Application of Nanotechnology in Biomedical Sciences [1st ed.] 9789811556210, 9789811556227

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Faheem A. Sheikh Editor

Application of Nanotechnology in Biomedical Sciences

Application of Nanotechnology in Biomedical Sciences

Faheem A. Sheikh Editor

Application of Nanotechnology in Biomedical Sciences

Editor Faheem A. Sheikh Department of Nanotechnology University of Kashmir Srinagar, Jammu and Kashmir, India

ISBN 978-981-15-5621-0 ISBN 978-981-15-5622-7 https://doi.org/10.1007/978-981-15-5622-7

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

I would like to dedicate this book to my PhD supervisor Prof. Hak Yong Kim of Chonbuk National University, South Korea. Furthermore, for some persons in my life, I have deep respect and regard for the completion of this book. The direct involvement from them through professional and personal dealings always motivate me to move forward and to perform excellent research. These include Prof. Javier Macossay of University of Texas Rio Grande Valley, United States of America; Prof. Chan Hum Park of Hallym University, South Korea; Gilson Kang of Chonbuk National University, South Korea; Gary L Bowlin of the University of Memphis, United States of America; Cheol Sang Kim of Chonbuk National University, South Korea; and last but not the least, the great person Prof. Hern Kim of Myongji University, South Korea. It would be highly unfair if I do not thank Prof. Nasser Barakat of Minia University, Egypt, for teaching basics of research during the time when I was a young researcher and Ph.D. scholar. All these professors taught me how to be a good person and motivated me to help society by

doing excellent research. I do respect them, not from the bottom of my heart, but from the core of my soul, and I wish to contribute what they have given to society through science. Finally, I would like to dedicate this book to my wife Er. Iqra Shafi and my son Sheikh Mohmmad Sabik without whom this book would not have been completed much earlier. However, they, along with my parents, were a great source of inspiration for completing this manuscript, for which I am very grateful to them.

Preface

In the scientific literature, we can find a lot of articles which are preferably written after investigating on materials resulting in diverse application from regenerative medicine to environmentally feasible solutions to obtain clean water while using approaches based on principles of basic nanotechnology. This book, which I published under the title of Application of Nanotechnology in Biomedical Sciences, will enable the readers with the most recent advancements happening in the field of biomedical sciences concerning the fabrication of different forms of nanomaterial and their applications. Knowing about the fact that the use of the word “nano” is widespread in every household, so a proper understanding of the knowledge originating from this book will impart a sense of utilization to general readers. Moreover, the prime focus of this book was to help inspiring researchers to understand the intricacies of nanotechnology in the field of biomedical sciences focusing on the needs to raise in the twenty-first century. This book comprises 8 uniquely written chapters, with each of them doing critical analyses on different applications. For instance, Chap. 1 covers the ability of various nanomaterials in terms of its speed, quality, effectiveness, and sensitivity for overall use in medical diagnosis, helping the healthcare system in general and diagnosis in particular by the inclusion of rapid testing, leading to early diagnosis. Chapter 2 introduces specially designed polycaprolactone-based nanofibers for bone regeneration, a historical and current perspective highlighting their importance in the repair of bone defects. Chapter 3 sheds light on nanomedicine while utilizing camptothecins loaded in the nanofibers to be used as anticancer agents. Next, Chap. 4 covers the functional nanofibers composed of chitosan for their applications as arterial grafts, artificial cartilage, delivery systems, therapeutics, wound regeneration, antibacterial, biosensors, analytic systems, diagnostic aids, 3D cell culturing, nerve tissue regeneration, and treatment of contaminated water. Chapter 5 brings us the approaches involved in the synthesis of gold nanoparticles by green and chemical methods. Their characterization, stabilization, and functionalization by various legends with a particular focus on biomedical applications are discussed. Chapter 6 involves the delivery of therapeutics using polymeric/ceramic nanoparticles and dendrimers through oral and nasal routes for the treatment of diabetics. Chapter 7 deals with the fabrication of nanorobotics to be used as a nano-drug delivery system. This chapter elaborates on the creation of nanomaterial with atom-by-atom precision, vii

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which can yield nanomachines. Finally, Chap. 8 focuses on fabrication titanium dioxide nanofibers incorporated with catalytically active nanoparticles for removing the environmentally harmful dye reflecting their role of nanotechnology in the purification of water bodies and treatment of industrial effluents. In this book, I have tried to cover only a few facets of nanotechnologies in the form of comprehensive essays and short protocols. Therefore, a lot remains to be discussed in coming editions of this book. This book titled as Application of Nanotechnology in Biomedical Sciences is genuinely multidisciplinary as it covers a wide range of scientific disciplines. We hope that this book will make a positive contribution to the future development of clinical applications using biomaterials for regenerative medicine. This book aims This journey was not easy and smooth; it needed a lot of courage, and we faced tremendous challenges during the writing of this book. I am grateful to all the contributors who participated in discussing different contents. I want to thank Dr. Bhavik Sawhney, who is the publishing editor of biomedical sciences of Springer Nature, for his guidance, suggestions, discussion, and pushing hard for the completion of this manuscript. My appreciation goes to Ms. Immaculate Jayanthi, who is the production editor of Springer Nature and has helped organize the book contents. This work would not have been possible to publish without their support. Srinagar, India

Faheem A. Sheikh

Acknowledgement

I am eternally thankful to all the scholars who without hesitation, participated and followed my instructions to shape this book. Although writing a book was harder than I thought; however, this was much more rewarding than I have ever imagined. All such efforts could set an example in our University and will globally catch the eye of the researchers who are working in the area of Nanotechnology for the betterment of society. My appreciations go to Dr. Bhavik Sawhney Editor—Biomedicine and Ms. Immaculate Jayanthi, Production Editor at Springer Nature. Furthermore, I would like to acknowledge the financial support given by the Department of Science and Technology, Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016, and Science and Engineering Research Board, Grant/Award-ECR/2016/001429. Faheem A. Sheikh

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Contents

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Advancements of Nanotechnology in Diagnostic Applications . . . . . . Zahid Rafiq, Pankaj Patel, Santosh Kumar, Hasham S. Sofi, Javier Macossay, and Faheem A. Sheikh

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Polycaprolactone-Based Nanofibers and their In-Vitro and In-Vivo Applications in Bone Tissue Engineering . . . . . . . . . . . . . . . . . . . . . Rumaisa Rashid, Hasham S. Sofi, Javier Macossay, and Faheem A. Sheikh

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Nanocamptothecins as New Generation Pharmaceuticals for the Treatment of Diverse Cancers: Overview on a Natural Product to Nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Touseef Amna, M. Shamshi Hassan, and Faheem A. Sheikh

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Smart Biomaterials from Electrospun Chitosan Nanofibers by Functionalization and Blending in Biomedical Applications . . . . . . . Hasham S. Sofi, Nisar Ahmad Khan, and Faheem A. Sheikh

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Unique Properties of the Gold Nanoparticles: Synthesis, Functionalization and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . Roqia Ashraf, Touseef Amna, and Faheem A. Sheikh

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Nanotechnology and Diabetes Management: Recent Advances and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rumaisa Rashid, Amreen Naqash, Ghulam Nabi Bader, and Faheem A. Sheikh

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Recent Advances in the Emergence of Nanorobotics in Medicine . . . Taha Umair Wani, Syed Naiem Raza, Nisar Ahmad Khan, and Faheem A. Sheikh

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Composites of Ceramic and Polymeric Nanofibers for Photocatalytic Degradation of Dairy Effluent . . . . . . . . . . . . . . . . . . Muzafar A. Kanjwal and Faheem A. Sheikh

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Editor and Contributors

About the Editor Faheem A. Sheikh is an Assistant Professor at the Department of Nanotechnology, University of Kashmir, India. Before this, he served as an Assistant Professor at the Department of Biotechnology at the Central University of Kashmir, India (2015– 2016); Brain Korea 21+ Research Professor at the Myongji University, South Korea (2014–2015); Assistant Professor Research at Nano-Bio Regenerative Medical Institute, Hallym University, South Korea (2012–2014); Postdoc/Research fellow at the University of Texas Rio Grande Valley, Texas, United States of America (2010–2012); and Research Professor at Myongji University, South Korea (2010). His research mainly focuses on fabricating 3D nanomaterials used in tissue engineering and regeneration. He has considerable expertise in the fabrication of polymeric, ceramic, and metal oxide nanofibers using electrospinning, as well as the production of porous scaffolds by solvent casting, salt leaching, 3D printing, gas forming, sol-gel synthesis, phase separation, freeze-drying, particulate leaching, and self-assembly for hard and soft tissue engineering. He has more than 16 years of research experience in nanotechnology, with a particular focus on new material fabrication, developing new techniques, tissue engineering and drug delivery. He has published over 100 peer-reviewed articles, contributed to 10 conferences, written 7 book chapters, and delivered 4 invited talks.

Contributors Touseef Amna Department of Biology, Faculty of Science, Albaha University, Al Bahah, Kingdom of Saudi Arabia Roqia Ashraf Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India Ghulam Nabi Bader Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India

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M. Shamshi Hassan Department of Chemistry, Faculty of Science, Albaha University, Al Bahah, Kingdom of Saudi Arabia Muzafar A. Kanjwal Mechanical Engineering, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Nisar Ahmad Khan Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India Department of Pharmaceutical Sciences, School of Applied Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India Santosh Kumar IIMT College of Pharmacy, Greater Noida, Uttar Pradesh, India Javier Macossay Department of Chemistry, The University of Texas Rio Grande Valley, Edinburg, TX, USA Amreen Naqash Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India Pankaj Patel University Institute of Pharmacy, CSJM University, Kanpur, Uttar Pradesh, India Zahid Rafiq Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, S.A.S Nagar, Mohali, India Rumaisa Rashid Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India Syed Naiem Raza Department of Pharmaceutical Sciences, School of Applied Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India Faheem A. Sheikh Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India Hasham S. Sofi Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India Taha Umair Wani Department of Pharmaceutical Sciences, School of Applied Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India

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Advancements of Nanotechnology in Diagnostic Applications Zahid Rafiq, Pankaj Patel, Santosh Kumar, Hasham S. Sofi, Javier Macossay, and Faheem A. Sheikh

1.1

Introduction

Diagnosis is the very first step towards the treatment and mitigation of diseases. The initial medical diagnosis involves the evaluation of symptoms that are corroborated with patient history and interpretation of test results. It has been the basis and will continue to be the future of medical treatment (McPhee et al. 2010). The efficiency of medical diagnosis is proportional to a fast, reliable, specific and accurate response, thus increasing the probability of providing an earlier treatment or in some instances of the survival of the patient. In regards to diagnostic methods, a high degree of sensitivity and specificity is desired for the early detection of disorders (Azzazy et al. 2006), and this has continuously improved based on technological development. Numerous techniques and assays, which include immunoassay, medical imaging and biosensing, are available for diagnosis. The initial and foremost run tests include gram and giemsa staining for the detection of particular bacteria and nucleic acid detection of some specific parasites.

Z. Rafiq Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, S.A.S Nagar, Mohali, India P. Patel University Institute of Pharmacy, CSJM University, Kanpur, Uttar Pradesh, India S. Kumar IIMT College of Pharmacy, Greater Noida, Uttar Pradesh, India H. S. Sofi · F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] J. Macossay Department of Chemistry, The University of Texas Rio Grande Valley, Edinburg, TX, USA # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_1

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Furthermore, some advanced strategies explore the use of molecular biology techniques, including polymerase chain reaction and enzyme-linked immunosorbent assays, that frequently are used for the detection of certain infectious viruses (Kumar 2007). Conventional diagnostic methods suffer from a few limitations, such as little specificity and low efficacy. Moreover, due to the genetic transformation in the case of bacterial species resulting in multiple drug resistance for different antibiotics, especially in developed countries, severely hinders the detection and treatment of diseases. These challenges led scientists to put countless efforts to improvise existing/additional advanced types of machinery for rapid and precise detection of diseases (McPhee et al. 2010). It is worth mentioning that one such revolutionary strategies are the application of nanotechnology in the medical system to increase pace, efficacy, reliability and sensitivity of diagnosis. Nanotechnology comes with tools such as nano-scale structured materials with dimensions of less than 1 μm (nanoparticles) and nano-devices that can be fabricated to achieve the potential for advanced diagnostics and biosensors. Owing to the unique properties, e.g., small size, biocompatibility, enhanced catalysis, surface plasmon resonance, optical and magnetic features, making them a desirable candidate for application in medical diagnosis. Mainly, nanotechnology involves the manipulation of matter at atomic, molecular and/or supramolecular scale, thereby interfering with the biological systems with many folds, which help to interpret the data for real-time detection of the infectious pathogens. Therefore, the inclusion of nanotechnology has revolutionized many fields, particularly medical diagnoses, to improve the health care system.

1.2

Augmenting Different Methods of Diagnosis with the Aid of Nanotechnology

Several modalities in medical diagnosis such as bioassay, biosensors and imaging tools are remarkably improved in terms of sensitivity, specificity, efficacy and reproducibility by integration with nanotechnology. Conventional methods of diagnosis suffer from limitations, for example, poor sensitivity, specificity and reproducibility. Moreover, the traditional diagnostic techniques often cannot identify life-threatening diseases such as early stages of cancers. However, modern tools taking the help of electronics and other related areas, are trying to modernize the current diagnosis in a shorter time and large samples can be investigated at one time (Satvekar et al. 2014).

1.2.1

Nanotechnology in the Method of Clinical Diagnosis

Currently, clinical diagnosis aims to provide point-of-care with particular emphasis on the fabrication of nano-scaled devices and nano-bio sensors, which can perform the tests at a much shorter time. These techniques, when available to the patient, can decrease the disadvantages of existing procedures and help to reduce the time for

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obtaining test results. In clinical diagnosis, especially in oncology, the detection of cancer markers in blood serum and cell lysates exemplifies the future of cancer diagnostics. The ability to investigate all biomarkers, present at a wide range of concentrations, will help clinicians to make an earlier diagnosis of malignant diseases. This otherwise would be difficult to detect during the initial or early stages of cancers. These so-called smart diagnosis kits are mostly combined form of devices that uses the samples for numerous kinds of biomarkers. Moreover, there is a clear-cut difference between the hospital and home diagnoses, and the former uses automated assays at large-scale, usually the PCR, and in some instances, the ELISA based techniques are frequently adopted. Whereas the home diagnosis kits work mostly on the ELISA technique. However, the patients performing the selftests should strictly observe the monitoring of the results. To prevent these problems, sensors with latest, smart and ultra-high sensitivity detection capacities are need of the hour for the scientist to manufacture for fast diagnoses. The resulting nano-based sensors will use less amount of the sample and combining the transducer with biorecognition, thereby enabling to transform biochemical reaction into a detectable signal. These smart sensors could be efficiently used for the detection of specific markers (e.g., thrombin, C-reactive protein, troponin). Additionally, the use of such devices/sensors could sharply decrease the huge expenses caused at hospitals, and this would ultimately reflect in the treatment of patients (Mascini and Tombelli 2008).

1.2.2

Nanotechnology and Medical Imaging

Modern-day science has involved the use of in-vivo imaging as the latest tool for disease diagnoses. The imaging can help to identify the symptoms within live tissue suspected of being infected. These imaging techniques include magnetic resonance imaging, positron emission tomography, photoacoustic imaging, computed tomography, optical coherence tomography, optoacoustic or photoacoustic tomography and near-infrared imaging (Rosen et al. 2011). In addition to the available diagnosis tools, the biomedical sciences have exploited properties of visualizing agents for in-vivo imaging. For instance, the nanoparticles distributed at a particular organ are capable of providing a desirable contrast in ultrasound and magnetic resonance imaging. Furthermore, the nanoparticles can be cross-linked with contrast agents to give a suitable resolution, which can favorably enhance the sensitivity. More specifically, the paramagnetic nanoparticles are extensively used as a contrast agent in a magnetic resonance imaging procedure. On the other hand, liquid perfluorocarbon nanoparticle and liposome are regularly used as a contrast agent in ultrasound machines. Optically active gold nanoparticles can be used as a contrast agent for photoacoustic imaging as well as in near-infrared imaging due to their unique luminescence properties. An additional advantage of using the nanomaterials is the human body cannot remove this material due to their nano-size; this perhaps gives ease of investigation at a long day as desired by doctors. Magnetic nanomaterial, for example, the iron oxide nanoparticles when functionalized using peptides capable of

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binding tumor cells, can significantly improve the results in imaging due to the magnetic properties. This allows the non-invasive evaluation of pancreatic inflammation for the diagnosis of autoimmune diabetes (Turvey et al. 2005).

1.2.3

Modulation of Electrodiagnosis by Nanotechnology

The basic principle involved in electrodiagnosis depends on recording the electrical impulse of the body, thereby interpreting the data to diagnose the diseases. It is the field of diagnosis which encompasses electrophysiology and electrical technology to understand the neurophysiology, neuro diagnosis, electromyography and evoked potentials. The most trusted and widely adopted strategy is the recording of electric signals spontaneously to keep an eye on the functioning of the heart and this technique is called electrocardiography. Other similar methods which revolve capturing the electrical signals from the brain and continuously assess these signals to form electroencephalography. Moreover, electromyography is used for monitoring nerve and muscle cell activity, tonometry used for measuring internal eye pressure and spirometry to perform the lung function test (Alzaidi et al. 2012). Figure 1.1 indicates the overall routes of diagnoses used currently in the biomedical sciences.

Fig. 1.1 Representation of various methods of diagnosis used in biomedical sciences

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1.3

Nanomaterial Based Techniques Used in the Diagnosis

1.3.1

Gold Nanoparticles and Their Role in the Diagnosis

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The gold nanoparticles are most commonly used for diagnoses purpose. The rationale is because of their easy synthesis process by physical or chemical routes, biocompatibility and nontoxic nature. Owing to these properties, the gold nanoparticles are regularly being used for labeling and biosensing applications. For labeling, certain features of the particles are exploited to generate contrast. Their nano-size and the possibility of functionalization with biomolecules mean that they also provide extremely high spatial resolution and specificity in specific applications. Moreover, properties such as optical properties, strong absorption, scattering and especially surface plasmon resonance make them the ideal candidate for a large variety of light-based techniques including combined schemes including photothermal and/or photoacoustic imaging (Sperling et al. 2008). Some of the applications of gold nanoparticles are discussed below.

1.3.1.1 Cancer Diagnosis with Surface-Modified Gold Nanorods Hetero-functional gold nanorods were fabricated using a wet chemical method. Further on, these nanorods were covalently conjugated with Herceptin (i.e., the antibody against tumor antigens) and poly(ethylene glycol) as a carrier and avoiding it from the reticuloendothelial system. The results indicated functionalized gold nanorods within HER2/neu overexpressing at tumor sites in the nude mouse model (Eghtedari et al. 2008). 1.3.1.2 Immunoassay and Electrochemical Method-Based Cancer Diagnosis Sensitive detection using a layer of gold nanoparticles on ELISA microplate was achieved by wet plating. The modified surface was able to bind biomarker carcinoembryonic more firmly. This slight improvisation with the existing ELISA resulted in cheaper and easier handing of early cancer diagnostic compared to the pristine microplates (Zhou et al. 2011). 1.3.1.3 Diagnostic Imaging in Cancer A general approach for the targeting and imaging of cancer cells using dendrimer entrapped with gold nanoparticles was found to link with targeting molecules covalently. These findings suggest that these nanoparticles help as a potential candidate for cancer imaging and therapeutic (Huang et al. 2007). 1.3.1.4 Role of Gold Nanoparticle-Based Biosensors Due to the desired optical properties of gold nanoparticle, allowing them to change the color after binding with specific molecules resulting in recognition of the analyte. Using these attributes, a small concentration of DNA can be elevated to figure out the levels of uric acid in diseases such as gout, hyperuricemia, pneumonia, and kidney

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damage that can be exploited by this property of gold nanoparticles (Kannan and John 2009).

1.3.1.5 Analysis of Biomolecules by Gold Nanoparticles Using surface-enhanced Raman spectroscopy, gold nanoparticles have been exploited to detect small amounts of biological molecules such as proteins and DNA. For instance, the gold nanoparticles were used to identify protein (low concentrations) molecules by functionalization. On the one hand, the thiol group cable to binding the gold nanoparticle was prepared. On the other hand, diazonium moiety was allowed to react with lateral chains of protein (Bizzarri and Cannistraro 2007). 1.3.1.6 Role of Gold Nanoparticle-Based Immunoassay in Diagnosis Using the surface-enhanced Raman spectroscopy, gold nanoparticles can be used to target tumors. Moreover, the PEGylated nanoparticles of gold are often conjugated with IgG and/or scFv antibody to get overexpressed in the cancer cells and then these PEGylated gold nanoparticles can be detected due to their surface-enhanced Raman effect. Gold nanoparticles functionalized with engineered scFv containing either a cysteine and/or histidine in its linker region, which were used to develop a colorimetric immunoassay, as shown in Fig. 1.2 (Liu et al. 2009).

Fig. 1.2 Representation of different applications of gold nanoparticles in clinical diagnostics. Reprinted with permission from Elsevier (Her et al. 2017)

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Magnetic Nanoparticles

Magnetic nanoparticles, including iron, nickel, cobalt and their derivatives, have specific properties that make them a promising candidate for diagnosis. These properties include superparamagnetism, high saturation field, blocking temperature, etc. In addition to these applications, nanoparticles used in particle imaging, defect sensors, immunoassay, medical imaging, resonance imaging, immobilization of DNA and proteins and isolation of pathogens (Mornet et al. 2004).

1.3.2.1 Applications of Magnetic Nanoparticles in Imaging Apart from using magnetic nanoparticles to treat cancers while allowing the hyperthermia, these nanoparticles can be used as contrast agents in magnetic resonance imaging. On the one hand, magnetic nanoparticles can provide cancer therapy. On the other hand, these nanoparticles can enable diagnoses. Moreover, the combined effect caused due to the external field beside the inherent permeability into tissue easily qualifies them for in-vivo imaging using magnetic resonance. Superparamagnetic iron oxide nanoparticles are the class of core-shell type nanoparticles (allowing to functionalize the organic and inorganic components) that can penetrate tissues with the influence of the external magnetic field. These nanoparticles can be used as treatment and diagnoses of cancers (e.g., breast, ovarian and cervical) and for atherosclerosis, calcium-sensing and cerebral ischemic lesions. Magnetic resonance imaging of intracranial tumors can be remarkably enhanced by loading dextran-coated iron oxide nanoparticles for more than 24 h (HofmannAmtenbrink et al. 2010). Figure 1.3. represents the use of magnetic nanoparticles in bioimaging of cancer.

Fig. 1.3 Representation of the use of magnetic nanoparticles for image diagnoses of cancers

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Quantum Dots in Diagnosis

Quantum dots that made up of semiconductor materials that are spherical and fluorescent nanocrystal substances whose size ranges from 2 to 8 nm. Primarily, the functionalized quantum dots with florescent properties can emit light when exposed to UV light of various colors. They also offer unique optical properties with size-tunable narrow emission, excellent photostability, less photobleaching, sensitivity and emit fluorescence without the use of laser. Therefore, quantum dots have entirely replaced the conventional fluorophores and are widely used in the fabrication of smart biosensors to identify the small molecules (e.g., antibodies, Nucleic acids, proteins, enzymes and amino acids) (Azzazy et al. 2007).

1.3.3.1 Quantum Dot-Based Fluorophore DNA Nanosensor In the quantum dot-based assays, a light source is excited to produce energy, which is accepted by the fluorescent molecules (acceptors energy). Further on, the energy excites “electrons” outer shell of the fluorescent carrier, is then released in the form of light (i.e., fluoresce); this occurs when the electron reaches to its ground state, referring to as Förster resonance energy transfer. This energy transfer is only possible if the electron donor and acceptor are extremely proximal to each other. Generally, the quantum dots are used to ascertain the presence and/or absence of fluorophores at the prefer of its surface. The preferred role of fluorophores can be to conjugate it to a high-affinity polymer such as cDNA and then analyze the detection of other matching DNA from the pool (Frasco and Chaniotakis 2010). This förster resonance energy transfer-based assays system detect other biological specimens such as the presence of methylated DNA, measuring the distance between the domain in a single protein, location of specific genes, educating the structure of integrin and membrane proteins and study the lipid raft in the cell membrane. Bio-conjugated quantum dots are capable of detecting the cancer cells due to bright and stable fluorescent light emission and sensitivity of fluorescence imaging. Moreover, quantum dots are the ideal candidate for biosensor application as it supports continuous monitoring of signals. Luminescent and stable quantum dot bioconjugates enable visualization of cancer cells in living animals. Moreover, labeling of cancer markers such as human epidermal growth factor receptor 2 using specific antibodies to functionalize of quantum dots covered by polyacrylate cap are also prepared (Karakoti et al. 2015). 1.3.3.2 Multiplexed Optical Coding The narrow emission spectra of differentially-sized quantum dots can leverage as distinct individual labels that provide multicolor visual coding for biological assays. In this regard, Han et al. demonstrated that a massive multiplex essay could be created by encapsulating varying quantities of different quantum dot colors within optically encoded polymer microbeads. This and other multiplexing schemes have been designed and employed in several notable works, such as in single-nucleotide polymorphism, single DNA molecule detection via multiplexed color colocalization, mutation analysis using fluorescence coincidence detection and magnetic quantum

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dot multiplexed gene expression analysis, respectively (Xu et al. 2003; Pisanic Ii et al. 2014).

