chemical properties of TMDCs are similar to those of graphene, implying potential use in electronic devices, energy storage devices, catalysis, and biological applications.

15.4.2.1 Molybdenum disulfide (MoS2) MoS2 has a hexagonal crystal system and conventional two-dimensional layered structure. Two sulphur (S) atoms and a molybdenum (Mo) atom are sandwiched together in each layer, making a S–Mo–S sandwich plate. The weak van der Waals force connects the layers [25]. The exceptional surface-area-tomass ratio of MoS2 nanosheets, which is due to their atomically thin 2D structure, allows for very effective therapeutic loading. PEG-functionalized MoS2 nanosheets were synthesized as a multifunctional drug delivery system (MoS2-PEG/DOX). Doxorubicin was loaded on the MoS2 nanosheets and synergistic effect of chemo­ therapy and PTT was observed in mice model using murine breast cancer 4T1 cells. The dosages of MoS2-PEG/DOX agent employed in this work were significantly lower than those required in earlier studies that used nano-graphene [26]. Folic acid targeted MoS2 nanosheets were designed to tumor-specifically deliver DOX against breast cancer cells MDA-MB-231 cells. The DOX delivery was enhanced at pH 5 under IR laser irradiation, making the system as dual responsive. The MoS2 nanosheets were further functionalized with PEG to increase biocompatibility. The synergistic effects of chemotherapy and PTT prove that MoS2-based nanoplatform might be a viable therapeutic agent carrier [27]. MoS2 has also been utilized in combating drug-resistant breast cancer (Figure 15.3b). MoS2 was conjugated with hyaluronic acid (HA) using polyethyleneimine (PEI) and loaded with doxorubicin. HA is applied to target CD44 which is a cell-surface glycoprotein. CD44-specific targeting of the HA-modified MoS2 nanosheets resulted in increased nanosheet uptake. HA targeting paired with modest

266 Nanomaterials in Healthcare NIR laser stimulation can downregulate the expression of drug-resistance-related P-glycoprotein, that results in considerably increased intracellular drug accumulation and hence drug resistance reversal. The nanosheets was also labeled with 64Cu to perform the positron emission tomography imaging. This proves that the MoS2 with a suitable targeting agent can be used to overcome drug resistance as well as can be used for both diagnosis and therapy [22]. HA targeting was utilized to achieve delivery of the anticancer drug camptothecin. Conjugation of MoS2 and HA was done via a disulphide bond, which not only improved the durability of MoS2 nanosheets in a physiological setting but also facilitated drug release via a Glutathione-mediated redox reaction. Furthermore, the heat produced during NIR irradiation caused the release of medication from the nanocomposite and promoted photothermal killing of tumors. A MoS2-based nanocomposite proved to be excellent cancer theranostic agent with its excellent targeting capacity, dual-stimulus-responsive drug release, and synergistic cancer therapeutic efficiency [28]. A tantalum oxide nanoparticle decorated; chitosan-coated MoS2 (TaO2-CS-MoS2) hybrid NS was designed for cancer PTT [29]. TaO2-CS-MoS2 hybrid NSs have negligible effect on cell viability as HBL-100 cells remained 89.1% viable even after a 24-hour exposure to a very high concentration of TaO2-CS-MoS2, thus demonstrating biocompatibility of these hybrid NSs. This may be due to the chitosan coating on these NSs. These hybrid NSs demonstrated a concentration dependant photothermal effect with a photothermal conversion efficiency of 47.6%. This photothermal efficacy may be due to the TaO2 attached on the NSs. Upon NIR irradiation, the viability of TaO2-CS-MoS2 treated MCF-7 cells drastically reduced, demonstrating the high PTT efficacy of these hybrid NSs. ROS generation was increased in the TaO2-CS-MoS2 hybrid NSs compared to free MoS2 as higher fluorescent intensity was displayed in MCF-7 breast cancer cells treated with the hybrid NSs than that in normal cells. This ROS generation increase is also due to the TaO2 decoration on the hybrid NSs. Thus, the chitosan coating and the TaO2 decoration significantly improved the properties of MoS2 NSs.

15.4.2.2 Tungsten disulfide (WS2) Tungsten disulfide (WS2) has become one of the most prevalent TMDCs, garnering a lot of attention in recent years because of its exceptional carrier mobility, chemical functionality, and optoelectronic characteristics [30]. According to geometric crystallization, the monolayer WS2 has a sandwich-like structure made up of three layers of atoms, with the sulfur atoms on the external layer and the tungsten atom on the internal layer [31]. WS2 nanosheets displaying substantial absorbance in the NIR range have been synthesized. The nanosheets were applied as bimodal contrast agent for photoacoustic (PA) imaging, and X-ray com­ puterized tomography (CT) imaging, and as a photosensitizer for PTT. X-ray CT imaging enables for whole-body imaging. Positron emission tomography (PET) imaging has significantly greater spatial resolution and sensitivity, gives useful information for understanding tumor microstructures and intratumoral distribution of respective theranostic nano-agents. The WS2 nanosheets surface was func­ tionalized with (PEG) using the thiol chemical approach, which considerably improved their physio­ logical stability and biocompatibility [32]. WS2 nanosheets have also been synthesized via a bottom-up approach assisted by polyvinyl pyrrolidone (PVP). The PVP functionalized WS2 nanosheets showed excellent colloidal stability and photothermal conversion. Furthermore, the great X-ray attenuation ability and NIR absorption enabled sensitive CT and PA imaging in vitro and in vivo. Further tests in cells and animals revealed that the WS2-PVP360kDa nanosheet had high biocompatibility and anti-cancer activity in vitro and in vivo [33]. Lipid-modified WS2 nanosheets were synthesized by electrostatic adsorption of phospholipids on WS2. The nanocomposite showed improved stability under physiological conditions and efficient drug delivery agent. The drug delivery rate was dependent on pH and NIR light irradiation. The nano­ composite showed excellent chemo-photothermal combination therapy, both, in-vitro against breast cancer cells MCF-7, and in-vivo using murine-derived breast cancer cells (4T1) tumor model [34]. WS2 NS and phosphocholine (DOPC) hybrid liposomes were synthesized and compared it with GO. DOX and

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calcein were loaded on the WS2 and liposomes respectively to achieve co-delivery. Thus, WS2 nanosheets loaded with DOX and calcein achieved successful tumor ablation in combinatorial PTTchemotherapy [35].

15.4.2.3 MXenes MXene, pronounced as “maxenes,” are carbides, nitrides, and carbonitrides of transition metals. The development and discovery of the MXene family, named for their structural similarities to graphene, has progressed rapidly. The parent material for MXene synthesis is represented as MAX, where M is an early transition metal, A represents group 13 and 14 elements, and X is carbon or nitrogen [36,37]. After etching of A from MAX phase using suitable method results into synthesis of MXene with formula Mn+1XnTx. The surface terminations, such as O, OH, F, and/or Cl, are represented by Tx in the formula [36]. MXenes occur in a variety of compositions and topologies. There are millions of potential combinations of transition metals (such as niobium, chromium molybdenum, or titanium), carbon, and nitrogen that might make up this material class, but all are not stable enough. Since the advent of titanium carbide (Ti3C2) etched from the MAX phase titanium–aluminum carbide in 2011, Ti3C2 has gained popularity and is the most explored among MXenes [37]. Ti3C2 MXene nanosheets modified with soybean phospholipid (Ti3C2-SP) were designed (Figure 15.3c). The enormous surface area of Ti3C2-SP helped in the efficient loading of DOX on the surface of the nanosheet. At a DOX/Ti3C2-SP nanosheets ratio of 4, the drug-loading amount reached 211.8%, which is significantly greater than most spherical drug-delivery nanosystems, which have drugloading capacity of 10% to 30%. The drug release was also pH dependent and showed an increase in drugs released from 33.9% to 58.0% and the pH was decreased from 6.0 to 4.5. With external NIR irradiation using 808 nm, drug-loaded Ti3C2-SP nanosheets accomplished synergistic chemotherapy and photothermal ablation of tumors for very effective cancer treatment. The nanosheets have also been shown to provide useful contrasts for PA imaging, demonstrating their theranostic potential. The great in vivo histocompatibility and ease of excretion have been also investigated and demonstrated by IV administration at 50 mg kg−1, indicating good biosafety for prospective clinical use [23]. Chemo-PTT involving iron chelation was also developed. They synthesized PVP functionalized Ti3C2 nanosheets. They loaded DOXjade on the sheets, which is a combination of deferasirox (ExJade®) and doxorubicin. The key highlight of the work was that the Ti3C2-PVP@DOXjade prevented the recurrence of tumor in-vivo when compared with Ti3C2-PVP [38]. This showed the potential of MXene as a better theranostic agent. A nanocomposite consisting of cobalt nanowires and Ti3C2 was synthesized (Figure 15.4a). The resultant composite showed magnetic properties, dual stimuli responsive drug delivery, and synergistic chemo-photothermal therapy in-vitro. DOX was loaded on Ti3C2-CoNWs with 225.05% loading effi­ ciency (DOX@Ti3C2-CoNWs). The results showed Ti3C2-CoNWs nanocarrier heterojunction might be a potential option for enhancing cancer therapy efficiency [39].

15.4.2.4 Xenes Unlike other dichalcogenides materials, Xenes are monoelemnetal in nature comprising group-IV, group-V, or group-VI elements. The examples of Xene include, silicene, stanene, arsenene, germanene antimonene, and bismuthine, borophene phosphorene. Their easy functionalization, degradability, and surface-to-volume ratio to load drug molecules are beneficial for drug-delivery application. Xenes are regarded as potential reagents for biosensors and biological applications due to their strong optical stability and photothermal conversion efficiency [40]. Germanene (Ge)-based Xene was designed for surgical adjuvant treatment, combining hyper­ thermia, and anti-bacterial properties (Figure 15.4b). Xene was combined with hydrogel consisting of agarose and chitosan (Ge@hydrogel) and loaded with DOX. It was used as a wound healing gel that was applied after tumor removal. When irradiated with NIR, the gel worked as anti-bacterial, wound healing

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FIGURE 15.4 (a) Illustration of the process by which Ti3C2 nanosheets are synthesized. The amalgamation of Ti3C2 nanosheets with CoNWs, and a display of their DOX releasing ability in response to a dual stimulus comprising of pH and 808nm laser. Reprint with permission [ 39]. (b) Flowchart representing the drug delivery utility of 2D Ge as well as its amalgamation with a hydrogel for topical tumour surgical treatment. Reprint with permission [ 41] (c) Diagrammatic representation of the preparation of ultrathin B.P. nanosheets as well as their morphology [ 42] (d) Depiction of the SEM image of BCNNSs in they show bending and curling morphology as well as vertical growth. TEM image of BCNNSs confirming the curling morphology. Images of EDX mapping demonstrate the uniform distribution of the B, C and N. HRTEM image revealing the good crystallinity of BCN NSs [ 43].

material and NIR light triggered drug delivery also helped in removing residual tumor cells. NIR irradiation aided the hydrogel in penetrating deeper into the tumor to release the drug, which resulted in better photothermal tumor ablation [41].

15.4.3 Black Phosphorus (BP) Black phosphorus (BP), an emergent member of the 2D nanomaterials family, has sparked interest in cancer PTT due to its unique structure and remarkable physicochemical features. BP nanoparticles possess high biocompatibility and degradability compared to other 2D materials. In a physiological environment, BP NSs degrade into a harmless products like phosphates and phosphites. BPs have excellent capability to form 1O2 under the full visible light spectrum, implying that they might be used in PDT. Ultrathin BP nanosheets were designed to be utilized as photosensitizer for PDT to treat cancer (Figure 15.4c). The nanosheets showed high quantum yield of 0.91, which is greater than the majority of

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PDT agents reported. The in-vivo study was done on Balb/c nude mice after inducing a tumor using MDA-MB-231 cells. After being injected with ultrathin BP nanosheets and exposed to a 660 nm laser, tumor development was considerably slowed [42]. The selective degradation of BP nanosheets in cancer cells when compared to normal tissues results into high concentration of phosphate in tumor cells. This leads to anti-proliferation of cancer cells while remaining highly biocompatible in normal cells, as well as selective delivery of DOX to tumor. Exposure of BP nanosheets to different cells revealed that the cell cycle arrest occurs at G2/M phase, which is generally associated with apoptosis. While the cell cycle arrest takes place within 24 hours, the decrease in healthy cells and cells undergoing apoptosis were observed in 48 hours. The anti-proliferation effect was observed at a minimum in normal A549 cells when compared to cancer cells HeLa and MCF-7. The tumor suppression effects were tested in vivo by utlilizing tumor-bearing nude mice xenografted with HeLa cells as models. The BP nanosheets treated mice showed a significant decrease in the tumor when compared to the DOX and PBS treated group. This showed that the BP nanosheets can be applied for reducing the tumor and as an agent for cancer treatment better than DOX [44]. In another study, a nanocomposite consisting of BP and Cu2+ was synthesized to combine PET and PTT for simultaneous imaging and therapy. Cu2+ ions not only help in photothermal stability of the BP nanosheets but also enhance the degradation process under NIR light irradiation. Along with this, the Cu2+ ions help in the generation of ROS like hydroxyl radical, which further leads to a combined effect of chemo dynamic therapy (CDT) and PTT. BP@Cu nanostructures was further functionalized with RGD-conjugated PEG to enhance tumor targeting. The acute toxicity of the material via I.V. administration was evaluated on healthy C57/BL6J mice using hematology, blood biochemical, and histological analysis and showed no adverse effect. Furthermore, this work lays the foundation for the possible application of BP in PET-imaging-guided combination cancer therapy, given the remarkable potential of PTT in the clinic [45]. NIR-II responsive carbon dot and BP (NIR-IICD/BP) NS hybrids were designed for synergistic PTT and chemotherapy for cancer. Upon 1,064 nm laser irradiation, the temperature of these hybrid NSs increased by 25.7°C, while that of NIR-II-CDs and BP only showed an increment of 20.2°C and 16.3°C, respectively, confirming that these hybrid NSs are photothermally very efficient. The photothermal conversion efficiency of NIR-II-CD/BP at 1,064 and 808 nm was 61.4 and 77.3%, respectively. These hybrid NSs are biocompatible as no obvious cytotoxicity was observed in NIR-II-CD/BP-treated HeLa cells and normal cells (LO2 and NIH-3T3). MTT assay demonstrated that cells treated with NIR-II-CD/BP and upon 1,064 nm laser irradiation were wiped out, showing that NIR-II window is more efficient in PTT. In vivo PTT efficacy evaluations showed that mice treated with NIR-II-CD/BP and 1,064 nm laser irradiation showed complete tumor ablation while also eliminating lung metastasis formation. DOX was loaded onto these hybrid NSs photothermal chemo combination therapy (PCT). Upon 1,064 nm laser irradiation, there was a 75% release of DOX from the hybrid in acidic pH conditions. Similar to the in vitro studies done on NIR-II-CD/BP, NIR-II-CD/DOX/BP showed high biocompatibility and complete tumor irradiation. In vivo studies showed that NIR-II-CD/DOX/BP exhibited complete tumor ablation even when the tumor was wrapped with an additional tissue, demonstrating the deep tumor targeting efficiency of NIR-II-CD/DOX/BP [46].

15.4.4 Boron Nitride (BN) Boron nitride (BN) is a covalent solid having the same number of boron and nitrogen atoms. It exists in diverse crystalline forms like hexagonal boron nitride (h-BN), diamond-like cubic BN (c-BN), and wurtzite BN (w-BN). h-BN has a chemically and biologically stable 2D structure; therefore, it is the most widely researched of the BN nanomaterials [47]. BN nanomaterials are structural analogs of graphene, differing only in the nature of the bonds in between the atoms as the C-C bond in graphene is of purely covalent nature while the B-N bond in BN has a partial ionic character. Owing to this slight ionic

270 Nanomaterials in Healthcare character, BN nanomaterials have improved mechanical, thermal, and antioxidant properties that make them suitable for use in biomedicine. The use of h-BN NSs in the treatment of prostate cancer was investigated by comparing the anti­ proliferative and metastatic effects of h-BN on androgen independent prostate cancer cells and normal prostate cells. The size of the NSs was 50 nm, which significantly increased upon sonication in distilled water and Dulbecco’s Modified Eagle Medium (DMEM). This showed that h-BN NSs were highly stable due to their high tendency of aggregation. Cellular uptake studies were first performed on normal prostate cell lines (PNT1A) and then compared to prostate cancer cell lines (DU145 and PC3). Flow cytometry analysis showed that h-BN is 10% more internalized by DU145 cells than PC3 and PNT1A cells. h-BN treated DU145 cells showed a change in morphology on increasing the concentration of boron containing h-BN. Cellular viability studies showed that the DU145 prostate cancer cells were most sensitive to h-BN and BA treatments. Since the production of ROS is critical for apoptosis of cancer cells, the h-BN treated cells were investigated for their level of ROS production. The study demonstrated there is significantly more ROS production from DU145 prostate cancer cells than from the normal PNT1A cells. Therefore, this study determined that h-BN NSs were an effective therapeutic tool for prostate cancer as they are degraded slowly, that ensured that they are retained for a longer lasting therapeutic effect and they also significantly enhanced intracellular ROS production and mitochondrial dysfunction in prostate cancer cells [48]. Palladium (Pd)-decorated hydroxy BN NSs loaded with DOX (Pd@OH-BNNS/DOX) was devel­ oped as a drug delivery system for PTT/chemotherapy. The nanocomposite was extremely stable under 808 nm laser irradiation and showed a drug loading capacity of 32%. Maximum DOX release was observed at pH 4.5 that is similar to the intracellular pH. This release dramatically increased upon laser irradiation at pH 7.4. In vitro cytotoxicity studies using the MTT assay revealed that Pd@OH-BNNS/ DOX inhibited MCF-7 cell proliferation in a dose-dependent manner when irradiated with an 808 nm laser. In vivo studies in mice demonstrated that tumor size was drastically up to 87.2% when laser irradiated. Thus, Pd@OH-BNNS/DOX was an effective PTT-chemotherapeutic weapon against can­ cerous tumors, while also being effective at drug concentrations as low as 250 µg/mL and exhibiting no biosafety problems [49]. Boron carbonitride NSs (BCNNSs) modified by PEG were synthesized to be used as a chemo­ therapeutic carrier for the breast cancer drug paclitaxel (PTX) (Figure 15.4d). This nanocomposite had an excellent photoluminescence ability at 380 nm. BCNNSs-PEG-PTX had a pH-dependant release profile exhibiting increased release when there was a pH change from 7.4 to 5.5. In vitro CCK-8 assay shows that the viability of MDA-MB-231 cells, L02 cells and Hep G2 cells exhibited no obvious variation even after a 72-hour incubation in BCNNSs-PEG, which spoke volumes for the bio­ compatibility of this nanocomposite. BCNNSs-PEG-PTX shows a time dependant anti-tumor effect than free PTX, even at a low dose. The evidence of the efficacy of this nanocomposite was further strengthened by staining of live and dead cells using the ReadyProbes™ Cell Viability Imaging Kit, which demonstrated that BCNNSs-PEG-PTX induced more cell death than free PTX for the same incubation time. Thus, BCNNSs-PEG-PTX can not only be used for cancer chemotherapy, but can also be utilized for cellular imaging [43]. Tannic acid functionalized and Fe-coordinated BN NSs (TA-Fe/BNNS) were synthesized for combined PTT/imaging of cancer cells. TA-Fe/BNNS had a photothermal conversion efficiency of 44.6%, that showed an increase in temperature to 54.6°C after 180 seconds of 808 nm NIR laser irradiation. In vitro photothermal studies proved that cells treated with TA-Fe/BNNS demonstrated a temperature increase of 46.9 ± 1.0°C after 7 minutes of NIR irradiation in comparison to plain BNS, TA/ BNS, or Fe/BNS, that showed negligible temperature changes. The viability of the tumor cells was dramatically decreased in TA-Fe/BNNS, which demonstrates the material’s anticancer effect. TA-Fe/ BNNS also demonstrated notable MRI properties in vitro and in vivo environments. In vitro studies on KB cancer cells demonstrated that tumor cells absorbed these NSs and exhibited higher MRI contrast than BNNS alone. MR images of mice xenografted with KB tumous revealed the gradual accumulation of TA-Fe/BNNS at the tumor’s site within 24 hours of administration and also showed no significant

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accumulation in normal tumor-free organs. In vivo studies proved that TA-Fe/BNNS demonstrated an efficient photothermal heating effect and successful tumour ablation. Thus, this biocompatible nanocomposite demonstrated excellent photothermal therapeutic efficiency when used in combination with MRI [50].

15.4.5 Metal Oxide Nanosheets Metal oxide nanosheets (MONS) have a 2D structure; therefore, they have been widely investigated in their potential biomedical applications. Nanosheets are ultrathin and have strong in-plane bonds, a consequence of which they show high mechanical strength, flexibility and optical transparency [51]. MONS possess a high specific surface area which gives them the ability of high chemical activity, and widened bandgaps, which allows for high responses to ultraviolet light which leads to an enhanced heating effect in the tumor cells, leading to tumor regression [52]. Also, MONS have high stability, can be prepared by simple processes and can be easily modified and functionalized, which further cements their status as a promising candidate for biomedical applications [53].

15.4.5.1 Manganese dioxide (MnO2) MnO2 NSs are composed of MnO6 octahedra wherein Mn2+ ions occupy the centers and are coordinated to six oxygen atoms. These NSs possess a large surface area, favorable redox behavior in acidic con­ ditions, fluorescence, and a good biocompatibility that make it a suitable material for use in cancer nanomedicine [54]. Ultrathin MnO2 NSs were synthesized for cancer PTT (Figure 15.5a). The photothermal conversion efficiency of MnO2 NSs was evaluated to be 21.4%, with the NSs showing the maximum temperature of 52.7°C upon NIR irradiation for 15 minutes. MnO2 NSs on being modified with surface SP demonstrated high sensitivity to acidic environments as these NSs collapsed and disintegrated, releasing Mn2+ ions on being kept in acidic solution for a mere 3-minute duration. This result demonstrated that MnO2-SP NSs was highly responsive to a pH similar to that in the tumor microenvironment (TME). This fact was further cemented by in vitro and in vivo experiments that showed that MnO2-SP NSs exhibited sig­ nificantly (significant difference at P ˂ 0.01) higher MRI contrast and brightening effect in acidic and reducing conditions. MnO2-SP NSs upon NIR irradiation, demonstrated high cytotoxicity toward 4T1 breast cancer cells and tumor ablation in 4T1 xenografted mice in in vitro and in vivo studies, respectively [55]. MnO2 NSs anchored to up-conversion nanoprobes (UCSMs) were designed for high-resolution up-conversion luminescent (UCL) imaging and synergistic PDT/Radiotherapy(RT). These UCSMs caused an increased production of 1O2 and H2O2 that increased the efficiency of PDT. In vitro studies on hypoxic murine breast cancer cells (hc-4T1) showed an efficient uptake of the NSs with relatively low cytotoxic effects of UCSMs. In vivo studies demonstrated a considerable tumor regression due to a high level of ROS production [56].