1.3.4

Carbon-Based Nanomaterials in Diagnostics

Carbon-based nanomaterials, including nanodiamond, carbon nanotubes, graphene, carbon nanofibers, and its derivatives, graphene oxide, etc., are highly versatile and applicable owing to their structural, thermal, optical, chemical, mechanical, electrical characteristics. Carbon-based nanomaterials have a diverse type of medical applications, including imaging of cells and tissues and their biocompatibility and ease of functionalization have made them as excellent candidate for imaging different tumors. The utilization of the carbon nanomaterials in diagnostic applications are shown in Fig. 1.4. To date, broad-range one-photon property of carbon based-nanomaterials has been extensively used for the fabrication of biosensors, resulting in imaging-based diagnostics. The carbon nanotubes are 1D nanomaterials having large hollow cylindrical structures allowing them to possess large surface area, optical property, and small sizes, which potentially favor the binding of the large number of biomolecules on it. The carbon nanotubes due to their uniqueness can penetrate cell membranes, which pave the road for using as a delivery agent of drug, nucleic acid and protein into the cytoplasm and some time into the nucleus. Furthermore, it is assumed that prospective applications are encouraged by their competence to penetrate the biological membranes and with relatively low toxicity (Zhang et al. 2010).

Fig. 1.4 Diagnostic and other forms of carbon-based nanomaterials, including CNTs and graphene oxide nanoparticles. Reprinted by permission from the Royal Society of Chemistry (Patel et al. 2019)

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1.3.4.1 Role of Carbon Nanotubes in Photoacoustic Imaging In the past, photoacoustic imaging technology was vigorously tested in different biomedical fields. Later on, after improvisation in a photoacoustic imaging system that led to the invention of pulse laser, which is delivered in the biological sample. During the process, some laser energy is absorbed and converted into heat, leading to produce acoustic waves, which is detected by the ultrasonic detector to form the 2D or 3D image. The main advantage of this system is that it provides spatial resolution with deeper penetration into tissues, which otherwise is difficult with other techniques. Due to the strong near-infrared photoluminesce of multi-walled nanotubes (MWNTs) and single-walled nanotube (SWNTs), they are continuously used as photothermal agents (Moon et al. 2009). Therefore, carbon nanotubes, as they possess strong NIR, are going to be routinely used as contrast agents for photoacoustic imaging (Hong et al. 2010).

1.3.5

Role of Graphene Oxide in Diagnosis

Graphene oxide is another versatile carbon-based transparent nanomaterial with the least capital investment for its production. Due to its highly conductive nature, it is used in electrochemical biosensors. Moreover, the graphene is thin 2D material in the form of sheets and comprises sp2 and sp3 hybridized carbon atoms. It has high thermal conductivity, high electron mobility, high elasticity and other properties such as tunable bandgap, high mechanical strength and very high room temperature quantum Hall effect make them highly desirable for various applications. Graphene oxide is a transparent material and internally, it is in the form of sheets that can be made to attach the antibody, which in turn binds to the cancer cells and tag them with fluorescent molecules to visualize the cancer cells in the specimen. It can also satisfactorily detect various analytes such as ascorbic acid, dopamine, cholesterol, glucose and other heavy metals (Pandit et al. 2016).

1.4

Nanodevice Based Platforms in Nanodiagnostics

1.4.1

Nanoparticle-Based Biochips

Biochips represent a collection of microarrays arranged on a surface of a solid substrate onto which a large number of biological molecules are attached. The term biochip represents all surfaces onto which tiny spots, each one being formed by specific capture probes. These capture probes complement the target sequence to be detected. Each capture probe will bind to its corresponding target sequence. In a high throughput screening mode, a biochip enabled simultaneous analyses of thousands of biological reactions, such as decoding genes, in a few seconds. Biochips consist of glass or silicon surface bearing printed circuits combining many processes for DNA analysis. Biochips are formed by in situ (on-chip) synthesis of oligonucleotides or peptide nucleic acids (PNAs) or spotting of DNA fragments. Hybridization of RNA- or DNA-derived samples on chips allows the

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monitoring of expression of the coding transcriptome (mRNAs) or the occurrence of polymorphisms in genomic DNA. The devices are mainly designed to interact with cellular components with a high level of specificity. Each biochip has hundreds to thousands of gel drops on a glass, plastic or membrane support, each about 100 μm. A segment of a DNA strand, protein, peptide or antibody is inserted into each drop, tailoring it to recognize a specific biological agent or biochemical signature. These drops are in known positions, so when a sample reacts, the reaction position can be detected, identifying the specimen. The biochip system can identify infectious disease strains in less than 15 min when testing protein arrays and in less than 2 h when testing nucleic acid arrays (FitzGerald et al. 2005).

1.4.1.1 Role of DNA Biochips as Nanofluidic Microarrays The surface of DNA biochips is having nanofluidic microarray in nature and are usually made up of microfluidic channel that provides paths for biomolecules to stream in individual biosensors. These are considered as small laboratories able to perform multiple biochemical tests. These chips can quickly screen nano-liter DNA concentration from diagnosis to identify biological terrorism. The nanofluidics chips smoothly allow the stretching of a single DNA molecule, its separation and detection at the endpoint. Using this technique, scientists can observe the image of a single molecule on a bench-top fluorescent microscope. Systems biology, personalized medicines, the discovery of disease-causing organisms and the development of drugs are unique possibilities of the nanofluidic technique (Bahadorimehr et al. 2010).

1.4.2

Role of Protein Nanobiochips

The protein nanobiochips are the recently developed devices that can detect proteins consuming small quantities of samples and reagents that earlier were challenging to analyze using microarray. They seem to be highly promising and efficient; however, they are in their intial developmental stage. These chips can capture the desired protein molecule from a pool of other protein molecules, and the reaction predominately can occur on a layer of silica nanoparticles. The robotic arm runs all these reactions in a predefined set of instructions. Furthermore, the antigen-antibody complex can be formed and then analyzed by a suitable method of detection. Protein biochips facilitate diagnostics of cancer by biochip readout activity levels of many proteins. For instance, the 12 tumor marker biochip system (C12 system) has been proven useful in some studies (Chen et al. 2008).

1.4.3

Role of Microelectromechanical Systems in the Diagnosis

It is noteworthy to mention here that this type of diagnostic does not require a fluidlike substrate as a base (i.e., the system is not based on microfluidics). Microelectromechanical systems are generally exploited in the delivery of expansive drugs. However, these days these systems are employed in detection resulting in

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excellent results with high reproducibility. These are “smart capsular pills,” intended to be swallowed to generate images of the gastrointestinal tract, which are then monitored to visualize internal ulceration and/or source of bleeding to estimate the cause (Satvekar et al. 2014). In brief, these are designed in the form of a capsule equipped with light-emitting diode, battery, a transmitter and complementary metaloxide-semiconductor recorder. The image of the gastrointestinal tract is captured using the capsular camera, which presents as moving pictures. This allows the visualization of gastric ulcers and tumors at different locations even at the stage of minute development, thereby allowing to diagnose early stages of cancers. A specific diagnosis of the condition of the internal structures can be non-invasively detected (Satvekar et al. 2014).

1.4.4

Potential of Nano Biosensors in Medical Diagnosis

Nano biosensors are the devices that are widely used to detect individual biological molecules (e.g., antibodies, nucleic acids, pathogens, metabolites and protein). Currently, the nano biosensors are carving their ways to identify specific cells and/or particular areas of the body. How it works is that nanomaterials can bind particular analytes through the receptors, which successively moderate the physiochemical signal associated with the binding. Later on, a transducer imprisonments these signals and converts them to an electric signal. In brief, the variation in the message such as electrical current, conductance, impedance, intensity/phase of electromagnetic radiation, mass, temperature and viscosity is supervised (Fig. 1.5). Analysis of the variation in one or more of these parameter quantifies the presence and/or absence of biological agents. The nanomaterial acts as an intermediate layer between biological agents and detector or biological agents and the transducer is combined with nanomaterials to construct a biosensor. In some cases, using fluorescence properties of quantum dots (e.g., cadmium selenide and zinc sulphide), the tumors within the body could be located by finding the fluoresced nanodot. The nanobiosensors can have different modes of detection (electrical, optical or mechanical). It is noteworthy to mention that early detection of tumors is possible by the individual difference between cell morphology and/or biochemical abnormality from normality. Furthermore, genetic defects can easily be identified using the specific sequence of DNA (Kumar 2007).

1.5

Conclusion

Nanotechnology-based tools and devices have markedly renovated the healthcare sector, particularly in medical diagnostics. Advanced and safer diagnostic tools are available, which enable the medical practitioners to diagnose a disease at its early onset. The conventional methods of diagonsis suffer limitations of low sensitivity and reduced efficacy, which have been effectively managed by nano-scale tools. The exceptional characteristics of nanomaterials (e.g., gold nanoparticles, carbon-based

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Antibody Potentiometric Enzyme Amperometric Nucleic acid Cell

Clinical analysis

Impediometric

Microparticles

Analyte

Sensing substrate

Electrochemical measurement

Fig. 1.5 Diagnostic potential of a nano biosensor. Reprinted by permission from Springer Nature (Singh et al. 2016)

nanomaterials like nanotubes and graphene oxide, several magnetic nanoparticles, quantum dots) have tremendously augmented the advancement in the medical diagnostics. The field of nanotechnology is expanding, and thus, the opportunities are widely increasing in the diagnosis of many life-threatening illnesses such as neurodegenerative, diabetes and cancer. In the coming decades, nano interventions to create new diagnostic tools will continuously improvise, which will open new avenues for patient treatment. However, there is a need for more proper attention and global coordination for establishing and maintenance of international standards to make this sphere of nanotechnology reaching its new heights. Acknowledgments The Department of Science and Technology Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016, supported this work.

References Alzaidi A, Zhang L, Bajwa H (2012) Smart textiles based wireless ECG system. In: Proceedings of the Systems, Applications and Technology Conference (LISAT), IEEE Long Island, Farmingdale, NY Azzazy HM, Mansour MM, Kazmierczak SC (2006) Nanodiagnostics: a new frontier for clinical laboratory medicine. Clin Chem 52:1238 Azzazy HM, Mansour MM, Kazmierczak SC (2007) From diagnostics to therapy: prospects of quantum dots. Clin Biochem 40:917 Bahadorimehr A, Yunas J, Majlis BY (2010) In: 2010 International Conference on Electronic Devices, Systems & Applications (ICEDSA 2010), Kuala Lump, Malaysia, p 242

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Bizzarri AR, Cannistraro S (2007) SERS detection of thrombin by protein recognition using functionalized gold nanoparticles. Nanomed Nanotechnol Biol Med 3:306 Chen C, Chen L, Yang G, Li Y (2008) The application of C12 biochip in the diagnosis and monitoring of colorectal cancer: systematic evaluation and suggestion for improvement. J Postgrad Med 54:186 Eghtedari M, Liopo AV, Copland JA, Oraevsky AA, Motamedi M (2008) Engineering of heterofunctional gold nanorods for the in vivo molecular targeting of breast cancer cells. Nano Lett 9:287 FitzGerald SP, Lamont JV, McConnell RI, Benchikh EO (2005) Development of a high-throughput automated analyzer using biochip array technolog. Clin Chem 51:1165 Frasco MF, Chaniotakis N (2010) Bioconjugated quantum dots as fluorescent probes for bioanalytical applications. Anal Bioanal Chem 396:229 Her S, Jaffray DA, Allen C (2017) Gold nanoparticles for applications in cancer radiotherapy: mechanisms and recent advancements. Adv Drug Deliv Rev 109:84 Hofmann-Amtenbrink M, Hofmann H, Montet X (2010) Superparamagnetic nanoparticles - a tool for early diagnostics. Swiss Med Wkly 140:w13081 Hong G, Tabakman SM, Welsher K, Wang H, Wang X, Dai H (2010) Metal-enhanced fluorescence of carbon nanotubes. J Am Chem Soc 132:15920 Huang X, Jain PK, El-Sayed IH, El-Sayed MA (2007) Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine (Lond.), 2:681 Kannan P, John SA (2009) Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode. Anal Biochem 386:65 Karakoti AS, Shukla R, Shanker R, Singh S (2015) Surface functionalization of quantum dots for biological applications. Adv Colloid Interf Sci 215:28 Kumar CS (2007) Nanomaterials for medical diagnosis and therapy. Wiley, Hoboken Liu Y, Liu Y, Mernaugh RL, Zeng X (2009) Single chain fragment variable recombinant antibody functionalized gold nanoparticles for a highly sensitive colorimetric immunoassay. Biosens Bioelectron 24:2853 Mascini M, Tombelli S (2008) Biosensors for biomarkers in medical diagnostics. Biomarkers 13:637 McPhee SJ, Papadakis MA, Rabow MW (2010) Current medical diagnosis & treatment 2010. McGraw-Hill Medical, New York Moon HK, Lee SH, Choi HC (2009) In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes. ACS Nano 3:3707 Mornet S, Vasseur S, Grasset F, Duguet E (2004) Magnetic nanoparticle design for medical diagnosis and therapy. J Mater Chem 14:2161 Pandit S, Dasgupta D, Dewan N, Prince A (2016) Nanotechnology based biosensors and its application. Pharma Innov 5:18 Patel KD, Singh RK, Kim H-W (2019) Carbon-based nanomaterials as an emerging platform for theranostics. Mater Horiz 6:434 Pisanic Ii T, Zhang Y, Wang T (2014) Quantum dots in diagnostics and detection: principles and paradigms. Analyst 139:2968 Rosen J, Yoffe S, Meerasa A, Verma M, Gu F (2011) Nanotechnology and diagnostic imaging: new advances in contrast agent technology. J Nanomed Nanotechnol 2:1 Satvekar R, Tiwale B, Pawar S (2014) Emerging trends in medical diagnosis: a thrust on nanotechnology. Med Chem 4:407 Singh P, Pandey SK, Singh J, Srivastava S, Sachan S, Singh SK (2016) Biomedical perspective of electrochemical nanobiosensor. Nanomicro Lett 8:193 Sperling RA, Gil PR, Zhang F, Zanella M, Parak WJ (2008) Biological applications of gold nanoparticles{. Chem Soc Rev 37:1896

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Turvey SE, Swart E, Denis MC, Mahmood U, Benoist C, Weissleder R, Mathis D (2005) Noninvasive imaging of pancreatic inflammation and its reversal in type 1 diabetes. J Clin Invest 115:2454 Xu H, Sha MY, Wong EY, Uphoff J, Xu Y, Treadway JA, Truong A, O’Brien E, Asquith S, Stubbins M (2003) Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res 31:e43 Zhang Y, Bai Y, Yan B (2010) Functionalized carbon nanotubes for potential medicinal applications. Drug Discov Today 15:428 Zhou F, Yuan L, Wang H, Li D, Chen H (2011) Gold nanoparticle layer: a promising platform for ultra-sensitive cancer detection. Langmuir 27:2155

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Polycaprolactone-Based Nanofibers and their In-Vitro and In-Vivo Applications in Bone Tissue Engineering Rumaisa Rashid, Hasham S. Sofi, Javier Macossay, and Faheem A. Sheikh

2.1

Introduction

Tissue engineering is a specific combination of desired cells capable of growth/ regeneration, biomaterials that are biocompatible and biodegradable, the necessary growth factors, and required load-bearing capability to sustain its mechanical integrity appropriately. In this direction, numerous approaches are put forward by physicists, chemists, biologists and engineers to cover the remaining unsolved issues related to the generation of new tissues and organs using a small number of cells. However, it is still unclear how much positive impact these approaches will have on actual clinical practice. Historically, there are various examples from early times when surgeons used crude or undesirably painful methods to amputate and/or to rejoin body parts. For instance, a famous painting depicted by Italian painter Fra Angeliaco in 1438, where the homograft transplantation of a limb onto a wounded soldier is described, and this is perhaps the first example of “tissue engineering” (Vacanti 2006). Archival evidence suggests that Urist in 1965, was the first one to regenerate bones artificially. However, he called this process an autoinduction in which a formation of distinct bone can occur in decalcified bone (Urist 1965). It was until the 1980s when the term “tissue engineering” was inappropriately used for the manufacture of prosthetics and/or surgical intervention of tissues, or sometimes to

R. Rashid Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India H. S. Sofi · F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] J. Macossay Department of Chemistry, The University of Texas Rio Grande Valley, Edinburg, TX, USA # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_2

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indicate the use of tools in plastic surgery. The first articles directly related to modern tissue engineering were published in 1991, and the term in the same context continues to be used today (Chaignaud et al. 1997). Significant success has been achieved in bone tissue engineering in recent years, but the resulting therapeutic interventions, like artificially constructed grafts, present a lot of shortcomings that are highlighted in this book chapter. The bone is a highly dynamic, most durable, and deeply vascularized tissue, which can efficiently support and protect vital organs present in the body. The bone is mainly composed of organic (forms flexible framework) and inorganic components (hardens soft framework). The former comprises collagen fibers (type I of ~90%) and other structural proteins. For example, bone sialoprotein (~8%), osteopontin, osteocalcin and other compulsory growth factors (Florencio-Silva 2015). The inorganic component is mainly composed of calcium/phosphate in the ratio of 10:6, which upon nucleation forms the crystals of hydroxyapatite (Ca10(PO4)6(OH)2) (Barakat et al. 2008). Fibrous proteins and bone salts (i.e., collagen and hydroxyapatite) along other aforementioned structural proteins impart bone a structural organization and mechanical support concerning resistance towards fracture toughness, high tensile and compressive strength (Sofi et al. 2019a). Bone is regarded as the second most transplanted tissue worldwide after the blood, with an estimate of 6.9 million orthopedic surgical procedures by 2020 (Campana et al. 2014). Therefore, bone tissue engineering has tremendous industrial potential as far as the job market need is concerned. It is estimated that the global market for bone grafts and/or its substitutes was initially valued to $2690 million in the year 2017 and is expected to increase by $3912 million by 2025, subject to the stability of the global economy. Therefore, this area of research have avenues for future jobs in the orthopaedic industries for biomedical engineers. Generally, major bone defects are due to the loss from osteoporosis, fractures, overuse, trauma and/or infections during the surgery. Among these, the fracture is one of the significant contributors leading to a loss in continuity of the bone, notably it is common in all age groups. According to an annual report published by the orthopaedic industry in 2013 volume, ~51% of the fractures occur in the young population, which notably is the productive age group, thereby emanating in economic losses (Piuzzi et al. 2019). The bone repair and regeneration cascade is a multistep and overlapping process that begins with inflammation leading to the enforcement of stem cells to the fracture site. However, if the inflammation prolongs, it delays the process of regeneration to occur naturally. Therefore, modulation of inflammation is a necessary step towards the restoration of bone defects. In continuation of this, later on, the newly recruited cells are differentiated into osteocytes at the site of fracture. In the meantime, the blood vessels innervate the regenerating tissue to reconnect it with the body leading to functional bone formation (Clines 2010). The modern bone tissue engineering approaches recommend the intervention of biomaterial in the form of a scaffold that could recapitulate all these natural processes occurring without any flaws, and this has been gaining notoriety in the last 20 years (Amini et al. 2012). However, recapitulating all these multiple processes utilizing a single bone regeneration

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scaffold is still a challenging task for researchers. In the case of regenerating the bone, three basic components are considered necessary (i.e., cells, scaffolds and growth factors) for the expansion of neo-tissue at the site where bone loss has occurred. Ideally, the aim is to create a 3D biomaterial which will favorably facilitate the attachment of cells on the small pores and penetration through the large size pores, this finally leads to proliferation of the osteo-inductive cells on the scaffold. In this interest, many studies have been attempted to enhance dual processes in a coupled manner that will ultimately lead to “engineered” bone. For instance, the coupling of osteogenesis and angiogenesis is the most commonly explored approach. The bone inducing scaffolds have been efficiently utilized to control inflammation for enhanced bone regeneration in a coupled manner (Rather et al. 2019). Moreover, and despite of the fact that biomedical science has reportedly worked on understanding and final improvements in the field of bone substitution medicine, no adequate bone substitute has been flawlessly developed (Qu et al. 2019). Therefore, most of bone related defects/injuries still are unrecoverable and are not adequately treated. In the past decades, quite a few implants fabricated for bone restoration resulting in repair of diseased and/or damaged tissues. The primary goal of bone tissue engineering is that after implant placement, the osseointegration should eventually occur. This process is often referred to as new stability and is directly associated with anchorage, design of implants and bone structure (Chan et al. 2015). Further on, the event of secondary anchorage occurs, which is characterized by a biological bonding at the interface of bone tissues and implant surface. However, to achieve biological inter-bonding, the initial stability of the implant, which will result in osseointegration, is a necessary step (Agarwal and García 2015). For this target, studies focused on augmenting the osseointegration by different surface modifications and/or by fabricating nano-composites. The main goal of these modifications is to provide mechanical stability of implants with a desirable biological property for the adsorption of proteins, the adhesion, and differentiation of various cells and, finally, leading tissue osteointegration (Chen et al. 2017). These natural possessions are related to physio/chemical composition, including the surface wettability and roughness of the scaffold materials. However, the control of these properties at the protein and cell levels is difficult. The modification of implants at a nano-scale level has produced acceptable surfaces that can have a controlled topology and chemistry, helping us to understand the biological interactions occurring with artificial scaffolds (Hidalgo-Bastida and Cartmell 2010). This can result in developing implants that are possessing a desirable surface for appropriate tissue integration (Seidi et al. 2011). Not only the surface morphologies of implants influence to formation of new bone tissue, but the selection of the material is also equally important (Pérez-Sánchez et al. 2010). Electrospinning is a unique and preferred technique than other strategies to produce nanofibers at lab and industrial-scale (Persano et al. 2013). This technique has taken intersect from renowned researchers because of its versatility and,

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importantly, to create the micro/nanofibers, which are somewhat similar to the collagen fibers present in the human body. Owing to their fibrous morphology, nanofibers are utilized as a scaffold for growing the tissue artificially (Sofi et al. 2020). During the electrospinning process, a charged polymer droplet gets ejaculated in the form of fibers under the influence of strong electric current. This principle is successfully exploited for the production of nanofibers at large-scale industries, mainly via the use of multiple-jet nozzle electrospinning. Herein, the abundant amount of polymer is feed through multiple needles, so the maximum number of fiber is deposited on continuously rolling numerous collectors (Varesano et al. 2010). On the other hand, needleless electrospinning is another intervention in which the nanofibers from the large container of the solution are deposited directly on the collector in an uninterrupted manner, with a high possibility of producing fibers at large quantity (Wang et al. 2009). With the aid of electrospinning, the nanofibers offer advanced porosity, large surface-area-to-volume ratio, interconnected fibers (due to conglomeration) and possible routes for surface functionality, therefore, are widely used in biomedical applications (Barakat et al. 2011; Lee et al. 2013, 2016). Despite these properties, only countable numbers of works have applied to uncover the potential of PCL nanofibers as a suitable candidate for bone substitutes. It is necessary to mention here that as-spun nanofibers, as such, have initially been used in bone regeneration studies; however, later on, these nanofibers were restricted to be used without modifications. The rationale for not to use in its pristine form is that PCL nanofibers are hydrophobic, have a low melting point, long in-vivo degradation, lacks desired mechanical properties and has low bioactivity (Mondal et al. 2016). In order to overcome limitations, various modifications has been adopted to make the PCL nanofibers well desirable. In the next sections of this chapter, we will elaborate on how scaffolds based on PCL nanofibers were modified to utilize them in the reconstruction of bone tissues flawlessly. Furthermore, modern science suggests that growth factors play an essential role, while acting as unique precursors for the development of bonny tissue. It has been concluded that promising growth factors can significantly influence the development of bone. These growth factors include bone morphogenetic protein (BMP), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), basic and acidic fibroblast growth factor (bFGF and aFGF) and insulin-like growth factor (IGF) are considered to signal, proliferate and help in differentiation of bone cells (Udomluck et al. 2020). The interplay of processes such as angiogenesis, vascular maturation and sustenance of the vasculature, which come into existence due to the presence of growth factors, results in bone regeneration (Tayalia and Mooney 2009). Moreover, the nanofibers composed of medical-grade PCL along with growth factors such as recombinant human bone morphogenetic protein-7 (rhBMP-7), which is a member of TGF-β superfamily, was noticed to form complete bone in the scaffold architecture indicating bone remodelling (Reichert et al. 2012). In addition to this, inter-connected porous networks of the biomaterial are primarily essential to augment the delivery of growth factors at the site of bone replacement. Although all these growth factors are used either in pure form and/or along with

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scaffolds, however, the BMPs are the only growth factors that are currently approved by US-FDA for bone regeneration (Kowalczewski and Saul 2018). With all this, bone tissue engineering represents an emerging field of regenerative medicine to tackle many problems associated with bone trauma or injury that usually require surgical intervention. Since surgical interventions are often associated with pain or immunological rejection, bone tissue engineering represents a novel strategy to overcome bone tissue injury by making use of scaffolds that allow remodelling of the injured bone by providing an artificial extracellular matrix (ECM). The scaffolds are constructs that represent a combination of materials with tunable properties to allow cell seeding, proliferation and differentiation resulting in early regeneration of the injured tissue (O’Brien 2011; Hutmacher 2000; Sofi et al. 2018). The basic framework of a scaffold is fabricated from natural or synthetic polymers and/or a blend of both along with different growth regulatory factors. Among those, the natural polymers for use in bone construction have unique properties of mimicking the biological niche, biocompatibility and biodegradability. However, they lack the requisite mechanical strength needed in bone regeneration and are challenging to fabricate into scaffolds (due to low molecular weight and use of undesirably toxic solvents to dissolve) by techniques like electrospinning (Coombes et al. 2002). Contrarily, synthetic polymers, however, offer advantages of secure processing and excellent tensile strength, but lack the benefit of mimicking the bone tissue (Gunatillake and Adhikari 2003). Therefore, in utility, a combination of the natural and synthetic polymers is needed for the development of a working scaffold. Among the synthetic polymers, PCL has shown promising results in various in-vitro, in-situ and in-vivo pre-clinical proof studies either alone or in combination with natural polymers (Reed et al. 2009; Cipitria et al. 2011). Essentially, the PCL is one of the commercially available and has high adaptability when used in biomedical applications. This polymer is biocompatible, biodegradable (when blended with polymers, e.g., starch), having a low melting point and glass temperature of 60, owing to these properties PCL is molded easily using a variety of available non-toxic solvent into desirably shaped biomaterial (Averous et al. 2000). Notably, this polymer in its pristine form has a long degradation in water (up to 3–4 years). However, it they are easily degraded using simulated body fluids, which is equal to human body plasma and/or in physiological conditions by the hydrolysis of its ester linkages (Chouzouri and Xanthos 2007). In addition to its natural degradation under physiological environment, it is observed that some phylum of bacteria (e.g., firmicutes and proteobacteria), specific fungal species of penicillium and aspergillus can enzymatically degrade the PCL, this tips off for care be taken while avoiding contamination of the polymer during its handing (Fields et al. 1974; Sanchez et al. 2000). Moreover, the PCL has got approval from US-FDA for its use in the manufacture of sutures and drug delivery devices. PCL belongs to the linear ester group of polymers synthesized either by radical ring-opening polymerization of 2-methylene-1-dioxapen (Woodruff and Hutmacher 2010) or directly by catalytic ring-opening polymerization of monomers (Woodruff and Hutmacher 2010). The polymer is semi-crystalline, with a melting point of 59–64  C (Cipitria et al. 2011;

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Woodruff and Hutmacher 2010; Kweon et al. 2003). PCL is classified among the biodegradable polymers and has been listed in the approved biocompatible polymers by the US-FDA (Kweon et al. 2003). The polymer is capable of undergoing degradation in physiological conditions by either hydrolytic cleavage or enzymatic degradation (Pitt et al. 1981). The degradation products of the polymer are either removed via the tricarboxylic acid cycle or directly by the kidneys. However, in the polyester class of polymers, the degradation rate of PCL is slow compared to other polymers such as polyglycolic acid and poly (l-lactic acid) due to repeated hydrophobic -CH2 moieties (Hao et al. 2002). However, the gradual degradation rate of PCL is not as advantageous for bone tissue engineering. Other loopholes include its hydrophobic nature, poor cellular adhesion due to low wettability, and some reported interactions with the biological niche (Tiaw et al. 2005). The shortcomings of this polymer are avoided by using surface grafting/functionalization (Sofi et al. 2019b), plasma treatment (Yan et al. 2013), chemical treatment (Zhang et al. 2010), coating with ECM proteins and blending with biologically active polymers or materials (Siri et al. 2010). PCL can be easily transformed into nanofibers by various techniques including electrospinning (Gautam et al. 2013), 3D printing (Temple et al. 2014), porogen leaching (Mondrinos et al. 2006), phase separation (Liu et al. 2014), force spinning (McEachin and Lozano 2012) and spin coating (Yoshimoto et al. 2003). Owing to their web-like structure and ECM mimicking properties, PCL based scaffolds have been demonstrated by various studies to aid in the bone regeneration process (Yeo and Kim 2012; Ren et al. 2017). The PCL in nano-micro form can potentially enhance cell infiltration, improve cell migration and boost the proliferation and differentiation of the cells. As aforementioned, growing osteoclasts, osteoblasts, osteocytes and chondrocytes essential for regeneration of hard tissue, using pristine PCL nanofibers, is a hard task (Arafat et al. 2011). Although PCL nanofibers demonstrated to have appropriate features in favoring the growth of soft tissues; however, to develop a scaffold that holds special importance in the conversion of a soft-to-hard interface, a series of modifications are required. Moreover, the current challenges faced by bone tissue engineering is to bridge the barrier to vascularize at the injured site, provide excellent mechanical strength for adhesion and promote host immune integration. Therefore, research is more focused on the creation of functional nanomaterials, which can overcome the gaps due to above-cited barriers. This requires in-vitro tests, pre-clinical proof of concept generated via high throughput animal studies, high-cost research grants and approval from regulatory authorities to conduct clinical trials. In this book chapter, we will explain various techniques used to fabricate either pristine or composite PCL scaffolds for bone tissue engineering. Further, we will enlighten the readers about the latest literature regarding in-vitro or in-vivo applications of the PCL or PCL composites in the area of bone regeneration.

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23

Methods of Fabricating PCL Scaffolds

Various methods in literature have reported fabricating PCL scaffolds that have shown potential applications in bone tissue engineering. Each method used for the fabrication of the PCL scaffold has its advantage and limitations. The commonly employed techniques include phase separation (Liu et al. 2014), electrospinning (Yoshimoto et al. 2003), gas foaming (Salerno et al. 2012), porogen leaching (Mondrinos et al. 2006), melt molding (Oh et al. 2011), 3D printing (Temple et al. 2014), force spinning (McEachin and Lozano 2012) and rapid prototyping (Park et al. 2012). Some of the methods commonly used to produce PCL scaffolds are discussed briefly here.

2.2.1

Electrospinning

This technique is the most of its kind to produce fibers with ultra-fine structure. Principally, based on the electrostatic deposition of the polymer threads on the collector after the application of very high static voltage (Doshi and Reneker 1993). The formation of micro/nanofibers out of a viscoelastic polymer droplet via spinneret results due to attraction from the oppositely charged high-voltage collector, connected to another terminal of the positively charged high-voltage static electricity (Doshi and Reneker 1993). As the polymer jet emancipates from the Taylor cone, the solvent evaporates in the due process, leaving the polymer fibers solidified in on the collector screen (Yarin et al. 2001). The electrospun technology has been used to produce nanofiber scaffolds from various natural and synthetic polymers (Gautam et al. 2013; Sofi et al. 2019c). This technique has been used by the researchers worldwide to fabricate woven and non-woven PCL scaffolds (Bölgen et al. 2005; Ye et al. 2011). The method has also been used for fabrication 3D scaffolds by modification of the primary collector device or process (Lee et al. 2013, 2016; Sheikh et al. 2014). PCL scaffolds have been developed while fabricating the polymer along with other bone regenerating materials (e.g., nano-hydroxyapatite and ceramics) (Kanjwal et al. 2011; Sheikh et al. 2011). The bone regenerating scaffolds have also been manufactured by co-spinning or blending the PCL along with other polymers such as silk fibroin (Bhattacharjee et al. 2015), gelatin (Ren et al. 2017), chitosan (Dong et al. 2017), poly (L-lactic acid) (Yao et al. 2017) and poly (Lglycolic acid) (Thi Hiep et al. 2017), etc. By altering the different process parameters of the electrospinning can influence the behavior of the scaffolds towards biological properties such as cell morphology, cell viability, adhesion to the matrix, proliferation on the scaffold, infiltration of the cells, gaseous/nutrient exchange and differentiation into a particular lineage. These process factors include fiber diameter (Yang et al. 2005), fiber elasticity (Johnson et al. 2009), fiber alignment (Ma et al. 2005), porosity of the nanofiber membranes (Pham et al. 2006) and pore size distribution (Pham et al. 2006). The other factors of significance include surface geometry (Dosunmu et al. 2006; Rumpler et al. 2008), surface curvature (Rumpler et al. 2008) and surface chemistry modifications as a result of different functionalization

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(Sofi et al. 2019b). Various bio-inspired materials have been fabricated using PCL as a scaffold base for the regeneration of critical-size bone defects. For instance, polydopamine templated nano-hydroxyapatite surface-functionalized with BMP-7 by catechol chemistry was spun with PCL into a fibrous bioactive scaffold for the reconstruction of the critically sized bone defects in rats (Gao et al. 2016). Similarly, many other studies reported where PCL has been functionalized to impart the hydrophilicity in the scaffold. In another study, before electrospinning, the PCL granules were treated with ice cold plasma of helium to generate functional groups sufficiently available to graft carboxymethyl chitosan and later on this grafted PCL was electrospun into nanofiber mats (Shapourzadeh et al. 2020). An interesting experiment observed was the use of synergistic inducers of osteoinductivity viz., (β-carotene a strongly red-orange pigment) as a biochemical signal modulator and electromagnetic field as a physical simulator. Dye staining and immunochemistry assays revealed that these super modified PCL scaffolds could self-differentiate the stem cells into functionally working osteoblasts.

2.2.2

Solvent Casting

This method is more commonly known as solvent casting/porogen leaching and is used to fabricate porous biomaterials for tissue engineering applications (Thadavirul et al. 2014). The technique involves pouring a solution of the polymer onto a bed of the porogen (e.g., ammonium bicarbonate, salts, glucose and emulsions) to generate scaffolds of well-defined porosities (Thadavirul et al. 2014). This is followed by the evaporation or lyophilization of the solvent resulting in the solidification of the scaffolds around the porogen. Later on, the porogen is removed by placing a framework in a bath of a suitable solution in which they are soluble. For example, sodium chloride used as a porogen can be leached out by the water, whereas paraffin can be leached out by an organic solvent (e.g., hexane) (Hou et al. 2003). This process results in the development of well defined, interconnected porous networks suitable for nutrient exchange, cell growth, cell migration, proliferation differentiation and known to support vascularization (Thadavirul et al. 2014). Different polymers have been fabricated into porous scaffolds by this method for use in tissue engineering application and PCL is not an exception. Many reports are available in the research demonstrating the use of PCL in the fabrication of porous scaffolds by this method. For example, PCL was fabricated into porous, 3D structures using sodium chloride and polyethylene glycol as porogens (Shapourzadeh et al. 2020). The frameworks have shown encouraging results as far as the growth and proliferation of pre-osteoblasts is considered. Fan Wu et al., also reported the fabrication of the PCL scaffolds using sodium chloride as a porogen by the same method (Wu et al. 2012). However, another study reported that the incorporation of the Zein along with PCL not only improved the hydrophilicity of the scaffolds but also improved the degradation rate of the PCL scaffold, which is known for its slow degrading nature (Bonadies et al. 2019).

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Spin Coating

This method of fabrication involves the formation of smooth and thin films by placing a polymer solution on a flat surface (usually with negligible roughness, e.g., silicon wafer or mica substrate). Later on, a uniform centrifugal force is applied to rotate the polymer solution to spread out a thin fibrous film. The spinning process eventually leads to evaporation of the solvent and then subsequent solidification of the polymer occurs to form a biomaterial. The thickness of the scaffold can be accustomed by changing the centrifugal force, the concentration of the polymer and/or by changing the time of spinning. However, this method of fabrication is limited by choice of the solvent used to solubilize the polymer to be tasked for spin coating. Having this noticeable difficulty with PCL, the spin coating method for scaffold fabrication has used to create scaffolds for various biomedical applications. For instance, the spin coating method was used to coat different layers of PCL and gelatin and then reinforced with bioactive glass by intercalation (Yazdimamaghani et al. 2015). This helped to prevent the degradation rate of magnesium, used in this study to promote bone growth. The in-vitro studies suggested that the degradation rate of magnesium was retarded as a result of its embedment in the intercalated layers of the scaffold.

2.2.4

Phase Separation Technique

This method of fabricating scaffolds involves the fabrication of biomaterial by precipitation from a polymer-lean phase vs. a polymer-rich phase requiring minimum apparatus. Thermal changes in the solvent system generally induce the precipitation. On the one hand, removal of the solvent from the polymer-rich phase results in the formation of a matrix of the scaffold. On the other hand, the removal of the solvent from the polymer-lean phase results in the creation of pores. In this process, a temperature-dependent removal of the solvent results in the gelation of the polymer and this step is most crucial in controlling the morphology and porosity of the scaffolds. The process is followed by solvent exchange with the deionized water to remove any traces of the organic solvent and finally freeze-dried to remove the entrapped water molecules in the pores (Ghalia and Dahman 2016). In this connection, the PCL has been fabricated into porous scaffolds using this technique. Using a ternary system consisting of PCL, dioxane and water mixed with fabricating scaffolds using the thermally induced phase separation method (Liu et al. 2014). The process parameters such as gelation temperature, phase ratio of water: dioxane and PCL concentration were affecting the final morphology and porosity of the scaffold. It was observed, carbonate hydroxyapatite crystals were formed in porous cavities of the scaffolds, indicating their potential in supporting bone growth.

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In-Vitro and In-Vivo Applications of PCL Scaffolds

Various reviews are available in the literature, citing the use of PCL based scaffolds in various branches of tissue engineering (Saveleva et al. 2018; Martin et al. 2015; Yeo et al. 2008). However, we will discuss some of the latest developments in the fabrication of PCL scaffolds with their utility in those applications which are not covered in those reviews. Briefly, we will also address the shortcomings of these reported studies and what could be done to present a strong pre-clinical proof of concept to make a strong appeal for phase-1 clinical studies to regulatory authorities. Bone injuries and trauma are most common in the age group of 18–40 years. This alarming situation often requires clinical intervention, especially in the elderly population, where the healing process is prolonged. It is roughly estimated in the United States alone, nearly eight million people develop fractures out, of which 5–10% fail to recover fully after standard treatment, or clinical intervention are allowed (Holmes 2017). Nowadays, in contrast with the past, strategies provide a personalized medicine in which bioengineered materials are combined with stem cells or osteoprogenitor cells, bioactive molecules, etc., and the final biomaterial can be customized to perfectly suit the needs of a patient. The process can be upgraded by isolating tissue-specific cells from the patient, thus paving the way for an autologous approach and hence preventing the risk of immune rejection or inflammation (Neves et al. 2016). In this context, Joao C. Silva et al. reported an in-situ method of preparation of ECM from the mesenchymal stem cells (MSCs) (Fig. 2.1) (Silva et al. 2020). The same material was then fabricated into a scaffold using PCL as a base material by 3D printing. Further, testing of this material revealed that it supported both the attachment and proliferation of MSCs without the addition of any osteogenic inducer. Besides, the gene expression studies revealed the upregulation of mRNA levels of markers of osteogenesis such as osteopontin (OPN), runt-related transcription factor (Runx), collagenase (COL1) and alkaline phosphatase (ALP). The results from these studies also provide evidence of higher calcium deposition, but unfortunately, an animal model has not been evaluated to this date. Likewise, more substantial evidence needs to be collected and enough data generated in assessing this material in a clinical setting. Similar and improvised results are reported by using trophic factors (e.g., BMP-2) along with PCL (Gadalla and Goldstein 2020). The scaffold has been fabricated by first covalently immobilizing BMP-2 with PCL and then spinning the composite into a porous network using the electrospinning apparatus. The need for novel materials to generate an ideal scaffold for bone tissue engineering is explored on an extensive-scale. Advances in material science exclusively to support bone regeneration need to be tested, mainly using a related animal model to evaluate scientifically valid data allowing us to figure out the competence of biomaterial. Ceramics like calcium phosphate (Mondrinos et al. 2006), hydroxyapatite (Shor et al. 2007) and tricalcium phosphate (Lohfeld et al. 2012) considered as bio-inspired materials have shown promising results in bone tissue engineering. However, ceramics are fragile, brittle, inflexible and lack mechanical strength; this probably restricts them from being used clinically, espacially at the load-bearing part

Fig. 2.1 (a) Schematic representation of ECM generated using a personalized patient-tailored bone tissue engineering approach. The in-situ generated ECM is then fabricated into a PCL scaffold using computer-aided design (CAD) and, finally, 3D printed. (b) Representation of the further experimental plan for promotion of attachment and osteogenic differentiation of human bone-derived mesenchymal stem cells (hbMSC). Reprinted with permission from John Wiley & Sons (Silva et al. 2020)

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of the skeleton. On the other hand, many new clay-based ceramics have been reported, which have shown better performance in bone tissue engineering. Attapulgite (i.e., oxides of magnesium, aluminium phyllosilicate and iron), an industrially used adsorbent and decolorizer. Originally, it is known to bind toxic substances present in the stomach to neutralize diarrhoea has recently demonstrated to have bone regeneration potential (Zhao et al. 2020). This ceramic was used as composite material along with PCL and gelatin to produce scaffolds by the salt leaching method. The scaffolds were highly porous and mechanically durable with higher degrees of hydrophilicity. Multipotent mesenchymal stem cells proliferated in a higher number on this scaffold compared to the pristine PCL/gelatin only scaffold as found during immunofluorescence and CCK-8 assay. Post 7 days of incubation, these scaffolds revealed more expression values of Runx, COL 1, OPN and Osterix (osteogenic markers). Furthermore, this study also demonstrated the in-vivo application of these scaffolds by implanting in a 15 mm rabbit radius defect model. X-ray radiography results taken account after 8 weeks of implantation revealed that defects were filled in completely, however with a little density as compared to the original bone. The similar results were demonstrated by examining the bone by micro-CT, showing less mineral density of the healed bone after 8 weeks of implantation. However, after 12 weeks of implantation, the mineral density of the defected bone was similar as that of the original and significantly higher than pristine or control groups. Another interesting finding (histological staining) was the presence of fibrous connective tissue in the control group, signifying no new bone had formed yet. In contrast, as in the attapulgite scaffold, fibrous connective tissue was entirely replaced by the new bone. PCL based scaffolds obtained by electrospinning are a promising candidates for bone tissue engineering. Being a synthetic biomaterial, PCL has a hydrophobic surface and lacks hydrophilic functional groups required for cellular adhesion. To improve the hydrophilicity of the PCL scaffold, various surface modifications such as plasma treatment (Ko et al. 2015), ion sputtering (Manso et al. 2004), oxidation (Song et al. 2015) and corona discharge (Song et al. 2017) have reported. A study has shown that plasma-treated (either by acrylic acid or oxygen) PCL nanofiber scaffolds have shown to improve the wettability and cellular proliferation of pre-osteoblast (MC3T3-E1) cells (Ko et al. 2015). Further, it indicated that plasma treatment with acrylic acid results in a decrease in fiber diameter. The ALP activity of MC3T3-E1 cultured on plasma-treated (acrylic acid and oxygen) PCL-nanofiber scaffolds increased as compared to the ALP activity on pristine scaffolds. These results have a direct correlation with the improvement in the wettability of the PCL scaffolds upon plasma treatment. The plasma treatment results in anchoring of functional groups over PCL-nanofiber surfaces and as such enhances MC3T3-E1 cell proliferation and their differentiation into osteoblast. Repair of tendons and ligaments, directly connected with bone, has become a common clinical problem. To provide the best possible solution in therapeutics, nanofiber scaffolds using electrospinning techniques have become famous for the repair of the tendon-bone interface (Cai et al. 2018). To accelerate the tendon-bone healing, an in-vitro study has shown that a bioactive PCL nanofiber combined with

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tendon stem/progenitor cells promoted osteogenic regeneration at the tendon and bone interface, hence gives a clue about possible repair or regeneration of tendons once injured. The efficient osteointegration between the tendon graft and bone tunnel was also observed (Lin et al. 2019). This study further provides a platform for conducting future animal model experiments to explore the possibility of using these nanofibers. Magnesium phosphate-based biomaterials have shown to improve the proliferation and differentiation of osteoblast cells for bone regeneration (Wang et al. 2014). These biomaterials have enhanced biocompatibility, degradability and osteoconductivity. For bone tissue engineering, nanoflakes of magnesium phosphate (prepared by one-step microwave irradiation method) was incorporated in a composite of PCL, nano-hydroxyapatite and hyperbranched polyglycerol. The study has shown that the addition of hydrophilic polyglycerol improves the mean diameter, biomineralization, wettability, swelling, degradation and cytocompatibility of PCL scaffolds. The in-vitro study of these scaffolds exhibited non-cytotoxicity towards MG63 osteosarcoma cells and hMSCs. These scaffolds thus can be used as a potential biomimetic material for bone tissue regeneration applications (Perumal et al. 2020). Gene delivery through nanofibrous PCL scaffold has shown a promising outcome. In this regard, the microfluidic-assisted synthesis of plasmid DNA (encoding BMP-2) were placed in PCL nanofibers for bone tissue regeneration (Malek-Khatabi et al. 2020). It was reported that PCL based scaffolds showed osteogenic differentiation of MSCs using both in-vitro and in-vivo models. Calcium deposition during in-vitro osteogenesis by Alizarin staining was observed. For in-vivo experiments, the rat calvarial defect model demonstrated a significant increase in the bone regeneration volume and dense bone-like structures. The data indicated the enormous potential of PCL in pre-clinical studies of bone regeneration, which can be highly expandable in nanomedicine. Similar studies on bone formation using PCL nanofibrous scaffold have reported, wherein MSCs incorporated in the PCL scaffold was incorporated in female Lewis rats (Shin et al. 2004). The results found were encouraging in depicting the potential of osteoblast-seeded electrospun scaffolds for new bone formation. The fabrication of PCL scaffolds incorporated with gelatin has also shown great benefit. Blended nanofibers of PCL composed of different gelatin concentrations of 0, 30%, 50% and 70% were differently labelled and crosslinked with cross-linker genipin (Ren et al. 2017). The composite containing 30–40% of gelatin with a solvent system had optimum hydrophilicity, degradability and appropriate bio-functionality for protecting hMSCs. The in-vitro experiments using MC3T3E1 cells indicated that PCL/gelatin/genipin composite nanofibers significantly increased the osteogenic capability. Furthermore, the in-vitro assay carried with alizarin red in normal and osteogenic medium confirmed the bone formation with composite nanofibers scaffolds. PCL scaffolds have been fabricated along with carbon nanotubes (CNT), including both single and multiwalled carbon nanotubes (SWCNTs and MWNTs) (Pan et al. 2012). CNTs due to their high Young’s modulus (~270 to 950 GPa) and tensile strength (~11 to 63 GPa) are suitable candidates for fabrication an artificial bone

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Fig. 2.2 Schematic representation of fabrication of 3D PCL scaffolds using thermally induced selfagglomeration process coupled with freeze-drying. Reprinted with permission from John Wiley & Sons (Xu et al. 2015)

material with desirable mechanical properties. Among these, MWNTs have been reported to be osteoproductive in nature. CNTs enhanced conductivity, mechanical and gas barrier properties of the PCL scaffolds. Furthermore, PCL biomaterial incorporated with a low concentration of MWNTs were assessed for in-vitro using rat bone-marrow-derived stromal cells. With the incorporation of the low density of MWNTs (0.5%), the PCL scaffolds show enhanced bone regeneration along with higher expression levels of bone markers like ALP. To improve the mechanical properties of CNT-PCL scaffolds, a study used varied quantities of CNT in the matrix of PCL scaffolds (Mattioli-Belmonte et al. 2012). By changing the ratio of CNTs, the elastic modulus of composite was changed from 10 to 75 MPa, thus indicating the improvement in the mechanical strength. The in-vitro study on human osteoblast-like cells (e.g., MG-63) revealed that PCL-CNT nano-composites show promising potential in bone tissue engineering through sustained osteoblast proliferation and cell morphology. A very novel 3D material using PCL was prepared by thermally induced selfagglomeration (TISA) phase separation method and subsequent freeze-drying for supporting bone formation (Fig. 2.2) (Xu et al. 2015). The prepared material was soft, highly elastic and contained hierarchically structured interconnected pores as compared to the PCL materials fabricated by electrospinning and thermally induced porogen leaching technique. The electron microscopy study revealed that the morphology and the porosity of the material were similar to the ECM of the bone. Usually, in bone tissue engineering, the scaffolds are designed in such a way that it leads to direct differentiation of the progenitor cells into osteoblasts by providing a surface similar to the ECM of the bone. Moreover, it depends on the type of the

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matrix of the scaffold, that it may either support osteogenic differentiation (if stiff) or chondrogenic differentiation (if soft). Since the scaffold fabricated by TISA is soft and flexible, it was observed that mouse-derived bone marrow MSCs cultured on these scaffolds resulted in chondrogenic differentiation with higher cell density. Early markers of bone differentiation, such as high ALP activity, were also reported in these scaffolds as compared with electrospun or porogen leached scaffolds. These results were in agreement with reports as far as calcium deposition and expression of osteogenic genes like Runx are considered. This study also reported in-vivo transplantation of BMP-2 expressed in the cells incorporated in TISA scaffolds in a mouse endochondral bone formation model. Encouraging results were reported demonstrating that TISA scaffolds with interconnected micropores were highly suitable for bone growth and BMP-2 expression by the transplanted cells in the scaffold induced endochondral bone formation. Such studies should be conducted further with add on materials to report very earlier bone formation. Moreover, relatively comparable data were reported by Hangody et al. (2008) and Martin et al. (2007), which concluded that PCL scaffolds would show promising results in bone and cartilage repair. Various drugs are known for the potential to induce bone formation and are incorporated into tissue scaffolds for early regeneration. PCL scaffold has also integrated with resveratol (i.e., an antioxidant mainly present in the skin of red grapes and partly in blueberries, raspberries, mulberries and peanuts) to study its prolonged release effects, leading to the bone regeneration (Kamath et al. 2014). In brief, the resveratrol loaded into albumin nanoparticles (RNP) was encased in PCL scaffold by the solvent casting and leaching method. Fourier transform infrared spectroscopy indicated no chemical transformation occurred during the entrapment process. The evaluation of PCL-RNP scaffold on bone formation potential was achieved using in-vitro hMSCs. The prolonged-release of resveratrol from the scaffolds has shown increased ALP activity and bone mineralization. PCL-RNP, due to its osteo-conductive, osteoinductive, and osteogenic potential, can be highly desirable in bone tissue engineering. The addition of the nano-hydroxyapatite (nHA) with PCL has been reported in many studies to modify the process of bone regeneration. This addition not only increases the hydrophilicity of the scaffold but increases the diameter of the nanofibers as compared to pristine PCL nanofibers. The composite PCL/nHA fibers are expected to have beneficial effects in osteoblast growth and hence in bone tissue engineering. However, nowadays, nHA is usually composed of other bone-forming materials like silicates. In this connection, 3D biodegradable PCL hybrid scaffold with silicate containing HA has shown potentiality in bone tissue regeneration (Shkarina et al. 2018). In-vitro studies using hMSC on PCL-Silica-HA scaffolds have shown improved cellular penetration and bone growth depicting its use in bone tissue regeneration. It is expected that this hybrid scaffold of PCL will improve the bone tissue structures in-vivo. PCL/HA scaffolds implanted in mouse calvarial defects have shown better support for the new bone tissue regeneration due to high osteoconductivity. Moreover, the amount of new bone formation in PCL/nHA

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Fig. 2.3 Schematic representation of various events in the experimental demonstration of bone regeneration using PCL based scaffolds with either ceramics or bioactive glass. (a–d) Represent different steps in in-vivo implantation of PCL composites containing bone marrow stromal cells subcutaneously in a mouse model. Reprinted with permission from Elsevier (Poh et al. 2016)

scaffolds was higher in pristine PCL scaffolds, making it a right candidate for repairing bone-related defects. Recent use of calcium phosphate-based ceramics with bioactive glasses (bioglass) in PCL scaffolds has enhanced the bioactivity of the scaffolds. These biomaterials can be easily modified in terms of porosity, mechanical properties, degradation rate using various in-vitro and in-vivo bioactivity studies. To check bone regeneration capabilities, Poh et al., conducted both in-vitro and in-vivo studies, using three different composite materials (Poh et al. 2016). The materials used in the study include PCL, PCL/50-(45S5) Bioglass®, PCL/50-SrBG (strontium-substituted bioactive glass) and PCL/CaP-coated (calcium phosphate) (Fig. 2.3). The additive manufacturing technique was additionally done to form 3D scaffolds. The in-vitro studies using sheep bone marrow-derived stromal cells under non-osteogenic and osteogenic conditioned media. In non-osteogenic media, only PCL/CaP, PCL/50(45S5) and PCL/50-SrBG scaffolds showed an up-regulation of osteogenic gene expression. Further, for the evaluation of osteoinductivity, in-vivo studies were carried out subcutaneously using nude rat models. Host tissue infiltration was seen in the scaffolds, without any mature bone formation. This is the first study; we came across where the authors reported no osteogenic potential during the in-vivo studies by various PCL based scaffolds.