15.4.5.2 Molybdenum oxide (MoOx) MoOx NSs show a great promise in PTT as they have high NIR absorption and pH-dependant oxidative properties. A combined chemotherapy/PTT platform was designed which included MoOx NS conjugated with FA modified BSA and a lipoic acid-PEG conjugate (FA-BSA-PEG/MoOx). PEG and BSA improved stability of the NS while FA acted as a targeting ligand to target the breast cancer cells. FA-BSA-PEG/ MoOx showed a photothermal conversion efficiency of 43.41% as well as an alkaline pH-dependant degradability. Docetaxel (DTX) was loaded onto FA-BSA-PEG/MoOx NSs with a drug loading effi­ ciency of 76.49%, and upon NIR irradiation, FA-BSA-PEG/MoOx@DTX NSs showed an efficient in

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FIGURE 15.5 (a) Flow chart depicting the process of synthesis of MnO2-SPs nanosheets and their utility in tumor theranostics with sensitivity to TME, encompassing T1-weighted MRI and efficient anti-tumor PTT, both being triggered by acidic/reducing conditions. Reprint with permission [ 55]. (b) The diagrammatic represen­ tation of the design and use of DOX-FA-ZnO NS as a functional drug delivery nanoplatform for targeted and Combined Chemo-PTT for breast cancer. Reprint with permission [ 60] (c) Six in vivo thermal images of mice that have been injected with saline (top) and FeMnLDH/MB (bottom) taken at 0 min, 5 min, and 10 min intervals under 808nm laser irradiation at 1.0 W cm-2 density. Photographic representations of different sets of tumor containing mice. Graphical representation of relative tumor volume and mean tumor weights of dif­ ferent sets of tumors bearing mice after their respective saline or FeMnLDH/MB with PTT/PDT treatment. Reprint with permission [ 61].

vitro photothermal effect and increase in cell apoptosis of MCF-7 cells. In vivo studies proved that direct tumor injection had a markedly higher photothermal effect than tail vein injection method, and these NSs not only exhibited breast cancer tumor ablation but also inhibited lung metastasis [57]. MoOx-PEG NSs loaded with drugs like 7-Ethyl-10-hydroxycamptothecin (SN38) and chlorine e6 (Ce6) were also developed. These NSs were efficient in vitro photothermal agents in acidic TME due to their ability to absorb NIR. They also showed an in vivo photothermal tumor ablation effect. These NSs are rapidly cleared from the body which signified their low toxicity. Even though these NSs are rapidly cleared from the body, they exhibit a higher tumor retention time, which can be effective for its use as a PA imaging contrast agent. Thus, MoOx-PEG NSs are effective NIR absorbers for tumor irradiation and also demonstrated biosafety, which shows their effectiveness as a combined chemo/PTT agent [58]. A single-layer indocyanine green (ICG) conjugated Mo-Se hybrid NSs (sMoSe2-ICG) was developed for PA image guided PTT for cancer. This hybrid NS had a broad NIR

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absorption spectrum. ICG conjugation significantly increased the PA performance of sMoSe2 NSs. The sMoSe2-ICG NSs show a significant temperature rise after NIR irradiation, demonstrating its high photothermal efficiency. No cytotoxicity was observed in cultured 4T1 cells, indicating that this hybrid is biocompatible. Upon laser irradiation, CCK-8 assay showed a decrease in cell viability in cells incubated with sMoSe2-ICG NSs, showing that its efficient PTT efficacy. In vivo PA imaging in mice showed that the PA signal increased almost sevenfold upon sMoSe2-ICG NSs treatment as compared to the pre-treatment state. In vivo PTT studies in 4T1 tumor bearing mice demonstrated complete tumor regression in mice treated with a laser and sMoSe2-ICG NSs. These NSs were biocompatible due to no changes in the weight of the mice and histological sections showing no damages to normal organs [59].

15.4.5.3 Zinc oxide (ZnO) ZnO NSs have an abundance of valence band spaces and conduction band electrons on their surface due to crystal defects; therefore, electron-hole pairs get trapped by dissolved oxygen in the cell, resulting in ROS generation that leads to cell death. Upon light irradiation, ZnO NSs produce more electrons, leading to more ROS generation, which makes it useful in targeted cancer theranostics. FA-PEG-coated ZnO NSs (FA-PEG-ZnO NS) were designed for combined chemo-PTT against breast cancer (Figure 15.5b). FA-PEG-ZnO NSs showed a much higher and dose-dependant photo­ thermal heating effect than plain ZnO NSs. These NSs, on being loaded with drug DOX, showed enhanced but slow drug release upon NIR irradiation at an acidic pH, which means that laser irradiation significantly increased DOX release from the NSs. Studies done in-vitro on MDA-MB-231 breast cancer cells showed the cytotoxic effect of these NSs. Upon NIR irradiation and in vivo injection in mice, this system was found to be highly cytotoxic against breast cancer cells and also had a high rate of tumor internalization with minimum toxicity [60].

15.4.5.4 Iron oxide (IO) IO can exist in forms such as magnetite (Fe3O4) or maghemite (Fe2O3), or a combination of these two. IO have the properties of biocompatibility, biodegradability, facile synthesis, and ease of func­ tionalization, which makes IO a desirable candidate for biomedical applications. The intrinsic ability of IO NSs to generate heat upon a short irradiation by NIR light has led to their use as a PTT agent. PEG functionalized-reduced GO (RGO)-IO NSs (RGO–IONS–PEG) were developed that could be used as a probe in PAT and MRI imaging. When compared to as-made GO NSs, RGO–IONS–PEG demonstrated a remarkedly higher photothermal effect. In vitro MTT assay showed no cytotoxicity of these NSs towards 4T1 cells. These NSs had a high tumor retention and longer circulation times. To prove the ability of these NSs as an effective in vivo tumor imaging agent, mice were injected with RGO–IONS–PEG labeled with Cy5. It showed a strong signal upon MR imaging, proving its high tumor uptake. Under this imaging guidance and upon low power NIR irradiation, RGO–IONS–PEG successfully ablated tumors, demonstrating effective PTT in vivo. This evidence throws light on the fact that RGO–IONS–PEG can be used for multimodal imaging guided PTT for cancer [62]. Chitosan-dextran stabilized GO-IO NSs were synthesized and loaded them with DOX. Chitosan and dextran significantly improved the stability of these NSs. DOX release from these NSs was enhanced in acidic environments. These NSs demonstrated excellent in dose dependant vitro cyto­ toxicity to A549 cells. Under NIR irradiation, these NSs demonstrated decrease in cell survival rate indicating an efficient photothermal effect. The ability of enhanced drug release in acidic environment and upon irradiation indicated that these NSs can be used in targeted therapy of solid tumor that have an acidic environment. The DOX-loaded magnetic nanocomposite material had an efficient internalization and a strong in vitro cytotoxicity, which proved that it can be used in combined chemo-PTT [63].

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15.4.6 Layered Hydroxides (LDH) Layered hydroxides or layered double hydroxides (LDH) nanomaterials consist of positively charged brucite-like layers containing M(OH)6 octahedral interlayers, the electrostatic interaction and hydrogen bonds between which form a layered structure [64]. LDH NPs have good biocompatibility, high stability, pH-dependant solubility, and high surface area that make them good candidates for biomedical appli­ cations, specifically cancer therapy [65]. Cancer cell membrane (CCM) camouflaged ICG-incorporated and Abraxane (PTX-BSA)-loaded LDH nanosheets were developed for combined PTT-chemotherapy of colorectal cancer. These NSs showed an efficient photothermal effect upon 808 nm laser irradiation that increased in an acidic pH of 6.6, which is similar to the TME. MTT assay revealed cytotoxicity of the NSs was in a dose-dependant manner and the ICG coating led to enhanced ROS production. In vivo studies showed that upon NIR irradiation, tumour progression was severely hampered, even at low drug dose. Thus, the synergistic effect of PTT-chemotherapy via targeted accumulation in the tumor tissues minimized clearance by macrophages, indicating that these NSs are biocompatible. This NS was especially suitable for combined PTT/PDT-chemotherapy as the CCM cloaking provided LDH nanosheets with increased tumor targeting efficiency that led to increase in their tumor accumulation and also shielded them from the immune system. This ensures that the NSs are kept in the blood circulation and are not seen as foreign entities by the memory B-cells, providing the NSs a higher possibility of acting on the target tumor cells. [66]. FA-MgAl-LDH nanosheets were synthesized as a drug carrier in cancer therapy. These NSs were loaded with drug DOX, which was released in when the system pH was 5.5 i.e., acidic. The in vitro fluorescence exhibited by FA-DOX-MgAl-LDH NSs when incubated with KB cells was significantly higher than DOX/GO and FA-DOX/GO NSs. The in vitro cytotoxicity investigated on KB cells and normal liver L02 cells shows up to 99.2% and 93.2% cell viability, respectively, indicating that these NSs are bio­ compatible. MTT assay showed that FA-DOX-MgAl-LDH NSs were more anticancerous towards KB cells than DOX-MgAl-LDH NSs indicating that the tumor targeting ability is enhanced by FA, while L02 cells showed remarkable tolerance to these NSs. PI staining of dead KB cells proved that FA-DOXMgAl-LDH NSs had a strong anticancer effect due to the observation of an intense PI signal, which is similar to that observed in cells treated with free DOX. Therefore, even though there was a stark sim­ ilarity between the anticancer behaviors of FA-DOX-MgAl-LDH NSs and free DOX, the LDH NSs showed an enhanced biocompatibility and lower cytotoxicity indicating that it was a better agent for chemotherapy [67]. Cerium (Ce) doped Cu-Al LDH ultrathin NSs were synthesized and loaded them with ICG (ICG/ CAC-LDH) for combined CDT and PDT. ICG/CAC-LDH NSs behaved like a catalyst wherein it reduced GSH in the cancer cells leading to production of Cu+ and Ce4+ ions. These ions further reduced H2O2, leading to ROS generation that led to cancer cell apoptosis. These NSs had a photothermal conversions efficiency of 57.2%. In vitro studies on HepG2 cells demonstrated significant increase in ROS generation upon laser irradiation and subsequent cell death. Due to the intrinsic magnetic properties of Cu (II), the in vitro PA intensity increased in a concentration-dependant manner. Based on these findings, ICG/CAC-LDH NSs were an efficient cancer therapy agent as they can be used not only for PTT but also for CDT as well as MR/PA imaging of tumors [68]. Mn-Fe LDH NS (MnFe-LDH) was developed for combined PTT-PDT of cancer. MnFe-LDH NSs demonstrated a photothermal conversion efficiency of 47.6% and could also produce a large amount of oxygen (Figure 15.5c). When the MnFe-LDH NSs were loaded with methylene blue (MB), the oxygen production was dramatically increased. This suggested that MnFe-LDH NSs exhibited a catalase-like activity wherein it catalyzed the intracellular H2O2 to produce oxygen, thus enhancing the effect of oxygen dependant PDT. MB acted as a photosensitizer to increase PDT. MnFe-LDH NSs showed a high bio­ compatibility in HeLa cells. In vitro photothermal effect was synergistic with oxygen production wherein cells treated with MnFe-LDH/MB NSs exhibit almost complete cell death. In vivo studies in mice showed that MnFe-LDH/MB NSs treated tumours showed complete necrosis after being subjected to PTT-PDT. The NSs did not show any adverse reactions in any of the normal tissues indicating its biosafety [61].

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15.4.7 Metal Organic Framework (MOF) Metal organic framework NPs (MOFN) are basically nanoscale coordination networks composed of crystalline porous scaffolds strongly bound by coordinate bonds linking the positively charged metal ions with the negatively charged organic linkers. MOFs have a remarkably large surface area and easily tuneable crystalline structures and chemical properties [69]. They also have high porosity, selective absorption due to high surface area, presence of strong metal–ligand interactions, and structural diversity, making them good candidates for research in cancer therapy [70]. Zr-Fc MOF NSs were synthesized for synergistic PTT-CDT for cancer. These Zr-Fc MOF NSs had a photothermal conversion efficiency of 53% upon NIR irradiation. Temperature increments of 20°C and 30°C was observed for Zr-Fc MOF NS concentrations of 100 μg/mL and 200 μg/mL, respectively, which was in stark contrast to the mere 3°C increment observed in the control sample. These NSs were also stable in acidic conditions that are uncannily similar to the microenvironment in the tumours. Zr-Fc MOF NSs also showed ROS production upon laser irradiation as it reduces GSH in the tumour. MB addition enhanced this ROS generation effect. The cell viability was almost 100% in the normal 7702 cells, even at NSs concentrations of 160 μg/mL. This indicates that Zr-Fc MOF NSs are not toxic to non-tumorous cells. The enhanced photothermal and catalytic behaviour of Zr-Fc MOF NS was attributed to the presence of Fc (COOH)2 ligand in its framework. In vivo studies showed that upon NSs injection and NIR irradiation, there was a significant uptick in tumor temperature and decrease in tumor size in contrast to the PBS injected control group of mice that showed negligible temperature rise. Thus, Zr-Fc MOF NS demonstrated effective PTT-CDT due to its ability to act as a catalyst and also absorb laser irradiation without the need to use any extra drugs or photo agents [71]. Ultrathin copper-tetrakis (4-carboxyphenyl) porphyrin (Cu-TCPP) MOF NSs were developed for MR image guided PTT and PDT for cancer. Cu-TCPP MOF NSs showed photothermal conversion efficiency of 36.8%, as it demonstrated a 9–34°C increase in temperature upon 808 nm laser irradiation. These NSs acted as good MRI contrast agents as the tumor site demonstrated a sharp color contrast upon NSs injection. In vivo studies showed that Cu-TCPP MOF NSs could absorb NIR irradiation efficiently as demonstrated by the high contrast in the thermal images. When the mice bearing Saos-2 tumors were evaluated for their tumor size, the group of mice treated with Cu-TCPP MOF NSs plus PDT and PTT showed complete regression in tumor size, which indicated that synergistic treatment is the best option [72]. Additionally, ultrathin Samarium coordinated tetrakis-4-carboxyphenyl porphyrin (Sm-H2TCPP) MOF NSs were synthesized for PDT for breast cancer. Sm-H2TCPP MOF NSs demonstrated remarkable photostability in UV visible absorption spectra, which indicated its ability for PDT. When compared to H2TCPP NSs, the Sm-H2TCPP NSs demonstrated a threefold increase in ROS generation which is critical for efficient PDT. This was mainly attributes to the ultrathin framework of these NSs that facilitated diffusion of ROS radicals. In vitro MTT assay showed that Sm-H2TCPP NSs negligible cytotoxicity, indicating its biocompatible nature. Flow cytometry studies showed that the Sm-H2TCPP NSs had an enhanced ROS generation ability than H2TCPP NSs. In vivo studies on mice with breast cancer showed that Sm-H2TCPP NS treatment in cognizance with laser illumination displayed 78.5% inhibition in tumor growth. Histological staining of the tumors revealed signs of obvious apoptosis or necrosis owing to cell shrinkage and nuclear condensation, while the histological analysis of liver and kidneys indicated no toxicity [73]. Gd-based porphyrin paddlewheel framework (PPF-Gd) MOF NSs were developed for bimodal cancer theranostics and imaging. PPF-Gd NSs had an efficient DOX loading capacity due its large surface area. DOX was released from the NSs in an acidic environment, with 49% release at pH 5.5 as against only a 15% release at pH 7.4. This pH-dependent behavior makes these NSs a good candidate for intratumoural drug delivery as tumors generally have an acidic environment. PPF-Gd/DOX-treated mice showed maximum tumor inhibition efficiency and cell apoptosis, with no acute toxicity in the normal tissue and organs [74].

276 Nanomaterials in Healthcare

15.5 CONCLUSION 2D nanosheets are novel materials that have emerged as the dark horse in cancer nanomedicine as they have some exceptional properties. These include a large surface area, ease of surface modification, high drug loading capacity, high PTE, and biocompatibility. They have been investigated to date in the PTT, PDT, CDT, and chemotherapy for a number of cancers. These materials have also been used for tumor imaging purposes in combination with its therapeutic usage and have demonstrated marvellous effects. They have demonstrated an effective photothermal tumor ablation and regression in such murine experiments. But these materials suffer from a number of disadvantages like low yield, untargeted toxicity, low clinical translation, and so on. Therefore, the need of the hour is to devise synthesis pro­ tocols that give a high yield of these nanosheets. Controlling the biological fate of these particles is a very tedious process. Many of these nanomaterials have shown an inflammatory response in lungs of patients. Many times, the kidney’s filtration mechanism acts as an obstacle in the way of the nanodrug carrier, hampering drug efficacy as well as indirectly increasing toxicity to normal cells. Clinical translation of these materials is still a distant dream, as most of the research in this area has been conducted on cells and animal models. The effects on these cellular or animal systems are bound to be differing from the therapeutic effects actual human organs. The aim should be to achieve a clinical translation of these nanomaterials to be effective in treating humans without any side effects such that PTT becomes a mainstream therapeutic strategy to treat any type of cancer.

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Solid Lipid Nanoparticles Towards Emerging Cancer Nanomedicine

16

Amreen Khan1,2, Vaishali Pawar1, Rupali Bagale1, Shruti Pendse1, and Akshara Adapa1 1

NanoBios Lab, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India 2 Centre for Research in Nanotechnology and Science, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 16.1 16.2 16.3

16.4

16.5 16.6

Introduction Characteristics Methods of Preparation of Solid Lipid Nanoparticles for Cancer Nanomedicine 16.3.1 High Shear Homogenization 16.3.1.1 Hot homogenization 16.3.1.2 Cold homogenization 16.3.2 Solvent Emulsification Technique 16.3.3 Ultrasonication or High-Speed Homogenization 16.3.4 Double Emulsion Method 16.3.5 Spray Drying Method 16.3.6 Supercritical Fluid 16.3.7 Microemulsion-Based SLNs’ Preparation Routes of SLNs’ Delivery 16.4.1 Transdermal/Topical 16.4.2 Oral 16.4.3 Parenteral 16.4.4 Pulmonary 16.4.5 Brain Toxicology and Clearance Applications

DOI: 10.1201/9781003322368-16

282 283 283 285 285 285 286 286 286 286 286 287 287 287 288 288 288 289 289 290 281

282 Nanomaterials in Healthcare 16.6.1 Breast Cancer 16.6.2 Lung Cancer 16.2.3 Colon Cancer 16.7 Conclusion References

290 291 292 292 292

16.1 INTRODUCTION Lipid materials for a long time have been the choice of materials for pharmaceutical industries, serving as important components of multiple formulations [1,2]. In different dosage forms, whether capsules, ointments, lotions, or emulsions, lipids enhance the safety and stability when incorporated in nanoparticle preparations [3]. The solid matrix supports the controlled release of the drug and its non-toxic and biodegradable nature provides added advantage over other biomedical applicable materials in terms of reduced toxicity [1,3,4]. The nanovesicle dissolution in biological fluid and rapid uptake are also important. This classical concept has been prioritizing beneficial components that can cross the cell membrane and facilitate cellular uptake [5]. Here is when the concept of lipid nanoparticles as delivery vehicles was developed, engaging both their advantages [6]. Additionally, among various delivery systems present, the versatile feature of lipid bilayer has gained interest recently in the formation of lipid nanoparticles. Solid lipid nanoparticles (SLNs) as lipid carriers have been widely used for multiple applications [7]. SLNs range from submicron to a few diameters in the nano range. Consisting of the lipid matrix with melting point above body temperature, SLN also contains surfactants and occasionally cosurfactants [8]. For various nanoparticles, with high productivity chances of parameter optimization also increases, unlike for SLNs which don’t require toxic chemicals or high-energy equipment, scale-up can be planned and implemented even at a laboratory scale [9]. As a successful drug delivery system (DDS), the advancement of SLNs for nanomedicine is emerging continuously. The applicability has increased mostly due to an enhanced SLN delivery system for both hydrophilic and lipophilic molecules [10]. With different types of lipids and surfactants that can be included as a component in the preparation of SLNs, even the possibility to carry biomolecules like genes and proteins are forthcoming [11]. Various types of lipids, like cationic lipids, have been used for different purposes depending on the charge of the drug. Like nucleic acids, because of their high negative charge, are more compatible with cationic lipids; hence, providing stability to the complex reaches the site of action. The lipid-nucleic acid interaction with other negatively charged components present on the cell membranes helps the cargo to cross the membranes more effectively. The composition of lipid amphiphiles consists of hydrophilic, hydrophobic, and linker domain that forms the bridge between the hydrophobic head and hydrophilic tail of lipid amphiphiles that are prone to carry diverse molecules with themselves [10]. For the drug to solubilize in the SLNs lipid matrix, the lipid core can be designed to have mono-, di-, and triglycerides; fatty acids; and their derivatives for an efficient entrapment [12]. Sometimes supercooled structures of solid lipid nanoparticles are also formed, which tend to coalesce and form large droplets, causing particles to aggregate and so the necessity of adding liquid lipid increases, subjecting them to appear flowy. The crystal structure can also reduce drug entrapment, leading to drug expulsion. Thus, polymorphic transformations can hamper the stability, size, and shape of lipid nanoparticles [8]. And so, the major disadvantage of SLNs is related to recrystallization and polydispersity, which might limit their applications in industries [8]. Cancer, along with the uncontrolled division of cells and cell death resistance, also retains the ability to invade other organs while entering the circulation and is the cause of death worldwide [13]. As mostfollowed treatment options currently available are chemotherapy, the issues faced include drug solu­ bility, high toxicity, low specificity, therapeutic index, and drug resistance. Overcoming these challenges and along with the benefits of nanosize particles, lipid nanocarriers penetrate through several biological

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283

barriers with minimal toxicity [14]. While dealing with nanomedicines’ pharmacokinetics, absorption of the drug is very important. Drug absorption happens through the small intestine, where they have to enter the blood capillaries and surpass vigorous metabolic cycles of the liver that also include metabolic enzymes [15]. Later in the systemic circulation, the drug has to reach the minimal plasma concentration required while getting distributed in tissues where they have to maintain themselves within a therapeutic window. Since absorption takes place mostly in the gastrointestinal medium, SLNs provide significant improvement in the permeability and absorption of the drug due to its lipophilic nature, while protecting the compound against metabolism before reaching the systemic circulation [15]. Of the two types of solid tumor targeting by nanoparticles, namely active and passive, passive mostly occurs by enhanced penetration and retention effect (EPR) [16]. The EPR effect involves the nanoparticle extravasation into the tumor region from leaky vasculature. The concept and mechanism of EPR follow a universal phe­ nomenon where even the macromolecules can be accumulated inside the solid tumor and serve for the delivery of anticancer agents. Many factors affect the accumulation of drugs inside the tumor; a few include the location of the tumor and the physical properties of macromolecules. Similarly, for SLNs, the EPR effect also plays an important role in drug accumulation as it takes advantage of the tumor micro­ environment. The retention has also been linked to oxygen and nutrient supply for growing tumors [16]. The active targeting is accomplished by ligand targeted interaction, involving receptors. Anticancer nanomedicines of lipid-based DDS offer improved treatment for chemotherapeutics [17]. In this chapter, we will come across the characteristics of SLNs, their preparation methods, and the routes of the delivery system. Description of different cancers in which SLN delivery has been effective is also stated. Further, toxicity and clearance of SLNs will be elaborated with regards to the concise overview.

16.2 CHARACTERISTICS Apart from the ideal characteristics of nanocarriers that include biodegradability, non-toxicity, stability, versatility, and negligible immune response, a good delivery system should also provide high en­ capsulation efficiency. Easy modification to attain target specificity and controlled release has also been explored in SLNs systems [14]. The benefit of carrying both hydrophilic and lipophilic compounds is another feasibility with SLNs. Small size and high surface area add to another unique property helpful in carrying higher amounts of the drug [10]. SLNs have the beneficial properties of liposomes, polymeric nanoparticles, and microemulsions. The characteristics are further enriched by surface modifications to improve stability, pharmacokinetic acceptability and internalization, and encapsulation and release of the therapeutic agent [14]. There are several physicochemical characteristics of the SLNs that are essential in their definition. Table 16.1 contains various characterization techniques and alternatives typically performed for a SLN to deduce crucial characteristics.

16.3 METHODS OF PREPARATION OF SOLID LIPID NANOPARTICLES FOR CANCER NANOMEDICINE The exponential increase in various nanoparticle designs and synthesis has allowed the exposure to different ways of preparation methods. For SLNs preparation, obtaining homogenous nanosize particles requires consistent parameter optimization [24]. The following describes the different methods of SLNs’ synthesis and has also been represented in Figure 16.1.

Particle size and zeta potential

Degree of crystallinity and lipid modifications

Dynamic phenomena and coexistence of other structures

Drug-loading efficiency and entrapment efficiency (%EE)

Drug release

Stability test

Biocompatibility assay

2.

3.

4.

5.

6.

7.

PARAMETER/ INFORMATION DEDUCED

3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay

Centrifugation/ microcentrifugation techniques followed by UV spectrometry or high-performance liquid chromatography Side-by-side diffusion cells with membrane Dialysis bag diffusion Reverse dialysis bag diffusion Size, zeta potential, and polydispersity index

Nuclear magnetic resonance Electron spin resonance

Dynamic Light Scattering Laser diffractometry Static Light Scattering Acoustic methods Nuclear Magnetic Resonance Electron microscopy including scanning electron and transmission electron microscopy Atomic Force Microscopy Powder X-ray diffraction Differential Scanning calorimetry

CHARACTERIZATION TECHNIQUES AND ASSAYS

Evaluation of particle size, drug content, and other factors with time are observed. Nontoxicity on normal cells is determined.

1. Interactions between surfactant and lipid matrix 2. Drug loading and release kinetics depend on crystallinity, gelation, and melting properties of lipid. 1. Significant for drug release and stability 2. Confirms the presence of additional supercooled melts, micelles, drug, and liposomes 1. Measures the amount of drug-loaded, as it affects the release characteristics. 2. Maximize the drug-carrying capacity. For monitoring the efficient release of the drug in the circulatory system before the carrier’s clearance.

1. Clearance and toxicity. 2. Storage stability of the colloidal system. 3. Crucial for SLNs with charged surface modifications

IMPORTANCE

80–100% cell viability is desirable

Negligible change is preferred.

1. Ideal value of drug release is 100% 2. 60–80% in an optimum time interval, is considered good.

1. Ideal value of drug release is 100% 2. 60–80% in an optimum time interval is considered good.

Appropriate values for optimum SLNs stability and drug release from the carrier.

Polymorphism should be appropriate to prevent drug spillage before reaching the target site for efficient release

1. Mean size is typically between 50–1,000 nm. 2. Zeta potential should be high to avoid aggregation of the colloidal particles. 3. Smooth morphology expected.

IDEAL VALUES

[ 20, 21, 23]

[ 18– 20]

[ 1, 22]

[ 1, 22]

[ 21]

[ 18, 20, 21]

[ 18– 21]

REFERENCES

Characterization techniques and assays to determine several characteristics of the solid lipid nanoparticles with their importance and ideal values

1.

S.NO.

TABLE 16.1

284 Nanomaterials in Healthcare

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FIGURE 16.1 Various methods of solid lipid nanoparticle preparation.

16.3.1 High Shear Homogenization This technique is widely employed for the synthesis of SLNs, nanoemulsions, and nanostructured lipid carriers (NLCs), providing effective means of synthesizing NLCs and SLNs on a large scale [25]. High shear homogenization is a method wherein an acceptable range of increased pressure usually between 100 to 2,000 bar is used to forcibly push the dispersion or liquid via a few micrometers’ gap to synthesize particles of submicron size. The gravitational force and high shear stress produced eventually break the particles, leading to a reduction in particle size [26]. This technique is further characterized into two based on the temperature.

16.3.1.1 Hot homogenization Hot homogenization is usually carried out where the temperature is above the melting point of the lipid particle and is equivalent to the homogenization of an emulsified solution. Pre-emulsion form of the drug is loaded into lipid melts and the aqueous phase of the emulsifier at the same temperature is acquired using a high-shear blending device [27]. Pre-emulsion quality is known to affect the final product quality and is advantageous in gaining a few micrometers size ranged droplets. Due to the decreased viscosity of the lipid phase, usually, small-sized particles are achieved at high processing temperatures. However, this might escalate the degradation of the drug and the carrier. Finer products are achieved after a large number of passes via a high-pressure homogenizer, approximately between 3–5 passes [26].

16.3.1.2 Cold homogenization The procedure is carried out on solid lipids and is equivalent to grinding suspension at higher pressure takes place in cold homogenization. Effective regulation of temperature is required to establish lipids in the solid state during the process of homogenization. The emergence of the cold homogenization technique has enabled to curb some of the issues of hot homogenization methods mentioned below [26]: i. Temperature-mediated escalated degradation of drug payload ii. Segregating and therefore, drug loss in the aqueous phase during the process of homogenization

286 Nanomaterials in Healthcare iii. Undetermined lipid polymorphic transitions because of the nanoemulsion step of complex crystallization which leads to super-cooled melts and/or several modifications.

16.3.2 Solvent Emulsification Technique In the solvent emulsification method involving nanoparticle dispersions through precipitation in oil/water emulsions, the lipophilic compound is dissolved in an organic solvent that can be cyclohexane or any other water-immiscible compound and is emulsified in an aqueous state [28]. Nanoparticle solvent dispersion evaporation is established via lipid precipitation in the aqueous phase [26]. The purification of SLNs’ dispersion can be achieved with the use of a dialysis membrane [29]. The benefit of this method is the avoidance of any heat and hence thermolabile agents can be used. Additionally, the suspensions are moderately dilute because of restricted lipids solubility [30].

16.3.3 Ultrasonication or High-Speed Homogenization Ultrasound radiation is a novel and unique technique used for the synthesis of nanoparticles [31]. SLNs can also be synthesized by sonication or high-speed stirring technique [26]. The principle is based on the reduction of particle size with the use of sound waves. In this technique, homogenization with ultra­ sonication and high pressure are simultaneously applied for the synthesis of SLNs with a ranging size of 80–800 nm [29].

16.3.4 Double Emulsion Method A novel technique established, based upon solvent evaporation-emulsification, is used for the synthesis of hydrophilic-loaded SLNs. In this case, the drug is entrapped along the stabilizer to obstruct the partitioning of the drug in response to outside water (liquid phase). This happens during the process of solvent evaporation in response to the outside water phase (w/o/w double emulsion) [26]. This is also used for SLNs’ preparation, carrying some biological molecules like insulin and peptides [29].

16.3.5 Spray Drying Method The spray drying method involves a one-step process wherein there is the conversion of liquid feed to dried particulate form [32]. A replacement technique utilizes lyophilization to convert SLNs aqueous dispersion into the actual product-containing drug. Spray drying is cheaper compared to lyophilization. A major drawback of this technique is that it may lead to the aggregation of particles because of shear forces, partial melting, and high temperature of the particles [26].

16.3.6 Supercritical Fluid Any material, if kept above its critical pressure and temperature, can persist in its supercritical form. A supercritical fluid is an emerging technique due to its inert, environment-friendly, economical, and non-toxic traits [33]. This novel technique for the preparation of SLNs has the benefit of solvent-less processing. The SLNs synthesis also uses the rapid expansion of supercritical carbon dioxide [26]. Supercritical carbon dioxide is known to disperse lipophilic drugs, which also in combination with ultrasonication, can be used to produce SLNs [29].

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16.3.7 Microemulsion-Based SLNs’ Preparation Microemulsion technique is based on the dilution of microemulsions [34]. SLNs are formed by the constant stirring of the optical transparent mixture which comprises low melting temperature fatty acids such as stearic acid, emulsifier (polysorbate 60, soy phosphatidylcholine), co-emulsifier (such as sodium mono octyl phosphate), and water with a temperature maintained at 65–70°C. This hot microemulsion is then dispersed into the cold water at approximately 2–3°C under constant stirring. The ratio of hot microemulsion to cold water is typically between 1:25 to 1:50. The process of dilution is determined by the constitution of microemulsion [26].

16.4 ROUTES OF SLNS’ DELIVERY In various routes of drug delivery, SLNs have been effectively administered to achieve plasma con­ centration, bioavailability, and therapeutic threshold. One of the common characteristics is the ability to get tuned by surface engineering as desired. For cancer drugs to deliver effectively, it’s important to find a compatible delivery system that can treat tumors by penetrating even deep tissues [14]. For this purpose, to achieve the anticancer effect, various routes for delivery of active ingredients have been employed for SLNs, as shown in Figure 16.2. The most common ones are discussed below.

16.4.1 Transdermal/Topical The consistency in the form of a semi-solid system is acceptable for ease of application on the skin. However, for transdermal delivery, the amount of lipid content is a limiting factor in many formulations [35]. The correlation of lipid content with particle size in solid lipid nanoparticles is proportional as a smaller size is favorable to achieve maximum surface area. With less lipid content, the size of solid lipid nanoparticles is also low leading to decreased viscosity of the dispersion. Hence, it becomes important to incorporate the SLNs into a thick base like ointment or gel for topical administration effectively [36]. Solid lipid nanoparticles and nanostructured lipids have been used in various delivery systems to carry active ingredients. Sunscreen and ultraviolet blocker formulations prefer SLNs for controlled release as

FIGURE 16.2 Major routes followed for delivery of solid lipid nanoparticles.

288 Nanomaterials in Healthcare well as a better-localized effect over the conventional system; hence, protecting from skin cancer. In addition, the system keeps skin hydrated for a long time. One such formulation with vitamin A carrying glyceryl behenate lipid-based SLNs has been studied to provide an enhanced local effect [37].

16.4.2 Oral Multiple lipids have been used to prepare SLNs for oral delivery. Enzymes and pH of the gastrointestinal tract influence the degradability of SLNs to a large extent [38]. The aqueous dispersion of SLNs and further coating with acidity protectant is one approach while other approaches are being studied to get better absorption from the stomach and increase the bioavailability of active ingredients. Particle aggregation and ionic strength are considered to be crucial when considering the administration of SLN through the oral route [39]. The ability to attain high saturation solubility and sustain drug release by maintaining a constant plasma level makes SLNs preferable for the oral route. Improved dissolution rate due to more surface area is another advantage of SLNs oral intake which helps to regulate drug onset of action [14]. However, there are certain drawbacks of oral delivery of SLNs as gelation and drug release during storage that limit their use. In an anticancer study, hyaluronic acid-lipid nanoparticles were used to deliver vincristine sulfate delivered through the oral route. An improved bioavailability, sufficient uptake, and cytotoxicity were seen in breast cancer cells in turning to be a good drug carrier system [17].

16.4.3 Parenteral SLNs has been widely used for the administration of small molecules even FDA-approved drugs and peptides for various applications. Likewise, doxorubicin a potent anticancer drug has been used in various formulations for parenteral delivery by SLN [40]. As the drug gets distributed in many organs like the lung, brain, spleen, liver, and kidney, the bioavailability of active agents in the blood is also more. The intravenous route of drug delivery is considered appropriate as most of the in vivo studies carried out in mice for toxicity or distribution have gained importance with time [41]. Studies have stated the success rate of the SLN-carrying drug system to be more efficient than the plain drug alone. The most common advantages of SLNs’ delivery by the parenteral route are ease of scale-up and prevention of degradation as no harsh conditions are faced by them during delivering of cargo. Constant blood plasma concentration of the drug can also be achieved due to direct delivery of the drug in the circulating system of the body [42].

16.4.4 Pulmonary Pulmonary delivery systems are very sensitive and need precaution both during manufacturing and administration. As directly the drug enters the respiratory system, the allergic reaction is frequently observed and seeks attention in many cases. Hence, utmost safety is required with the type of lipid and surfactants being used for preparing lipid nanoparticles [43]. Faster entry into the respiratory route also activates the target site by accelerating the onset of action. High amounts of drugs are accumulated and permeate at the target site, especially in the alveolar area due to the thin epithelium. Another advantage is reduced toxicity to the undesired site as no other body system comes in direct contact but only the pulmonary attains a high local concentration of the drug [44]. Drawbacks that need to be overcome by the SLNs through the pulmonary route of delivery include burst release, not suitable for deep lung tissue delivery, and fewer safety data available on humans [45]. SLNs loaded with epirubicin was nebulized and given to rats to study characteristic potential cytotoxicity on lung cancer. The effect on A549 cells showed enhanced deposition of drug and increased plasma area under the curve as compared to plain drug with inhaled SLNs formulation [46].

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16.4.5 Brain The most challenging route for the delivery of drugs is into the blood-brain barrier (BBB). Surpassing the reticuloendothelial systems (RES), the nanoparticle can easily cross the BBB due to its smaller size and high surface area [47]. Along with this increased entrapment efficacy and lipid content makes SLNs, seemingly reliable for drug delivery to the brain. Penetration through BBB and efflux of the drug by brains into blood circulation are major hurdles however, the increased retention time of drugs by SLNs allows the drug to act efficiently by loosening the junction facilitating transcytosis [48]. In addition, both the hydrophilic and lipophilic drug-carrying capacity of lipid nanoparticles has led to significant delivery to the brain by SLNs [10]. A study conducted in vitro and in vivo by an anticancer drug edelfosine showed a high accumulation rate and decline in brain tumor growth when delivered through SLNs [49].

16.5 TOXICOLOGY AND CLEARANCE The excretion or the clearance of the drug carrier from the system is as essential as having the drug released from the carrier into the system. Excretion is listed as one of the most critical aspects in the pharmacokinetics of the drug, commonly referred to as ADME: absorption, distribution, metabolism, and excretion. A prolonged accumulation, or use of non-biodegradable drug carriers, due to non-clearance can lead to toxicity. Hence, developing a drug nanocarrier with the ideal property of 100% clearance (after the release of the drug) and complete non-toxic property has been a crucial challenge for several decades [50]. In comparison to several types of nanoparticles, SLNs have proved to be a near-ideal mode of drug delivery, which possesses both these ideal properties of negligible toxicity and maximum clearance. In conventional chemotherapy, the chemotherapeutic drugs are exposed to several hindrances, such as low specificity, instability, and drug resistance. These obstacles can be partially overcome by delivering these therapeutic agents with SLNs. Chemotherapeutic drugs delivered through SLNs have shown less drug resistance susceptibility and proven to be more toxic to the cancerous cells than when in free form [51]. To minimize the phagocytic uptake, the surface of the nanoparticles is modified. Better versions of SLNs in the form of NLC, polymeric lipid hybrid nanoparticles, long-circulating SLNs, and lipid drug conjugates have been developed to improve the circulation time and versatility to carry various chemotherapeutic agents. Also, various SLNs’ preparations have been extended to treat other conditions such as tuberculosis and parasitic infections [52]. SLNs have a wide range of applications due to their non-toxic nature. The nature of specificity of the target site is due to the lipid nanoparticles’ ability to pass through the crucial biological barriers, and they also overcome cell internalization, degradation by nucleases, and intracellular trafficking [53]. Additionally, the omission of organic solvents gives an added advantage for SLNs to be non-toxic. Also, as the drug carrier can deliver high amounts of the drug due to its high surface area, the materials used to deliver them is low, further contributing to its low/non-toxic nature. To increase stability, 0.5–5% surfactant is added. Natural surfactants are used; hence, they are not toxic too [54]. Typically, the size of lipid nanoparticles is such that they cannot be cleared by renal filtration; hence, they are usually opsonized by the serum proteins [55]. They are then taken up by the organs such as kidney, lungs, spleen, liver, and lymph nodes, through their respective RES for clearance from the body. These nanoparticles can deform and squeeze back through the fenestrations of the spleen, which usually clears out larger particles (>200 nm), and remains in the bloodstream. After the successful targeted release of the drug, the SLNs are bound to be cleared from the system through the RES [56]. Based on the size and biodegradability of the nanocarriers, to analyze the potential toxicity, they are categorized into four by Keck and Müller, described as a nanotoxicological classification system (NCS) [57]:

290 Nanomaterials in Healthcare Class Class Class Class

1. 2. 3. 4.

Biodegradable nanoparticles of > 100 nm size Non-biodegradable nanoparticles of > 100 nm Biodegradable nanoparticles of < 100 nm size Non-biodegradable nanoparticles of < 100 nm

– No/low risk size – medium risk – medium risk size – high risk

Since the material used to prepare SLNs is biodegradable and biocompatible lipids, for example, glycerides, fatty acids or fatty acid esters, waxes, PEGylated lipids, sterols, etc., they fall into the cat­ egories of classes 1 and 3. Also, the toxicity of the nanoparticles mostly depends on the route of their administration. Orally administrated SLNs are prone to erosion and degradation by pancreatic lipase and bile salts. Differently administered SLNs undergo different environments and hence meet different ultimate fates [58]. A nanoparticle is phagocytosed by the RES when in the bloodstream after it is opsonized. The phagosome needs to be developed, by a sequence of interactions between endosomes and liposomes, into a phagolysosome, which has a special membrane that can resist harsh environments of acid and deg­ radation [59]. Several chronic cytotoxic effects, such as progressive tissue injury and inflammation are observed due to the internalization of the non-biodegradable classes of nanoparticles [60]. The NCS classification does not consider the effect of the surface charge of nanoparticles on toxicity and clear­ ance. Based on the need, nanoparticles may be fabricated with a positive charge on the surface for a nonspecific uptake by negatively charged plasma membranes of cells, which further enhances endocytosis. Although the positively charged nanoparticles possess several advantages and have potential applications in various delivery systems, they are toxic. As per a few studies, cationic SLNs have proven to be more toxic than their neutral and anionic counterparts [61].

16.6 APPLICATIONS The utilization of SLNs has been emerging exponentially for the treatment of cancer therapy. SLNs’ wide availability due to their low toxicity, bioavailability, and feasibility of synthesis is diversifying drastically. Many novel drugs developed could not be commercialized due to poor solubility in their natural form, and permeation into the cell membrane [62]. To subdue this problem, high doses of drugs used eventually cause high toxicity and many unwanted side effects. Hence, drugs need to be encapsulated into drug carriers before delivery into the systemic circulation. Further, SLNs overcomes many physiological barriers like multidrug resistance (MDR) mechanisms which hinder the drug delivery to specific tumor cells. Thus, SLNs has expanded in the field of nanotechnology for developing an efficient DDS [63]. The use of SLN is emerging in the most common cancers like breast, lung, and colon, which provided promising results (Figure 16.3).

16.6.1 Breast Cancer The prevalence of breast cancer is increasing over the years. SLNs are widely being used for the treatment of breast tumors with good outcomes. For instance, tamoxifen-loaded SLN can be potentially used for the treatment of breast cancer. These nanoparticles have small sizes and therefore reduce the uptake of macrophages by the mononuclear phagocytic system [64]. Paclitaxel, when incorporated into SLN, has shown improved anticancer activity in breast cancer cell line MCF7/ADR. This study showed that MDR breast cancer can be treated using Paclitaxel [65]. The MDR cells can be treated with doxorubicin as well. Doxorubicin is a water-soluble anticancer drug. The efficacy of doxorubicin can be increased by incorporating it into SLN [63]. The newly improved nano-formulation of SLNs

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FIGURE 16.3 Scheme showing application of solid lipid nanoparticles.

made up of cholesterol and curcumin into the core of cholesterol developed by Box Behnken design holds great capacity in cancer treatment [66]. Cisplatin is an effective anticancer drug that has limiting factors of non-specificity and dosage. Stearic acid-functionalized SLNs as the cisplatin carrier has augmented anticancer efficacy in MCF-7 cancer cells. Additionally, it has been reported to also be within the nano-size range with entrapment efficiency of 60–70% and loading capacity of 3.6–4.6% (w/w) [67].

16.6.2 Lung Cancer Lung cancer is one of the biggest killer types of cancer worldwide seen over the last century. It is reported that over 1.2 million people died due to lung cancer in 2012. Epirubicin (EPI) is an anthra­ cycline anti-cancer drug for many types of tumors and also a stereoisomer of doxorubicin. For lung cancers, drug delivery via inhalation has attracted the scientific and biomedical committee. Delivering drugs via lung has an advantage since it can provide a non-invasive route of delivery. EPI-SLNs is used for its potential carrier for pulmonary delivery of anticancer drugs. In a study, it was revealed that the drug concentration of EPI-SLN after inhalation was found to be higher than in only epirubicin solution [68]. The stability of SLN-DTX (Docetaxel) prepared using the high-energy method is for a longer time with uniform distribution and high encapsulation efficiency (~86%). These SLN-DTX prevent lung metastasis [69]. Gemcitabine (GmcH) a potent anti-cancer drug is used in chemotherapy for lung cancer. But it obstructs many problems like low blood residence time, poor penetration in the surrounding of lung cancer cells, etc. To overcome this, GmcH-loaded mannosylated SLNs has been developed which improved drug uptake in the cancer cells. This study shows the small size of GmcH-SLN (~100–200 nm), improved targeted delivery in lung cancer cells can be achieved with the required therapeutic effect [70].