2.4

Conclusion

In conclusion, nano-based PCL scaffolds have tremendous potential in the repair of injured bone tissue. The properties of slow degradation rate limit its utility; however, the versatile techniques of fabrication and blending with other natural polymers and/or bioactive material qualifies it for one of the extensively used polymers as

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implant materials. Additionally, the hydrophobicity of the PCL limits its use in bone tissue engineering. Still, surface modifications and the use of hydrotropic substances blended with PCL ideally make it a promising material. Besides, it does not encounter immune rejection and is soluble in most of the organic solvents. The goal is to provide additional pre-clinical proof of studies that will lead to the final development of industrially scalable materials for bone regeneration and treatment of severe bone injuries. Acknowledgments The Department of Science and Technology, Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016, supported this work.

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Kanjwal MA, Sheikh FA, Nirmala R, Macossay J, Kim HY (2011) Fabrication of poly (caprolactone) nanofibers containing hydroxyapatite nanoparticles and their mineralization in a simulated body fluid. Fibers Polym 12:50 Ko Y-M, Choi D-Y, Jung S-C, Kim B-H (2015) Characteristics of plasma treated electrospun polycaprolactone (PCL) nanofiber scaffold for bone tissue engineering. J Nanosci Nanotechnol 15:192 Kowalczewski CJ, Saul JM (2018) Biomaterials for the delivery of growth factors and other therapeutic agents in tissue engineering approaches to bone regeneration. Front Pharmacol 9:513 Kweon H, Yoo MK, Park IK, Kim TH, Lee HC, Lee H-S, Oh J-S, Akaike T, Cho C-S (2003) A novel degradable polycaprolactone networks for tissue engineering. Biomaterials 24:801 Lee JM, Sheikh FA, Ki CS, Ju HW, Lee OJ, Moon BM, Park HJ, Kim JH, Park CH (2013) 3D electrospun silk fibroin nanofibers for fabrication of artificial skin. J Nanoeng Nanomanuf 3:269 Lee JM, Chae T, Sheikh FA, Ju HW, Moon BM, Park HJ, Park YR, Park CH (2016) Three dimensional poly(ε-caprolactone) and silk fibroin nanocomposite fibrous matrix for artificial dermis. Mater Sci Eng C 68:758 Lin Y, Zhang L, Liu NQ, Yao Q, Van Handel B, Xu Y, Wang C, Evseenko D, Wang L (2019) In vitro behavior of tendon stem/progenitor cells on bioactive electrospun nanofiber membranes for tendon-bone tissue engineering applications. Int J Nanomedicine 14:5831 Liu S, He Z, Xu G, Xiao X (2014) Fabrication of polycaprolactone nanofibrous scaffolds by facile phase separation approach. Mater Sci Eng C 44:201 Lohfeld S, Cahill S, Barron V, McHugh P, Dürselen L, Kreja L, Bausewein C, Ignatius A (2012) Fabrication, mechanical and in vivo performance of polycaprolactone/tricalcium phosphate composite scaffolds. Acta Biomater 8:3446 Ma Z, He W, Yong T, Ramakrishna S (2005) Grafting of gelatin on electrospun poly(caprolactone) nanofibers to improve endothelial cell spreading and proliferation and to control cell orientation. Tissue Eng 11:1149 Malek-Khatabi A, Javar HA, Dashtimoghadam E, Ansari S, Hasani-Sadrabadi MM, Moshaverinia A (2020) In situ bone tissue engineering using gene delivery nanocomplexes. Acta Biomater 108:326–336 Manso M, Valsesia A, Ceccone G, Rossi F (2004) Activation of PCL surface by ion beam treatment to enhance protein adsorption. J Bioact Compat Polym 19:287 Martin I, Miot S, Barbero A, Jakob M, Wendt D (2007) Osteochondral tissue engineering. J Biomech 40:750 Martin JT, Milby AH, Ikuta K, Poudel S, Pfeifer CG, Elliott DM, Smith HE, Mauck RL (2015) A radiopaque electrospun scaffold for engineering fibrous musculoskeletal tissues: scaffold characterization and in vivo applications. Acta Biomater 26:97 Mattioli-Belmonte M, Vozzi G, Whulanza Y, Seggiani M, Fantauzzi V, Orsini G, Ahluwalia A (2012) Tuning polycaprolactone-carbon nanotube composites for bone tissue engineering scaffolds. Mater Sci Eng C 32:152 McEachin Z, Lozano K (2012) Production and characterization of polycaprolactone nanofibers via forcespinning™ technology. J Appl Polym Sci 126:473 Mondal D, Griffith M, Venkatraman SS (2016) Polycaprolactone-based biomaterials for tissue engineering and drug delivery: Current scenario and challenges. Int J Polym Mater Polym Biomater 65:255 Mondrinos MJ, Dembzynski R, Lu L, Byrapogu VK, Wootton DM, Lelkes PI, Zhou J (2006) Porogen-based solid free-form fabrication of polycaprolactone-calcium phosphate scaffolds for tissue engineering. Biomaterials 27:4399 Neves LS, Rodrigues MT, Reis RL, Gomes ME (2016) Current approaches and future perspectives on strategies for the development of personalized tissue engineering therapies. Expert Rev Precis Med Drug Dev 1:93 O’Brien FJ (2011) Biomaterials & scaffolds for tissue engineering. Mater Today 14:88

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Nanocamptothecins as New Generation Pharmaceuticals for the Treatment of Diverse Cancers: Overview on a Natural Product to Nanomedicine Touseef Amna, M. Shamshi Hassan, and Faheem A. Sheikh

3.1

Introduction

The clear-cut definition of cancer is the abnormal division of cells resulting in undesired spreading anywhere through blood and lymph systems. In other words, anything that affects healthy cells to develop these peculiarities may finally result in stage 4 cancer that is metastatic cancer, during which the cells migrate from tumor and enter into the bloodstream, thereby spreading to other body parts. Briefly, the causative agents of the disease can be tobacco smoke, environmental factors (UV radiation), alcohol consumption, polyaromatic hydrocarbons emitted by barbecuing, bacterial and viral agents and genetic factors, etc. (Glinsky et al. 2005). However, the actual cause of cancer in human populations is still a topic of debate. Recent research indicates that more than 200 types of different cancer are predominating. It is interesting to note that the natural products, mainly have been utilized for the management of an assortment of diseases without knowing that these extracts can be potential anticancer agents (Atanasov et al. 2015). The prehistoric era (painting from stone walls) indicates the plant and plant products utilized as traditional medicines in Egypt, China, India and Greece. Nevertheless, the prominent fractions of current drugs/medication for treating the different cancers are mainly isolated from the plant-based extracts (Atanasov et al. 2015; Cragg and Newman 2005). It is factual that the quantity of natural medicines T. Amna Department of Biology, Faculty of Science, Albaha University, Al Bahah, Kingdom of Saudi Arabia M. S. Hassan Department of Chemistry, Faculty of Science, Albaha University, Al Bahah, Kingdom of Saudi Arabia F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_3

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Fig. 3.1 Role of camptothecin against diverse types of cancers

(i.e., lead molecules in them) produced from medicinal plants is always not up to the mark for having a desired level. It may be noteworthy to mention that these pro-drugs are formed during a particular stage of development and/or under specific environmental conditions, stress, as well as minerals accessibility. Even so, some plants are very slow growing; therefore, it takes several years to arrive specific growth phase appropriate for accumulation and extraction of desired lead molecules. Keeping into consideration the limitations coupled with the output of plants-extracts of desired pharmaceuticals, the endophytic microbes serve as the vital and inexhaustible resource of novel metabolites possessing attractive medicinal potential. Notably, the endophytic fungi have been considered a source of active metabolites with promising drug capabilities (Chandra 2012; Schulz et al. 2002). Here in, our focus is on anticancer pro-drug (e.g., camptothecin), which is a plant alkaloid capable of inhibiting the DNA topoisomerases I. Camptothecin has been considered a powerful cytotoxic molecule and has displayed exceptional properties for the treatment of solid malignancies. Moreover, camptothecin and its analogs are useful for treating different colon, breast, liver, lung, prostate, ovarian and pancreatic cancers (Fig. 3.1). The manner of working is by binding the DNA Topoisomerase I and DNA complex, thereby restricting the DNA re-ligation leading to damage to DNA and apoptosis in the cancer cells (Redinbo et al. 1998; Staker et al. 2002).

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Molecular Perception of Cancer

After cardiovascular ailments, the tumor is a second major cause of death in the world. Till this date, the competent treatment for various cancer is a big challenge for the researcher around the globe. The exact reason and causes are uncertain, as the disease can affect any tissue/part of the body, and few times it is quite debatable. At present, we know that cancer mainly develops when various genes experience mutations, it may occur by activation of oncogenes and/or by inactivation of suppressor genes, that can significantly influence the cause of cancers in different individuals. This results in the uncontrolled growth of cells and finally leading to malignant tumors (Fig. 3.2), tissue infiltration, ultimately, the metastasis and dysfunction of the organs (Sarkar et al. 2007). Also, the poor lymphatic drainage contributes to the superior permeation and retention effect (Byrne et al. 2008; Iyer et al. 2006). Moreover, oncogenes are unstable and frequently display gene variation. The symptoms and signs of cancer are being ruled by the specific type and grade of cancer that ultimately can reflect in the treatment. Generally, the intrinsic complexity of the tumor microenvironment and the presence of P-glycoproteins obstruct conventional chemotherapy (Wiradharma et al. 2009). Furthermore, the performance of drugs has limited due to their low solubility, narrow therapeutic window, as well as severe side effects on the healthy tissues (Pulkkinen et al. 2008). Therefore, to get off the hook is to develop novel therapeutic methods that are

Fig. 3.2 Graphical presentation showing the proliferation of cancer cells through healthy cells

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competent in distributing the anticancer drugs exclusively at the specific tumor site (Panyala et al. 2009). In this direction, nanotechnology has introduced drug delivery systems allowing the presentation of drugs at specific target sites. In particular, the use of nanomaterials into therapeutics is one of the promising areas in the medical sciences, which can revolutionize cancer treatment.

3.3

Celebrated Examples of Model Anticancer pro-Drugs

Since ancient times, a plethora of pharmaceuticals derived from medicinal plants that were utilized in traditional medicines and still are used due to their unique health benefits (Atanasov et al. 2015). For hundreds of years, the medicinal plants exploited for the isolation of drugs that are useful and simultaneously possessed negligible and/or no side effects compared to synthetic drugs. As aforementioned, the cancer is a significant health concern in developing as well as in the developed nations. Worldwide the various plant-derived anticancer drugs such as taxol, vinblastine, vincristine, camptothecin-derivatives, topotecan, irinotecan and etoposide are in clinical use. In addition to the above-mentioned molecules, several other promising cell cycles arresting agent and tumor growth inhibitors such as flavopiridol, roscovitine, combretastatin A-4, betulinic acid and silvestrol are also in clinical or at pre-clinical developmental stages (Cragg and Newman 2005). However, in this chapter, our goal is to provide the impression of the history and current state of knowledge of cancer and anticancer pro-drug camptothecin.

3.4

Camptothecin

Approximately in 1950s, it was being established that the Chinese tree (i.e., Camptotheca acuminate) possesses outstanding anticancer properties against mouse leukemia. Later on, after proper scientific investigations by National Cancer Institute, USA, the active component (i.e., Camptothecin) was found in the bark and stem of Camptotheca acuminate known to inhibit the cancers. In the early 1970s, camptothecin was clinically tested on high-grade cancer patients at the National Cancer Institute in Washington, DC, and it is being established to possess anticancer potential. However, it couldn’t be used practically during that period due to its acute toxicity causing healthy cells. This drug and its several other analogues are a prospective group of antitumor molecules that typically binds the Type I topoisomerase, blocking the active site and hence resulting in cell death. Furthermore, the analogues of camptothecin are chemically similar concerning the planner aromatic five-ring system with a common lactone moiety. The screening and activity result established that the preservation of the lactone ring of camptothecins is essential for their antitumor action. Nevertheless, the two analogs of camptothecin (e.g., Hycamtin and Camptosar) have obtained authorization from the US Food and Drug Administration (US-FDA) for use in ovarian, lung and colorectal cancers (Li and Adair 1994; Oberlies and Kroll 2004). Although the

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camptothecin possesses excellent antitumor activity, however, its therapeutic exploitation is inadequate due to the following reasons: (1) poor aqueous solubility (2) serious side effects (3) opening of lactone ring at physiological pH (Fassberg and Stella 1992; Chourpa et al. 1998). To overcome the problems as mentioned above, camptothecin is clinically administered as a sodium salt of carboxylate form, but this requires a high dosage, which results in toxic reactions. Considering solubility and stability issues, a series of camptothecin analogs are prepared, and few of them are clinically used (Venditto and Simanek 2010). However, it has been regrettably specified that all camptothecin derivatives experience pH-dependent quick and reversible hydrolysis from closed lactone ring to carboxylated form, which results in the loss of activity. To overcome low water solubility and instability at biological pH, various delivery systems are being tried, which is being reflected in the next section of this chapter.

3.4.1

Attempted Nano-Vehicles for Delivery of Camptothecin

Nanotechnology presents the innovative techniques used for curing cancers. Notably, the nanomaterials possess novel characteristics; for instance, the drug-coated polymer nanoparticles can amplify the intracellular accumulation of anticancer drugs (Lv et al. 2008). Therefore, nowadays, substantial attention has been dedicated to designing new drug delivery systems that can specifically target the drug to a tumor site and is released at a controlled rate. To address the problems associated with insoluble lactone forms of camptothecin and its derivatives, some modified drug delivery systems are developed (Lalloo et al. 2006; Zhang et al. 2007). In recent times, an important consideration has been paid in the preparation of therapeutic formulations consisting of biocompatible nanocomposites (e.g., micellar structures, liposomes, nanocapsules, conjugates and nanofibers) (Fig. 3.3). All the delivery routes, as mentioned earlier systems, generally are polymeric and are micro-to nanometer in dimensions; therefore, exploited to offer targeted drug delivery and improved bioavailability. Fig. 3.3 Different nanotechnology approaches attempted to deliver the camptothecin

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For the time being, it is acknowledged that nanocarrier systems have multifold applications then the crude formations. These include: protection to the recombinant proteins and genes (Ko et al. 2009), enhance the solubility of inadequately soluble drugs (Liu et al. 2008), circulate in the bloodstream for prolonged periods without being noticed by macrophages, programmed liberation of drugs at preferred sites (Husseini et al. 2002). Conclusively, it has been proved that nanocarrier systems and nanomaterials possess superior potential than the competence to business formations; therefore, they provide innovative approaches to combat the cancers. Nevertheless, different types of nano-vehicles have been synthesized for delivery of camptothecin and these systems may be classified according to their chemical nature, structure, morphology, etc. (Botella and Rivero-Buceta 2017). Currently, the electrospinning process has gripped attention due to the fascinating ability to generate fibers ranging from micro-to-nanometer range and these fibers have amazing biomedical applications (Reneker and Chun 1996). Besides their extensive biomedical uses, electrospun nanofibers can also be utilized as drug carrier systems. These high aspect nanofibers offer an efficient dissolution rate of the drugs. Unlike other encapsulation methods that need sophisticated techniques, the pharmaceuticals compounds can be easily incorporated into the carrier polymers utilizing the electrospinning process. Nonetheless, these nanofibers possibly are favorable as cancer tools through passive tumor targeting owing to improved permeability and retention effect (Matsumura and Maeda 1986). Moreover, polymer-carriers can stabilize amorphous drug dispersion by specific molecular interactions and propensity of the drug to re-crystallize to make a kinetically unfavorable environment for an extended period; therefore, makes material pharmaceutically beneficial (Leuner and Dressman 2000). Conclusively, the advantage of using nanomaterials by the aid of nanotechnology are as follows: (a) drugs in the nanoformulation can enhance the solubility and biocompatibility (b) tolerance time can be prolonged in body by surface modified nanoparticles (c) accurate accumulation of chemotherapeutics at cancer spot by targeting strategy (d) toxic side effects can be reduced as the load of drug will be mininm. Considering the remarkable uses of nanofibers, our group previously attempted to encapsulate camptothecin into polymeric nanofibers (Fig. 3.4) to develop nano-camptothecin for better performance (Amna et al. 2013, 1659). Therefore, we hypothesize that a combination of conventional chemotherapeutics along with nanotechnological interventions, will benefit from achieving efficient treatment for cancers.

3.4.2

Endophytes as an Alternative Source of Camptothecins

Plants and their by-products used since ancient times for fitness as well as aromatic value. However, the natural wealth of medicinal plants is steadily exhausted. Moreover, plant-based therapeutic compounds confront problems such as insufficient concentration at which these products accumulate and uprooting of 30–40 years old trees for bioactive compounds makes the process economically and environmentally unfeasible. However, it recognized that the endophytic fungi inhabiting plants

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Fig. 3.4 Schematic illustration describing the use of high electrical charge helping to ejaculate the polymeric solutions containing camptothecin in the form of nanofibers. The top image shows the picture of nanofiber mats when illuminated to UUV light. The middle (low magnification) and bottom (high magnification) photos are the scanning electron microscopy results of the nanofibers when encapsulated with camptothecin. Obtained with permission from Elsewhere (Amna et al. 2013)

are competent to biosynthesize pharmacologically active compounds that are the same and/or similar to host plants. The endophytes are relatively unstudied but can be prospective sources of new lead molecules to utilize in the fields of agriculture, medicine and pharmaceutical industry. All vascular plants, such as mosses, algae, and ferns, are accounted for the anchorage of endophytes. Additionally, it has also been reported that biosynthetic pathways of plants and endophytic fungi are distinct but share some similarities (Chandra 2012). In this regard, our group in past said the synthesis of the quinoline alkaloid camptothecin by an endophytic fungus (Puri et al. 2005, 2008). Later on, we performed the kinetic (Amna et al. 2012, 2006; Amna 2006) and fermentation studies (Amna et al. 2006) of camptothecin accumulation in suspension culture and confirmed that endophytic fungi could be a prospective alternate source of anticancer pro-drug camptothecin (Fig. 3.5). In continuation of our scientific journey from plant-based drug to nano-camptothecin, we tried to encapsulate camptothecin in polymeric nanofibers (Amna et al. 2013, 1659) for the better dissolution and to keep lactone ring intact for efficient anticancer activity.

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Fig. 3.5 Schematic illustration for isolation of camptothecin from plant and endophytic fungi and encapsulation in the poly(ε-caprolactone) and poly(lactic-co-glycolic acid) nanofibers. Moreover, in the case of poly(lactic-co-glycolic acid) nanofibers simultaneously, they are loaded with Fe2O3 nanoparticle for possible multimodal cancer therapy. Obtained with permission from Elsewhere (Amna et al. 2012)

3.5

Conclusion

In conclusion, the nanomaterials possess multipurpose uses owing to their exceptional structures, especially in cancer therapy. Camptothecins have acknowledged general consideration in the area of pharmaceutical formulation. However, the nanodrugs are also facing a lot of challenges from laboratory to clinical translations. Although camptothecin drug delivery systems have been extensively studied, a good number of nanodrug dependent pharmaceuticals is limited due to their side effects, and in-vivo metabolism has uncontrollable issues. The drug camptothecin has a wide range of antitumor effects in cancers, including gastric rectal and colon, liver and lung cancer. Camptothecin-based drugs are currently used in treatment. However, the major problem with cancer therapy is the systemic toxicity of conventional anticancer drugs, their low accumulation at the site, and low bioavailability. Nanopharmaceuticals have been turned out to be alternatives to overcome drug development problems.

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3.6

47

Future Scope

Although the nanotechnology-based drug carriers have provided several medical applications, particularly in the field of cancer therapy, however, this area still needs comprehensive research to eradicate cancer. Currently, need is an improved understanding of the drug delivery systems, which will aid in the development of effective methods for clinical cancer therapy. Using nanotechnology will assist in delivering anticancer drugs to the exact spots of the body, especially the nanofibers will be used as transdermal patches providing the anticancer drugs to only cancer cells. It is believed that when all technological barriers are crossed, the next step towards a successful substitute for the authentic anticancer product is marketing and commercialization. Acknowledgments The Department of Science and Technology, Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016, supported this work.