292 Nanomaterials in Healthcare

16.2.3 Colon Cancer Colon cancer is the third most common cancer among developed countries and it affects majorly in elderly people, but minor cases were seen in younger ones as well. The rate of people being diagnosed has reduced over the past decades but it is still the leading cause of cancer deaths [71]. COX-2 enzyme expression is seen in cancer cells and celecoxib (CXB) is a selective inhibitor of this enzyme showing significance in treating colon cancer. The CXB-SLN was prepared by the melt emulsification method which yields high-quality SLN. This SLNs does not show burst release and targeted delivery to the colon [72]. To target colon cancer, resveratrol (RSV) and ferulic acid (FER) loaded SLNs have also been synthesized. This complex was coated with chitosan and stearic acid for the conjugation of folic acid to make SLNs for folate receptors that will be used as a drug delivery vehicle. The C-RSV-FER-FA-SLNs cancer cell targeting and apoptosis studies revealed that they induced cell death in HT-29 cell lines [73]. Topotecan (TPT) loaded with SLNs has shown positive results against colorectal cancer. These TPTSLNs have been developed into a thermoresponsive hydrogel system for controlled release and reduced toxicity of the drug. The microemulsion technique and cold method have been used for the preparation of TPT-SLN-TRHS. These particles have shown good gelation time and antitumor activity in mice [74].

16.7 CONCLUSION SLNs have been considered an efficient DDS as a nanocarrier in incorporating and accumulating drugs in tumor sites for almost all cancer cells. The active and passive mechanisms can also be easily achieved with SLNs overcoming biological barriers. Various methods for the preparation of SLNs provide the advantage of easy-to-follow and process different parameters for nanosize optimization. The rationale of design and tailoring can be better understood by studying the recent updates on SLNs formulations. With diverse applications in cancer treatment, SLNs has gained scope in exploring highly useful therapies for different tumors. Surface modification and the ability to carry hydrophilic and lipophilic molecules have made SLNs the nanoparticle of choice for enhancing the stability and solubility of the drug. The toxi­ cological studies performed so far also show good results focusing on the development of SLNs to be of great interest in the future.

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Janhavi Devrukhkar and Jasmeen Kaur NanoBios Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 17.1 17.2 17.3

Introduction Synthesis of Gold Nanoparticles Gold Nanoparticles for Cancer Therapeutic Application 17.3.1 Gold Nanoparticles for Drug Delivery and Nucleic Acid Delivery 17.3.2 Photodynamic Therapy 17.3.3 Photothermal Therapy 17.3.4 Gold Nanoparticle-Based Combined Cancer Therapy 17.4 Application of Gold Nanoparticles in Cancer Diagnosis 17.4.1 Bio-Imaging 17.4.1.1 Computed tomography (CT) 17.4.1.2 Magnetic resonance imaging (MRI) 17.4.1.3 Nuclear imaging 17.4.1.4 Fluorescence imaging (FI) 17.4.1.5 Photoacoustic imaging (PA) 17.4.2 Biosensing 17.5 Gold Nanoparticles as Theragnostic Agents 17.6 Clinical Status of Gold Nanoparticle Formulations 17.7 Safety Concerns and Challenges for Application of Gold Nanoparticle in Healthcare 17.8 Conclusion References

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DOI: 10.1201/9781003322368-17

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17.1 INTRODUCTION In the year 2020, the estimated number of cancer cases and cancer-related deaths globally were 19.3 million and 10 million, respectively, with 1 in 5 people being at the risk of developing cancer at least once in their lifetime [1]. Surgery, chemotherapy, and radiation therapy are being widely used to treat and improve survival rates of cancer patients. Surgery and radiation therapy are suitable for earlystage benign tumors but ineffective in malignant tumors and there is a risk of recurrence. The efficacy of chemotherapy is limited due to side effects, non-specificity, toxicity, poor bioavailability, and insuffi­ cient drug distribution in the tumor area. In modern cancer diagnosis and therapy, nanosized contrast and therapeutic agents i.e., nanoparticles are used to accurately locate and treat cancer. High surface area carrier systems viz. liposomes, meso­ porous silica, polymers, quantum dots, biological macromolecules like proteins and DNA as well as metallic particles including gold, silver, and iron oxide have been extensively applied in nanomedicine [2]. The primary advantage of these nanostructures is their small size (between 10–300 nm), mor­ phology, cargo carrying capacity, targeting ability, stimuli responsive smart drug delivery, etc. The use of nanoparticles in diagnosis and treatment of cancer relies mainly on passive targeting attributed to the accumulation of nanoparticles in the tumor region due to the enhanced permeability and retention effect (EPR). FDA-approved nanoformulations viz. Myocet liposomal (liposome and Doxorubicin), Doxil (PEGylated doxorubicin encapsulated in liposomes) [3] and Abraxane (albumin paclitaxel nanoparticle) [4] are being used to overcome some shortcomings of traditional chemotherapy. Strategies for localized therapy like thermal and magnetic hyperthermia, photodynamic therapy (PDT, oxidation in the target region by generation of singlet oxygen species leading to cellular damage and death), and photothermal therapy (PTT) where photothermal agents exhibiting strong extinction in the near infrared region (NIR) between 650–900 nm have recently gained popularity in comparison to the traditional therapeutic approaches. Gold nanoparticles (GNPs) have garnered more attention in recent times as ideal candidates for cancer theragnostics (therapy + diagnosis) due to the ease of synthesis of GNPs of different shape and sizes and the possibility of modifying the physicochemical properties according to the change in mor­ phology. Large surface area increases the cargo carrying capacity, ease of surface modification, and allows for different functionalization. GNPs can be conjugated with a host of molecules like drugs, dyes, peptides, nucleic acid, ligands, peptides, etc. Gold can bind hydrophobically, covalently through thiols or chemical crosslinking, or electrostatically if the surface is rich in amines. Noble metal NPs such as gold nanostructures are efficient photothermal agents due to their excellent optoelectronic properties and high atomic number. The extinction coefficient of gold nanoparticles is orders of magnitude stronger than that of organic dye molecules and other nanoparticles and require lower laser energies. Absorption or scattering of light varies with size and increased scattering is observed with an increase in size. Photoexcitation over the NIR region causes electrons in the conduction band of gold nanoparticles to oscillate and release their energy in a phenomenon called localized surface plasmon resonance (LSPR), which can be utilized for localized tumor destruction due to the localized heating. These optical properties make them promising candidates in imaging and PTT applications [5]. Moreover, gold nanoparticles are inert, biocompatible, and stable in physiological conditions making them ideal candidates for nanomedicine applications. This chapter explores the therapeutic and diagnostic potential of gold nanoparticles as vectors for drug and nucleic acid delivery, as therapeutic agents in PTT and PDT, as contrast agents in computed tomography, MRI, fluorescence, nuclear, and photoacoustic imaging, as well as detection agents in biosensing. The present status of preclinical research and clinical trials is discussed in detail along with the safety concerns and challenges faced that are restricting the translation of promising research on GNPs into practical health care for patients and towards complete cancer elimination.

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17.2 SYNTHESIS OF GOLD NANOPARTICLES Gold nanoparticles are synthesized by several techniques to obtain various shapes and sizes. Spherical gold nanoparticles, gold nanorods, nanocages, nano stars, nano clusters, hexagons, and nano urchins are some of the shapes in which gold nanoparticles have been synthesized. The Turkevich method [6] is a classic synthesis method using sodium citrate to reduce chloroauric acid using heat. The need for heating can be eliminated by using sodium borohydride as a reducing agent instead of citrate. Brust-Schiffrin synthesis [7] technique takes advantage of the gold thiol affinity for preparation of spherical nano­ particles by reduction gold salt with NaBH4 in the presence of tertraoctylammoniumbromide (TAOB) and dodecanethiol while tuning the size of GNPs by changing ratio of dodecanethiol and TAOB. The seed growth method of Jana et al. using CTAB for synthesis of gold nanorods and the Biswal method using ascorbic acid as the reducing agent are other well-established chemical synthesis methods of GNPs [8]. Recently, several green synthesis methods using organic extracts like amines, proteins, and phenols for reduction of Au to improve biocompatibility and sustainability of GNPs have gained momentum. Organic biological agents like bacterial cultures and plant extracts have been used for reducing gold salts to nanometallic form in a one-pot eco-friendly manner such that they form a coating on synthesized GNPs to improve stability and reduce aggregation while retaining the anti-cancer activity. Nanoparticles prepared from flax seed (Linum usitatissimum) extract and dragon fruit (Hylocereus undatus) extract showed remarkable effectiveness against breast cancer cells MCF-7. Similarly, gold nanoparticles synthesized from marine bacterium (Vibrio alginolyticus) showed dose-dependent apoptosis in colon cancer while Thymus vulgaris leaf extrat GNPs tested on myeloid leukemia were proven to be more effective than the classic anti-cancer drug Doxorubicin [9]. Gold nanoparticles synthesized using Curcuma longa extract have shown anti-cancer activity against multiple cancer cells including glioma, breast cancer, colon cancer, and cervical cancer. Biocompatible multifunctional gold nanoparticles synthesized with turmeric as a reducing agent in the presence of paclitaxel and quercetin were able to inhibit angiogenesis and cell proliferation. Curcumin-reduced gold nanoclusters have also been shown to possess fluorescence, thus having application in fluorescence-based cancer imaging [10]. Thus, GNPs can be synthesized by chemical and green synthesis methods and the concentration of different com­ ponents can be varied to obtain nanoparticles of different sizes and thus properties.

17.3 GOLD NANOPARTICLES FOR CANCER THERAPEUTIC APPLICATION The optical, physical, and thermal features of gold nanoparticles make them ideal candidates for cancer nanomedicine applications. In this section, we discuss the complete and ongoing research on GNPs in cargo delivery, photodynamic therapy (PDT), and photothermal therapy (PTT). The multifunctionality of GNPs is explored in terms of combined and synergistic therapeutic advances.

17.3.1 Gold Nanoparticles for Drug Delivery and Nucleic Acid Delivery Gold nanoparticles are non-toxic and passively accumulate in the tumor region by the enhanced per­ meability and retention (EPR) effect or may be targeted actively through functionalization by

300 Nanomaterials in Healthcare

FIGURE 17.1 Schematic design for targeted drug delivery system procedure based on AuNPs [ 11]. International Journal of NanoMedicine 2021 16 7891-7941‘ Originally published by and used with permission from Dove Medical Press Ltd.’

conjugating with ligands, antibodies (Figure 17.1). These established fundamental features make them excellent cargo carriers for delivering therapeutic drugs, nucleic acids, peptides, phytochemicals, and coordination compounds. Gold nanoparticles are negatively charged and require positive charge for binding. Gold can bind hydrophobically, covalently through thiols or the use of chemical cross-linking, or electrostatically if the surface is rich in amines or carboxyl groups. Widely used anticancer drug Doxorubicin has been delivered using gold nanoparticles to reduce p-glycoprotein-based drug efflux [12]. Methotrexate, 5-flurouracil [13], bombesin peptide, and trastuzumab antibodies have been shown to improve the targeting ability and uptake of drug-loaded GNPs via receptor mediated endocytosis. Various targeting receptors like hyaluronic acid, galactose, transferrin, folic acid [14], etc. have been chosen to improve intracellular uptake of GNPs. Thus, GNPs have been used to reduce multidrug resistance, improve specificity and cancer cell cytotoxicity due to conjugation with targeting moieties, reducing drug dosage and non-specific cytotoxicity (Table 17.1). DNA- and RNA-based gene therapy is evolving as a novel treatment strategy for cancer. Nucleic acids are however easily degraded by enzymatic, chemical, immunogenic, or physical processes making their delivery more difficult than drug delivery. Therefore, appropriate vectors need to be chosen. Viral vectors have been a popular choice for this purpose; however, there is a risk of triggering immune response affecting future therapy attempts. Gold nanoparticles help prevent these problems by not only offering a compatible and easily modifiable surface but also improve the chances of transfection. Gold nanoparticles can protect unstable nucleic acid fragments from degradation by nucleases. Tunc et al. found morpholino oligonucleotide embedded DNA tile GNP complex to be superior to liposome-based delivery systems for silencing HER2 and Erα gene in breast cancer [16]. FOXM1 aptamer and Doxorubicin were simultaneously delivered in to cancer cells through a guanaosine rich single-stranded

PEG, C225 and BSA

ZnD

HepG2 cells

CELL LINE

MAIN OUTCOME

ANTICANCER APPLICATION

Stronger cytotoxicity compared to Targeted delivery of DOX to free DOX. hepatocarcinoma cells AuNPs-PEG- 5-FU-FA MI39 and M2I3 cells Higher cytotoxic effects as compared to Targeted delivery of 5-FU free 5-FU and FA. and targeted therapy of cholangiocarcinoma cells DTX@HA-cl- AuNPs HeLa and MCF-7 cells Higher cytotoxicity and tumor inhibition Targeted anticancer therapy efficacy than free DTX under nearin combination with laser infrared laser irradiation. treatment LIN-AuNPs- CALNN MCF-7 cells Higher antioxidant activity and Human breast cancer anticancer activity as compared to therapy Linalool and AuNPs alone. K-AuNPs MCF-7 cells Higher cell apoptosis, antiproliferative Human breast cancer ability and inhibition of angiogenesis therapy compared to pure kaempferol. PI -AuNPs HT-29 and MDAHigher DNA disintegration in both cells Human colon cancer and MB- 231 cells and subsequent cell apoptosis breast cancer therapy compared to AuNPs and PI alone. TargetNanoTS265 A549 and HCT I 16 Elevated tumor cytotoxicity and tumor Targeted delivery of cells inhibition efficacy as compared to the anticancer agents free TS265. NanoZnD DOX- resistant HCT I 16 Enhanced antiproliferative potential and Targeted drug-resistant cells reduced tumor growth when cancer therapy compared to free ZnD.

DOX-PEC- AuNPs

NANOCOMPLEX NAME

Abbreviations: 5-FU, 5-fluorouracil; anti-EGFR D- II, monoclonal antibody D-1 I against epidermal growth factor receptor; AuNPs, gold nanoparticles; BSA, bovine serum albumin; C225, cetuximab; CALNN, Cys-Ala-Leu-Asn-Asn; DOX, doxorubicin; DTX, docetaxel; FA, folic acid; GFLGC, Gly-Phe-Leu-Gly-Cys; GSH, glutathione; HA, hyaluronic acid; K, kaempferol; LIN, linalool; PI, Boc-L-DP-L-0Me; PEC, pectin; PEG, polyethylene glycol; TS265, CoCI(H20) (phendione)2][BF4]; ZnD, [Zn(DION)2]C12.

PEG, anti- EGFR D-I I and BSA

TS265

–

CALNN and GSH

LIN

PI

HA and GFLGC

DTX

–

PEG and FA

5-FU

K

PEC

MODIFYING MOLECULE

Gold nanoparticles for chemotherapeutic drug delivery in different cancers [ 15]

DOX

ANTICANCER DRUG

TABLE 17.1

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302 Nanomaterials in Healthcare DNA, nucleolin aptamer AS1411 [17]. Gold nanoshells have been used for combined release of Docetaxel and complementary dsDNA using laser-triggerd Au-S bond clevage [18]. Similarly, microRNA delivery with a hyaluronic acid (HA) and polyethylene glycol (PEG)–based targeted GNP delivery system showed effective cell death in liver cancer cells [19]. Crispr/Cas9 system spCas9 was loaded into gold nanoclusters for nuclear delivery. It induced apoptosis in cervical cancer cells and also successfully knocked out E6 oncogene to enable correct functioning of p53 [20]. Moreover, the photothermal effect has been coupled to enhance the therapeutic effect and to release DNA at target site dur to thermal cleavage of conjugate bonds.

17.3.2 Photodynamic Therapy Since its advent, PDT has evolved as a minimally invasive effective therapeutic approach for effectively treating cancer. The main components are a photosensitizer, a laser source and molecular oxygen. The photosensitizer is excited to the singlet state by irradiation with specific wavelength light. This activation of photosensitizer results in generation of reactive oxygen species. This excess production of free rad­ icals causes oxidative stress, leading to cellular toxicity that kills cancer cells by apoptosis or necrosis (Figure 17.2A). Some of the photosensitizers are photofrin, temoporfin, verteporfin, methylene blue, toluidine blue, etc. [21]. However, PDT poses some limitations as most photosensitisers have low water solubility, tendency to aggregate in physiological media, undesirable side effects, non-specific biodis­ tribution in vivo, ineffectiveness under hypoxic conditions, and risk of immune reactions of the pho­ tosensitizers. Hence, specificity and stability are two important features essential when designing an ideal photosensitizer. Several methods have been adopted to address the above limitations by conjugating photosensitizers to gold nanoparticles. Hydrophobic photosensitizers like porphyrin derivatives [22] and mesotetrahydroxyphenylchlorine have been attached to gold nanoparticles [23]. Hydrophilic molecules do not

FIGURE 17.2 Schematic illustration of A) photodynamic therapy and B) photothermal therapy [ 30].

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have the issue of solubility; however, they cannot cross the bilipid cell membrane easily. To overcome this, hydrophilic photosensitizers like 5-Amino levulinic acid (5-ALA) have been bound to GNPs pre­ viously conjugated to RGD peptide, which facilitate its easy entry into the cancer cells by receptormediated endocytosis [24]. Similarly, transferrin peptide-coated PEGylated gold nanoparticles were used to deliver photosensitiser drug Phthalocyanine for targeted delivery to glioblastoma cells overexpressing transferrin receptor [25]. Enhanced cell death was observed in the case of targeted drug delivery due to PDT as compared to non-targeted delivery. Similar improvement in photodynamic efficacy was observed in Pc-loaded protoporphyrin IX GNP nanostructures tagged with folic acid, indicating that targeting improves the phototoxicity in HeLa cells [26].

17.3.3 Photothermal Therapy Photothermal therapy (PTT) is a novel mode of cancer therapy, in which an electromagnetic radiation incident on a photothermal agent is converted to heat to moderately increase the temperature locally leading to destruction of cancer cells (Figure 17.2B). Typically, a tuneable wavelength selected laser is used as a light source which acts as an external stimulus causing photothermal conversion agents accumulated in the tumor region to convert the incident light to heat. Being minimally invasive PTT is gaining popularity for treatment of superficial and moderately deep-seated tumors [27]. It is best suited for the ablation of solid tumors using hyperthermia and the generated heat can also trigger cargo release from nanovesicles [28]. PTT is effective in cancer, as cancer cells are more susceptible to temperatures in the range of 40–44°C, owing to hypoxia and low pH in the tumor environment. Cell death occurs in PTT due to apoptosis or necrosis, depending on the laser type, power, and irradiation time. In addition to heatinduced cell damage, the heating of photothermal agents such as gold nanoparticles cause formation of cavitation bubble around the nanoparticle, resulting in cellular destruction due to mechanical stress and the cancerous cells are eventually cleared by macrophages, causing shrinkage of tumors and even complete ablation [29]. When NIR light is incident on a tissue surface, a portion of light is reflected and the photons entering the tissue could either be absorbed, scattered, or transmitted. A biological window exists in the NIR region (wavelengths 650–950 nm), where the absorbance of light by water and hemoglobin is lowest and this could be exploited for its application in PTT as these wavelengths induce minimal heating in sur­ rounding healthy tissues. Laser irradiation with power intensity between 0.2 and 1.5 W/cm2 are used to retain cell and tissue viability while effective hyperthermia induces tumor regression. The therapeutic efficacy of the PTT relies significantly on the conversion of NIR light to heat and this is achieved by employing GNPs as photothermal agents. By changing their morphology and size, GNPs can be tuned to absorb at different wavelengths in the NIR region giving them an advantage over other photothermal agents. For example, the absorption spectrum of gold nanorods is dependent on its aspect ratio while that of spherical nanoparticles changes with different core-to-shell ratio and surface morphology. Several reports of gold nanoparticle-based PTT have shown tumor regression and even complete tumor ablation.

17.3.4 Gold Nanoparticle-Based Combined Cancer Therapy Synergistic effect to enhance the efficacy, control adverse side effects, and reduce the dosage of chemotherapeutic or photothermal agent can be achieved by combination therapy involving PDT, PTT, radiotherapy (RT), and chemotherapy. A subtherapeutic dose is sufficient when chemotherapy is used in conjunction with gold nanoparticle-based PTT as any remaining cells post-chemotherapy can be killed by PTT. Also, drug-loaded GNPs limit the interaction of free drugs with surrounding normal tissue significantly reducing side effects. Combination therapy simultaneously employs several different methods of tumor regression, hence reducing the chances of chemoresistance, radio resistance, or

304 Nanomaterials in Healthcare chemoresistance while increasing the chances of complete tumor ablation by potentially killing any leftover cells that the other methods may have missed. Several gold nanocomposites involving GNPs and polymers, lipids, and biomolecules loaded with drugs have shown noteworthy difference in tumor regression as compared to a single therapy approach. Gold nanoparticles tagged with aptamer AS1411 and loaded with Dox showed excellent collaborative chemo-photothermal therapy on 808 nm laser irradiation on colon cancer cells [31]. Chemotherapeutic drugs cause DNA damage and hyperthermia slows down natural DNA repair mechanism. Hyperthermia also improves EPR effect due to the localized increase in temperature-causing drugs to accumulate in the tumor region for a longer duration. Moreover, hyperthermia also helps inhibit drug efflux caused by p glycoprotein due to increased expression of heat shock proteins [32]. Combined PDT and PTT is also gaining interest as light used for PDT is less than 700 nm and cannot penetrate deep into the tissues. Also, PDT requires ample oxygen which is sparse in the hypoxic tumor microenvironment. PTT can help overcome these challenges as longer wavelength lasers are used in PTT and improved blood flow due to hyperthermia bring in oxygenated blood. A single gold nanoparticle serving as both PDT and PTT agent is therefore desirable for enhanced therapeutic effect without the need for a separate photothermal agent and photosensitizer. This was achieved by Liu and group, who irradiated captopril stabilized gold nanoclusters with a 808 nm laser and observed instant temperature rise as well as sustained O2 generation [33]. Glioblastoma multiforme was treated with oral administration of choline e6 (photosensitizer) and glutathione-coated GNPs that have the ability to cross the blood-brain barrier and show effective combined photothermal and photodynamic therapy [34]. A combination of PTT with RT too has been reported wherein PTT before RT results is effective therapy with low X-ray dose requirement and PTT after RT prevents radiosensitivity by restricting DNA repair mechanisms. Triple combination therapy, although in the nascent stage, seems to be the promising novel approach. pH-responsive gold nanorods loaded with DOX and 5-ALA were reported for combined chemo-photothermal and photodynamic therapy [35].

17.4 APPLICATION OF GOLD NANOPARTICLES IN CANCER DIAGNOSIS An early or timely diagnosis of cancer is the most effective means to improve its prognosis and reduce the mortality rate. The routine cancer diagnosis involves multiple cancer screening tests, including different imaging tests to visualize the tumor, and laboratory-based tests for the detection of specific tumor biomarkers. The screening tests are often followed by a tissue biopsy and its histological analysis for a confirmatory cancer diagnosis and to rule out false positives. While the screening tests are inefficient and not sensitive enough to detect the early stages of cancer, a biopsy is highly invasive, timeconsuming, expensive, and involves patient discomfort [36]. Hence, efforts are being made by researchers around the world to make the screening tests more sensitive to accurately identify early-stage cancers and distinguish the benign forms from the life-threatening malignant forms. Advancements in the area of nanotechnology have allowed the incorporation of nanomaterials such as metal nanoparticles, carbon nanotubes, graphene, etc. for different bio-imaging and biosensing applications. In particular, gold nanoparticles have been studied extensively in the last decade for these applications. Their unique characteristics such as inertness, versatile surface properties, outstanding biocompatibility, and ease of functionalization allow them to be used for multiple applications including biomedical imaging, ther­ apeutics, and biosensing systems [37]. The different bio-imaging and biosensing techniques commonly used to diagnose cancer have been discussed in greater detail in the following sections with the major focus on the incorporation of gold nanoparticles for better sensitivity.

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17.4.1 Bio-Imaging Bio-imaging is done with the sole purpose of clearly and accurately visualizing and identifying the tumor area, not only for diagnostic purposes but also for image-guided therapeutic applications to demarcate the tumor target areas. Some of the most commonly used imaging techniques are computerized tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography scan (PET), etc. However, classical techniques are inefficient and not sensitive enough to detect the early stages of cancer. Therefore, to enhance the differentiation between a healthy and a diseased tissue during imaging, several imaging contrast agents (CA) have been employed to highlight the anatomical or functional features at the cell or tissue level [38]. Gold nanoparticles have piqued great interest as contrast agents for multiple imaging techniques owing to their extraordinary light scattering properties, surface plasmon resonance (SPR), biocompatibility, enhanced permeability and retention (EPR) effect, and easy bioconjugation for active targeting of tumor cells. In this section of the chapter, we will briefly describe the principle of various imaging techniques and focus on the application of gold nanoparticles as contrast agents in these techniques.

17.4.1.1 Computed tomography (CT) Computed tomography (CT) is an imaging method that employs X-rays to non-invasively obtain crosssectional images. The higher and more efficient absorbance of X-ray irradiation by gold nanoparticles due to their stronger X-ray mass attenuation and high electron density, has rendered them superior to conventional iodine-based CT contrasting agents [39]. In addition, gold nanoparticles offer multiple advantages, such as biocompatibility, low toxicity, and longer circulation time, thereby enabling pro­ longed imaging. The ability of gold nanoparticles to passively (via EPR or actively (via surface func­ tionalization with specific target moieties) target the tumor, facilitates precise tumor accumulation and avoids off-target accumulation, thereby allowing a more sensitive imaging diagnosis [40]. Numerous studies have exploited the passive and active targeting ability of gold nanoparticles as CT contrast agents for their application in cancer diagnostics. For instance, Luo et al. prepared AuNPs decorated with prostate-specific membrane antigen (PSMA) targeting ligand, PSMA-1, to and reported their increased uptake and higher accumulation in PSMA-expressing PC3pip tumors that was confirmed using a microCT [Figure 17.3(i)] [41]. Lately, efforts are being put into the development of ultrasmall gold nanoclusters (AuNCs) as CT contrast agents that have good renal clearance, unlike gold nanoparticles [42]. Basilion and group reported that the PSMA-targeted gold nanoclusters showed higher tumor affinity with excellent renal clearance and much lower liver uptake as compared to their counterparts, PSMA-targeted gold nanoparticles [43].

17.4.1.2 Magnetic resonance imaging (MRI) MRI is a non-invasive imaging technique that provides high-resolution, three-dimensional anatomical images of soft tissues. It works on the principle of nuclear magnetic resonance (NMR) and employs a strong magnetic field to excite and detect changes in the direction of the rotational axis of protons present in different tissues [37]. MRI has an added advantage over CT in that they do not require harmful ionizing radiations like X-rays. Yet, it is still not sensitive enough to effectively diagnose cancers, as a result of which several MR contrast agents are required to improve sensitivity. Superparamagnetic iron oxide nanoparticles (SPIO) and gadolinium (III) [Gd (III)]-based paramagnetic agents are the most commonly used and extensively investigated MR contrast agents [45]. However, SPIOs can produce reactive oxygen species (ROS) causing oxidative stress and have limited in vivo stability [46,47], and the toxicity of Gd (III)-based contrast agents is of major concern [48]. Gold nanoparticles, on the contrary, are biocompatible and are free of ROS-induced toxicity and therefore have been studied extensively for in vivo applications. Jean Debouttière et al. synthesized small-sized gold nanoparticles (2–2.5 nm) functionalized with a dithiolated ligand [diethylenetriaminepen-taacetic acid (DTPA)] and gadolinium

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FIGURE 17.3 i (a) Schematic representation of PSMA-targeted AuNPs based targeted radiotherapy of prostate cancer. (b) 3D CT images depicting selective tumour accumulation of untargeted (top panel) and PSMA-targeted (bottom panel) AuNPs for PC3pip (blue circle) and Pc3flu (green circle) tumor-bearing mice at 4 h post-injection. ii (a) Schematic representation of a SPAuNC along with its synthetic procedure. (b) Timecourse T2-weighted MR images after injection of affibody conjugated SPAuNC in the healthy mice and hepatic tumor bearing mice (MDA-MB-468) (liver is indicated by yellow dotted circles) [ 44].

(Gd) chelates as an MRI contrast agent [49]. The ligand-anchored gold nanoparticles allowed the for­ mation of a multi-layered shell that could hold much more Gd chelates and ensured good colloidal stability as contrast agents. The Au@DTDTPA-Gd nanoparticles as in vivo contrast agents for MRI and X-ray imaging and showed appropriate circulation without undesirable accumulation in organs [50]. More recently, Lee and co-workers used a virus like particle as a synthetic scaffold for targeting tumor to develop superparamagnetic AuNP clusters (SPAuNCs) that were less than 3 nm [Figure 17.3(ii)] [44]. These SPAuNCs show a typical superparamagnetic behaviour and T2-weighted magnetic resonance, along with excellent biocompatibility and renal clearance.

17.4.1.3 Nuclear imaging Nuclear imaging involves positron emission tomography (PET) and single-photon emission computed tomography (SPECT). These techniques use radioactive substances linked to biological targeting ele­ ments that are introduced into the patient’s body. While the gamma rays are emitted and detected by gamma camera detectors in SPECT, photons produced as a result of positron-electron reaction are measured in PET, both to create three-dimensional images to detect subtle molecular changes and determine if a tumor is benign or malignant. Traditionally used radiotracer molecules such as 64Cu are usually stabilized with macrocyclic chelators that are potentially toxic and cause unwanted radiation burden [51]. In comparison, the recent use of gold nanoparticles in combination with the conventionally used radiotracer molecules such as 64Cu has helped improve their stability and biodistribution along with systemic clearance, greatly reducing the potential toxicity [52]. For instance, Zhao et al. synthesized

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pegylated 64Cu alloyed gold nanoclusters (64CuAuNCs) for PET imaging in a mouse prostate cancer model that showed significant renal clearance with low non-specific tumor retention evidencing that it can passively target the prostate cancer [53]. The same group later modified the probe by fuctionalization of AMD3100ligand for active targeting for PET-based detection of primary breast cancer and lung metastasis [54].

17.4.1.4 Fluorescence imaging (FI) Fluorescence imaging (FI) utilizes fluorophores to visualize molecular signatures/processes specific to cancer to improve the early detection of neoplasia. In comparison to other imaging modalities such as MRI and CT, it is much safer, portable, relatively inexpensive, and can be used for real-time imaging [55]. Different sources such as fluorescent dyes, quantum dots, gold nanoparticles, etc. have been used as fluorophores for FI. The surface plasmon resonance (SPR) property of gold nanoparticles has been widely used in conjunction with the fluorophores to enhance their fluorescence, commonly known as plasmon-enhanced fluorescence (PEF). The maximum enhancement occurs when there is an overlap between the SPR of gold nanoparticles and the molecular absorption and emission spectra of the fluorophore [56]. For instance, Ayala-Orozco et al. synthesized nanomatryoshk, an Au@SiO2@Au multiple-shelled structure and placed near-infrared (NIR-1) dyes Cy7 and IR800 in between the gold core and shell [Figure 17.3(ii)]. The group reported fluorescent enhancements by ~16 times as a result of strong interaction between the plasmon resonances of the gold core and gold shell such that hybridized modes are created in the NIR [57]. Also, gold nanoparticles by themselves start exhibiting photo­ luminescence, when reduced to the sub-nanometer scale, including nanocrystals and nanoclusters [58]. In fact, a study by Liu et al. showed that glutathione stabilized gold nanoclusters (~2.5 nm) are more suitable than IRDye 800CW for rapid detection of tumors owing to their longer retention time and faster renal clearance, indicating superior signal-to-noise ratio for FI [59].

17.4.1.5 Photoacoustic imaging (PA) Photoacoustic imaging (PA) uses laser-generated ultrasound waves that are detected and analyzed to produce images. It provides real-time information about the anatomical, functional, and molecular content of the diseased tissues with a high spatial resolution [60]. It is advantageous over other imaging techniques as the radiation used is non-ionizing and it has a greater penetration depth (up to 5–6 cm) due to lower scattering of ultrasonic signals in tissue than light. Gold nanoparticles have been explored as contrast agents for PA owing to their strong and tuneable optical absorption as a result of the SPR effect. The ability to precisely control their shape and size during synthesis to exploit their absorbance over a specific wavelength range, for instance, biological window (650–1,100 nm) or NIR spectral window (1,100–1,350 nm) has allowed the targeting of areas deep within the tissues. For instance, Li and the group used PEG-coated hollow gold nanospheres (HAuNS) as a contrast agent for the photoacoustic tomography of the mouse brain vasculature [61]. The SPR of the fabricated gold nanospheres tuned at the NIR wavelength (∼800 nm) allowed deeper penetration of laser with low intrinsic background noise and provided high-resolution images of the mouse brain vasculature with capillaries detected of diameter as little as 100 μm [61]. The use of other nanoparticle shapes such as gold nanocages in PA imaging has also been reported for the detection of B16 melanomas [62] and U87 brain tumors [63].

17.4.2 Biosensing Biosensing systems have recently gained a lot of attention to detect cancer biomarkers through liquid biopsy. Liquid biopsies involve the analysis of specific cancer biomarkers in non-solid tissues such as blood and are non-invasive, relatively fast, and patient-friendly compared to routine tissue biopsies. The

308 Nanomaterials in Healthcare different cancer-specific biomarkers that are analyzed via liquid biopsy include proteins, circulating tumour cells (CTCs), cell-free DNA (cfDNA), mitochondrial DNA (mtDNA), microRNA (miRNA), exosomes, vesicles, etc. Biosensing systems are analytical devices that integrate bioreceptors, trans­ ducers, and signal detectors to measure these cancer biomarkers. Bioreceptors such as enzymes, anti­ bodies, nucleic acids, etc. interact with the target biomarkers to generate signals via transducers that can be optical, electrochemical, piezoelectric etc. Finally, the generated signals are measured and provided as a digital output. The use of gold nanoparticles in the classical biosensing systems has allowed highly sensitive biomarker testing owing to its different physicochemical and optical properties such as light absorption and scattering, localized surface plasmon resonance (LSPR), surface-enhanced Raman spectroscopy (SERS), stability, etc. In optical biosensing systems, gold nanoparticles are generally used as nanocarriers, allow easy functionalization of bioreceptor molecules, and produce signals based on LSPR, SERS, or chemilum­ inescence (Table 17.2). An LSPR biosensor for the detecting prostate-specific antigen (PSA), an earlystage prostate cancer biomarker, was developed by covalently immobilizing anti-PSA antibodies onto the gold nanoparticles (probe) [65]. The binding of the analyte (PSA) to the probe resulted in small changes in the dielectric medium around the probe which further led to a shift in the LSPR peak. The fabricated biosensor was highly sensitive and could achieve a limit of detection of 0.2 ng/mL [65]. Trau and group fabricated a multiplexed extracellular vesicles phenotype analyzer chip (EPAC) that integrates the nanomixing-enhanced microchip with the multiplexed SERS nanotag system [66]. Gold nanoparticles functionalized with specific Raman reporters and antibodies were used as SERS tags to detect four melanoma biomarkers in EVs that are associated with melanoma progression, namely, melanoma chondroitin sulphate proteoglycan (MCSP), low-affinity nerve growth factor receptor (LNGFR), receptor tyrosine-protein kinase (ErbB3), and melanoma cell adhesion molecule (MCAM). Complex biological fluids were directly analyzed to detect low-abundance, tumor-specific EVs without requiring EV puri­ fication and enrichment. SERS mapping was used to read signals and generate SERS spectral image based on the characteristic peaks of the different Raman reporters used. The observed signal intensity corresponded to the number of EVs and expressed biomarker levels [66]. In electrochemical biosensing systems, gold nanoparticles are generally used as a suitable electrode material (either alone or in composite with other conducting materials) because of their high chemical stability, outstanding electrical conductivity enhancement, and excellent catalytic ability [52]. They can also be employed as labels for further amplification of the electric signal by mediating efficient electron transfer. Bare gold nanoparticles were developed for labelling and electrochemical detection of breast cancer gene BRCA1 [67]. They exploited the unique interaction of gold nanoparticles with ssDNA immobilized on an electrode surface for enhanced electron transfer that reduces in the presence of target DNA. The target DNA binds to the ssDNA making it a dsDNA to which gold nanoparticles cannot adsorb anymore and thus the enhancement in charge transfer is altered. The signal difference is thus monitored to quantify the target DNA and a limit of detection of 1 pM could be achieved [67]. There are numerous studies that employ gold nanoparticles-based optical and electrochemical biosensing systems for the detection of circulating cancer biomarkers. However, their analytical performances are still in question to accurately diagnose early-stage cancer. Additional research is necessary to employ these biosensing systems in clinical applications.

17.5 GOLD NANOPARTICLES AS THERAGNOSTIC AGENTS More recently, multifunctional gold nanoparticles have been incorporated as agents for simultaneous diagnosis and therapy, an integrated field called theragnostics [68]. The term theragnostics implies

PSA—ACT AFP AFP

PSA—ACT mAb Antihuman AFP—MBA—GNPs Capture antibody/target AFP/ fluorophores labeled secondary antibody Capture antibody PSA (PSA 10), tracer antibody PSA (PSA 66) First antibody/AFP/GNP conjugated secondary antibody MaCEA. polyclonal rabbit anti-CEA IgG. goat anti-rabbit IgG conjugated to 1R800 Anti-AFP antibody Gold nanostar conjugated with Raman label malachite green and detection antibody. capture antibody Anti-PSA antibodies. alkaline phosphatase labeled secondary antibody PSA mAb PSA mAb Anti-HER2 primary antibody. antirabbit IgGs secondary antibody f-PSA PSA—ACT HER2

PSA

AFP VEGF

CEA

AFP

PSA

VEGF 165 PSA—ACT

ANALYTES

VEGF 165 aptamer PSA—ACT mAb

PROBE MOIETIES

100 fg/mL I aM

25 ng/mL

0.2 ng/mL in PBS and 2 ng/mL in bovine serum 5 fM

I pg/mL

0.1 pg/mL 100 pg/mL 0.1 ng/mL

I pg/mL

LIMIT OF DETECTION

Optical fiber Glass Glass

Glass

Quartz slides Glass

Glass

Optical microfiber

Optical fiber

Glass Glass Optical fiber

Colloidal Colloidal

SUBSTRATE TYPE

LSPR LSPR SERS

LSPR

LSPR SERS

SEF

LSPR

LSPR

Rayleigh light scattering SERS LSPR

SEF LSPR

DETECTION MECHANISM

Abbreviations: GNP, gold nanoparticle; VEGF, vascular endothelial growth factor; SEF, surface-enhanced fluorescence; PSA, prostate-specific antigen; PSA—ACT, PSA complexed to R-1-antichymotrypsin; PSA—ACT mAb, PSA—ACT monoclonal antibody; LSPR, localized surface plasmon resonance; MBA, mercaptobenzoic acid; AFP, alpha-fetoprotein; SERS, surface-enhanced Raman scattering; MaCEA, monoclonal mouse anti-CEA IgG; CEA, carcinoembryonic antigen; GNR, gold nanorods; f-PSA, free PSA; HER2, human epidermal growth factor receptor 2; Au@Ag NRs, Gold@silver core-shell nanorods; HE4, human epididymis secretory protein 4; GNS, gold nanosphere; IgG, immunoglobulin G.

Gold nanodisk array GNR GNR

Elliptical Au nanodisk arrays

Gold film island Au triangle nanoarray

Gold-on-gold films

GNPs

Spherical GNP

GNP GNP/PSA—ACT/ magnetic microbeads Spherical GNP GNPs GNS

NANO-STRUCTURE TYPE

TABLE 17.2 Gold nanoparticle-based plasmonic biosensors for cancer diagnosis [ 64]

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310 Nanomaterials in Healthcare therapeutics and diagnostics through a single nanoparticle system. Gold nano stars linked to FeSO3 nanoparticles and targeted to cancer cells via quantum dot-antibodies showed diagnostic potential in MRI and fluorescence imaging while simultaneously being used for image-guided photothermal therapy [69]. Doxorubicin-loaded radio-labeled gold nanoparticles were used for PET scanning as well as chemotherapy in cervical cancer [70]. Combined chemo-photothermal therapy for breast cancer along with multi-modal imaging (CT, photoacoustic) was reported with mayransise, mPEG, and trastuzumab conjugated gold nanoshells [71]. Gold nanoshells coated with superparamagnetic iron oxide and decorated with anti EGFR antibodies were used for MRI-based detection and treatment of oral cancer. Gold nanoparticle-loaded liposomes were examined for trimodal therapy viz PDT, PTT, and chemotherapy as well as bimodal imaging viz. fluorescence and computed tomography through a single system [72]. F. Silva et al. recently reviewed radiolabelled gold nanoparticles for cancer theragnosis [73].

17.6 CLINICAL STATUS OF GOLD NANOPARTICLE FORMULATIONS AuroLase therapy, consisting of PEGylated Au nanoshells of diameter 150 nm, with absorption max in the range of 780–820 nm were administered intravenously for treatment of primary as well as metastatic lung cancer [74]. This formulation was also tested clinically for treatment of recurrent and refractory head and neck cancer. Phase 1 clinical trials of PEGylated gold-TNF nanoconjugate (Aurimune) showed excellent results in patients with advanced stage cancer with only mild side effects like fever. Traces of the nanoparticle were detected in liver and tumor region without any adverse effects on patients. Hence, it has progressed to Phase 2 clinical trials [75]. Another silica-gold nanoformulation called Auroshell was tested on 22 men with prostate cancer. Only minor side effects of itching and burning sensation were reported and overall excellent tolerability was observed in humans [76]. Photothermal therapy with NIR laser in combination with MR/ultrasound imaging showed promising tumor ablation potential in 87.5% patients in a period of 1 year [77]. Nano Swarna bhasma composed of gold nanoparticle and ayurvedic phytochemical compound mangiferin was tested for human metastatic breast cancer therapy [78]. A gold nanomaterial-based breath analyzing nano­ sensor for indirect detection of gastric cancer by detection of other gastric diseases was developed and is currently under clinical trial [79]. Despite these advances, no GNP formulation has been success­ fully used for diagnosis or treatment of cancers.

17.7 SAFETY CONCERNS AND CHALLENGES FOR APPLICATION OF GOLD NANOPARTICLE IN HEALTHCARE Despite decades of research, toxicity and biocompatibility remain the most challenging for GNP application. Being a noble metal gold is inert in the bulk state but toxicity must be evaluated at the nanoscale. Toxicity can be attributed to mainly size and surface modification. Biological processes like endocytosis, localization and accumulation are size dependent; hence, important in determining tox­ icity [80]. Chemicals used in the synthesis of gold nanoparticles render the final particle toxic due to the bound or free excess chemical residues. CTAB used in gold nanorod preparation and sodium citrate used as a reducing agent in gold nanoparticles are known to cause cellular toxicity. Repeated washing, coating with PEG or phosphotidylcholine significantly reduce the toxicity [81]. To evaluate the impact

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of particle shape on the toxicity of gold nanoparticles, Wang et al. synthesized different-shaped gold nanoparticles i.e., rods, cages, and hexapods and modified them with polyethylene glycol (PEG). In vitro cytotoxicity studies on MDA-MB-435 cells, after incubation of 48 hours revealed that hexapods were least toxic in the case of unmodified as well as PEGylated nanoparticles [82]. In vivo toxicity of gold nanoparticles is influenced by dose, route of administration, particle size, surface charge, metabolism, and excretion. Farhat et al. evaluated the systemic bio-distribution and toxicity of gold nanoparticles and found gold to be accumulated mostly in spleen. Serum levels of creatinine, alkaline phosphatase, aspartate aminotransferase, bilirubin, alanine aminotransferase, urea, uric acid, calcium, glucose, albumin, tissue histological studies, weight, hair color, and occurrence of mortality revealed no significant toxicity for a period of 90 days indicating that gold nanoparticles are safe to use for biomedical applications [83]. Surface charge too plays a role in determining toxicity as negatively charged nanoparticles have been found to be less toxic that positively charged. Surface modification like PEGylation and attachment of targeting moieties as well as drug loading also changes the toxicity of nanoparticles and these changes could be different for different mice species. Choice of animal models, biodistribution of nanomaterial and related toxicity, dosage, and route of administration and heterogeneity among tissues and cells lead to diverse results which are difficult to comprehend. There is also a lack of universal standardized toxicity evaluation method so that data from different studies can be compared. Before clinical translation, it is also important to determine the cost of production of nanoparticle formulations. Addition of drug, targeting ligands, antibodies, and imaging agents offers advantages but increases the cost of production. Hence, the trade-off between advantages and cost needs to be evaluated. Gold nanoparticles can offer both therapy (PDT, PTT) and imaging (CT) without any additional moieties and hence are in the forefront for several translational studies.

17.8 CONCLUSION Cancer continues to affect quality of life and mortality rates in patients worldwide. While taking full advantage of available therapeutic options like surgery, chemo and radiotherapy, there is a need for research on new treatment modalities. Newer anti-cancer drugs are being tested and alternate therapies like PTT and PDT are being researched. Due to the complexity of pathophysiology in cancer and the unique physicochemical properties of gold nanoparticles they are being extensively researched for application in both cancer diagnosis and therapy. They are being used as drug carrier vehicles to reduce side effects and dosage, they are being tagged with targeting moieties for specific and selective drug delivery. Apart from drugs gold nanoparticles also deliver peptides and nucleic acids and hydrophobic photosensitizers. Gold nanoparticles are a promising nanoplatform for cancer theranostics by combining therapeutic and diagnostic moieties in a single particle. Also, there is a possibility of combining therapies like chemotherapy, radiotherapy, photodynamic therapy, and photothermal therapy for synergistic effect. Apart from the extensive in vitro and in vivo pre-clinical research on gold nanoparticles, promising results have also been observed in clinical trials. But most studies are in phase 1 clinical trials and none of the products have been marketed. Also, a number of clinical trials of gold nanoparticles remains small in comparison to liposome and polymeric nanoparticles. The main limitations are due to biodistribution, toxicity, and stability of gold nanoparticles that can be attributed to either the core nanoparticle or the modifications. Hence, cost effective and stable nano formulation that can show optimum imaging and therapeutic ability is desired. Although many improvements are needed before gold nanoparticle for­ mulations can be marketed, the current research data proves that on addressing the current drawbacks, gold-based nanoparticles will play key role in all stages of cancer theranostics from early detection, solid tumor ablation, to metastatic cancer treatment.