References Amna T (2006) Studies on Endophytic fungi harbored in Nothapodytes Foetida plants. Aligarh Muslim University Amna T, Amina M, Sharma P, Puri S, Al-Youssef HM, Al-Taweel AM, Qazi G (2012) Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus. Braz J Microbiol 43:1476–1489 Amna T, Barakat NA, Hassan MS, Khil M-S, Kim HY (2013) Camptothecin loaded poly (ε-caprolactone) nanofibers via one-step electrospinning and their cytotoxicity impact. Colloids Surf A Physicochem Eng Asp 431:1–8 Amna T, Hassan MS, Nam K-T, Bing YY, Barakat NA, Khil M-S, Kim HY (1659) Preparation, characterization, and cytotoxicity of CPT/Fe2O3-embedded PLGA ultrafine composite fibers: a synergistic approach to develop promising anticancer material. Int J Nanomedicine 7(2012) Amna T, Khajuria RK, Puri SC, Verma V, Qazi GN (2006) Determination and quantification of camptothecin in an endophytic fungus by liquid chromatography–positive mode electrospray ionization tandem mass spectrometry. Curr Sci:208–212 Amna T, Puri SC, Verma V, Sharma JP, Khajuria RK, Musarrat J, Spiteller M, Qazi G (2006) Bioreactor studies on the endophytic fungus Entrophospora infrequens for the production of an anticancer alkaloid camptothecin. Can J Microbiol 52:189–196 Atanasov AG, Waltenberger B, Pferschy-Wenzig E-M, Linder T, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S, Heiss EH (2015) Discovery and resupply of pharmacologically active plant-derived natural products: a review. Biotechnol Adv 33:1582–1614 Botella P, Rivero-Buceta E (2017) Safe approaches for camptothecin delivery: structural analogues and nanomedicines. J Control Release 247:28–54 Byrne JD, Betancourt T, Brannon-Peppas L (2008) Active targeting schemes for nanoparticle systems in cancer therapeutics. Adv Drug Deliv Rev 60:1615–1626 Chandra S (2012) Endophytic fungi: novel sources of anticancer lead molecules. Appl Microbiol Biotechnol 95:47–59 Chourpa I, Millot J-M, Sockalingum GD, Riou J-F, Manfait M (1998) Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy. Biochimica et Biophysica Acta (BBA)-General Subjects 1379:353–366 Cragg GM, Newman DJ (2005) Plants as a source of anticancer agents. J Ethnopharmacol 100:72–79

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Fassberg J, Stella VJ (1992) A kinetic and mechanistic study of the hydrolysis of camptothecin and some analogues. J Pharm Sci 81:676–684 Glinsky GV, Berezovska O, Glinskii AB (2005) Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest 115:1503–1521 Husseini GA, Rapoport NY, Christensen DA, Pruitt JD, Pitt WG (2002) Kinetics of ultrasonic release of doxorubicin from pluronic P105 micelles. Colloids Surf B: Biointerfaces 24:253–264 Iyer AK, Khaled G, Fang J, Maeda H (2006) Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov Today 11:812–818 Ko YT, Kale A, Hartner WC, Papahadjopoulos-Sternberg B, Torchilin VP (2009) Self-assembling micelle-like nanoparticles based on phospholipid–polyethyleneimine conjugates for systemic gene delivery. J Control Release 133:132–138 Lalloo A, Chao P, Hu P, Stein S, Sinko PJ (2006) Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly (ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins. J Control Release 112:333–342 Leuner C, Dressman J (2000) Improving drug solubility for oral delivery using solid dispersions. Eur J Pharm Biopharm 50:47–60 Li S, Adair KT (1994) Xi Shu, a promising antitumor and anti-viral tree for the 21st century. College of Forestry, Stephen F. Austin State University, The Tucker Center Liu B, Yang M, Li R, Ding Y, Qian X, Yu L, Jiang X (2008) The antitumor effect of novel docetaxel-loaded thermosensitive micelles. Eur J Pharm Biopharm 69:527–534 Lv G, He F, Wang X, Gao F, Zhang G, Wang T, Jiang H, Wu C, Guo D, Li X (2008) Novel nanocomposite of nano Fe3O4 and polylactide nanofibers for application in drug uptake and induction of cell death of leukemia cancer cells. Langmuir 24:2151–2156 Matsumura Y, Maeda H (1986) A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 46:6387–6392 Oberlies NH, Kroll DJ (2004) Camptothecin and taxol: historic achievements in natural products research. J Nat Prod 67:129–135 Panyala NR, Peña-Méndez EM, Havel J (2009) Gold and nano-gold in medicine: overview, toxicology and perspectives. J Applied Biomedicine (De Gruyter Open) 7:75–91 Pulkkinen M, Pikkarainen J, Wirth T, Tarvainen T, Haapa-aho V, Korhonen H, Seppälä J, Järvinen K (2008) Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin–biotin technology: formulation development and in vitro anticancer activity. Eur J Pharm Biopharm 70:66–74 Puri SC, Verma V, Amna T, Handa G, Gupta V, Verma N, Khajuria RK, Saxena AK, Qazi GN, Spiteller M (2008) Endophytic camptothecin and camptothecinoid producing fungi and process of producing the same. Google Patents Puri SC, Verma V, Amna T, Qazi GN, Spiteller M (2005) An endophytic fungus from Nothapodytes f oetida that produces Camptothecin. J Nat Prod 68:1717–1719 Redinbo MR, Stewart L, Kuhn P, Champoux JJ, Hol WG (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279:1504–1513 Reneker DH, Chun I (1996) Nanometre diameter fibres of polymer, produced by electrospinning. Nanotechnology 7:216 Sarkar FH, Banerjee S, Li Y (2007) Pancreatic cancer: pathogenesis, prevention and treatment. Toxicol Appl Pharmacol 224:326–336 Schulz B, Boyle C, Draeger S, Römmert A-K, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004 Staker BL, Hjerrild K, Feese MD, Behnke CA, Burgin AB, Stewart L (2002) The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci 99:15387–15392 Venditto VJ, Simanek EE (2010) Cancer therapies utilizing the camptothecins: a review of the in vivo literature. Mol Pharm 7:307–349

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4

Smart Biomaterials from Electrospun Chitosan Nanofibers by Functionalization and Blending in Biomedical Applications Hasham S. Sofi, Nisar Ahmad Khan, and Faheem A. Sheikh

4.1

Introduction

Electrospinning is a unique technique to form ultrathin fibers from viscous polymeric dispersions under the influence of the external electric field. This engineering field gained significant importance after the Reneker demonstrated the features of electrospinning experimentally as a fabrication technique to form nanofibers with distinct morphology (Doshi and Reneker 1995). The primary instrument to fabricate nanofibers by this process comprises mainly of four components viz., power supply with high voltage, a pump for the flow of viscoelastic fluids, a spinneret system and a collector (Sheikh et al. 2016). The fundamental of the technique is based on the fact that when a static charge of suitable high electric voltage is subjected to a polymeric droplet. It promotes the surface of the charged droplet and results in repulsion between the similar charges on the droplet and counteracts surface tension. This leads to destabilization and, consequently, in fiber formation after dragging of opposite charge from the collector end. As the repulsion between polymer chains grows strong enough, the droplet loses its integrity and changes into a conical shape, usually referred to as Taylor cone and emancipates as a jet (Doshi and Reneker 1995; Yarin et al. 2001; Taylor n.d.). The jet then enters a whipping instability regime resulting in decrease to the diameter of the fiber. This process is subsequently followed by evaporation of the solvent, which may also be facilitated by the use of heat (Shin et al. 2001). The resulting nanofiber mats produced by electrospinning

H. S. Sofi · F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] N. A. Khan Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_4

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Table 4.1 Different process variables of the electrospinning process Solution parameters Viscosity Conductivity Surface tension The molecular weight of the polymer Dipole moment Dielectric constant Polymer glass transition temperature

Controlled parameters Flow rate Electric field strength Distance between tip and collector Design of the needle tip

Ambient parameters Temperature Humidity Air velocity

The geometry of the collector Geometry of electrodes Vapor pressure of the solvent

have a diameter range of 60 to 1000 nm or can be more than a few microns (Thompson et al. 2007; Frenot and Chronakis 2003). The following parameters tabulated below (Table 4.1) affect the process of electrospinning and characteristics of formed fibers. According to Reneker, who is highly regarded and pioneer in demonsrating the electrospinning stated that parameters controlling this process are mainly categorized as solution properties, controlled variables and ambient parameters (Doshi and Reneker 1995). Among them, solution properties such as conductivity, viscosity, molecular weight, dipole moment, surface tension and dielectric constant play a significant role in fiber formation by electrospinning (Deitzel et al. 2001). It is noteworthy to mention here that it is complicated to isolate a single parameter and study its effects independently; instead, the impact of one variable is dependent on the other (e.g., a change in the conductivity results in shifting the viscosity) (Geng et al. 2005). The controlled variables affecting the electrospinning process include the electric field strength, flow rate, distance between tip and collector, needle tip design and collector composition/geometry (Doshi and Reneker 1995). Ambient parameters include humidity, temperature and air velocity (Doshi and Reneker 1995). Overall, the effect of all these factors on the process of electrospinning are tabulated (Table 4.2). Chitosan, the de-acetylated derivative of chitin is a polysaccharide found abundantly in the shells of crustaceans. This polymer has shown utility in different fields of tissue-engineering and as a drug delivery carrier. Recent studies revealed that chitosan exhibits anticancer and antimicrobials properties (Smith et al. 2013). Chemically the polymer is similar to glycosaminoglycans (Griffon et al. 2006), making it an excellent candidate for tissue-engineering applications (Jayakumar et al. 2010). The polymer is also reported to possess low immunogenicity and has an excellent biodegradability (Adams 2009). However, the solubility of chitosan is limited in almost all organic solvents used to process the fibers and therefore requires harsh solutions for processing (Ignatova et al. 2013). These problems are dealt with by blending chitosan with other polymers and/or functionalization of chitosan/ blends by different techniques, such as covalent or non-covalent crosslinking or

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Table 4.2 Summary of the effect of different process variables affecting nanofiber morphology Process parameters Concentration/ viscosity of the solution Conductivity

Surface tension The molecular weight of the polymer Dielectric constant and dipole moment Field strength and voltage A flow rate of solution Distance between tip and collector Design of needle tip Collector composition Collector geometry Temperature

Humidity

Morphological changes Increasing viscosity increases nanofiber diameter while decreasing the viscosity leads to bead defects Increasing conductivity leads to bead-free and uniform nanofiber diameters. Higher conductivities lead to smaller fibers Increase in surface tension leads to a smaller diameter of fibers An increase in molecular weight increases viscosity and reduces the number of beads and droplets formation up to a saturation level. Higher dipole moment favors spinning process

Too high voltage leads to beaded nanofibers High flow rate produces fibers that are not dry enough and low flow (possibly the controlled rates) produce fibers with small diameters Beading can arise if the collector distance is too close or is too far away from the tip. An optimum length is required to form dried fibers Hollow fibers can be produced using a two-capillary spinneret. Moreover, multiple needle tips can increase fibers throughput Metalic collector results in smooth fibers whereas porous/hollow fibers can be produced on perforated collectors Aligned fibers can be obtained on rotating drums, conductive frames and wheel-like bobbin collectors Increasing temperatures decrease the solution viscosity resulting in fibers with a smaller diameter Increasing humidity can result in the formation of small pores over surface topography

Ref. (Doshi and Reneker 1995; Deitzel et al. 2001) (Angammana and Jayaram 2011; Son et al. 2004) (Deitzel et al. 2001; Fong et al. 1999) (Doshi and Reneker 1995; Fong et al. 1999) (Doshi and Reneker 1995; Deitzel et al. 2001) (Tan et al. 2005) (Tan et al. 2005; Sheikh et al. 2015) (Doshi and Reneker 1995) (Li et al. 2004; Zhou et al. 2009) (Doshi and Reneker 1995; Li et al. 2005) (Baji et al. 2010)

(Deitzel et al. 2001; De Vrieze et al. 2009) (De Vrieze et al. 2009; Casper et al. 2004)

thermal methods (Austero et al. 2012; Sridhar et al. 2015; Schiffman and Schauer 2007). In this chapter, we will review the recent advancements in the electrospun nanofibers of chitosan. Furthermore, the novel applications of chitosan nanofibers ranging from complex problems of drug delivery to the detection of tumor cells have been discussed.

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Functionalized Nanofiber Scaffolds Blended with Other Polymers

Recently, studies conducted on the functionalization of chitosan and its blend have found applications in various fields like general tissue-engineering (Fukunishi et al. 2016), bone-regeneration (Raftery et al. 2017), nano-catalysis (Karakas et al. 2017), analytics (Asiabi et al. 2017), filter aids (Li et al. 2017), drug delivery (Irani et al. 2017), antibacterial (Gupta et al. 2017), 3D cell culturing (Shamosi et al. 2017) and wound healing materials (Li et al. 2017). The detailed description of the electrospinning conditions utilized in the fabrication of these scaffolds is given (Table 4.3).

4.2.1

Chitosan Scaffolds: From Arterial Grafts to Artificial Cartilage

In general tissue engineering, the nanofiber constructs have gained tremendous importance due to nano-topography. Therefore, different materials have been synthesized for applications ranging from bone remodeling to arterial stents. Recently published articles have attracted significant attention for the development of novel materials, which not only can mimic the bio-interface, however, can those scaffold materials provide suitable a mechanical strength to support tissue development. For instance, electrospun constructs of chitosan and polycaprolactone have been used as vascular grafts in arterial tissue-engineering. These have been claimed to be used as prosthetic grafts for the replacement of small diameter arteries (Sell et al. 2009). Compared to traditional stents usually composed of slow degrading materials, these artificial grafts resulted in rapid host cell infiltration and remodeling. Ultrasonography of implanted grafts in a sheep model after 6 months revealed that the stunt mimicked the arterial bio-structure. Analysis of the tissue by histological examination revealed the deposition of ECM like constituents resembling elastin and collagen. Interestingly, these artificial constructs have mechanical properties comparable to the human carotid artery. Significant positive correlations can be drawn between the wall thickness of these grafts and CD68+ macrophage infiltration. This can be accredited to the rapid degradation rate of chitosan grafts (Fukunishi et al. 2016). Composites of chitosan, poly(vinyl alcohol) and graphene oxide fabricated by electrospinning revealed enhanced tensile strength, possibly due to the addition of graphene oxide. Biocompatibility of mouse chondrogenic cell line (ATDC5) was studied using two different concentrations of chitosan in the composite. It was found the composite containing a higher proportion of chitosan (6%) was appropriate for the growth of these cells as compared to chitosan (4%). These constructs are thus claimed to have good potential in cartilage regeneration (Cao et al. 2017). In another instance, a composite nanofiber of chitosan/gelatin/polycaprolactone cross-linked by glutaraldehyde was tested for growth, adhesion and topographical behavior of human fetal fibroblasts (HFFF2). These composites, due to excellent hydrophilicity, supported the growth and proliferation of these cells (Gomes et al. 2017).

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Table 4.3 Summary of electrospinning conditions and applications of different Electrospun scaffolds of chitosan fabricated either by functionalization and/or blending with other polymers. In the table, Voltage is designated as “V,” Tip to collector distance as “D” and flow rate as “F” Electrospun system Chitosan/poly(vinyl alcohol) /zeolites

Chitosan/poly(vinyl alcohol)/Fe3O4/ bovine serum albumin

Chitosan/poly(vinyl alcohol)/ampicillin

Chitosan/poly(vinyl alcohol)/ Acetylcholinesterase

Composite of chitosan/gelatin/ polycaprolactone

Chitosan/poly(vinyl alcohol)/Geneistein

Chitosan/poly(vinyl alcohol)/levofloxacin

Electrospinning conditions Chitosan (7% w/v) in acetic acid Poly(vinyl alcohol) ¼ (7% w/v) in DIW V ¼ 10 kV D ¼ 10 cm F ¼ 0.1–0.4 ml/hr. Chitosan (2% w/v) in acetic acid Poly(vinyl alcohol) ¼ 10% w/v in DIW V ¼ 18 kV D ¼ 15 cm F ¼ 8 ml/hr. Chitosan (3% w/v) in acetic acid Poly(vinyl alcohol) ¼ 10% w/v in DIW V ¼ 15 kV D ¼ 15 cm F ¼ 1 ml/hr. Chitosan ¼ 3% w/v in acetic acid Poly(vinyl alcohol) ¼ 8% w/v in DIW V ¼ 18 kV D ¼ 10 cm Chitosan ¼ 2% w/v in acetic acid Polycaprolactone ¼ 2% w/v in DIW Gelatin ¼ 2% w/v in DIW V ¼ 18 kV D ¼ 25 cm F ¼ 0.3 ml/hr. Chitosan ¼ 3% w/w in acetic acid Poly(vinyl alcohol) ¼ 20% w/v in DIW V ¼ 25 kV D ¼ 10 cm Chitosan ¼ 5% w/v in acetic acid

Characteristics and applications The higher adsorption capacity for Cr, Fe and Ni ions. Potential for reusable filter aid

Ref. (Habiba et al. 2017)

Controlled release of therapeutic proteins (BSA) combined with targeted delivery due to magnetic properties

(Nicknejad et al. 2015)

Transdermal delivery of ampicillin sodium. There is an initial burst release of the drug and then sustained over a period. Drug release is by Fickian diffusion

(Cui et al. 2017)

Prompt detection of pirimiphos-methyl in olive oil. Possible lower limits of detection than existing international standards Glutaraldehyde crosslinked fibers for skin tissue regeneration

(El-Moghazy et al. 2016)

Improved loading and delayed release of geneistein from crosslinked fibers of chitosan

(Ibrahim et al. 2016)

Delayed release of the formulation with 90%

(Jalvandi et al. 2017)

(Gomes et al. 2017)

(continued)

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Table 4.3 (continued) Electrospun system

Chitosan/ Polycaprolactone/ nickel decorated nanoparticle scaffolds

Chitosan/poly (e-caprolactone)/ Tinidazole scaffolds

Chitosan/poly (ethylene oxide)/ silver decorated nanofibers

Chitosan/silk fibroin scaffolds

Chitosan/poly (ethylene oxide)/ insulin buccal scaffolds

Chitosan/poly(lactic acid) scaffolds

Electrospinning conditions

Characteristics and applications

Poly(vinyl alcohol) ¼ 10% w/v in DIW V ¼ 12 kV D ¼ 10 cm Chitosan ¼ 2% w/v in acetic acid Polycaprolactone ¼ 6.5% w/v in formic acid Chitosan: Polycaprolactone ¼ 1:1 V ¼ 15 kV D ¼ 10 cm F ¼ 0.5 ml/hr. Chitosan ¼ 2% w/v in acetic acid Polycaprolactone ¼ 8% w/v in formic acid V ¼ 18–22 kV D ¼ 10.5 cm F ¼ 0.8 mL/hr. Chitosan + poly(ethylene oxide) ¼ 4% w/v in 50% v/v acetic acid/water Chitosan: Poly(ethylene oxide) ¼ 75:25 V ¼ 21 kV D ¼ 10 cm F ¼ 1 ml/hr. Chitosan + silk fibroin ¼ 20.5% w/v in TFA: DCM (7:3 w/w) Chitosan: Silk fibroin ¼ 8:12.5 V ¼ 18 kV D ¼ 12 cm

of the drug released after initial burst release

Chitosan ¼ 8% w/v in HFP Poly(ethylene oxide) ¼ 2–8% in HPF V ¼ 15 kV D ¼ 17 cm F ¼ 3 ml/hr. Chitosan ¼ varying concentrations with poly (lactic acid)

Ref.

Nano-catalyst for the reduction of nitrophenols under milder conditions

(Karakas et al. 2017)

Mucoadhesive nanofibers membranes containing tinidazole for the treatment of periodonitis

(Khan et al. 2017)

Antibacterial activity with sustained release of bioactive silver nanoparticles

(Kohsari et al. 2016)

Differentiation of human bone marrow mesenchymal stem cells into osteoblasts along with mineralization of inorganic phosphate at the middle of the differentiation period Oral delivery of insulin via buccal mucosa

(Lai et al. 2014)

Cardiac tissueregeneration by cell -scaffold interaction as

(Liu et al. 2017)

(Lancina et al. 2017)

(continued)

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Table 4.3 (continued) Electrospun system

Chitosan/ phospholipid scaffolds

Chitosan/gentamycin scaffolds

Chitosan/poly(vinyl alcohol)/Uricase scaffolds

Chitosan/poly(vinyl alcohol)/chitosan wiskers scaffolds

Chitosan + poly (ethylene oxide) nanofibers with cellulose nanocrystals

Chitosan/poly(vinyl alcohol)/curcumin nanofibers crosslinked by Si and

Electrospinning conditions

Characteristics and applications

Poly(lactic acid) ¼ 25% w/v in TFA/DCM (80:20 v/v) V ¼ 20 kV D ¼ 10 cm F ¼ 10 ml/hr. Chitosan (2% w/v) in TFA/DCM (70:30) V ¼ 25 kV D ¼ 10 cm F ¼ 1.2 ml/hr. Chitosan ¼ 6% w/v in TFA: DCM (7:3) V ¼ 15–20 kV D ¼ 12 cm F ¼ 0.8 ml/hr. Chitosan ¼ 3% w/v in 90% v/v acetic acid Poly(vinyl alcohol) ¼ 9% w/v in DIW Chitosan: Poly(vinyl alcohol) ¼ 40:60 V ¼ 15 kV D ¼ 15 cm F ¼ gravity driven Chitosan ¼ 3% w/v in acetic acid Chitosan wiskers ¼ 6.5% w/v Poly(vinyl alcohol) ¼ 10% w/v in DIW V ¼ 18 kV D ¼ 15 cm F ¼ 0.5 ml/hr. Chitosan ¼ 1.2% w/v Poly(ethylene oxide) ¼ 20% w/v Chitosan: Poly(ethylene oxide) conc ¼ variable V ¼ 15 kV D ¼ 5 cm F ¼ 0.2–1.0 ml/hr. Chitosan (2% w/v) in acetic acid Poly(vinyl alcohol) ¼ 5–8% w/v in DIW

well as growth enhancement by acting as supporting ECM

Ref.

Transdermal delivery of diclofenac sodium, Vit B12 and curcumin

(Mendes et al. 2016)

Sustained antibacterial effect of the aminoglycoside and gentamycin

(Monteiro et al. 2015)

Amperometric detection of uric acid in serum samples

(Numnuam et al. 2014)

Improved growth and proliferation of osteoblasts and hydroxyapatite induced mineralization for applications in bone tissueengineering

(Pangon et al. 2016)

Supporting material for the culture of 3 T3 fibroblasts

(Ridolfi et al. 2017)

Antibacterial and anticancer properties of curcumin delivered in sustained release profile

(Sedghi et al. 2017)

(continued)

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Table 4.3 (continued) Electrospun system functionalised by graphene oxide

Chitosan/gelatin scaffolds

Chitosan/poly (ethylene oxide)/ Cinnamaldehyde scaffolds

Chitosan/poly (ethylene oxide) / Chloehexidine/silver nanoparticle scaffolds

Chitosan/ poly (ethylene oxide) / poly(carboxybetaine methacrylate) (pCBMA) functionalised brushes Chitosan/ poly (ethylene oxide) / henna leaf extract scaffolds

Chitosan/sericin nanofiber scaffolds

Thiol chitosan/ chitosan acetamide scaffolds

Electrospinning conditions fGO-si-Curcumin ¼ 9% w/v V ¼ 22KV D ¼ 14 cm F ¼ 0.6 ml/hr. Chitosan ¼ 2% w/v in acetic acid Gelatin ¼ 18% w/v in DIW V ¼ 15 kV D ¼ 15 cm F ¼ 0.4 ml/hr. Chitosan + poly(ethylene oxide) (1:1) ¼5% in glacial acetic acid V ¼ 25 kV D ¼ 12.6 cm F ¼ 0.6 ml/hr. Chitosan: Poly(ethylene oxide) (75:25) ¼ 3% w/v in 35% w/v acetic acid V ¼ 27 kV D ¼ 15 cm F ¼ 4 ml/hr. Chitosan: Poly(ethylene oxide) (9:1) ¼ 2.5% w/v in 70% acetic acid V ¼ 18 kV D ¼ 20 cm F ¼ 0.5 ml/hr. Chitosan ¼ 3% w/v in 50% acetic acid Poly(ethylene oxide) ¼ 4% wt% in DIW V ¼ 5–25 kV D ¼ 10–20 cm F ¼ 0.1–1.5 ml/hr. Chitosan/Sericin (2.5: 1) ¼ 3% w/v in TFA V ¼ 18 kV D ¼ 15 cm F ¼ 0.8 ml/hr. Thiol chitosan ¼ 3% w/v distilled water Chitosan acetamide in different blends V ¼ 30 kV D ¼ 20 cm F ¼ 18 μl/hr.

Characteristics and applications

Ref.

Differentiation of endometrial stem cells into endothelial cells for blood vessel repair

(Shamosi et al. 2017)

Enhanced antimicrobial activity of chitosan nanofibers by incorporation of cinnamaldehyde

(Rieger and Schiffman 2014)

Antibacterial delivery of chlorhexidine and nano active silver. Antibacterial activity against S. aureus

(Song et al. 2016)

A substrate capable of capturing circulating tumor cells with high efficiency

(Sun et al. 2016)

Wound healing applications and antimicrobial properties

(Yousefi et al. 2017)

Wound healing and antibacterial properties

(Zhao et al. 2014)

Excellent candidate for wound healing applications

(Abdelgawad et al. 2017)

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Fig. 4.1 Schematic diagram showing the process for the fabrication of random or aligned chitosan/ PLA nanofibrous composites. The cardiomyocytes taken from the mice are then seeding and cultured over these scaffolds. Reprinted with permission from Elsevier (Gomes et al. 2017)

Loss of cardiomyocytes or injury to cardiac musculature results due to myocardial infarction (Cesselli et al. 2017). Cardiac tissue being anisotropic is challenging to reproduce because of its characteristic alignment of myocardia (Celikkin et al. 2017). Related to this concept, various approaches have been put forward to fabricate materials that can are in the regeneration of cardiomyocytes. In this regard, varying ratios of chitosan and poly(lactic acid) cross-linked by glutaraldehyde have been fabricated by electrospinning into fibrous scaffolds, as demonstrated in Fig. 4.1. In-vitro studies have shown that completely-aligned nanofibers elicit growth of cardiomyocyte along the longitudinal axis. The process is crucial and the very first step for the formation of cardiac tissue. Further, studies have demonstrated that a ratio of 7:1 between poly(lactic acid) and chitosan promotes cell scaffold interaction as well as the growth of supporting ECM, as evidenced by the production of sarcomeric α-actinin and troponin I proteins (Liu et al. 2017). A composite of de-polymerized chitosan and polycaprolactone polymers fabricated by electrospinning has been found to mimic the respiratory airway. The scaffold formed into an elastic and ductile fiber mimicking the upper respiratory tree. Using the air-liquid interface culture technique, tracheobronchial epithelial of porcine origin were seeded on these scaffolds. The cells were found to adapt to this scaffold-like its natural niche. Therefore, these scaffolds are hypothesized to have immense potential in respiratory tissue regeneration and can be used to treat pulmonary fibrosis in the future (Liu et al. 2017). Furthermore, chitin whiskers were produced from chitosan/poly(vinyl alcohol) nanofibers. These nanofiber scaffolds were able to promote the growth of hydroxyapatite crystals when subjected to treatment with simulated body fluid. The Young’s modulus values suggest improved tensile strength while as the cell culture experiments signify excellent growth and proliferation of osteoblasts (Pangon et al. 2016). Fibroblast growth factor-2 (FGF-2) was impregnated into chitosan by the formation of water-in-oil emulsion with poly(vinyl alcohol). The emulsion was then spun into nanofibers using electrospinning. The complex was stabilized utilizing heparin. The technique allows the incorporation of solvent affected fragile growth factors into the network of fibers for use in bone and tissue-engineering (Place et al. 2016). The composites of chitosan and poly(ethylene oxide) incorporated with leaf extracts of henna were

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fabricated into the nanofibers by electrospinning. These composites demonstrated significantly better healing properties than commercially available henna-based ointments during the in-vivo studies conducted on Wistar rats. These scaffolds were porous in nature, hydrophilic in character, and demonstrated significant antimicrobial activity. The highly porous nature of these scaffolds is attributed to the hydrogen bonding between the amine group of chitosan and the hydroxyl group of lawsone. During a period of the 2-week study, apparent differences were noticed in the wound healing process, the difference was due to the presence of a high surface area of the nanofibers and its ability to absorb exudates and, more importantly, the antibacterial properties of the henna extract (Romero et al. 2017).