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Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment

18

Pallavi Kiran, Vibha Kumari, Baishali A. Jana, and Prachi Kulkarni Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Contents 18.1

Introduction 18.1.1 Self-assembly of Peptide 18.1.2 Targeting Peptides 18.2 Peptide-Based NPs in Cancer Therapeutics 18.2.1 Peptide-Based NPs for Gene Delivery/Cytotoxic Drug 18.2.2 Peptidomimetics with Chemotherapy 18.2.2.1 Peptide hormones-based drug conjugates 18.2.2.2 Peptide-based NPs vaccines for immunotherapy 18.3 Peptide-Based NPs in Cancer Theragnostics 18.3.1 Targeting Peptides 18.3.2 Environment Responsive Peptides 18.3.3 Cell-Penetrating Peptides (CPPs) 18.3.4 Peptide Receptor Radionuclide Therapy (PPRT) 18.4 Peptide-Based Nanoparticles 18.5 Cell-penetrating Particles 18.6 CPPs: Protein Delivery in Cancer 18.7 Conclusion and Future Prospects References

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DOI: 10.1201/9781003322368-18

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18.1 INTRODUCTION Globally, cancer is one of the leading causes of death. Current treatments of cancer have noteworthy adverse effects and lack effective elimination from progression of the disease [1,2]. Synthesis of multidrug resistance-reduced therapeutic ability of targeting and insufficient cellular uptake are some of the major limitations [2,3]. These can result in elevated likelihood and reduced efficacy of metastatic dis­ ease. Increase in treatment efficacy while circumventing off-target side effects are vital factors to develop more advantageous therapeutics as well as a better system for therapeutic drug delivery [2,4]. Nanomaterials ranging from 10 to 100 nm in size, have novel physico-chemical attributes differing from those of conventional bulk materials. Mainly, their size, which is ultra-small and surface-to-volume ratio, which is very high, provides advantages in production of bioengineered materials that can thereby assist in interaction with different micro- and nano-sized biomaterials. To fabricate peptides-based nanoparticles, the direct approach can be self-assembly. On the contrary, in the thermodynamic process, spontaneity does not allow formation of nano-sized constructs with precise and regulated compositions, size, and shape. Conjugation of peptide-NP offers better control in terms of structural attributes of nanostructures, which allows superficial modification to overall dimension, size, and shape of the conjugates by engineering nanoparticle scaffolds that are altered for particular applications [5]. Due to the variety of functions offered by peptides, the nonviral carrier has swiftly achieved popularity and exposure as a potential delivery vehicle [2,6]. Peptide comprises of amino acids which have the ability to assemble on their own into a variety of nanostructures (structure with more than one dimension, usually measured in nanometre range between 10–9 m). They exhibit good biological activity and biocompatibility because of which they are very advantageous. Significantly, peptide-based nanoparticles being adaptable and versatile in nature have a wide application in the arena of material sciences. However, orderly structured supramolecules (molecular assembly and intermolecular bond present between the molecules) are considered fascinating “bottom-up” biomaterials, which can have applications in the fields of nanomedicine and nano­ technology. Remarkably, the peptide-based nanoparticles are fascinating and significant due to varying reasons, enlisted below: A. Peptides that are produced by the method of solid-phase can be altered at molecular level, resulting in peptide-based nanomaterial with adjustable properties. B. Additionally, functionalization for peptide-based nanomaterials can be done by introduction of exterior molecules like enzymes, to the existing peptide nanostructure. C. The process of self-assembly can be designed by altering the building blocks of secondary structures of peptides which are α-helices and β-sheets [7].

18.1.1 Self-assembly of Peptide Occurrence of self-assembly can be decided by dissolving peptide in a solvent, which usually adopts a special conformation. Secondary structures that are preferred for self-assembly of peptides are β-hairpins, α-helices, and β-sheets. Out of these, in proteins, α-helix is the key secondary structural motif. It is because of its intrinsic thermodynamic instability, that linear-shaped peptides having an α-helix eventually lose their helical conformation when added in solution after their separation from their indigenous environment. Hence, it is very crucial to stabilize α-helix in order to trigger self-assembly of peptides. Some basic approaches for stabilization of α-helix are formation of salt-bridge, hydrogen-bond surrogates, metal-coordination, and cross-coupling of side-chains [7]. Assemblies of peptide molecules can act as better nanoscale ranged scaffolds in comparison to individual peptides and the vital difference between assemblies of peptide molecules and individual

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 319 peptide is diffusivity. For instance, diffusion of peptide assemblies is slower than monomeric peptides. The formation of peptide assemblies is controlled by managing the diffusive kinetics of peptide monomers [8]. The conception of EISA (enzyme-instructed self-assembly) is based on this feature of diffusivity and means that peptide assemblies in an aqueous solution can be yielded by bond cleavage or formation. In biological terms, formation and transformation of vital molecules is usually catalysed by enzymes under certain physiological factors. Additionally, the variation in expression levels of enzyme correlates with the occurrence of diseases. EISA might occur within the abnormal cells or at the location of disease. Henceforth, use of EISA for triggering in situ self-assembly of nanomaterials and treating of disease is feasible [7,9]. Biomacromolecular condensates could be mimicked by the assemblies of peptides synthesized by EISA in the cellular conditions. Molecular self-assembly controlled by enzymes play a pivotal role in many cellular processes, which triggers synthesis of small-sized molecule enzymatic hydrogelation. Enzymatic hydrogelation of small molecules exists in three steps, mentioned as follows: Firstly, the precursor is converted by enzyme into hydrogenator via cleavage of bond. Then the hydrogenator self-assembles itself into nanofibers and lastly entangles to perform as hydrogel matrix. Additionally, the small molecules also have the ability of self-assembly to become supramolecular hydrogels. They exhibit three features: • Within the nanofibers, molecular arrangement demonstrates proper orderliness, even though the nanowires are entangled randomly; • Within the nanofibers, molecular order can be replenished by simple structural alteration of small molecules; • Small-sized molecules can be easily transformed into hydrogelators. [7] By introducing o-[bis(dimethylamino)phosphono] tyrosine molecules as protection strategy in the motif of self-assembly, the programmed precursor can resist the process of hydrolysis by phosphatases outside and inside of a cell, since the enzymatic cleavage site exposure occurs particularly in the lysosome’s acidic environment. Here, the acid phosphatase commands hydrogel’s self-assembly spatiotemporally in the lysosome [10]. The study of factors that impact self-assembly of peptides is important for designing required peptide-based nanostructures. These can be subdivided into intrinsic and extrinsic factors (Figure 18.1).

18.1.2 Targeting Peptides For synthesis of targeting peptides, liquid or solid phase is utilized as an advanced technique for fab­ rication. Earlier, the most commonly used technique was solid-phase synthesis. It is based on the dis­ solution of the group that shields the carboxyl group that is exposed. Solid-phase synthesis is only performed if the shielding group is not soluble inside the synthesis-reaction medium. However, synthesis of solid-phase can be attained by attachment of first amino acid to the resin, which is usually 1–2 % divinylbenzene-cross-linked polystyrene followed by the peptide elongation to its ultimate product [2,11]. Varieties of resins can preferably be used to immobilize the first amino acid molecule of a structured peptide block, which can then be expanded through a side-chain backbone like C-terminus and N-terminus. After the development of targeting peptides, they can be combined with the nanoparticles carrying therapeutic agents or can conjugate with it, which can be achieved with the help of linkers. Usually cleavable linkers are used, as they can release drugs after reaching the targeted site [2]. They can be categorized into two vital groups namely; enzyme-cleavable and chemically cleavable. Cleavable linkers can function as pro-drug conjugate activators, which causes activated release of chemotherapeutics at their targeted site. However, as per a previous study, when daunorubicin was combined with residues of leucine, they were responsible for TME (tumor macro-environment) retention, liner cleavage, and accumulation. It was then dissociated through aminopeptidase cleavage and distributed to leukemia cells,

320 Nanomaterials in Healthcare

FIGURE 18.1 Intrinsic and extrinsic factors affecting self-assembly. Referred from [ 7].

which showed elevated levels of anti-cancer effects and TME accumulation as compared to intra­ venously delivered free daunorubicin to tumor-bearing mice [2,4]. This book chapter focuses on peptide-based nanoparticle-based drug delivery approaches as an emerging and potential field for theragnostic effect of cancer. (Figure 18.2)

FIGURE 18.2 The schematic representation showing various examples of the peptides-based nanoparticles used for both cancer theragnostics and therapeutic applications of various tumor targeted treatment.

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 321

18.2 PEPTIDE-BASED NPS IN CANCER THERAPEUTICS 18.2.1 Peptide-Based NPs for Gene Delivery/Cytotoxic Drug Peptide-based NPs are in high demand for self-assembling nanostructures in a building blocks form with varied chemical structure and simple methodology. They have huge advantages due to skills to react in biological environment to the causing changes for developing peptide-based nanomaterials. In one literature, therapeutic peptide assembled nanoparticles i.e., the nanoformulation was synthesized for stimuli responsive delivery by combined amphiphilic peptide- 3-diethyl aminopropyl isothiocyanate (DEAP) molecule, a peptide substrate (MMP-2) matrix metalloproteinase-2 and antagonist peptide diphenyl phosphorazidate (DPPA-1) and indoleamine-(2,3)-dioxygenase (NLG919) [12,13]. 3-diethylaminopropyl isothiocyanate (DEAP) molecule protonation and the peptide substrate cleavage showed release of DPPA-1 and NLG919 in a controlled manner thus enhancing the antitumour immune response with minimal toxicity. Here the drug stability and bioavailability was also improved. In another literature, doxorubicin (DOX) was selected as prodrug nanovehicle using dendritic arginine-rich peptide which activated the tumor microenvironment delivery (TME) across the tumor and cell barriers. This self-assembled nanoparticle once stimulated in the weakly acidic pH of tumor tissue demonstrated excellent penetration; thus, showing effective results in SKOV3/R ovarian cancer bearing nude mice mainly extending the blood circulation and efficient tumor suppression. P51 is considered as a multifunctional peptide combined with nanoparticle-loaded pirarubicin, which showed excellent stimuli responsive drug delivery consequently reducing the systemic toxicity and higher tumor inhibition. Novel research has shown that highly based cationic peptide nanovehicle, a dual nanoparticle system combining cationic peptide nanocomplex and plasmid DNA containing tumor necrosis factor (TNFα); further encapsulated in polylactic acid- polyethylene glycol combined (PLA-PEG) poly­ meric nanoparticles thus providing serum stability, preventing nanocomplex degradation, and leading to effective anti-tumor movement due to the cytokine expression of the TNFα for cancer therapy [14].

18.2.2 Peptidomimetics with Chemotherapy Synthetic molecules are peptidomimetics that are designed mainly to imitate a natural peptide or a protein. The limitation such as poor bioavailability and proteolytic stability has been improved by designing the natural peptides preserving the capability to interact with envisioned targets that are biological. These can be produced by altering the prevailing natural peptide or designing structurally the same systems like peptoids [15,16]. USFDA have approved some prominent peptidomimetics such as bortezomib, carfilzomib, and ixazomib as single agents for cancer therapy. These three are also approved in combination for cancer therapy with lenalidomide with DOX and with dexamethasone. Several studies have shown enhanced efficacy in combination with nanoparticles and approved peptidomimetics. Zein nanoparticles is currently being explored; a dual-loaded nanoparticles using bortezomib and histone deacetylase inhibitor vorinostat showed improvement of threefold in tumor inhibition for treating metastatic prostate cancer. Studies also showed a good synergistic action in in-vitro lung cancer model encapsulating carfilzomib and survivin siRNA in mesoporous silica nanoparticles [17]. Peptidomimetics seems to be an attractive option in combination therapy for future research due to their substantial number of these and their combinations are approved by the FDA for the treatment of cancer. These combination-based nanoformulations proved enhanced tumor inhibition, biodistribution, and effective holding potential for rapid clinical translation in cancer treatment [18–20].

18.2.2.1 Peptide hormones-based drug conjugates Overexpressed markers such as bombesin; luteinizing hormone releasing hormone (LHRH); and somatostatin of prostrate, ovarian, and breast carcinoma, are being explored based on the peptide

322 Nanomaterials in Healthcare hormones for targeted therapy. Peptide hormones are prepared up of polypeptides ranging from three to few hundred amino acids in length released by pituitary glands. The main peptide hormone approved for an anticancer therapy mainly for prostate cancer treatment was a LHRH agonist. Several other LHRH agonist and antagonists have been accepted by FDA for breast and prostate cancer such as leuprolide, nuserelin, and goserelin and antagonists like cetrorelix, degarelix, and abarelix advances treatment. Many researches have shown excellent targeted therapy conjugating both cytotoxic or radionuclide drugs and these peptides hormones [21–23]. However, many experiments and clinical trials are ongoing for achieving successful targeted therapy for cancer treatments using these peptide hormones. In 2018, lutathera, a peptide hormone antagonist, has set a prominent example; it is approved by the FDA and has been related to a radionuclide mainly a combination radiotherapy in somatostatin receptor tumors. Low solubility drugs and biomolecules with low stability problems can be solved and have shown positive possibilities using nanoparticles in peptide hormone conjugates systems. Lipid solid nanoparticles for melanoma, PEG nanoparticles for ovarian cancer, and others conjugated with peptide hormones have shown significant tumor targeted delivery and better antitumor efficacy as compared to only drug delivery alone. Still in-depth studies are required to prove the potential in the nanoformulation form for combinatorial cancer therapy [24,25].

18.2.2.2 Peptide-based NPs vaccines for immunotherapy Peptide vaccines in the form of nanoparticles act as non-viral delivery vectors that are currently being explored widely by pharmaceutical companies to develop and solve mainly the confined essentials in peptides for betterment of future and targeted delivery of drugs [26,27]. The current trend of formulating nanoparticles looks promising for peptide vaccine delivery has many advantages and also prevents further degradation thus increasing the opportunity of also aiming dendritic cells that are antigen pre­ senting cells for overall refining the efficiency of the vaccine. In one research, the design of the for­ mulation of bi-adjuvant neoantigen nanovaccine; consisting of two synergistic adjuvants, e.g. toll-like receptor 6 (TLR6) and nod-like receptor (NLR) and peptide neoantigen Adpgk in the same nano­ formulation stimulated both adaptive and innate immunity in a colorectal mouse model. Significant tumor inhibition was observed where neovaccine enhanced the neoantigen specific cytotoxicity Tlymphocyte when used in combination with anti-PD1. ADP dependent glucokinase (Adpgk) neoantigen is very commonly used for a combination immunotherapy-based nanovaccine for the treatment of colorectal cancer. In another literature, curcumin-loaded nanoparticles were formulated along with neovaccine consisting of unmethylated cytosine–guanine dinucleotide (CpG) oligodeoxynucleotide, antigen epitope peptide E75, and self-assembled cationic peptide in a thermo-responsive injectable hydrogel form was performed on postoperative breast carcinoma mice model. The combination results showed significant increase in T-cell response, immunogenic cell death thus preventing recurrence and lungs metastasis. Thus, this area of nanoparticles-based research shows attractive substitute of peptide vaccines and their combinations for drug delivery [28,29]. Though many preclinical studies proved the decrease in the local tumor reoccurrence; the limitation in the field of clinical translation for optimization of the antigen presentation and provides adequate immune response that still needs to be explored more.

18.3 PEPTIDE-BASED NPS IN CANCER THERAGNOSTICS 18.3.1 Targeting Peptides To provide safe and effective treatment mainly in precision medicine and modern nanomedicine, specific site targeting plays a vital role and is a very basic need. In comparison to the healthy tissue,

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 323 lesions i.e., the tumor sites, have the tendency in showing prominent different characters. Peptide library screening technologies had mainly discovered the targeting peptides and its various kinds for cancer treatment. The high affinity and targeting capability were mainly ensured by this screening process. Targeting peptides are based on nanoparticles (NP) consisting of three major types, cell targeting, subcellular organelle targeting, and lesion microenvironment targeting (cell nuclei, en­ doplasmic reticulum, mitochondria, etc.). Cancer possesses a distinct mechanism leading to the tumor microenvironment including some markers and receptors being overexpressed. The overexpressed receptors and marker include the asialoglycoprotein receptor (ASGPR), integrin receptor, epidermal growth factor receptor (EGFR), neuropilin-1 (NRP-1) receptor, transferrin receptor (Tf-R), protein tyrosine phosphates receptor type J (PTPRJ), vascular endothelial growth factor (VEGF), low-density lipoprotein receptor-related protein 1 (LRP1), the insulin-like growth factor 1 receptor (IGF1R), etc. During the past decades, a screening technique was developed where peptides precisely bind to these markers and receptors that can be immensely used for tumor-targeted diagnosis and treatment. Currently, a large quantity of receptor targets detected in cancer vasculatures are precisely expressed or overexpressed. Peptides have constantly shown excellent biocompatibility and diversity compared to other targeting ligands [30]. The subclass in the cellular cytoskeleton of cell adhesion molecules are the integrins that play a key role in targeting the cancer cells, vastly expressed on various endothelial cancer cells and tumor new blood vessels including breast, glioblastoma, melanoma, ovarian, and prostate cancer cells. Adhesive interactions take place by mediation mainly through integrin between cells and extra­ cellular matrix. Alpha-v beta-3 (αVβ3) and αVβ5 integrins are the common integrins that are used for anti-angiogenesis targets and others are used for therapeutic targets. The widely used target ligands are a membrane-bound enzyme associated with angiogenic tumor vessels i.e., aminopeptidase N (CD13), which is also the class of peptide containing asparagine–glycine–arginine (NGR) sequences are been explored in the form of nanoparticles with drugs for targeting deep tumor tissues; another one known for antitumor homing peptides are vascular endothelial growth factor receptor 1 (VEGFR1/Flt-1) and vascular endothelial growth factor receptor (VEGFR-3) and are commonly tumor-related blood vessel endothelial cell targets, which help the high internalization in the form of nanoparticles leading to accurately targeting. For example, the colorectal cancer neovasculature considerably elongate with no obvious toxicity of the mouse survival. F56 peptide binds with VEGFR-1 showing high affinity furnished similar results; a new tumor-vessel-targeting nanoparticle form loaded with vincristine for colorectal cancer treatment. KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK peptide, well known as F3, is usually grafted onto a nanoparticle surface and is a 31 amino-acid peptide; thus enhancing in the nanoparticles the tumor angiogenesis [31,32]. This peptide is considered for those drug-resistant cancers that require metabolic activation, conjugated for chemo-photodynamic com­ bination therapy in a nanoparticle form, and thus have shown to exhibit at the tumor site more favored enrichment and prolong existence time. Another known peptide i.e., Esbp peptide (DITW­ DQLWDLMK) mainly binds to E-selectin; both have high affinity and when equipped with a chemotherapeutic drug such as doxorubicin drug (DOX) in a nanoparticle form have shown excellent results in in-vivo model; more significantly reducing the Lewis lung carcinoma tumors’ growth in mice rather than only free DOX. Targeting tumor cells requires sufficient strategies; a general rule is that expression of the targeted protein should have at least threefold increase present on the cancer cells compared to normal cells to deliver the anticancer agents, thus delivering the targeted therapeutic dose in a proper period of time with less or nil toxicity. One of the commonly known cell membrane protein targets is epidermal growth factor receptor (EGFR), mainly overexpressed in several tumors including breast cancer cells of ductal or lobular origin. EGFR contains four members: EGFR (ErbB1, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). In regulating differentiation, cell proliferation, survival, and migration, these receptors play vital roles [33,34]. However, the highly expressed especially in some breast cancers and in many cancer cells other than the three HERs is HER2. Peptide-drug conjugates and antibody-drug

324 Nanomaterials in Healthcare conjugates are known cell targeting peptides (CTP). Among them, peptide-drug conjugates have many therapeutic advantages. Phage display library screened GE11 (YHWYGYTPQNVI) are being widely explored for photodynamic therapy, breast cancer treatment, and radioactivity study. Many studies hence proved that GE11 show a high affinity to effectively target EGFR expressing tumor cells, thus enhancing the antitumour efficacies and specificities in targeting and treating many different cancer cells. Transferrin receptor (Tf-R), a transmembrane protein, VEGFR-1 and VEGFR-3; in humans glioblastoma has the main gene expression ratio than normal cells, CD44 specific peptides binding CD44 showed high affinity and specificity on the surface of many cancer cells and tissues including gastric cancer cells and Pep42 located at the plasma membrane of malignant cancer is a member of heat shock protein family; conjugated containing DOX drug identified to show high efficiency targeted delivery for lung cancer treatment; are likewise another equitable target widely used for antitumor homing peptides. Apart from these, many other peptides have been also introduced, discovered, and explored by scientists onto the surface of varied nanoparticle formulation like silica, metal, liposomes, ruthenium-loaded selenium nanoparticle (SeNP), gold, and other theragnostic nanomaterials for intranasal delivery and cancer initial diagnosis and therapy [34].

18.3.2 Environment Responsive Peptides Environment responsive, especially the physiological tumor microenvironment, plays a very important role. In nanoparticle formulation, all these factors are taken care of. The environment-responsive factors involve enzyme concentration, partial pressure of the oxygen, pH, redox, and native tissue temperature [35,36]. Peptide development mainly responds to many factors such as hypoxia condition, high redox gradient, pH variation, and enzymes that are upregulated. These circumstances play a vital role for detailed diagnosis and drug release in a controlled manner responding according to the bond cleavage and structural changes. However, for cancer therapy, the perfect targets and precise drug release are mostly lipases, proteases, and phosphatases. Matrix metalloproteinases (MMPs) are responsive peptides discovered by peptide library screening methods that have been introduced into theragnostic nanoma­ terials and these can be used for triggering the signal change and drug release. These are mainly a zincdependent endopeptidases family that degrades protein during cancer invasion process and metastasis. In one of the literatures, for imaging, gastric tumor, and ablation, the MMPs were loaded onto the lipo­ somes with photothermal nanodisks that were inorganic in nature. MMPs had also been used for pho­ tothermal therapy as imaging guides [37]. Cysteine cathepsin proteases are also a similar form used for imaging in photodynamic therapy. This also helps in the release of DOX leading to apoptosis of cancer cells and significant antitumor efficacy. pH-responsive peptides could efficiently enter the cancer cell targeting the tumor environment and release the drug for tumor imaging and chemotherapy. In addition to this, environment-responsive peptides can also be combined with other factors for aggravating several pathways to confine several cellular factors for tumor suppression and targeted tumor penetration, facilitates cellular uptake, and controlled delivery of hydrophobic drugs that are encapsulated at various sites of tumors. Thus, the above unique design in self-assembled nanoparticles formed have huge promising aspects for cancer therapy [38,39].