4.2.2

Chitosan Scaffolds: As Sustained Delivery Systems for Drugs and Other Therapeutics

Chitosan, as a polymeric scaffold, has been tried to study the release pattern of various medications. Surprisingly, the polymer being hydrophilic can result in sustained release of the hydrophobic drug molecules from the scaffold architecture. In this context, nanofibers of chitosan and poly(vinyl alcohol) cross-linked by glutaraldehyde were studied for release and wound healing efficacy of tetracycline hydrochloride, especially in wounds which take time to heal. Tetracycline was released in bursts initially for a period of 2 h, sufficient to maintain the minimum inhibitory concentration in the case of S. epidermis and S. aureus. Furthermore, the presence of the drug in the nanofibers resulted in decreasing the fiber diameter (Alavarse et al. 2017). Pristine chitosan nanofibers prepared by electrospinning were studied for in-vivo delivery of donepezil for possible treatment of Alzheimer’s (Fig. 4.2). During release studies, the nanofibers released donepezil (up to 97%) instantaneously within 10 min in comparison to chitosan cast films. The pharmacokinetic parameters observed during in-vivo studies of nanofibers in an animal model

Fig. 4.2 Schematic representation of the preparation process for the synthesis of chitosan/poly (vinyl alcohol)/donepezil scaffolds using electrospinning. Reprinted with permission from Elsevier (Anji Reddy and Karpagam 2017)

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showed the maximum absorption rate of the drug during the early time (Anji Reddy and Karpagam 2017). An in-situ cross-linking technique incorporating Fe3O4 nanoparticles in the chitosan and poly(vinyl alcohol) nanofibers for loading bovine serum albumin (BSA) was utilized. The iron oxide was added because of its inherent magnetic properties for the targeted delivery of BSA. The cross-linkers (e.g., tripolyphosphate and glutaraldehyde) were utilized to bind these nanoparticles. Nanoparticles of the average size of 45 nm were deposited on this composite. Encapsulated BSA showed a slow release profile initially, with higher amounts released to 60% after 30 h (Nicknejad et al. 2015). These cross-linked composite scaffolds have been fabricated to study the transdermal drug delivery of ampicillin. The ampicillin was released from the scaffold in bursts, followed by a sustained peak over time (Cui et al. 2017). Moreover, the functionalized carboxymethyl derivative of chitosan has been electrospun while incorporating doxorubicin as anti-neoplastic agents. This study found that increasing the doxorubicin concentration resulted in fibers with larger diameters. The drug distributed into the fibrous matrix as crystallites and is released in a controlled manner (Fakharmanesh et al. 2017). The in-vivo studies of antibacterial wound dressing materials of chitosan and poly(ethylene oxide) were conducted on incised female Wistar rats. The composite polymers contained cefazolin in the form of nanoparticles of fumed silica. A dressing nanofiber mat containing 2.5% cefazolin was found to be 100% bactericidal against both S. aureus and E. coli. The in-vivo studies revealed that the inflicted wounds skin in rats healed entirely after ten days while using these nanofiber dressings. Additionally, there was no sign of infection or scar formation in the wounded area (Fazli and Shariatinia 2017). A conjugated system of chitosan where fluoroquinolone antibiotic is attached via a cleavable amide bond and then electrospun with poly(vinyl alcohol) as a composite nanofiber scaffold has been studied to evaluate the behavior of drug release. The formulation has a characteristic of releasing only 27% of the drug during the initial phase (burst release), which is 90% less as compared to other sustained release formulations (Jalvandi et al. 2017). The delivery of insulin via the oral route is not advised due to the enzymatic degradation of the drug in the gastrointestinal tract (Caffarel-Salvador et al. 2017). There are, however, many means for insulin to bypass the gastrointestinal tract and reach to systematic circulation. For example, delivery via the subcutaneous route can prevent the peptide from enzymatic degradation. An efficient method is to deliver the insulin via the buccal mucosa, which is highly vascularized and permeable to this peptide. However, the tight junctions in the epithelial cells of the buccal system make diffusion a limiting process (Richard 2017). In this very context, insulin permeation was studied across buccal mucosa in an ex-vivo pig model. The insulin release was 16-folds higher as compared to free-insulin administered via the same route, making nanofiber materials as ideal candidates for oral-transmucosal delivery (Lancina et al. 2017). Chitosan-phospholipid nanofiber composite developed as a transdermal delivery system by electrospinning was used to study the combined release of diclofenac, vitamin B12 and curcumin. The kinetic study demonstrated the significantly higher release of vitamin B12 as compared to the diclofenac or curcumin (Mendes et al.

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2016). In another study, liposomes of cholesterol were loaded with gentamicin and surface-immobilized on the electrospun nanofibers of chitosan. Surface immobilization of liposomes was achieved by functionalizing the amine group of chitosan with thiol groups and then covalently immobilizing the gentamicin-loaded liposomes with –SH groups using maleimide. The in-situ release from these nanofibers demonstrated the continued release of the drug. The delivery system has promising performance in terms of reducing the drug dosage and toxicity, hence increasing the therapeutic efficacy of the formulation (Monteiro et al. 2015). The composite nanofibers of chitosan and poly(vinyl alcohol) supplemented with gold nano-rods have found applications in imaging using optical microscopes. This is attributed to the plasmon-enhanced absorption and high light-scattering effect of the gold nanorods. This system by combining dual electronic and optical microscopies can provide information of the intracellular locations. Experiments were conducted to deliver doxorubicin (an antineoplastic drug) to nucleus by incorporation within these fibrous scaffolds (Yan et al. 2016).

4.2.3

Chitosan Nanofiber Scaffolds: In Wound Regeneration

In the field of wound regeneration and healing, various scaffolds containing pristine chitosan nanofibers or chitosan composites have developed to create efficient scaffolds. Herein, we will discuss some of the examples related to this section. For instance, the asiaticosides obtained from Centella asiatica known as triterpenoids possess wound healing and anti-inflammatory properties. These compounds were co-electrospun into core-shell nanofibers using chitosan and sodium alginate. The composite nanofiber scaffolds applied to Sprague-Dawley rats inflicted with circular burn injuries. The prepared scaffolds resulted in facilitating the wound healing process. This is evidenced as improvements in healing ratio, increased expression of serum markers like a cluster of differentiation, vascular endothelial growth factor, proliferating cell nuclear antigen, and downregulation of interleukin-6 (Zhu et al. 2016). Chitosan and arginine were co-electrospun to form wound dressing materials with properties of mirroring the human dermis. The schematic representation of arginine functionalized chitosan nanofibers is presented (Fig. 4.3). The human fibroblasts very well proliferated on these scaffolds suggesting the biocompatible nature of the material. These mats represented bacteriostatic properties when tested against E. coli DH5α and S. aureus. The application of these scaffolds on wounds in a rat model demonstrated faster-wound closure rate as compared to the chitosan only scaffolds. Necropsy of the regenerated tissue also demonstrated the regenerating potential of these scaffolds (Antunes et al. 2015). Chitosan, pullulan fabricated and tannic acid fabricated as a composite mat by electrospinning has shown a synergistic antimicrobial activity against E. coli. These fibrous scaffolds support the growth of fibroblasts as in a natural tissue besides supporting the interlayer growth of cells (Xu et al. 2015). Chitosan and sericin, both being natural polymers have been electrospun successfully using a mass ratio of 2.5:1. Continuous nanofibers with uniform distribution produced during the process

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Fig. 4.3 Schematic representation of the synthesis of arginine functionalized chitosan nanofibers using electrospinning. Reprinted with permission from Elsevier (Antunes et al. 2015)

and in-vitro cell toxicity studies revealed that these nanofibers supported the proliferation of L929 fibroblasts. The stability of nanofibers was indicated by their high zeta potential of +44 mV. Antibacterial activity was observed against E. coli and B. subtilis in the zone of inhibition studies, approving their role for wound healing management (Zhao et al. 2014).

4.2.4

Chitosan Scaffolds: As Antibacterial Materials

Chitosan scaffolds have been used for various antibacterial applications as either topical systems and/or in implant applications. In this section, we will discuss various examples of exploring different strategies. Periodontitis is a typical infection of the gums that proceeds with gingival inflammation (Nanci and Bosshardt 2006). This condition often requires mechanical interventions and the use of systemic antimicrobial administration, especially the higher oral dosages. The development of chitosan/polycaprolactone scaffolds containing tinidazole as a localized delivery system can deliver the antimicrobials to these tiny pockets. The drug release during in-vitro studies suggested that tinidazole released in a sustained manner until 18 days in therapeutic concentrations. The formulation was found to have antibacterial activity against S. aureus over 21 days (Khan et al. 2017). In another study, the herbal extract of Falcaria vulgaris used for depositing silver nanoparticles on the surface of chitosan-poly(ethylene oxide) nanofibers earlier fabricated by electrospinning. The presence of the extract resulted in the reduction of AgNO3 into silver nanoparticles. The bactericidal properties of the nanofiber scaffold were attributed because of bioactive silver nanoparticles (Kohsari et al. 2016). Cinnamaldehyde oil obtained from the bark of the cinnamon has reported antibacterial activity. Incorporation of this oil into chitosan and poly(ethylene oxide) nanofibers (0.5 to 5.0%) initiated to impart bactericidal activity to these fibrous materials. Cinnamaldehyde was released from these fibers for over 3 h and

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has demonstrated activity against both E. coli and P. aeruginosa. Furthermore, these scaffolds can provide broad-spectrum natural antibiotic delivery for the treatment of nosocomial infections (Rieger and Schiffman 2014). Similarly, chitosan and poly (ethylene oxide) were fabricated by electrospinning for delivery of two antimicrobial agents viz., chlorhexidine and silver nanoparticles. During the release studies, it indicated that chlorhexidine was released within 48 h while silver nanoparticle release sustained over 28 days. Moreover, antibacterial efficacy against staph was observed over four days while using 5% of AgNO3 in the reduction process (Song et al. 2016). Various derivatives thiol and iodoacetamide derivatives of chitosan are known to possess antibacterial activity against various species microbes. These were electrospun into cross-linked nanofiber mats using electrospinning. Mechanical testing showed that these fibers have high tensile strength as compared to pure chitosan fiber or chitosan composites. These nanofiber mats displayed bactericidal activity against E. coli at a minimum inhibition count of 400 μg/ml. The additional antibacterial activity is presumed to be due to the formation of a disulfide bond between the thiol groups and the bacterial proteins (Abdelgawad et al. 2017).

4.2.5

Chitosan Nanofibers: From Biosensors, Analytic Systems to Diagnostic Aids

Nanofiber systems are highly porous and have a large surface area. As such, they find utility in the fabrication of biosensors and other analytical systems. In this section, we cite different examples from the literature describing such illustrations. For example, iron complexed chitosan nanofiber systems prepared by electrospinning have shown to detect even trace amounts of 9-tetrahydrocannabinol from the blood samples. Tetrahydrocannabinol is a cannabinoid molecule present in the cannabis plant, and its identification in the blood used as an indication of marijuana use. The huge surface area of these composites promotes greater chances of interaction between the adsorbent and analyte. Comparison of this system with conventional C18 columns used in high-performance liquid chromatography has proven then better in terms of analytical performance parameters such as the limit of detection, reproducibility, precision and accuracy (Asiabi et al. 2017). Enzyme immobilization is another area where highly porous and large surface area materials can prove useful, especially in devising biochemical sensors. For example, cholinesterase’s have been immobilized onto chitosan and poly(vinyl alcohol) electrospun nanofibers for the rapid detection of primiphos-methyl, a component of olive oil. This system could detect the primiphos-methyl at values as low as 0.2 nM compared to the already existing international standard of 164 nM (El-Moghazy et al. 2016). Fluorescence-based sensing systems were developed by decorating rhodamine B over the surface of chitosan nanofibers to determine mercury in the samples. This system works on the principle that mercuric ions present in the open spirolactam ring of the rhodamine unit and the complex formed gives fluorescence, which can be detected. Such a system can also be designed to detect other elements and use in testing of water and measure of environmental indicators (Horzum et al. 2016) (Fig. 4.4).

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Fig. 4.4 Synthetic route for the preparation of modified Rhodamine B functionalized nanofibers of chitosan. Reprinted with permission from John Wiley and Sons (Horzum et al. 2016)

Fig. 4.5 Schematic representation of Ni decorated nanoparticle on the surface of chitosan/ polycaprolactone nanofibers, showing subsequent reduction of Ni from Ni (II) to Ni (0) by nitrophenols (green to brown). Reprinted with permission from Elsevier (Karakas et al. 2017)

In the field of catalysis, chitosan-based nanofibers have been efficiently utilized to carry out some demanding reactions in the industry, like the reduction of nitro-aromatic compounds. A study has shown the introduction of Ni nanoparticles on the composites of chitosan, and polycaprolactone efficiently catalyzes the reduction of nitrophenols even under mild conditions (Fig. 4.5) (Karakas et al. 2017).

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Nanofiber materials have been used in the field of tumor diagnosis to separate and identify tumor circulating cells considered as makers of cancer metastasis (Sun et al. 2016). One such study reported an exciting experiment whereby they conjugated poly(carboxybetaine methacrylate) (pCBMA) brushes with the amine groups of chitosan (Fig. 4.6). The pCBMA brushes are, in turn, attached to DNA aptamers. This results in specific cell capture and control over non-specific cellular adhesion. Once the circulating tumor cells bind to the target by immobilization, the attached cells are then released by binding to the complementary DNA sequence of the aptamer (Sun et al. 2016).

4.2.6

Chitosan Scaffolds: For the Treatment of Contaminated Water

Chitosan nanofibers produced by electrospinning for Cr(VI) contamination in water. This scaffold had a maximum adsorption capacity of 20.5 mg/g and could individually adsorb copper, cadmium and lead ions. However, selectivity towards the adsorption of chromate ions was observed when it coexisted with other metal ions. Comparison to commercially available membranes, these nanofiber systems were more efficient in parameters like trans-membrane pressure, permeation flux and metal ion rejection (Li et al. 2017). Similarly, reusable nanofiber membranes of chitosan and poly(vinyl alcohol) fabricated by the electrospinning method demonstrated a higher adsorption rate for chromate, ferrous and nickel ions. However, less adsorption capacity at high concentrations was observed (Habiba et al. 2017). Moreover, water sources, including industrial wastes, are containing antibiotics (e.g., clindamycin as contaminants). To prevent this problem, various techniques like the ion-exchange filtration and adsorption-based filtration can prove detrimental to clean water bodies from these contaminants. An electrospun nanofiber mat of Ag2S containing chitosan has shown very promising results to remove clindamycin using adsorption technique (Gupta et al. 2017).

4.2.7

Chitosan Scaffolds: Supporting Structures for 3D Cell Culture

The nanofibrous structures due to their high porosity and 3D architecture act as supporting structures for different cell lines. These scaffolds allow penetration and mobility of the cell as well as the exchange of gaseous materials, nutrients and metabolites. For example, in the case of bone tissue engineering, polymeric scaffolds provide a suitable surface to stem cells to attach, infiltrate, and finally proliferate into bone cells. Electrospun scaffolds for the time being act as guiding constructs at applied places of bone damage. For instance, nanofiber constructs of chitosan/silk fibroin supported the growth and proliferation of human mesenchymal stem cells (hMSCs), followed by their differentiation into the osteogenic lineage. The ability of these scaffolds to act as cell supporting structures is advocated by cytoskeleton analysis and MTT assay (Lai et al. 2014). Cellulose nano-crystal added to the

Fig. 4.6 Illustrative scheme for the design of pBMA brushes/aptamer on chitosan nanofiber biointerface. Tumor cells are captured by binding to specific antibodies and then released by complementary base hybridization. Reprinted with permission from John Wiley and Sons (Sun et al. 2016)

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chitosan-poly(ethylene oxide) solutions allows high yield of nanofibers with uniform diameters and thermally stable state. The presence of cellulose in the nanofibers promotes the cytoskeletal organization of 3T3 fibroblasts. Culturing both fibroblasts and hepatocytes over these 3Ds scaffolds results in fibronectin adsorption giving the scaffold liver-like appearance. This 3D system was found to sustain well-formed colonies of cells individually and demonstrated significant cytochrome release (Rajendran et al. 2017). Similarly, the electrospun scaffolds of chitosan/gelatin composites along with bioactive glass powders supported differentiation of human endometrial stem cells (hEnSCs) when angiogenic factors were supplemented externally. The presence of bioactive glass in the scaffold provides a suitable 3D microenvironment for cellular differentiation. This was further supported by the upregulation of endothelial markers over the cell surface. These constructs can be thought to have a promising future in regenerative medicine (Shamosi et al. 2017).

4.2.8

Chitosan Scaffolds: Supporting Systems for Nerve Tissue Regeneration

Aligned nanofibers of chitosan and other biodegradable polymers have a huge potential in nerve tissue regeneration. Schwann cells form a column-like structure by aligning in a specific direction over the aligned chitosan nanofibers. These results are encouraging in nerve tissue regeneration as these structures resemble in a nerve like projections. These structures also are found to upregulate adhesion molecules on the surface of the fibers. The Schwann cells growing over these scaffolds exhibited electrophysiological properties, and functional recovery occurred on time. These studies have revealed that oriented chitosan constructs can serve as substitutes for autogenous nerve graft (Wang et al. 2009). Recently, work has been done on fabricating scaffolds for guided differentiation of human dental pulp stem cells (hDPSCs) into neuron-like cell structures using a microenvironment construct of intercalated chitosan montmorillonite (MMT) and poly(vinyl alcohol). These constructs were fabricated by an initial ion-exchange reaction between the MMT and chitosan, followed by electrospinning with poly(vinyl alcohol) solutions (Fig. 4.7). Neuronal induction in hDPSCs seeded on constructs was determined by a polymerase chain reaction. Moreover, the upregulation of neural markers like Oct-4, NF-M and βIII-tubulin was seen. These artificial nerve grafts can prove to be materials of significance for the regeneration of damaged neural tissue (Ghasemi Hamidabadi et al. 2017).

4.3

Conclusion

In conclusion, unique and smart biomaterials can be fabricated by electrospinning chitosan either by blending it with other polymers or by using surface-functionalized derivatives. Chitosan-based nanofibers have shown excellent results in various fields, including tissue engineering. They are very close in mimicking the

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Fig. 4.7 Representation for the synthesis of intercalated chitosan/montmorillonite/poly(vinyl alcohol) nanofibers meshes using electrospinning. Reprinted with permission from ACS (Ghasemi Hamidabadi et al. 2017)

bio-interface, providing natural topography and have unique degradability properties. This book chapter has summarized the fabrication schemes, electrospinning conditions and novel applications of chitosan-based biomaterials. This chapter, in brief, will provide readers an outlook of the recent advancement for the fabrication of novel nanofiber-based scaffolds and a future strategy to develop smart biomaterials. In brief, the reviewed literature provides a summary of the delivery of insulin through the buccal mucosa, the capture of circulating tumor cells, the development of diagnostic aids, fabrication of arterial stunts and respiratory tissue-regenerating materials. On the other hand, the recent applications, such as water purification systems, pharmaceutical analysis, 3D cell culturing, nerve tissue regeneration and antibacterial wound dressings, have also been discussed. We are sure that this chapter will provide insights into the recent advancements involved in the fabrication of chitosan nanofibers. This will enable young researchers to work in nanotechnology to develop strategies that can be considered for human welfare using nano-based approaches. Acknowledgments We thank the Department of Science and Technology, Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016 for providing lab funds and supporting the fellow.

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Unique Properties of the Gold Nanoparticles: Synthesis, Functionalization and Applications Roqia Ashraf, Touseef Amna, and Faheem A. Sheikh

5.1

Introduction

Nanotechnology deals with the design, production and application of materials and devices at the nano-scale level. Owing to their properties at nano-scale, which are designed while manipulating the matter at atomic, molecular and supramolecular scale results to create material and devices with ultra-small size and large surface to volume ratio, that displays enormous characteristic properties rendering them to be used in different applications (Dykman and Khlebtsov 2012). Due to recent advances in synthetic chemistry over the years, it is likely possible to synthesize the nanoparticles (NPs) with proper control over physio-chemical and optical properties (Zhang 2015). In this regard, several methodologies have been put forward for the synthesis of the NPs (Grimsdale and Müllen 2005; Snure and Tiwari 2007; Lang et al. 2011; Tiwari and Snure 2008; Yin and Alivisatos 2005). The metallic NPs, especially the Au and silver (Ag) NPs significantly have acquired the consciousness because of novel characteristics that provide them with an edge over other metallic NPs. Interestingly, the Au NPs due to their opto-electric properties have been explored to a greater extent in the biomedical field (Grzelczak et al. 2008; Pérez-Juste et al. 2005; Thaxton et al. 2006). On the one hand, Ag NPs are notable for their action against gram-positive and gram-negative bacteria along with different fungi (Kalwar et al. 2018; Aziz et al. 2016). On the other hand, Au NPs in current years are widely explored in imaging, diagnostics and theranostics applications.

R. Ashraf · F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] T. Amna Department of Biology, Faculty of Science, Albaha University, Al Bahah, Kingdom of Saudi Arabia # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_5

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Furthermore, their claim in cancer treatment is arising due to an improved permeability and retention effect of Au NPs with tumor cells (Her et al. 2017). The functionalization of the NPs with drugs, nucleic acids, fluorescent molecules have paved a way towards the novel approaches in targeted delivery, bioimaging and so on (Cheng et al. 2013; Ghosh et al. 2008; Butterworth et al. 2012). The interface at which the NPs interact with the biological molecules termed as a bio-nano interface; this boundary plays a significant role in defining the delivery of biomolecules to the target cells (Zhang 2015). These interactions are strictly dependent on colloidal forces, thermodynamic and physio-chemical interplays between the NPs and the biological ligands (Lynch et al. 2009). The Au NPs, when conjugated with antibodies and cell membrane specific antigens are capable of targeting different antigens and cell surface receptors (Sahay et al. 2010). The diverse applications of Au NPs due to their extraordinary physio-chemical and opto-electric properties have opened new ways in diagnostic and therapeutic fields. Moreover, keeping in consideration the unusual features of Au NPs, this chapter will highlight different protocols for the synthesis of Au NPs, including the eco-friendly, cost-effective and green route synthesis. In detail, characteristic properties of NPs will be summarized, followed by the importance of capping/stabilizing agents in size-controlled combination are discussed. Furthermore, the applications of Au NPs in the biomedical field are discussed in a detailed manner. For instance, the role of Au NPs in targeted delivery of drugs including anti-cancer drugs and DNA and non-coding RNA sequences are highlighted. The colorimetric assays that explore the submission of Au NPs in bio-sensing and their role in cell-imaging have been highlighted in the next sections.

5.2

Synthesis of Au NPs

The synthesis of Au NPs chiefly involves two approaches similar to as reported for the synthesis of other metal-based NPs. These two approaches are top-down and bottom-up approaches. The former approach is identical to the atomic fission, which involves the disintegration of bulk Au into nanometer-size particles. The bottom-up approach is similar to nuclear fusion, which involves the building up of particles with size at the nanometric-scale. The methods involved in the top-down approach include the disintegration methods such as laser ablation (Birtcher et al. 2004), sputtering, arc discharge (Lung et al. 2007), UV and IR radiation (Sakamoto et al. 2009; Zhou et al. 1999), etc. The bottom-up approach involves vapor deposition (Daniel and Astruc 2004), sol-gel (Kobayashi et al. 2001) and chemical methods such as reduction by using reliable reducing agents (Turkevich et al. 1951) and/or by biological extracts (Rafique et al. 2017) (Fig. 5.1).

5.2.1

Citrate Reduction Method

This is the most commonly adopted method that includes the chemical reaction using chloroauric acid (HAuCl4) in water by using trisodium citrate (Na3C6H5O7) as a

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Fig. 5.1 Different techniques (top-down and bottom-up approaches) used for the synthesis of Au NP

reducing agent (Turkevich et al. 1951). The synthesis process by this method involves the addition of Na3C6H5O7 to the aqueous solution of HAuCl4. After a few minutes of vigorous stirring, the resulting red colloidal solution confirms the reduction of the HAuCl4 into Au NPs and other byproducts CO2, HCl and NaCl. The resultant Ag NPs need to be separated and characterized using techniques such as SEM, TEM, AFM, EDS, UV and DLS. The size control during the synthesis is important so facilitate poly or mono-distribution of NPs. The stabilizing and capping agents (e.g., citrate, polyethyleneimine, poly(vinyl alcohol), sodium alginate, polyvinylpyrrolidone and glycol chitosan) are used to avoid the aggregation of the NPs in a suspension. In this method, the proper citrate/Au ion ratio plays a critical role. In detail, it has been observed that if the concentration of the citrate is increased, the NP size tends to decrease. Moreover, oxidation of citrate into dicarboxy acetone plays a significant role as a capping agent for particles, thereby prevents their aggregation during the synthesis process. Seeding of the Au NPs in the presence of precursor and the reducing agent is also beneficial in regulating the size. The appropriate amount of seed and growth solution allows significant control of the size of particles (Daniel and Astruc 2004).

5.2.2

Brust-Schiffrin Method

The method initially developed by Brust and colleagues in 1994 (Brust 1994), involves the reduction of the HAuCl4 in a two-phase system by the addition of C7H8 and NaBH4 as anticoagulant and the reducing agent followed by treatment with alkanethiols. Upon mixing, the reactant mixture is supported by the addition of tetraoctylammonium bromide (C32H68BrN) to transfer in the organic phase. Upon analysis, the morphologically distinct cuboctahedral and icosahedral particles in the

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range of 1-3 nm were obtained. Later on, several modifications were put forth by other research groups. This includes a one-phase system that utilizes the methanol as a solvent and p-mercaptophenol as a stabilizing agent (Brust et al. 1995). This method can be explored for the preparation of other metallic NPs (e.g., silver and copper) (Brust et al. 1995). Furthermore, the size of the particles and their stability can be enhanced and/or improved by using different reducing and stabilizing agents.

5.2.3

Green Synthesis Method

The reagents used in the chemical synthesis of Au NPs possess some limitations (e.g., use of toxic reducing agents) such as NaBH4 and Na3C6H5O7 (Kumar et al. 2008). Besides, the stabilizing agents that are used in chemical synthesis, to control the size, morphology and to keep the NPs dispersed. However, these toxic chemicals possibly lead to biocompatibility issues. To improvise the bioactivity and biocompatibility of the synthesized NPs, the green synthesis approaches are currently explored by several research groups (Salam et al. 2012; Shukla and Vankar 2012). It involves the reducing agents (e.g., oxidoreductase, hydrogenase and α-amylases) mostly isolated from plant sources (Mittal et al. 2013; Vankar and Bajpai 2010; Elia et al. 2014) and microorganisms such as algae, bacteria and fungi (Das and Marsili 2010; Raveendran et al. 2006) using one-step green synthesis (Fig. 5.2). Moreover, the synthesis process of Au NPs requires stabilizers (e.g., collagen, starch, gelatin, polysaccharide, and in some cases synthetic polymer or ligands), which are used depending upon the ease and availibility. Allowing the use of nontoxic reducing and

Fig. 5.2 The scheme represents the green synthesis of Au NPs from different biological sources and their applications in biomedical sciences

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stabilizing agents from plant-based extracts are becoming a common platform to overcome the issues associated with the chemical synthesis of NPs. However, the limitation with green synthesis is the low efficiency and tedious process involved in the extraction of herbal extracts. Intriguingly these plant extracts and biomass from microbes facilitate the presence of some unknown components, thereby helps in the conversion of HAuCl4 into NPs (Yu et al. 2016). Besides, their effects on Au NPs, these extracts possess a similar impact on other metal ion precursors such as AgNO3 (Rafique et al. 2017). Moreover, this approach for the synthesis of NPs is considered as simple, high-throughput, efficient, cost-effective, and, more importantly, eco-friendly.

5.3

Characteristics Features of Au NPs

The metallic NPs offer a diverse use in biomedical, bio-sensing, imaging, diagnostics, therapeutics and targeted delivery areas. Au NPs, among these, play a preferred role than other metal particles significantly due to their inherent characteristic properties (Eustis and El-Sayed 2006). For instance, the Au NPs are inert (and hence biocompatible) and have high stability. They have a unique optical and electrochemical characteristic that arises due to the phenomenon of Surface Plasmon Resonance. This phenomenon is an optical property usually observed when a beam of light encounters a metal surface and is used to monitor the interaction between two molecules (Jazayeri et al. 2016). It is significantly affected by any alteration in the size or shape of the Au NPs, and the corresponding change in the electron arrangement is reflected in the absorption maxima and color of the dispersion (Jain et al. 2006; McDonnell 2001). For example, the colloidal dispersion containing 13 nm Au NPs presents a characteristic peak at 520 nm and upon an increase in the size of NPs the Surface Plasmon Resonance peak shifts to the longer wavelength observed by the color change of the dispersion from red to the blue (Njoki et al. 2007) (Fig. 5.3). Moreover, the large surface area potentiates their sensitivity, hence contributes significantly towards the application of Au NPs as sensors and detectors (Zeng et al. 2011). The fluorescence studies, approaches such as femtosecond emission (Chen and Katz 2002) and study state investigation are also reported in the literature (Hu et al. 2001). The fluorescent capping groups such as pyrenyl (Thomas and Kamat 2000), fluorenyl (Dubertret et al. 2001), polyoctylthiophenyl (Xu and Yanagi 1999) have been explored to elucidate the possible applications of Au NPs.

5.4

Stabilization and Functionalization of au NPs

The basic problem faced in the preparation of Au NPs is that they tend to aggregate, which results in undesired precipitate during and/or after the synthesis process. However, this aggregation can be avoided by using appropriate stabilizing and/or capping agents. Additionally, these agents necessarily help to synthesize the NPs

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Fig. 5.3 Representation of the absorption spectrum of Au NPs. As the size of NPs increases, we can observe the shift occurring in the peaks from red to blue. Reprinted with permission from American Chemical Society Copyright (2007) (Njoki et al. 2007)

with the desired diameter. The stabilizing agents commonly used in the chemical synthesis process of particles are thiols, citrate, polymers and other protecting ligands (Zhao et al. 2013). These agents, besides preventing the aggregation of the NPs also enhance the properties of improved solubility, electron transfer efficiency and sensing property (Zhao et al. 2013). Moreover, these agents possess polar groups that surround the particles during the synthesis process and thus are responsible for the isolation of the particle from the adjacent NPs (Alex and Tiwari 2015). Furthermore, the charge stabilization and steric interaction strategies are the most common methods employed in the stabilization of the particles. On the one hand, the charge stabilization method invokes the adsorption of the charged moieties on NPs surface. As a result of repulsion among the charges, there is inhibition of the aggregation process. On the other hand, in the case of steric stabilization, the polymers prevent the aggregation by steric hindrance effects. Alkyl thiols one of the best stabilizing agents, self-assemble as a monolayer around the particle during the synthesis process (Thomas and Kamat 2003; Jackson et al. 2006). The polymers play a significant role in the stabilization of the particles. They conceal the surface of the NPs, thus prevents them from precipitation. Polymers such as poly(acrylonitrile) (Aziz et al. 2016) and poly(N-vinyl-2-pyrolidone) (Hoppe et al. 2006) had been explored for stabilization of steric hindrance. Moreover, polymers such as poly(vinyl alcohol), poly(ethylene oxide) and polyvinylpyrrolidone can function as both reducing and stabilizing agents in the synthesis of NPs. Especially, the poly(N-vinyl-2pyrolidone) which is a water soluble and biocompatible has played this dual role in one of the studies carried by Hoppe and co-workers (Hoppe et al. 2006). In this study, a simple one-step synthesis of Ag and Au NPs exploring this polymer was achieved. The ratio of the polymer poly(N-vinyl-2-pyrolidone) and metal precursor

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can be controlled for achieving the desired properties of the synthesized particles. Similarly, the poly(N-vinyl-2-pyrolidone) and poly(acrylonitrile) were explored by another group where the affinity for Au was potentiated by the polymers with cyano and sulfhydryl functional groups. In this study, the synthesized NPs possess a smaller size as well as narrow size distribution (Teranishi et al. 1998). The use of polymers as stabilizing agents affects the efficiency of NPs during the in-vivo experiments. The use of poly(ethylene glycol) as a stabilizing agent enhances the blood circulation time of NPs (Paciotti et al. 2016). The rationale is because the poly (ethylene glycol) is a highly hydrophilic polymer with no surface charge, this property avoids the uptake of Au NPs by macrophages. It prevents non-specific interactions with other biomolecules during circulation (Niidome et al. 2006; Panahi et al. 2017). Besides, the polymers the co-polymers have also been employed as stabilizing agents. For instance, the co-polymer composed of polystyrene-blockpoly(ethylene oxide) and polystyrene-block-poly(2-vinylpyridine) microemulsions have been explored. These block co-polymers have the tendency to form micelles in suitable solvents allowing the organization of particles into morphologies such as vesicles, rods, spheres etc. These preparations resulted in the particles with a wellcontrolled size and homogenous distribution of the particles into micelles with a single particle in each micelle (Möller et al. 1996; Spatz et al. 1996; Mössmer et al. 2000). The polysaccharides such as chitosan and starch have also been explored as efficient stabilizers for the synthesis of Au NPs. The shape and size distribution of the synthesized particles varies with concentration and molecular weight of the polymer. The bimodal size distribution with polygonal and spherical-shaped Au NPs can be obtained using chitosan as reducing and stabilizing agent (Huang and Yang 2004). Functionalization with desired reactive groups and/or biomolecules is the important feature that contributes towards the diverse applications of the NPs. Functionalization of the Au NPs was first reported by Mirkin and Alivisators (Mirkin et al. 1996; Alivisatos et al. 1996). These researchers used the property of selfassembly for adsorption of the oligonucleotides to bind the surface of Au NP for the detection of genomic DNA. Different strategies can achieve the process of functionalization. For instance, physical adsorption is a simple and quick process that involves the chemistry of electrostatic and/or hydrophobic molecular interactions. The physical parameters, such as a change in pH or ionic strength, effects these molecular interactions (Tan et al. 2009). The study has reported that upon addition of the lead (Pb2+) destabilizes the stability of the Au NPs functionalized with ssDNA. The fact behind this de-stability is the less binding affinity of dsDNA to NPs compared to the ssDNA molecule (Wei et al. 2008; Wang et al. 2008). Another approach of the functionalization of Au NPs is the adsorption of functional groups and biomolecules by covalent interactions. These functionalized NPs inherit a greater stability compared to the NPs functionalized with physical adsorption method. The rationale is because of disulfide bond formation between the Au particle and functional group or ligand. A number of molecules containing thiol groups have been reported in literature (e.g., glutathione, alkanethiolates, thioethers, thiocarbamates) for desired functionalization of NPs (Zhao et al. 2012; Priyadarshini and Pradhan 2017). Ligand-analyte specific interaction has employed for

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functionalization of Au NPs. This specific recognition approach has been explored further in biosensing application of Au NPs. For instance, Au NPs conjugated with different ligands such as allylmercaptan (Matsui et al. 2005), 11-mercaptoundecanoic acid (Yu et al. 2007) have been analyzed for the detection of dopamine and antibodies. This process of functionalization undoubtedly amplifies the diversified applications of NPs but also encounters some inevitable snags (e.g., costly two-step synthesis procedure and conjugation/adsorption of the functional groups and ligands). This process sometimes affects the intrinsic properties of Au NPs. For example, the chemical reduction method by citrate as a capping agent masks the negatively charged surface of the particles. However, the green synthesis rules out these pitfalls as the biological extract of plants and microorganisms are rich in natural capping/stabilizing agents and hence provide high specificity and high stability to the synthesized NPs (Yu et al. 2016).

5.5

Biomedical Applications of Au NPs

The aforementioned sections provided an insight into the diverse characteristics of Au NPs. The upcoming sections of this chapter will be focused on biomedical applications like targeted delivery, gene delivery, immunotherapy, biolabeling, biosensing, bioimaging and therapeutics.

5.5.1

Targeted Delivery of Au NPs

Au NPs can intracellularly enter the lipid bilayer by endocytosis although the underlying mechanism of permeation is not well understood. These NPs can enter the cells even after the process of functionalization with ligands, functional groups, drugs and antibodies. In this section, the targeted delivery of drugs and nucleic acids in conjugation with the NPs will be discussed. Chemotherapy is the most commonly used for the treatment of cancers. The major challenge faced by approach is the proper delivery of the drugs to tumor-specific sites without damaging the normal cells. Targeted delivery of magnetic NPs was later on introduced to overcome the non-specificity associated with conventional chemotherapy. The researchers synthesized the Au NPs with a size of about 22 nm. Further on, these particles were functionalized with the thiol terminated poly(ethylene glycol) and subsequently loaded with doxorubicin (i.e., an anti-cancer drug), with the amine group of the drug playing a role in attachment with particles (Elbialy et al. 2015). Following characterization studies, the in-vivo studies revealed excellent biodistribution and retention of the particles throughout the tumor in the presence of a suitable magnetic field. The histopathological analysis of tumor tissue showed extensive necrosis compared to tissue with doxorubicin only (Fig. 5.4) (Elbialy et al. 2015). Another study explored the doxorubicin-loaded Au NPs for targeted delivery in breast cancer cells. In this regard, the NPs were synthesized using fucoidan as a capping and stabilizing agent. The in-vitro analysis revealed the cytotoxic effects of

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Fig. 5.4 The figure depicts the histological staining of tumor tissue. (a) represents the control, (b) represents the tissue containing doxorubicin only, (c) and (d) represent the NPs conjugated with doxorubicin in absence and presence of the magnetic field. Reprinted with permission from reference (Elbialy et al. 2015). Copyright (2015) Elsevier

these NPs on breast cancer cells, and the apoptosis induced by these NPs was confirmed with the Annexin V-FITC/PI dual staining and DNA content analysis (Manivasagan et al. 2016). These studies indicate the potential of the Au NPs as efficient nanocarriers for therapeutic applications. Similarly, the doxorubicin was attached to the Au NPs with acid-responsive linker hydrazine and functionalization with low-density lipoprotein receptor-related protein angiopep-2. This provides the whole assembly to infiltrate the blood-brain barrier to target the glioma cells (Fig. 5.5) (Ruan et al. 2015). Upon in-vivo investigations, using mice as an animal model, it was revealed that functionalized NPs substantially got delivered to glioma and release of doxorubicin extended the median survival time by 2.89-fold (Ruan et al. 2015). Besides doxorubicin, other anticancer drugs and their analogs have been routinely employed in the functionalization of Au NPs (Paciotti et al. 2016) as we know that liposomes are comfortably endocytosed by the cells. In this regard, the researchers have explored these liposomes for release studies of drugs, but the problem

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Fig. 5.5 Transmission electron microscopy micrographs of glioma cells targeted with (a) PEGylated Au NPs conjugated with doxorubicin (b) represents the same NPs in treated mice. Reprinted with permission from reference (Ruan et al. 2015). Copyright (2015) Elsevier

associated was sub-optimal release. To counter this, approaches have been utilized in different studies such as pH-sensitive (Simoes et al. 2004), photosensitive (Shum et al. 2001; Yavlovich et al. 2009), thermo-sensitive (Paasonen et al. 2007, 2007, 2010), and other approaches to optimal release of drug to the target site. The absorption of the light by the Au NPs is released in the form of heat that induces phase transition in lipid bilayers that initiates the content release from the liposomes (Mulvaney 1996). Au NPs encapsulated by liposomes have been studied for their delivery applications, especially drug delivery. In one of such studies, the researchers have conjugated the calcein, a fluorescent probe with Au NPs followed by their encapsulation in liposomes. It was revealed that upon exposure of the liposome to light, the calcein was released into the cytosol. Moreover, the study has also analyzed the behavior of calcein release as a function of pH, where it was revealed that discharge was faster at acidic medium as compared to the neutral pH. The cells used in this study were human retinal pigment epithelial cells and

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Fig. 5.6 Confocal microscopic images (a-f) of retinal pigment epithelial cells (ARPE-19) and umbilical vein endothelial cells (HUVEC). Nuclei are shown in magenta, and calcein is shown in green. Bright field channel of fluorescent images (j-l) is represented in pictures (m-o). Both cell lines were exposed to light for 20 min. Scale bar 30 μm. Reprinted with permission from reference (Lajunen et al. 2015). Copyright (2015) Elsevier

umbilical vein endothelial cells (Fig. 5.6) (Lajunen et al. 2015). Besides the abovementioned drugs, the researchers have conjugated methotrexate, a folic acid analogue and anti-cancer drug with Au NPs. There it was reported that the cytotoxic effect of this drug conjugated Au NPs was 7-folds higher than the methotrexate alone when investigated on Lewis lung carcinoma cells (Chen et al. 2007).

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5.5.1.1 Gene Delivery The drug delivery systems offer numerous opportunities to improve the solubility, in-vivo stability, distribution and pharmacokinetics of drugs. They also prove to be excellent carriers of nucleic acids (Felnerova et al. 2004). The gene delivery employs the vectors for the delivery of genes of interest to the target cell. The vectors/carriers can be of viral origin or non-viral. However, the viral vectors possess some drawbacks like the stimulation of immune response, less specificity in targeting cells, irregular cytotoxicity, low carrying capacity and difficulties in production and packaging (Ghosh et al. 2008; Check 2002; Luo and Saltzman 2000). Thus, the non-viral vectors for gene delivery overcome these drawbacks and provide benefits such as low toxicity and improved carrying capacity. The NPs and liposomes display great potential as non-viral carriers for gene delivery (Boyer et al. 2010; Han et al. 2006; McIntosh et al. 2001). Au NPs owing their unique properties play a significant role in the gene-based delivery. The Au NPs can be conjugated with cationic quaternary ammonium groups and electrostatically bound to plasmid DNA. It was reported that this whole assembly was able to prevent the nuclease mediated degradation of DNA. These functionalized NPs were also able to regulate the transcription of T7RNA polymerase (McIntosh et al. 2001). The DNA release was also studied from Au NPs after treatment with glutathione (Han et al. 2005). Researchers have explored the Au NPs for co-delivery of DNA and siRNA simultaneously. This was possible with the layer-by-layer polymer coating using Au NPs as the core. This DNA delivery by hybrid NPs upon in-vitro analysis was successfully endocytosed by human primary brain cancer cells that were confirmed with exogenous gene expression and transmission electron microscopy. Also, the delivery of siRNA to the target cells was confirmed by siRNA mediated knockdown. The knockdown efficacy upon comparison with Lipofectamine® 2000 was found to be superior. These particles, upon appropriate functionalization with nucleic acids can prove as an excellent vector for gene therapy (Bishop et al. 2015). Researchers have also delivered the miRNAs into the cells by cysteamine functionalized Au NPs. These NPs, upon in-vitro studies were capable of releasing the conjugated miRNA and as consequence, was able to down-regulate the target genes when tested on two different cancer cell lines (i.e., neuroblastoma and ovarian cancer cells) (Ghosh et al. 2013). Phototherapy, in combination with conventional gene therapy, can offer an excellent possibility to improve the nucleic acid delivery to the cells (Umeda et al. 2005). For example, one of the studies has explored the exposure of the pulsed laser irradiation mediated release of plasmid DNA from Au NPs. The particles were functionalized with PEG-orthopyridyl-disulfide as a stabilizing agent. The plasmid was released successfully in the target cell after exposure to laser irradiation at a power density value of 80-mJ/pulse without any fragmentation (Niidome et al. 2006). Similarly, studies are reported where the phototherapy was used to initiate the release of the nucleic acid from the NPs. Furthermore, near-infrared radiations have been successfully utilized for the release of green fluorescence protein-DNA conjugates in HeLa cell lines (Takahashi et al. 2005). Another fascinating work in the gene delivery to stem cells is reported in the literature, where the conjugation of the arginine-glycine-aspartic acid (RGD) was explored. This sequence possesses

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Fig. 5.7 Von- Kossa staining (b-e) of human mesenchymal stem cells. Transfected with different vectors, the cells transfected with K4/DNA vector displayed the highest mineralization (f) vector/ pDNA polyplexes on day 21 and (a) represents the non-transfected cells. Reprinted with permission from reference (Kong et al. 2015). Copyright (2015) American Chemical Society

high affinity for the integrin receptor that is overexpressed in most of the cancers, e.g., glioblastoma, ovarian cells and breast cancer cells (Qi et al. 2009). The peptide modified dendrimers entrapped Au NPs for gene delivery to stem cells have been investigated. The dendrimers were used for entangling the Au NPs and used for the delivery of plasmid DNA to human mesenchymal stem cells. The plasmid DNA possess green fluorescent protein and luciferase reporter genes as well as human bone morphogenetic protein-2. These nano-vectors were capable of transfecting the cells and the studies were validated by both qualitative and quantitative fluorescence microscopy and Luc activity assay, respectively. The gene delivery efficiency was also validated by confirming the concentration of human bone morphogenetic protein-2 concentration and subsequent differentiation of mesenchymal stem cells into osteogenic lineage was confirmed by alkaline phosphate activity, calcium deposition, osteocalcin secretion and von Kossa staining assays, that confirmed the osteogenic differentiation of cells (Fig. 5.7) (Kong et al. 2015). Besides the conjugation of the nucleic acids with Au NPs, the researchers have also co-conjugated the antibacterial peptides and plasmid DNA for delivery to stem cells. For example, Peng and co-workers have functionalized cationic Au NPs with an antibacterial peptide HIV-1 twin-arginine translocation and PEP peptide sequence from lactoferrin (antifungal and antibacterial). The plasmid encoding vascular endothelial growth factor and reporter gene luciferase was used in the transfection process with TAT and PEP antimicrobial peptides. The expression of the luciferase and angiogenesis confirmed the transfection process. The results revealed the ability of these hybrid Au NPs to act as vectors for gene activation in tissue regeneration with the potential of both gene delivery and antibacterial properties (Fig. 5.8) (Peng et al. 2016).

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Fig. 5.8 Schematic illustration of NPs conjugated with the antimicrobial peptide and plasmid DNA with permission from (Peng et al. 2016)

5.5.2

Therapeutic Applications of au NPs

5.5.2.1 Immunotherapy Immunotherapy involves the active participation of the host immune system to recognize the external or unusual patterns of both endo and exogenous origins. Currently, the Au NPs have been explored for cancer immunotherapy. Keeping in view the fact of cancer cell behavior against immune cells like suppression of dendritic cells (Zhou et al. 2013), induction of apoptosis of T cells (Guo and Huang 2014), the sustained immunological response against cancer cells was recognized as the primary goal of immunotherapy. The researchers have carried out studies keeping into consideration the properties of Au NPs and explored these particles as nanocarriers of antigens specific to tumors (Ovais et al. 2017). For instance, CpG 1668 oligonucleotide conjugated with the Au NPs with a spacer of ten adenine nucleotides was investigated for the invoking of immunological response by recognizing the toll-like receptor 9. This whole assembly was successfully delivered to lymph nodes and taken by APCs. The triethylene glycol stabilized Au NPs functionalized with CpG was reported to have greater tumor inhibition capability than CpG alone (Lin et al. 2013). The tumor necrosis factor functionalized Au NPs upon intravenous administration displayed rapid uptake in prostate tumor in mice model (Kim and Jon 2012). The researchers have also explored the conjugation of monoclonal antibodies to Au NPs against different tumor antigens. In one such study, the researchers have conjugated the anti-epidermal growth factor antibody with Au NPs. The results demonstrate the homogenous and specific binding of these

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NPs with malignant oral epithelial cell lines compared to non-malignant cells (El-Sayed et al. 2005). Similarly, the monoclonal antibody trastuzumab against the HER-2 receptor that is overexpressed in breast cancers was conjugated with poly (ethylene glycol) stabilized Au NPs (Chattopadhyay et al. 2010). IgG immunoglobin conjugated with Au NPs and protein-G as co-factor revealed the disruption of the cell membrane upon exposure to laser irradiation with the power potential of 540 J/ cm2 in Breast cancer cell line SK-BR-3 (Sun et al. 2013). Moreover, the ability of the Au NPs to permeate the lipid bilayer and resulting accumulation in various tissue explores and provides the potential of NPs to act as adjuvants in antitumor therapies and vaccines (Bagheri et al. 2018). Au NPs conjugated with carbohydrates and proteins had studied for vaccine development. The carbohydrate-based antigens conjugated Au NPs that are present in tumor cells such as sialyl-Tn and Lewis -Y (Ojeda et al. 2007), Tn (Parry et al. 2013) and Thomsen-Friedenreich (Svarovsky et al. 2005) have been investigated for the stimulation of immune response and showed a higher response than unconjugated/free carbohydrates. The conjugation of the antibodies with NP can contribute towards the passive immunization. On the other hand, the conjugation with the antigens can significantly stimulate the immunological response and thus active involvement of the host immune system for ultimate protection against the same antigen (Bagheri et al. 2018; Ahiwale et al. 2017). Thus, the studies carried by the researchers and future work in this field will produce a promising tool against diseases.

5.5.2.2 Bioimaging Currently, bioimaging is considered the best tool for disease diagnosis and treatment. The principle imaging techniques include positron emission tomography, singlephoton emission computed tomography, magnetic resonance imaging, computerized tomography, optical imaging and ultrasound imaging. The researchers have employed the Au NPs in techniques like computerized tomography, magnetic resonance imaging, ultrasound and optical imaging (Dubertret et al. 2001). Computerized tomography is an imaging technique that produces the 3D image of the body, by combining X-ray images taken from different angles and processing it into cross-sectional images (slices) of a particular organ. In the case of positron emission tomography, the absorption of the radiation by tissues is detected by the X-ray sensors and the absorption difference is utilized by the computer to construct slices of the brain as tomograms (Pysz et al. 2010). Magnetic resonance imaging is a non-invasive technique used for the diagnosis of diseases and forms the images of the physiological process of the body. The image is generated due to the differences between the proton’s relaxation time within and between the samples (Padmanabhan et al. 2016). A study indicates that Au NPs coated with polyethylenime and conjugated with ligand DOTA for chelation of gadolinium for magnetic resonance imaging and computerized tomography applications. In this study, the results reveal that due to low toxicity, these synthesized hybrid particles can act as an efficient contrast agent for imaging of blood pool and a significant organ of animals (Zhou et al. 2016). Antibody conjugated Au NPs directed towards the CD4 receptor was seen to induce the specific contrast enhancement of peripheral lymph nodes (Eck

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et al. 2010). Another study conjugated the PEGylated Au NPs for the delivery of monoclonal antibodies against HER-2 receptor. The post-intravenous injection to mice confirms the preferential uptake of the particles by the tumor, and by enhanced contrast efficiency of particles, the millimeter size tumors were detected by microCT imaging (Hainfeld et al. 2011). Another study investigated the conjugation of the anti-epidermal growth factor receptor antibody with Au NPs against human squamous cell carcinoma of head and neck cancer developed in nude mice. They were successful in imaging the tumor-targeted by functionalized Au NPs by CT imaging (Reuveni et al. 2011). Another study has explored the properties of rare earth materials lanthanides in in-vivo imaging and therapy. Xiaoqian Ge and co-workers have functionalized the PEGylated 10 nm Au NPs with lanthanide ions Gd3+ and Yb3+. The synthesized NPs bear low toxicity both in-vitro and in-vivo, which was validated by MTT assay and histological and serum biochemistry analysis, respectively. The results further demonstrate that these NPs can successfully be applied to MRI and CT imaging, thus providing a powerful strategy for nano-bio imaging (Ge et al. 2016). The researchers have involved the Au NPs in other imaging techniques such as ultrasound and optical imaging. Ultrasound is useful in the diagnosis of internal body structures and is essential techniques in analyzing the physiological and anatomical images. One of such study have utilized the bovine serum albumin compressed fluorescent Au NPs for creating a contract in ultrasound and near infrared fluorescent imaging (Xie et al. 2009). Besides having the great potential to act as contrast agents in imaging techniques, these functionalized Au NPs possess a great potential to stain nucleus of cells. For instance, Venkatesh and colleagues synthesized the green fluorescent Au NPs utilizing 8 mercapto-9propyladenine. Post characterization, the uptake of the particles and biocompatibility was tested by MTT assay using four different cell lines HeLa, A498, Schwann and L929. Further on, the cellular uptake of the NPs was validated by confocal microscopy (Venkatesh et al. 2014). The above-mentioned imaging techniques can prove an important imaging tool for the early detection of diseases and can facilitate in-vivo the expression and behavior of disease markers especially tumor markers.