18.3.3 Cell-Penetrating Peptides (CPPs) A natural barrier to entry into the cell is the cell membrane. Cell membranes have two main components that are mainly liposomes as well as proteins. The short sequence peptides are the CPPs that improve targeted delivery of the impermeable drugs and can cross the plasma membrane. These are normally collective of fewer than 30 amino acids that can translocate to different cargoes into the cell and also enter the cell membrane. Arginine and lysine are the basic amino acids that are always rich in CPPs,

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 325 which enhances the CPPs to penetrate into the cell wall. Still, the exact mechanism for the internalization and transportation is unclear; there are three possible pathways for internalization of CPPs in each membrane: peptide sequence, peptide concentration, lipid component, endocytosis, and direct trans­ location [40]. Scientists have been exploring CPPs by modifying it on the external layer of the nano­ particles, mainly increasing the efficiency of the nanoparticles to enter the cell effectively. In past decades, peptides with penetrating or targeting have been broadly studied for mainly designing the targeted delivery system approach. Cell-penetrating peptides and tumor-targeting peptides are the two key groups classified for peptide development. Like cell targeting peptides (CTPs), CPPs have been used to deliver peptides, proteins, siRNA, plasmid DNA, and anticancer drugs. Cell penetration of the peptides are divided into two parts; peptide origin and the second is based on the physiochemical property. Peptide origin is subdivided into three types: chimeric, protein derived, and synthetic. Physiochemical properties are divided into three types: amphipathic, hydrophobic, and cationic. The positively charged CPPs are mostly cationic and the mostly commonly used cationic CPPs are Tat-derived peptides and poly-arginine. Hydrophobic CPPs comprises high lipid affinity and nonpolar amino acids with low net charge [41,42]. Amphipathic CPPs include penetratin, transportan 10, antennapedia (Antp), pVEC, M918, VP22, and SAP. This CPPs contain both nonpolar and polar amino acids; thus, they are both hydrophilic and hydrophobic in nature. A distinct collection of amphipathic CPPs includes MPG, S413-PV, and Pep-1 and are synthesized forming a stable complex via nonconvalent interactions; providing high efficiency once penetrated into the cell by fusing segments of reverse transcriptase of HIV type 1, HIV GP 41 protein, or S4 peptide. Micelles and liposomes in the form of nanoparticles loaded with drugs such as paclitaxel or DOX comprising both cell-penetrating and cell-targeting peptides showed excellent therapeutic effect with antitumor activity of dual-ligand liposomes. Selfassembled nanoparticles like TAT CPPs mainly enhance the nuclear localization and cellular inter­ nalization, leading to amended synergistic chemotherapeutic and photothermal/photodynamic effect for cancer therapy. Than CPPs; tumour homing peptides are TTPs are smaller, which interact with receptors and can be used as targets. Somatostatin receptors, integrins, folate receptors, transferrins, and EGFR receptors are common receptors used as targets for TPPSs. The combination of CPPs and TPPs benefits in drug delivery to the intracellular site as CPPs effectively cross the membrane barrier but lack target specificity, but TPPs have target specificity. This has gained lot of momentum at present [43]. This bioconjugate loaded with drug in the form of nanoparticles significantly increased the cytotoxicity, anti-angiogenic activity, and cellular uptake when compared to the sole targeted bioconjugate for cancer therapy.

18.3.4 Peptide Receptor Radionuclide Therapy (PPRT) In radionuclide therapy, the application of exploring nanoparticles has been largely restricted due to many limitations in cancer therapeutic drugs such as damage to surrounding normal tissue. The role of nanomedicine in oncology conjugating with peptide is widely studied and has gained considerable popularity [44–46]. In an in-vitro study, the results showed that the PPRT drug 177Lu-DOTATATE and poly lacto-glycolic acid based biodegradable nanoparticles loaded in nanoparticles provided significant, stable, and efficient release for longer duration, implying targeting capabilities of these particles. Neuroendocrine tumours treatment is mainly carried out by PRRT, which is a molecular targeted therapy. Molecular targeted therapies use drugs and other substances and recognize and attack the cancer cells without harming healthy tissue. The general procedure involves a dose of amino acid solution given to avoid a large amount of radiation once the patient qualifies for PRRT. Further synthetic cell-targeting protein or similar peptide like octreotide is given along with the small amount of radionuclide, which is injected in the form of radiopeptide into the bloodstream. The radiopeptide binds to the protein receptor, called a somatostatin receptor, and the high dose of radiation gets delivered to the tumor, which gives an

326 Nanomaterials in Healthcare advantage in avoiding major operations for tumor removal. In the case of nanoparticle formulation for radionuclide therapy of EGFR expressing tumours, formulated 1311-labeled anti-epidermal growth factor receptor targeted EGFR was experimented and showed excellent results in targeted cell killing and leading to cancer tumor supression. This therapy is becoming popular and playing a significant role in management of cancer, especially in case of unresectable tumors [47]. The unnecessary radiation exposure to normal tissues is one of the limitations observed in the case of using radionuclide therapy. Hence, nanoparticles play a crucial role and potent tool as drug delivery vehicles to overcome this limitation; this arena of translational research still needs to be explored in terms of radionuclide therapy and nanotechnology for various cancer treatments [48].

18.4 PEPTIDE-BASED NANOPARTICLES A short chain of amino acids is known as a peptide and these peptides are connected by a peptide bond in a particular sequence. Proteins, especially collagen peptides, are believed to have an anti-aging role, improving skin health and appearance. They have a therapeutic importance in diseases such as cancer. Peptide-based nanoparticles (PBNs) or carriers have gained popularity as colloidal systems for drug delivery in treating cancer. Protein carriers with low toxicity and a high amount of drug binding capacity and drug uptake by targeted tumor cells have a few advanced applications possessed with the PBNs system. PBNs can be formed by using protein moieties such as albumin, gelatin, legumin, fibroins, lipoproteins, and ferritin proteins, which can be synthesized by using several methods such as electro­ spray emulsion and desolvation methods. The protein nanoparticles that can be used in cancer therapy are as follows: (Tables 18.1 and 18.2) 1. Albumin: The highly abundant plasma protein, also known as globular protein with 585 amino acid moieties, can be used as a therapeutic agent in the form of biodegradable NP, especially in cancer treatments. It has a carboxylic and amino group that can be used for surface modification. These modified HAS-based NPs are proven to be well tolerated by patients without giving any serious side effects in clinical trials. Albumin NPs exhibited satisfactory binding efficiency and sustained drug release for many anti-cancer drugs [49]. 2. Gelatin: It is a denatured, versatile natural biopolymer used widely in pharmaceutical industries. Gelatin nanoparticles (GNPs) can be used for both types of anticancer drug- hydrophobic and hydrophilic, with multiple modification treatment/method to couple with cross-linkers and targeting-ligands for producing tumor-targeted drug delivery vehicles [49].

18.5 CELL-PENETRATING PARTICLES Cell-penetrating peptides (CPPs) consist of small amino-acid sequences with their ability to facilitate the cellular uptake and intake of nano-sized molecules to small chemical compounds and also large DNA fragments. CPPs being rich in amino acid such as lysine are able to translocate over cell membranes and can gain access to the cellular plasma. Various studies have been done on efficient use of CPPs for treatments of various diseases. Apart from the ability to penetrate, CPPs have significant efficacy results due to potent and rapid drug delivery with low toxicity compared to other drug delivery systems. Although having good efficacy and efficiency, they are very unstable as well as lack specificity to the

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 327 TABLE 18.1

Protein nanoparticles loaded with anticancer drugs

PROTEIN NANOCARRIER

THERAPEUTIC DRUG

METHOD/TECHNIQUE

Gelatin type A

Camptothecin Doxorubicin Paclitaxel

• Self-assembly • Coacervation-phase separation

Albumin (BSA) NPs

Doxorubicin

• Conjugation • Millard conjugation/ thermal gelation

Casein micelles

Celecoxib Flutamide Mitoxantrone Paclitexal

• • • •

Lyophilization Spray drying Dialysis Self-assembled micellization

APPLICATION

[ 50, 51] • High drug encapsulation efficiency • Improved cytotoxicity and escape from multidrug resistance mechanism • Enhanced therapeutic index, [ 52] reduced cardio-toxicity. • Increased survivability of hepatoma H22 tumor bearing mice • High drug encapsulation [ 53] efficiency • Decrease in relative weights of prostate tumour compared to free FLT

TABLE 18.2

Protein-based nanoparticles preparation techniques

S.NO.

TECHNIQUES

1

Desolvation

2

Coacervation-phase separation

3

Emulsification-solvent evaporation

4

Nano spray-drying

5

Self-assembly

REFERENCES

PROCESS AND EXAMPLES Alcohol or acetone, desolvating agent is added dropwise to protein solution (aq.) with constant stirring. Protein dehydration results in coil conformation changed from stretched protein conformation. E.g., Albumin and gelatin NPs with anticancer drug A protein-rich dense phase develops at the bottom by the principle of lquid-liquid phase separation while adding salt to protein solution, resulting in a transparent solution of desired NPs. E.g., Paclitexal-loaded GNPs BSA NPs loaded with HCPT anticancer drug can be prepared by adding castor oil w/ Span-80 to an aqueous alkali solution of BSA-HCPT. The solution then homogenized, followed by addition emulsion to castor oil for thermal cross-linking. It involves a vibrating mesh technology to develop fine droplets of NPs. This technique is very simple, affordable, and provides alternative approach to develop nanoparticles. The hydrophobically modified protein molecules selfassemble themselves into micelles-like nanoparticles in an aqueous solution whereas the hydrophobic micelle core reserve the poor watersoluble anticancer drug such as Paclitexal. E.g., development of Paclitexal NP drug loaded into Octyl-modified bovine serum Albumin (OSA).

REFERENCES [ 54]

[ 55]

[ 56]

[ 57]

[ 58]

328 Nanomaterials in Healthcare TABLE 18.3

Cell-penetrating peptide classification

PEPTIDE

TYPE

Antennapedia penetratin (43-58) HIV-1 TAT protein (48-60) MAP Azurin-p28 R6W3

Cationic and amphipatic Cationic Amphipatic Anionic Cationic

LENGTH 16 13 18 28 9

ORIGIN Protein-derived Protein-derived Synthetic Protein-derived Synthetic

REFERENCES [ 60] [ 61, 62] [ 63] [ 59] [ 64]

target [59]. CPPs can be classified on the basis of their ionic characteristics and their range of appli­ cations. A well-known list of CPPs based on their ionic characteristics is shown in Table 18.3 [59].

18.6 CPPS: PROTEIN DELIVERY IN CANCER As the repertoire of possible pharmaceutical agents and biologically active molecule are compromised because of their poor permeability and selectivity at the cell membrane, the recent developed method for delivery of cell-impermeable materials has come into the role. Cell-penetrating peptides (CPPs) are one of the novel developed strategies that can be used to deliver the bioactive cargos from intracellular pathways to live cells. CPPs are also known as protein transduction domains (PTDs), short cationic, and/ or anionic peptides (less than 30 residues). Protein therapy is an emerging strategy for cancer treatments after applying other therapies such chemotherapy, etc. TAT CPP is observed to deliver the large bio­ logically active compound i.e., 120kDa b-galactosidase, in several organs after injecting it into the intraperitoneal of mouse without crossing blood-brain barrier. Peptides are observed to induce multiple immune responses which delays mucin-1 tumors in mice. CPPs along with the modulation of proteinprotein interactions (PPI) that involve the cellular process and regulating signal pathways are evolving as a promising therapy for cancer treatment [59]. The other CPPs that are molecular carriers in cancer are cargo complexes internalization (CPP), delivery of chemotherapeutic agents, and delivery of nucleic acid.

18.7 CONCLUSION AND FUTURE PROSPECTS On the concluding note, we have focused on peptide-based nanoparticles as a potent drug delivery option and novel approaches such as CPP and PRRT, which are based on peptide-nanoparticle interaction, for targeting cancer. High specificity and affinity peptides can be analyzed via specific screening techniques followed by rational design. This leads to procurement of peptide-based nanomaterials with complex functions [65]. The effectiveness and the bioavailability of the peptides can be remarkably enhanced by an in-vivo self-assembly approach for the treatment of cancer [66]. Approaches that are peptide-based can be used to target specific site and they can be developed for various specific receptors, certain transcription factors, and tumor-suppressor proteins that tend to derange the regulation of time/rate of progression of cell cycle and gene expression [67]. In comparison to the traditional nanoparticles for targeting cancer, peptide-based nanoparticles have unraveled various advantages, such as high bio­ compatibility, enhanced biodegradability, and high bioactivity [68]. Peptide-based therapies are vastly used rather than using a traditional delivery system. They can be altered for particular applications leading to appropriate cellular uptake, specific cancer cell target, and

18 • Peptide-Based Nanoparticles for Theragnostic Application in Cancer Treatment 329 endosomal escapism of therapeutic cargo. Irrespective of the fact that peptide-based system is delivered independently or in conjugation with another moiety (other particulate system), peptide inclusions have demonstrated promising effects in improving efficacy of cancer therapeutics. However, with proper specifications, numerous threats to vehicle and therapeutic disruption before reaching the target site can be tackled [2]. The vast features of peptide-nanomaterials can remarkably affect our routine in the future. But many obstacles will be tackled by exploration by self-assembly of peptides and hence it is a sig­ nificant and achievable arena of nanotechnology [7].

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Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor

19

Safe Nanomedicines Fatemeh Salemizadehparizi1, Rajendra Prasad2, and Berivan Cecen3,4 1

Department of Biomedical Engineering, Binghamton University, Binghamton, New York, USA 2 School of Biochemical Engineering, Indian Institute of Technology-BHU, Varanasi, India 3 Department of Biomedical Engineering, Rowan University, Glassboro, New Jersey, USA 4 Department of Mechanical Engineering, Rowan University, Glassboro, New Jersey, USA

Contents 19.1

19.2

Cell-Inspired Systems 19.1.1 Exosomes 19.1.2 Cell-Derived Nanovesicles Lipid-Based Systems 19.2.1 Solid Lipid Nanoparticles (SLNs) 19.2.2 Coordination Micelles 19.2.3 Filomicelles

DOI: 10.1201/9781003322368-19

334 334 334 335 335 335 336

333

334 Nanomaterials in Healthcare 19.3

Bacteria-Inspired Systems 19.3.1 Cellular Ghost 19.3.2 Microbots 19.3.3 Recombinant Bacteria 19.4 Hydrogel-Based Systems 19.4.1 Alginate-Based Hydrogel 19.4.2 Interpenetrating and Semi-Interpenetrating Polymer Network (IPN) Hydrogels 19.4.3 Imprinted Hydroxyethyl Methacrylate (HEMA) Hydrogels 19.5 Virus-Inspired Systems 19.5.1 Viral Gene Vectors 19.5.2 Virus-Like Particles 19.5.3 Virosomes 19.6 Mammalian Cell-Based Systems 19.6.1 RBC 19.6.2 Stem Cells 19.6.3 Platelets 19.6.4 Macrophages 19.6.5 Lymphocytes 19.7 Application in Cancer Therapy 19.8 Biological Effects and Toxicity of Biomimetic Nanovesicles 19.9 Conclusions 19.10 Limitations and Future Research References

336 337 337 337 337 337 338 338 339 339 339 339 339 340 340 341 342 343 344 344 344 345 346

19.1 CELL-INSPIRED SYSTEMS 19.1.1 Exosomes Exosomes are vesicles with diameters of 30–120 nm commonly detected in bodily fluids such as blood, breast milk, urine, etc. [1,2]. They are produced by multivesicular bodies and released when they merge with the cell membrane, releasing intra-luminal vesicles that aid communication with neighboring cells [3,4]. These exosomes are made up of many biological parts, like nucleic acids and proteins and have properties that make them amphiphilic [5,6]. Exosomes, produced by dendritic cells and B cells, primarily mediate immune reactions to pathogens and tumors. Exosomes perform various functions depending on where they originate in cells or tissue [7]. Exosomes generated from tumor cells have a crucial role in tumor formation, metastasis, and treatment response via oncogene transfer between tumor cells since these exosomes include chemicals related to cancer and other metabolic or infectious illnesses [7,8]. Examining exosomal protein indicators in certain types of human cancer revealed that exosomal CD63 may be a protein marker for cancer since it was detected in higher amounts in carcinogenic cells than in regular cells [9–11]. Salivary exosomes are also studied as potential pancreatic cancer indicators [12,13]. Surprisingly, other physiological fluids may also perform as alternative sources of diagnostic exosomes.

19.1.2 Cell-Derived Nanovesicles Exosomes of 30–120 nm generated by eukaryotic cells have gained popularity for their diverse roles, most notably cell-to-cell interaction [14,15]. Furthermore, throughout the creation of exosomes, some

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 335 endogenous molecules such as mRNAs, proteins, and short RNAs are packaged and transferred throughout numerous cells. They are commonly employed in delivering medications like curcumin, paclitaxel (TAX), and others due to their capacity to act as biological carriers. Even though exosomes offer such promised benefits, their research is hampered since they are released from cells in small quantities in terms of protein content. Furthermore, isolating and purifying the appropriate number of exosomes is time-consuming [16,17]. The assembly of cell-derived nanovesicles suggests a potential solution to the issues encountered in exosome production. These are essentially nanoscale vesicles formed by subjecting cells to different physical processes [18,19]. Forcing cells down hydrophilic microchannels is an efficient artificial way of producing nanovesicles. The nanovesicles produced will have the structure of cell-secreted exosomes and will contain plasma membrane proteins, mRNAs, and intracellular proteins. This technology may be used to do more studies on functions in medication and gene delivery systems [20,21]. A micro-size porous filter and centrifugal force are used in a new method for the mass production of cell-derived nanovesicles [22–24]. This method produces nanovesicles with exosome-like size and membrane features. Plasma membrane proteins, intracellular RNAs (microRNA to mRNA), and intra­ cellular proteins are the major components of these nanovesicles. This device generates roughly 250 times the number of naturally released nanovesicles. Furthermore, the intracellular substances of these vesicles are almost twice those of exosomes. As a result, these nanovesicles have promise in medication delivery and cell-based treatment [25–27].

19.2 LIPID-BASED SYSTEMS There are several classifications of lipids, all of which include molecules having fatty acids as their primary constituents. Lipids have been categorized based on their interactions with water and behavior at the water-air interface. In addition to differences in structure and characteristics, lipids have different digestion and absorption mechanisms [24,28]. Lipids’ digestion and absorption pathways are as diverse as their structural and physicochemical features. Drugs are pre-dissolved in lipids, surfactants, or lipid-surfactant combinations. Since medication absorption and distribution are influenced by bio­ pharmaceutical properties such as lipid excipients and dose form, this selection is important for both in vitro and in vivo studies.

19.2.1 Solid Lipid Nanoparticles (SLNs) SLNs are round and have smooth surfaces. Their average diameter is between 50 and 1,000 nm. The physicochemical properties of SLN surfaces influence their behavior in the body. Active pharmaceutical ingredients (API) and an appropriate solvent solution for solubilizing the lipid and nonlipid phases comprise most of the components in a typical SLNs formulation. In the API-loaded shell model, the APIloaded shell model becomes firm when the hot liquid droplets quickly cool down because of phase separation. The reversal of the crystalline method described above applies in the API-loaded core model, and the API must crystallize before the liquid [29].

19.2.2 Coordination Micelles Drug distribution and bioimaging are two primary uses of bioinspired coordination micelles. Micelles contain a hydrophobic center and a hydrophilic outer shell and are spherical, amphiphilic structures.

336 Nanomaterials in Healthcare While the hydrophilic surface enables intravenous medication administration, the hydrophobic core provides a therapeutic payload. The nanoscale size (less than 50 nm) and hydrophilic shell of polymeric micelles protect them from reticuloendothelial system elimination, enhancing their circulation duration and capacity to transport medicine to the target [28]. In cells, phosphatidylcholine (PC) molecules play a critical role in maintaining cell membrane integrity. They are amphiphilic and may be made to selfassemble into micelles, bilayers, or other nanostructures by changing their chemical structure. Because different lipid types, fatty acids, and cholesterol can substantially impact the design and dynamics of PC products, such as micelles developing instead of bilayers, it is difficult to predict the fate of a single PC species when it is included in a combination [30].

19.2.3 Filomicelles Filomicelles are hydrophobic polymeric nanoparticles made of biodegradable block copolymers in a non-spherical shape. In comparison to other spherical micelles, they have the benefit of being able to hold more drugs and have a longer circulation duration. Filomicelles have been shown to transport nanocarriers better to tumors and solubilize more medications in the core than conventional carriers [31].

19.3 BACTERIA-INSPIRED SYSTEMS Exosomes produced from mammalian cells would be challenging to translate owing to the difficulty and expense of large-scale mammalian cell production [32]. However, exosomes from bacteria may have significant translational potential. A mass assembly of bacteria in bacterial growth tanks is simpler and cheaper than in mammalian cell cultures [33]. The simplicity of genetic engineering in bacteria makes it possible to create and generate bacterial membrane vesicles with functional moieties. Biofilms stop metastasis because different bacteria grow only on tumor cells or all tumor cells (Figure 19.1).

FIGURE 19.1 (A) Design of drug and gene delivery methods inspired by virus. (B) Illustration of drug and gene delivery methods inspired by bacteria. Created with Biorender.

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 337

19.3.1 Cellular Ghost Recently, cellular ghost structures have been recognized as versatile therapeutic carriers due to easy prep­ aration, good biocompatibility, natural surface biomarkers, etc. However, these cellular ghost structures have different surface protein biomarkers and are different from each other at the molecular level. Recently, these ghost structures have been produced from various cell sources such as cancer cells, stem cells, red blood cells (RBCs), healthy fibroblast cells, bacterial cells, etc. Among these are bacterial cells-derived ghosts (BGs), empty gram-negative bacterial covers produced by regulated expressions of the cloned gene E, resulting in a lysis tunnel structure. BGs include pathogen-associated molecular forms such as lipopolysaccharide, lipo­ protein, peptidoglycan, and fimbriae [34]. Identification receptors on immune cells are activated when BGs enter the host, releasing various immunological mediators that promote the development of antigenpresenting cells, such as dendritic cells [35,36]. Washing and centrifugation can purify nucleic acids, proteins, and chemical medicines [37]. Doxorubicin (DOX) was recently shown to be effective in vitro against human colorectal adenocarcinoma (Caco-2) cells by using M. haemolytica bacterial ghosts. According to the adhesion studies, M. haemolytica ghosts targeted Caco-2 cells and released the laden DOX inside. Cells treated with DOX-loaded ghosts displayed a two-log increase in cytotoxic and antiproliferative activity compared to DOX delivered directly to the culture medium in cytotoxicity assays [38].

19.3.2 Microbots Using the microbot technique, nanoparticles carrying therapeutic cargo might be attached to bacteria and delivered to cells at the same time. These bacteria have not been subjected to any genetic modification, which gives them an edge over invasive methods of delivering bacteria into cells [39]. The microbot’s negative charge may be leveraged to effectively conjugate positively charged metal-based nanoparticles for tumor-targeted drug delivery and imaging. The conjugated particle may be successfully directed to the tumor location by cells without damaging healthy cells. Tumor cell imaging using a quantum dot–based microbot arrangement is achievable. Microbots’ dose-limiting toxicity reduces therapeutic efficacy. They are genetically modifying bacteria before conjugation and minimizing toxicity. Overmodification may reduce bacterial obtrusiveness, delivery effectiveness, and applicability [40].

19.3.3 Recombinant Bacteria Through the production and release of large amounts of a variety of heterologous antigens, recombinant bacteria may serve as a vehicle for the delivery of vaccines. These are bacteria inserted with plasmid vectors. Because of a whole RNA polymerase complement, this recombinant bacterium can produce the required protein encoded by the human genome [23,41]. Recombinant bacteria that target cancers and contain imaging probes or marker genes may be used to assess the condition of a tumor, monitor the progression of therapy, determine how well the treatment worked, and even find metastases and tumors that are difficult to diagnose [42].

19.4 HYDROGEL-BASED SYSTEMS 19.4.1 Alginate-Based Hydrogel Alginate nanovesicles shield encapsulated chemicals from the environment while increasing bio­ availability [42,43]. Encapsulation allows for the continuous and local distribution of loaded materials.

338 Nanomaterials in Healthcare More significant concentrations are reached in specific body areas while having minimal side effects in undesirable areas. Encapsulated chemicals are liberated as a result of matrix breakdown or diffusion. When the alginate matrix swells, the delivery rate increases. For example, sodium alginate hydrogels cross-linked with CaCl2 expand in the presence of Na+ ions, causing ion exchange and an increase in electrostatic repulsive forces [44–47]. For cell-derived nanovesicles, alginate is the most commonly used biopolymer [48–50]. It functions as a semipermeable membrane, allowing molecules to move in both directions. Nutrients and oxygen are permitted to enter, while cellular waste is eliminated from the cellular environment. Once injected into an organism, encapsulated cells continually create active chemicals [51]. Cell therapy provides for a longer and more consistent supply of medicinal substances, but it also allows for more complicated release patterns. Small molecules and proteins, for example, can diffuse through the alginate layer. Alginate maintains the immunization of encapsulated cells and lowers the chance of rejection. As a result, immunosuppressive therapy co-administration may be unnecessary. There have been no reports of detrimental effects from using alginate hydrogels. Because they are biodegradable and compatible with the host’s body, they can’t be taken out [52–54].

19.4.2 Interpenetrating and Semi-Interpenetrating Polymer Network (IPN) Hydrogels The current section focuses on producing and characterizing novel IPN hydrogels for tissue engineering applications [55,56]. IPN hydrogels are polymer hybrids made up of two or more polymers that are physically or chemically cross-linked networks that are entangled. Because of the presence of highly entangled networks, adding two polymers strengthens and reinforces the scaffold. The hydrogel’s breakdown rate may be fine-tuned based on the properties of each polymer complex [56,57].

19.4.3 Imprinted Hydroxyethyl Methacrylate (HEMA) Hydrogels Imprinted HEMA hydrogels are made by polymerizing an interacting pair of precisely chosen monomeric residues and a template molecule to create a synthetic biomimetic network [58,59]. Noncovalent bonding, metal coordination, and covalent bonds can be used with the right components. Before polymerization within a solvent, these interactions between monomers and templates are tolerated. By decreasing the system’s conformational energy, polymerization of monomeric residues and cross-linking activities stabilize these linkages. Following the elimination of template molecules, a method was established to recognize and select the specific template molecule [60–63]. Developing HEMA hydrogels capable of recognizing and binding any physiologically important chemical is exciting [64,65]. HEMA hydrogels can then be utilized as standalone systems or included in strategies that help remove or transport analytes from body fluids. These advancements will significantly influence disorders like diabetes and arteriosclerosis, characterized by elevated levels of specific chemicals in the blood. The ability of polymeric systems to screen and remove such harmful substances on demand is very desirable, and this may be accomplished via biomimetic networks. The applications for which imprinted HEMA hydrogels are intended to determine their selectivity. Other properties, such as how easy it is for analyses to move into and out of the polymer complex, become essential in applications such as medication distribution, where fewer similar substances must be found [66,67].

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 339

19.5 VIRUS-INSPIRED SYSTEMS 19.5.1 Viral Gene Vectors Viruses are infectious microorganisms that contain nucleic acid molecules enclosed by a protein shell. They are ideal carriers for gene delivery due to their capacity to transmit genes into host cells [68,69]. Viral vectors replace undesirable viral genetic material with beneficial material, allowing for the longterm expression of target genes. They are also capable of viral transduction and self-replication. The combination of specific viral vectors with pharmaceuticals can have a synergistic impact. The combo treatment looks fresh with the development of tumor-targeted nanoparticles [70–72].

19.5.2 Virus-Like Particles Virus-like particles are self-assembled capsules made of viral capsids or envelope proteins that retain antigenicity [73,74]. They have inherent tropism and may be further modified to improve targeting. They are most commonly seen in vaccines, medication, and gene delivery purposes. Protein-DNA complexes have been shown to mimic an artificial virus and play an essential role in gene and medication delivery systems and other biological applications. A newly discovered bioinspired artificial virus-carrying DOX and DNA/RNA adduct the therapuetics response (Figure 19.2). The artificial virus is suited for anticancer treatment when supplied in a pH-responsive coat. A virus-like particle integrated into a single nano­ particle can be used to dine as an artificial virus [75,76].

19.5.3 Virosomes A virosome is a phospholipid bilayer vesicle that resembles a virion and contains an integrated glyco­ protein within an empty part [78,79]. They lack a capsid protein as well as genetic material. A detergentmediated solubilization process can be used to make them. The glycoproteins have outstanding adjuvant properties and the ability to transport a variety of therapeutic compounds. They oversee giving virosomes structural stability and uniformity. They also aid in receptor-mediated endocytosis and target [80,81]. Virosomes are used to deliver vaccines, genes, and drugs. They’re simple to make and have lower toxicity. They protect the active substance in endosomes against low pH and enzymatic breakdown. When utilized for gene and medication delivery, the danger of immunogenicity restricts their in-vivo utilization. Modifying polyethylene glycol or a targeted ligand can reduce the impact (Figure 19.1). As a result, virosomes are now a viable technique for medication delivery [69,82–84].

19.6 MAMMALIAN CELL-BASED SYSTEMS Everybody knows that cells are the fundamental building blocks of life and are involved in several processes, such as transferring nutrients and transmitting signals. Due to the assembly and function of the natural cell membrane, nanoparticles with cell membrane coatings are now being developed for cancer therapy and diagnostics (Figure 19.3). Nanocarriers with complicated functional surfaces can be made top-down by starting with the cell membrane (Table 19.1).

340 Nanomaterials in Healthcare

FIGURE 19.2 Nanovectors of many forms (e.g., nanoparticles, extracellular vesicles, viruses) can recover molecular carriage in circulation by aggregate solubility, half-life, and bioavailability, as well as improving in biological barrier crossing. Better targeting of the tumor microenvironment also enhances tumor delivery, resulting in the concentration of therapeutic molecules in tumors and increasing the efficacy of combination therapies. Copyrights Ref [ 77].

19.6.1 RBC In humans, the life span of red blood cells (RBCs) is 120 days, and they play a vital function in the movement of nutrients and waste products in the blood [103]. Encapsulated cargo is well-protected and may continue circulating in the bloodstream for a long time owing to these natural compartments. Numerous RBC-based or RBC-mimicking delivery structures are now under clinical or preclinical progress because of biomimetic methods for developing delivery systems. A hypotonic technique is often employed to load therapeutic substances into RBCs. Many of the cell’s biophysical and immu­ nological features may be preserved using this technique [104–106].

19.6.2 Stem Cells Stem cells are multipotent cells that can self-renew and self-replicate. When the right conditions are met, it may progress into a wide range of functional cells involving muscle, bone, fat, and other types of cells, producing tissues and organs [107]. The most attention has been paid to MSCs because they are easy to grow in vitro, have a low risk of causing an immune reaction, and raise less ethical questions than other types of stem cells. These stem cells are biocompatible, attract malignant tumors, and may adhere to developing tumors. MSCs’ homing capabilities aid in targeting these cells to the microenvironments of

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 341

FIGURE 19.3 A wide range of mammalian cell types may be employed as sources of membranes to load nanoparticles. Created with Biorender.

growing tumors. MSCs contain receptors on their surface for certain signaling chemicals released by tumors. Biomimetic stem cell membrane coverings have also been discovered to exhibit tumor-homing activity. The non-immunogenicity of stem cell membrane coatings makes them appealing as biomimetic drug delivery methods since they lengthen blood circulation periods and limit clearance by the reticu­ loendothelial system [108,109].

19.6.3 Platelets Platelets have a natural propensity to move to damaged sites or tissues, such as malignancies, since they are highly reactive and sensitive. Platelets’ distinct features are primarily due to functional receptors on the platelet membrane. They are small fragments of megakaryocytes that have developed in the bone marrow. Their primary role is cruor, hemostasis, and blood vessel integrity. Numerous diseases, including cancer and atherosclerosis, are acquired and worsened by platelet dysfunction and accumu­ lation. The high association level between these diseases and platelets may be attributed to the markers found on their surface. Immune thrombocytopenic purpura (ITP) is a common autoimmune disease. In ITP, autoantibodies produced by the disease cause the destruction of platelets, which may then lead to persistent bleeding if platelet counts fall below the critical level. Nanoparticles made from platelet membranes were used to bind and prevent pathogenic antiplatelet antibodies, which play a role in the disease process [110].

342 Nanomaterials in Healthcare TABLE 19.1 CELL TYPE RBC

Platelet

Characteristics and limitations of different mammalian cells as drug carriers CHARACTERISTICS • Capable of loading various agents • Large volume and high surface-tovolume ratio with great potential for drug loading • Controlled drug delivery • High accessibility to preparation • Reversible deformation • Long life span in vivo, > 100 days • Good biocompatibility • Homing to tumor cells • High responsiveness and sensitivity to the cell microenvironment • High transport and storage capacity • Long life-span in circulation, 7–9 days

Macrophage

• Actively target the injured endothelium area • Enhanced therapy and decreased adverse effects

Stem cell

• Transdifferentiation and self-renewal capacity • Natural anti-tumor properties • Strong ability to migrate to diseased areas and tumors • Excellent biocompatibility and lack of immunogenicity • Good capacity for loading various agents

LIMITATIONS

REFERENCES

• Environmental stimuli induce rapid particle detachment from RBCs • Only circulates in the bloodstream • Limited storage time • Limited shelf life in ex-vivo

[ 85– 89]

• Enhanced thrombosis and carcinogenesis • Difficulty in preparing • Difficulties in genetic engineering • Risk of thrombosis or bleeding upon undesirable activation • Limited shelf life in ex-vivo • Insufficient therapeutic stability • Insufficient mass production • Storage time is limited • Predisposed to adopt a proinflammatory phenotype • High heterogeneity • Lack of efficient mass manufacturing • Concerns about the safety of long-term effects • Preparation and quality control difficulties • Individualized therapy is costly

[ 90– 93]

[ 94– 98]

[ 99– 102]

On the surface of platelets, primary amine or thiol residues are plentiful and easily accessible for chemical ligation with nanoparticles or biomolecules. After surgical cancer treatment, monoclonal antibodies against programmed-death ligand-1 were attached to platelets as a delivery platform for checkpoint inhibitors. Receptors on the surface of blood cells, such as platelets, let them retain their selfrecognized states for long periods. Cell transport behavior can be imitated via cell membrane coating (Figure 19.4), which is critical for better targeting of tumor tissue and improved drug accumulation. If the nanoparticles are oriented correctly, platelet membrane proteins can be translocated or even enhanced onto the nanoparticle surface [85,111].

19.6.4 Macrophages Macrophages are immune cells that play a crucial role in innate and adaptive immunological responses. Macrophages mainly phagocytose and eliminate cell debris and pathogens when activated in inflammation. Many macrophages infiltrate the tumor microenvironment and significantly impact growth, metastasis, and therapeutic therapy. Macrophages may carry small and large materials, such as proteins and nucleic acids.

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 343

FIGURE 19.4 Initial generations of nanoparticles were biologically inert and had non-fouling layers to inhibit them from interrelating with the cells they encountered in vivo. The second generation evolved into functional targeting molecules, allowing the nanoparticles to reach the region of the ailment and interact with the surrounding environment. The third generation of cell membrane-based biomimetic nanoparticles emulates the surface structures of actual cells by using entire cell membrane or membrane protein functionalization onto synthetic carriers. Copyrights Ref [ 114].

Macrophages, membranes, and vesicles are used in biomimetic drug delivery because of their long cir­ culation time, vast number of surface receptors, and dynamic targeting capability [112,113].

19.6.5 Lymphocytes T cells are potentially valuable for modification since they play an essential role in the host’s immuno­ logical response to several diseases, including infections, cancer, and autoimmune disorders. Human cytotoxic T cells have more adhesion molecules than those obtained from other species, making them more efficient at targeting tumor sites [115–117]. Cancer-targeting nanoparticles were created in research using human cytotoxic T cells membrane. The T cell was chosen because it has a lengthy blood circulation period and can locate and remain in tumors [118]. The use of endocytosis to load drugs into T cells is one way the

344 Nanomaterials in Healthcare T cell-based drug delivery system is improving. T cells were loaded with Au-nanoparticles by Kennedy et al. via endocytosis without altering the cells’ viability or functionality. In vitro and in vivo studies revealed that Au-nanoparticle-loaded T cells retained their ability to migrate to tumor sites and accumulate tumor Au-nanoparticles, implying that Au-nanoparticle-based phototherapy could be improved [119].

19.7 APPLICATION IN CANCER THERAPY Cancer nanovesicles have significantly contributed to effective immune activation to improve cancer immunotherapy by delivering therapeutic antigens to tumor tissues, T cells, macrophages, and other immune organs such as lymph nodes. However, other obstacles must be overcome before cancer nanovesicles may be extensively used in clinical settings. For example, introducing exogenous materials may result in offtarget immunotherapy reactions. As a result, the technique for creating biomimetic nanovesicles utilizing autologous cell-based materials has attracted much interest in effective cancer immunotherapy [120–122]. Cell-derived nanovesicles with various molecular contents can be created spontaneously and slowly by nearly all types of cells, including T cells, B cells, tumor cells, and antigen-presenting cells [123,124]. Exosomes have been shown to merge with target cells and convey membrane proteins and cytoplasm, as well as nucleic acids like mRNAs and microRNAs, across two cell types. Exosomes with immunostimulatory or immunosuppressive properties generated by specific cells have significant promise in immunotherapy against malignancies or autoimmune diseases. For example, exosome nanovesicles produced by dendritic cells have been shown to stimulate T-cell priming. Because of the expression of costimulatory molecules MHC-I and MHC-II on the vesicle surface, the production of specific CD4+ and CD8+ T cells may limit tumor growth [125–127].

19.8 BIOLOGICAL EFFECTS AND TOXICITY OF BIOMIMETIC NANOVESICLES The use of nanovesicles as diagnostic agents has potential in tumor treatment. The body will exhibit several aberrant behaviors in the early stages of any disease. In the early stages of illness, vascular inflammation increases circulating leukocytes’ recruitment, adhesion, and trans-endothelial extravasation. The innovative biomimetic nanovesicles might be ideal for tumor-targeted imaging and treatment. These researchers’ novel nanodiagnosis and therapy technologies have potential implications for cancer. Biomimetic carriers derived from several fundamental nanostructures have unrivaled benefits in tumor therapy and diagnostics. For example, liposomes and nanovesicles have different physical and chemical characteristics; thus, the medications they transport during tumor treatment differ. Some nanovesicles are made to deliver drugs or be used in combination with chemotherapeutic drugs with different solubilities (such as hydrophilic, hydrophobic, and amphipathic agents), while others are made to make medications more stable [128–130].

19.9 CONCLUSIONS Biomimetic cell-derived nanovesicles are recognized as clinically possible therapeutic platforms. So far, various nanovesicles have been tested for site-selective tumor imaging and treatment in

19 • Biomimetic Nanovesicles for Targeted Imaging and Therapeutic of Solid Tumor 345 in vitro and in vivo models due to their excellent biocompatibility, minimum side effects, and inherent tumor-targeting ability. However, the tumor accumulation mechanism is yet to be achieved and is beyond the scope of this chapter. Moreover, low reproducibility and product yield remained critical challenges for these biomimetic therapeutic agents. In this chapter, we focused on how nanovesicles for selective multimode imaging of solid tumors are a boon for biomimetics break­ throughs. It is clear how vital it was to thoroughly examine natural nanostructures to replicate them in the lab properly. This was only made feasible by developing several nanotechnologies and microscopic and spectroscopic technologies. The approaches performed for the artificial development of each nanostructure and natural process were reviewed. Then, some of the novel applications of these bioinspired materials and how they are changing the shape of our future were discussed. Finally, it is crucial to underline that nature will constantly surprise us if we continue to look attentively and learn how it works.

19.10 LIMITATIONS AND FUTURE RESEARCH Protein-based biomimetic nanocarriers offer several better qualities as a nanovehicle, including out­ standing biocompatibility, reduced hazardous and side effects, and increased chemotherapeutic impact [131,132]. Most advanced nanocarriers based on a similar biomimetic approach might exploit the particular physicochemical features of proteins or chemotherapeutic medications to achieve selfassembly, avoid the compound manufacturing progression of conventional nanocarriers, and restore nanoparticle stability. Despite the various benefits of biomimetic nanovesicles, their clinical appli­ cability is limited by specific possible difficulties. For example, the protein may undergo specific undesirable alterations due to the solid physical or chemical processes utilized to manufacture the nanovesicles. To summarize, further research is required to overcome the abovementioned limitations [133–135]. We summarize recent findings on diabetes and cancer in this study using natural particles, syn­ thetics, and cellular nanoformulations in this chapter. By replicating or directly exploiting how cells interact and connect with living organisms, we seek to increase treatment efficacy while avoiding undesired side effects. Meanwhile, these categories include advances in bioconjugation chemistry, bionanotechnologies, and genetic engineering nanomedicines’ scope and design flexibility. Such designs, in some respects, give nanomedicines “life,” allowing them to “communicate” with the biological environment and respond to specific biological factors. These approaches are not mutually exclusive and can be combined to develop more effective nanomedicines. Exciting uses of bioinspired and biomimetic nanomedicines for healing various illnesses with various symptoms are anticipated due to distinct structural and functional designs and varied delivery channels, learning from nature to enhance human lives [69,75]. Furthermore, many bioresponsive drug delivery devices struggle to match the rapid dynamics of natural processes, indicating a time lag between detection and drug release [136,137]. Second, unlike well-defined synthetic systems, biologically membrane-derived biomimetic nanomedicines must address issues such as the repeatability of membrane protein amounts and scaling up production [138,139]. Third, while the viability and intended biological activities of genetically or chemically altered natural particles may be successfully preserved, the undesirable impacts on their other functions and long-term conduct in biological structures should be extensively examined. Before clinical trials, the total biodistribution of the controlled, designed parti­ culates and their influence on nontargeted tissues must be determined. As this discipline grows, future advancements will rely on a thorough understanding of natural particulates, improved synthesis tech­ niques, and collaborative efforts from several fields [140–142].

346 Nanomaterials in Healthcare

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Index Additive manufacturing, 27 Biosensor, 240, 307 application, 245–252 characteristics, 242 prospects, 253 Black phosphorus (BP), 268 Boron nitride (BN), 269 Cancer, 5, 24, 45, 168, 299, 322, 344 Carbon-based nanoparticles, 4, 9, 250 application, 90–93 prospects, 94 risk assessment, 89 synthesis, 86 type, 77–86 Ceramic biomaterials, 183 Communicable disease HIV/AIDS, 22 influenza, 22 tuberculosis, 21 Computed tomography, 305 Engineering of nanoparticles application, 180–182 shape, 178 size, 178 surface, 179 Exosome, 195, 334 absorption and distribution, 205 application, 201 challenges, 205 mechanism, 197 prospects, 206 source, 196 structure, 199 Fluorescence imaging, 307 Formulation, 3, 23, 28, 36, 40, 43, 47, 48, 60 Graphene, 263 Green synthesis, 3, 158, 162, 299 Gold nanoparticles application, 299 clinical status, 310 introduction, 298 safety concerns, 310 synthesis, 299 Hydrogel-based systems, 337

application, 64–68 characterization, 62–64 need, 56 preparation, 59–62 prospects, 70 Magnetic nanoparticles application, 108–110 introduction, 102 properties, 106–107 synthesis, 104–106 Magnetic resonance imaging (MRI), 110, 162, 181–182, 305 Metallic-based nanoparticles, 3, 8, 245 Metal-organic framework application, 122–126 functionalization, 121 introduction, 116, 275 synthesis, 118–120 Metal oxide nanosheets, 271 Non-communicable diseases autoimmune, 24 cardiovascular diseases, 23 diabetes, 23 neurodegenerative, 6, 23 Nanoemulsions, 3, 285 Nanofibers application, 227 introduction, 225 prospects, 235 Nanogels application, 213–219 challenge and perspective, 220 Nanoparticles advantages, 18 application, 4, 24–27 disadvantages, 19 prospects, 10 toxicity, 7 type, 3 Nanomedicine clinical trials and approval, 29 ethical concerns, 28 history, 30 trends, 20 Nanovesicles application, 344 prospects, 345 toxicity, 344 type, 334–343 Nuclear imaging, 306

Iron oxide, 273 Layered hydroxides, 274 Lipid-based nanoparticles, 3, 8, 335 Liposome

Peptides application, 321 cell penetration, 326 drug conjugates, 321–322

353

354 Index gene delivery, 321 introduction, 318 prospects, 328 Photodynamic therapy, 302 Photothermal therapy, 303 Photoacoustic imaging, 307 Polymer-based nanoparticles, 4, 10, 36 application, 44–47 prospects, 48 type, 38–43

preparation, 283–287 route of delivery, 287–289 toxicity, 289 Theragnostic, 308, 322 Two-dimension nanomaterials application, 262–275 properties, 260 synthesis, 261 Virus-inspired systems, 339

Silica-based nanoparticles, 4, 9, 188 application, 138–147, 190 clinical trials, 148 strategies, 135 surface structure, 189 Solid lipid nanoparticles application, 290 characteristics, 283

ZnO quantum dots application, 160 introduction, 156, 273 properties, 156–157 prospects, 170 synthesis, 158–159