5.5.2.3 Colorimetric Assays Due to aformentioned characteristics of Au NPs helps them to play a considerable role in the detection of biomolecules and ions. The interaction of the Au NPs with the analytes renders a color change in the dispersion that can be observed visibly. The different types of analytes such as proteins, nucleic acids and ions can be detected by means of the Au NPs. The NPs upon interaction with analytes tend to aggregate into nanoclusters that result in color of NPs. The technique called the sol-particle immunoassay is based on the agglutination of the NP functionalized with monoclonal antibodies (Dykman et al. 2005). Several biomolecules were analyzed using this approach. For example, the conjugation of the human chorionic gonadotrophin hormone antibody with Au NPs was investigated for the presence of human chorionic gonadotrophin hormone in urine. The antigen-antibody reaction here leads to agglutination, thus renders the change in the spectrum, which results in the color change of the solution from red to blue/purple (Leuvering et al. 1980). This method

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has also been applied to other analytes such as anti-rubella antibody (Wielaard et al. 1987), estrogen in urine (Leuvering et al. 1983) and selenocysteine protein P in serum (Tanaka et al. 2016). The color change reaction can be monitored and/or examined by both UV/Vis spectrophotometer. The Au NPs can rapidly detect the pollutants, especially the metal ion contamination in water. The ion-specific aptamer functionalized Au NPs have been utilized for the detection of arsenic in drinking water. The study investigated the use of Au NPs with arsenic specific aptamer and cationic polymer poly(diallyldimethylammonium) (PDDA). This polymer facilitates the aggregation of the NPs; in the absence of arsenic, the aptamer stabilizes the polymer. Thus prevents its binding and hence aggregation of the NPs. On the other hand, the presence of arsenic facilitates the binding of the aptamer with itself and allows the interaction of the polymer and Au NPs. This interaction leads to aggregation of the Au NPs and color changes from red to blue (Wu et al. 2012). Similarly, the researchers have employed the Au NPs for the detection of other metal ions such as mercury (Lee et al. 2007; Xue et al. 2008), copper (Nath et al. 2018), chromium (Zhao et al. 2012), lead (Li and Wang 2010), etc. Au NPs can also detect the presence of microorganisms in a contaminated sample. The detection of the genomic DNA of organisms in a sample can be detected by conjugation of the oligos/primers specific to the target genes with NPs (Soo et al. 2009) or used in combination with the NPs (Hussain et al. 2013). The color change can be detected by the interaction of the oligos with the target gene in case the sample is positive; otherwise, the oligos will stabilize the NPs by electrostatic interaction between the two, thus prevents the aggregation of the NPs (Hussain et al. 2013). This colorimetric detection has been employed for Mycobacterium tuberculosis (Tammam et al. 2017), Bacillus anthracis (Deng et al. 2013), Listeria monocytogenes (Fu et al. 2013), Escherichia coli (Jyoti et al. 2010), Leishmania (Niazi et al. 2013), etc. The Au NPs conjugated with antibodies against tumor-specific antigens have also been subjected to colorimetric detection (Jazayeri et al. 2018). For example, the detection of the prostatespecific antigen by conjugation of the Au NPs with the anti-prostate-specific antibody (Jazayeri et al. 2016). The other biogenic markers, such as diabetic marker HBA1c (Wangoo et al. 2010), toxins like aflatoxin (Wang et al. 2014) and enzymes like lysozyme (Chen et al. 2008) have been detected by Au NP-based colorimetric assay.

5.6

Conclusion

The Au NPs, can be used in targeted delivery of biomolecules, especially nucleic acids and drugs, emerged a few years ago. The advancements in synthetic chemistry and surface functionalization have provided diversity in the applications of Au NPs. As a result of selective functionalization different biomolecules through reactive groups, these NPs play a significant role in biomedicine, especially in the targeted delivery and therapeutics. The unique optoelectrical and physio-chemical properties of these NPs have provided the possibilities and explored the ways for better sensing, imaging, enhanced delivery of drugs, genes and efficient therapeutic methods. The

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Au being inert still has some drawbacks that can affect the healthy biological environment of an individual. The limitations such as clearance rate from circulation, the appropriate dosage must be standardized before application of the Au NPs for human use. Acknowledgments We thank the Science and Engineering Research Board (SERB) research grants (ECR/2016/001429) for providing lab funds and supporting the fellow.

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Nanotechnology and Diabetes Management: Recent Advances and Future Perspectives Rumaisa Rashid, Amreen Naqash, Ghulam Nabi Bader, and Faheem A. Sheikh

6.1

Introduction

A group of metabolic disorders in which the body is unable to respond and/or produce adequate levels of the hormone “insulin,” which is characterized by hyperglycemia (i.e., a high blood sugar level over a prolonged period), is being defined as diabetes mellitus (A. D. Association 2010). It is a relatively common disease in which the body fails to produce enough insulin or produces no insulin (Type 1 diabetes), or cells do not respond properly to the insulin (Type 2 diabetes), hence prevents the body from properly utilizing energy from the food. Common symptoms include polyuria (i.e., recurrent urination), polydipsia (i.e., increased thirst) and polyphagia (i.e., increased hunger) (Alanazi et al. 2017). Among the types of Type 2 diabetes accounts for 90% of worldwide occurrence. The rationale to have a predominance of this metabolic disorder is a perhaps hidden behind sedentary lifestyle, lack of physical activity, obesity and genetic predisposition (Ismail-Beigi 2012). Furthermore, the Type 2 diabetes is associated with resistance towards insulin in peripheral tissues (a condition when cells do not respond to insulin produced by β-cells leading to conditions called hyperglycemia) (Donath and Shoelson 2011). Additionally, the insulin deficiency and impaired glucose homeostasis initiate an exhausting process which leads to β-cell dysfunction resulting in increased morbidity, mortality and ultimately reduces treatment efficacy (Meier and Nauck 2008). Globally, it is presumed that several patients affected by diabetes are expected to increase from 280 million to 400 million by 2030. The global burden of disease report of 2015 has revealed a 30.6% increase in the incidence of diabetes (333 million R. Rashid · A. Naqash · G. N. Bader Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu and Kashmir, India F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_6

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in 2005 to ~435 million in 2015) (Vos et al. 2016). The conventional drugs such as sulphonylureas (tolbutamide and glipizide), meglitinide (repaglinide and nateglinide), biguanide (metformin), thiazolidinediones (TZD) and α-glucosidase inhibitors (acarbose) are used to treat Type 2 diabetes. Woefully, these treatment modalities have life-threatening side-effects owing to their safety concerns, intolerability, weight gain, edema and gastrointestinal intolerance (Drucker et al. 2010). However, insulin replacement is the only therapy prescribed for Type 1 diabetes. The treatment aimed to mimic natural fluctuations in insulin levels throughout the day (Berenson et al. 2011). Needless to say, syringes remain the most common vehicle for insulin administration even though it has many disadvantages (pain at the site of injection, non-compliance by the patient, cost constraints, and dosage errors). Experts in this area are trying to figure out other ways to deliver insulin protein (Babu et al. 2008). Typical treatment includes long-acting insulin (i.e., long plasma half-life than regular insulin) this provides a basal level of insulin supplemented with bolus injections of fast-acting insulin (with a shorter plasma half-life) at mealtimes (Berenson et al. 2011). Due to the unfavorable acidic environment in the gastrointestinal tract, insulin and other antidiabetic therapies (e.g., glucagon-like peptide 1) are not administered orally. Unfortunately, these antidiabetic peptides have to be injected subcutaneously, which is highly expensive, painful and time-consuming, thus inconvenient to the patient, leading to poor patient compliance (Heinemann 2011). Moreover, several methods, including recently devised pumps, continuous glucose monitors, and dermo-jets, have been developed to enhance patient compliance and reduce the economic burden associated with treatment (Arya et al. 2008). One of the recent examples is the fabrication of a dual hormone system (i.e., insulin and glucagon), a bionic pancreas and glycaemic control system, which has been evaluated in a phase II trial in patients with Type 1 diabetes. This control system has shown significant improvement in balancing hyperglycemia and has also decreased the frequency of hypoglycaemic episodes to occur in the patients, which is a common side-effect in most of the antidiabetic treatment systems (Russell et al. 2014). Various strategies proposed for non-invasive monitoring of blood sugar levels in the past two decades using approaches are based on nanotechnology. This has improvised both therapeutics as well as diagnostics methodologies in several fields of medicine, including cardiology and oncology (Weissleder and Pittet 2008). In biomedical sciences, nanotechnology may be defined as a cross-disciplinary area for monitoring, imaging, repairing, targeted therapy and control of human biological systems at the cellular level with the help of utilizing materials and structures engineered at the molecular-scale (Kralj and Pavelic 2003). It works on atomic, molecular, supra-molecular levels to understand, produce and use material structures, devices and systems with totally new properties and special functions as a result of their small structure (Roco et al. 2000). Nanotechnology, when applied to medicine, it is referred to as nanomedicine a discipline, where there are promises of revolutionary achievements to fight against infectious disease and control system (Logothetidis 2006). Nanotechnology has a significant impact on society and is anticipated as a human affair designed in the services of social problems (Schiemann

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2006). Various limitations of conventionally available drug delivery systems include lack of target specificity, altered effects and diminished activity (due to metabolism in the body), and cytotoxicity (due to some anticancer drugs). Biocompatible nanoparticles with optimized physical, chemical, and biological properties can overcome these limitations and serve as an effective drug delivery system. Recent advances in nanotechnology, molecular imaging, and biomedical imaging tools have created new possibilities for early diagnosis, monitoring of disease progression, and treatment for patients with diabetes (Naesens and Sarwal 2010) (Fig. 6.1).

6.2

Recent Advances in the Treatment of Diabetes

Various types of biomedical nanoparticles are currently available and studied for insulin delivery in the treatment of diabetes mellitus (Yih and Al-Fandi 2006; Attivi et al. 2005) (Fig. 6.2). These include: • • • • •

Polymeric biodegradable nanoparticles (nanospheres and nanocapsules) Polymeric micelles Ceramic nanoparticles Dendrimers Liposomes.

6.2.1

Polymeric Nanoparticles

These are particulate dispersions or solid, colloidal particles consisting of macromolecular substances that vary in size from 10 nm to 1000 nm (Brigger et al. 2012). Polymeric nanoparticles are considered to be effective and efficient over traditional oral and intravenous methods of drug administration. These nanoparticles are biodegradable polymers surrounded by a nanoporous membrane and are used as carriers for insulin. The pH changes in the body swell the polymer system, resulting in insulin release. Copolymers such as N, N-dimethylaminoethyl methacrylate, polyurethanes, polyanhydrides, polyacrylic acids and polyacrylamide are being investigated for the above applications (Harsoliya 2012; Cui et al. 2009). Depending on the methods of preparation, these nanoparticles can be nanosphere (Fig. 6.3a and b) or nanocapsule (Fig. 6.2) (Tsapis et al. 2002; Si et al. 2003). These nanostructures have completely different properties and release the encapsulated drug by physicochemical mechanisms. A nanosphere is a matrix device where the drug is physically and uniformly dispersed whereas, nanocapsule is a vesicular system where the drug is confined to a cavity or vesicle surrounded with the unique polymeric membrane (Sahoo and Labhasetwar 2003). These particles degrade in the body into biologically acceptable compounds by hydrolysis, thus delivering the encapsulated medication to the target site.

Fig. 6.1 Nanotechnology-based approaches to deal with challenges in the diagnosis and treatment of diabetes, obtained with permission from (Veiseh et al. 2015) (a) The loss of β-cells in the pancreas results in the progression of diabetes into three stages: primary, secondary and tertiary. During each stage, new types

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of therapies are necessary to prevent advancement to the next step. (b) The disease progression due to loss in β-cell mass can be controlled and treated with potential nanotechnology-based interventions which include: nanoparticle-based contrast agents to improve early diagnosis of the onset of Type 1 diabetes; nanoparticle-based glucose sensors for frequent monitoring of blood glucose levels; nanoparticles to improve the pharmacodynamics of insulin; and protection of transplanted pancreatic islet cells with the help of nanotechnology

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Fig. 6.2 Nanoparticles are the particles between 1 and 100 nm in size with a therapeutic agent either dispersed in the polymer matrix or encapsulated in a polymer. Polymeric micelles are nanoscopic shell structures, which in aqueous solution arrange to form an outer hydrophilic layer and an inner hydrophobic core. Liposomes are the spherical vesicular lipid structures that can be made ‘stealth’ by PEGylation and further conjugated to antibodies for targeting. Dendrimers are highly branched macromolecules built around a small molecule with an internal cavity surrounded

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Fig. 6.3 (a) Electron microscopic image of a hollow nanosphere obtained by spray drying of hydroxyl propyl cellulose (b) a magnified view of the particle surface. Obtained with permission from (Tsapis et al. 2002)

Fig. 6.4 A diagrammatic representation of calcium phosphate (CAP)-PEG-insulin-casein (CAPIC) oral insulin delivery system. Obtained with permission from (Morçöl et al. 2004)

6.2.2

Oral Delivery of Insulin Through Polymeric Nanoparticles

Enzymes present in the gastrointestinal tract are barrier for the orally administered insulin, as they degrade insulin in the stomach (Morishita et al. 1992). Therefore, insulin has to be enveloped in a matrix-like system to protect it from degradation. This can be achieved by encapsulating the insulin molecules in polymeric nanoparticles, e.g., a combination of calcium phosphate-polyethylene glycol-insulin with casein (a milk protein) (Morçöl et al. 2004). The casein coating protects the insulin from the gastric enzymes (Fig. 6.4). ä Fig. 6.2 (continued) by a large number of reactive end groups. Quantum dots are semiconductor nanocrystals that can be conjugated to a ligand and thus can be used for imaging purposes. Ferrofluids are a colloidal mixture of iron oxide magnetic nanoparticles surrounded by a polymeric layer, that attract the drug or ligand. Obtained with permission from (Sahoo and Labhasetwar 2003)

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Fig. 6.5 Insulin delivery system using chitosan nanoparticles. Obtained with permission from (Di et al. 2014)

Insulin is a hydrophilic drug and, as such, cannot diffuse across epithelial cells, which favor the absorption of lipoidal drugs. Chitosan nanoparticles have been used as permeation enhancers for the incorporation of insulin (Arya et al. 2008; Harsoliya 2012). As reported by Cui et al., in 2008, encapsulation of insulin in the shell of carboxylated chitosan grafted poly(methyl methacrylate) nanoparticles have improved the efficiency of insulin delivery (Cui et al. 2009) (Fig. 6.5).

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Inhalation Delivery of Insulin Through Polymeric Nanoparticles

Chitosan nanoparticles are one of the most promising delivery systems for the treatment of Type 2 diabetes. Chitosan is a biodegradable and biocompatible polymer. It is a copolymer of β (1-4)-glucosamine and N-acetyl-D-glucosamine derived by partial deacetylation of chitin from crustacean shells (Ilium 1998). Inhalable, polymeric nanoparticle-based drug delivery systems have been used earlier for the treatment of tuberculosis (Paul and Sharma 2008). Such approaches have been tried for insulin delivery through inhalable nanoparticles. Different inhalation systems are used to deliver inhaled products such as dry powder formulations and drug solutions. Inhalations, as compared to the oral route, offer potential advantages of the mild environment such as low level of metabolizing enzyme concentrations and neutral pH. The dry powder formulation of insulin can be inhaled by encapsulating the insulin in the nanoparticles. A nanoparticle should be small enough to avoid blocking up the lungs, but at the same time should also be large enough to avoid being exhaled in the air. Such a property of drug will allow direct delivery of insulin molecules into the bloodstream without undergoing any degradation. Thus, the inhalation method not only prevents insulin degradation, however, also allows immediate delivery of insulin molecules into the bloodstream for instant action. Studies by Paul et al., have revealed that guinea pig lungs with insulin loaded poly(lactic-co-glycolic acid) nanospheres (nanoparticles) presented a significant reduction in blood glucose levels with long duration of action (48 h) compared to insulin solution (Paul and Sharma 2001). Similarly, the study by Kawashima et al. has shown that insulin-loaded poly (butyl cyanoacrylate) nanoparticles, when delivered to the lungs of rats, show the extended duration of hypoglycemic effect (>20 h) compared to pulmonary administration of insulin solution (Kawashima et al. 1999). However, inhalation methods are not preferred because these require regular lung function tests, which are cumbersome and relatively expensive (Woldu and Lenjisa 2017).

6.2.4

Polymeric Micelles

Polymer-based macromolecular approaches are among various nano-carriers that have resulted in improved drug delivery for diseases such as diabetes, autoimmune disorders and cancers, etc. Polymeric micelles are nanoscopic core or shell structures that consist of hydrophilic exterior and hydrophobic core. They have established a record of anticancer drug delivery from the laboratory to commercial reality. Micelles are formed by amphiphilic copolymers, which self-assembled to nanosized aggregates above the critical micellar concentration. The hydrophilic moiety forms the corona in the shell of micelles, whereas the hydrophobic moiety forms the core of micelles. Micelles possess a dynamic structure where the unimers of the amphiphilic copolymer are interchangeable. The micelle complex method involves introducing a cross-linkable hydrophilic group into the hydrophobic polymer to form a stable

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micelle system (Plapied et al. 2011). For example, the multifunctional, poly(ethylene glycol) is conjugated with biodegradable hydrophobic polymers to form an amphiphilic block copolymer in which polyethylene glycol branches create a crosslinkable structure in the micelle system. The micelles responding to glucose are formed by the complexation of a phenylboronic acid-containing block copolymer, e.g., poly(ethylene glycol)-b-poly (aspartic acid-co-aspartamido phenylboronic acid) with a glycopolymer, e.g., poly (aspartic acid-co-aspart-glucosamine) (Yang et al. 2013). The sensitiveness to glucose (fast response to glucose change at the physiological pH) and stability against aggregation are the advantages of micelle complex. Other than polymeric and ceramic nanoparticles, there are also gold nanoparticles that have gained tremendous attention as a model drug delivery platform. They have also been tested as insulin carriers. Gold nanoparticles synthesized with chitosan as a reducing agent have been used as a carrier for insulin (Tao and Desai 2003). These nanoparticles have shown functional insulin loading capacity and long term stability in terms of aggregation. Similarly, the dextran nanoparticles-vitamin B12 combination has also been successfully tested to overcome vitamin B12 degradation in the gastrointestinal tract (Bhumkar et al. 2007). These nanoparticles have been found to protect the entrapped insulin against gut proteases. This vitamin B12-coated dextran has shown a release profile that is suitable for oral delivery systems of insulin.

6.2.5

Ceramic Nanoparticles

Ceramic nanoparticles, also known as nanoceramic, are composed of ceramics. A few studies have been conducted to test the potential use of ceramic nanoparticles as a drug delivery system (Douglas et al. 1987; Cherian et al. 2000). They have a wide range of applications owing to several beneficial properties, such as high heat resistance and chemical inertness. The development of new ceramic materials for many biomedical applications has grown at a tremendous pace. These materials are made up of oxides, carbides, phosphates and carbonates of metals and metalloids such as calcium, titanium, silicon, etc. (e.g., calcium phosphate). Nanoscale ceramics such as hydroxyapatite, zirconia, silica, titanium oxide, and alumina are made from new synthetic techniques to improve their physico-chemical properties so that their cytotoxicity in biological systems is reduced. The advantages of ceramic nanoparticles include easier preparative processes (of desired size, shape and porosity), high biocompatibility, ultra-low size (usually less than 50 nm), and excellent dimensional stability (Sarmento et al. 2007). Ceramic nanoparticles do not undergo swelling or porosity changes caused by changes in pH. Thus, these particles effectively protect the drug molecules against denaturation that is caused by changes in external pH and temperature. Ceramic nanoparticles have been extensively studied as particulate carriers in the pharmaceutical and medical fields. They are excellent platforms for drug delivery systems because of their controlled and sustained release properties. These nanoparticles serve as a potential tool in controlling drug delivery due to their high stability, different routes of administration (oral, inhalation, etc.),

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high load capacity, easy incorporation into hydrophobic and hydrophilic systems. Furthermore, their surfaces can easily be modified with different functional groups and can be conjugated with a variety of ligands or monoclonal antibodies to make them target specific (Roy et al. 2003). Ceramic based nanoparticle entrapping water-insoluble photosensitizing anticancer drugs have shown properties of a novel drug delivery system for photodynamic therapy (Vollath et al. 1997). In a recent study, it has been shown that tricalcium phosphate nanoparticles can be used for oral delivery of insulin (Corkery 2000). Self-assembled carbohydrate-stabilized ceramic nanoparticles have been tested for the parenteral delivery of insulin (Douglas et al. 1987; Cherian et al. 2000). In this method, calcium phosphate nanoparticle core is used as the insulin carrier and studied in vivo. The in vivo action of this drug delivery system has shown better results as compared to the efficacy of standard porcine insulin solution.

6.2.6

Dendrimers

Dendrimers are discrete nanoparticles with “onion skin-like” branched layers (Kesharwani et al. 2014; Kannan et al. 2014). They are highly branched, starshaped, three-dimensional globular macromolecules having a diameter on a nanometer-scale ~2–10 nm (Fig. 6.6). They are spherical in shape and highly lipophilic and can penetrate cell walls easily, which makes them an ideal drug delivery carriers (Tolia and Choi 2008). A macromolecular drug delivery moiety is used as the vehicle into which a drug is attached and is carried to a specific location. Dendrimers are composed of multiple, highly branched monomers that emerge radially from the central core. Their branched structure offers various advantages such as mono dispersion, controllable size, water-solubility, modifiable surface, multivalency, and an available internal cavity for drug delivery (Cho et al. 2008). The resultant spherical macromolecular structure has a format similar to albumin and hemoglobin, although it is smaller than multimers such as IgM antibody complex (Dykes 2001). Their characteristic architecture and flexibility in the modification of structure allow them to be used as biocompatible dendrimers for targeted drug delivery. Studies have been conducted on the use of biocompatible dendrimers for cancer treatment (cisplatin and doxorubicin) (Tekade et al. 2008). Karolczak et al., in his study, have reported that dendrimer, polyamidoamine generation 4.0 dendrimer reduces blood hyperglycemia and restores impaired bloodbrain barrier permeability in streptozotocin-induced diabetes in rats (Karolczak et al. 2012). Pre-clinical studies have also shown that dendrimers act as a potential therapeutic tool in the prevention or alleviation of diabetes-associated metabolic complications. Moreover, dendrimer based formulations are also available that target retinal microglia and macrophages by systemic and intravitreal injection. This provides new insights for developing dendrimer drug formulations as treatment options for retinal diseases associated with microglia or macrophage activation such as diabetic retinopathy and retinal degenerations etc. (Selvaraj et al. 2017). Dendrimers have several advantages, such as the encapsulation of a poorly water-

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Fig. 6.6 A schematic representation of dendrimers. Obtained with permission from (Kalomiraki et al. 2016)

soluble drug into the internal cavities, small size and targeting efficiency, etc. However, the disadvantage of dendrimers as anti-glycation agents is their uncontrolled toxicity, poorly established pharmacokinetics in living organisms and their high cost of production. These factors limit their application in chronic diseases (e.g., diabetes).

6.2.7

Artificial Pancreas

The idea of an artificial pancreas was first given and described by Albisser et al. (1974). These are also known as closed-loop control of blood glucose, is a device that controls/mimics the blood glucose level automatically by providing the substitute for endocrine functionality of a healthy pancreas. Artificial pancreas comprises a continuous glucose monitor, glucose meter and an insulin infusion pump for calibrating the monitor. It can serve as a permanent solution for diabetic patients. Another approach for artificial pancreas is the usage of tiny silicon box which contains pancreatic beta cells taken from the animal source. A material surrounds the small box with a specific nanopore size diameter of about 20 nm (Fig. 6.7). This approach offers the advantage of protecting transplanted cells from the immune system and allows sufficient diffusion of glucose, insulin, oxygen and other

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Fig. 6.7 Artificial Pancreas containing: (1) Glucose monitoring sensors (2) Receiver (3) Control device (4) Insulin pump to deliver insulin and to maintain standard glycemic control. Obtained with permission from (https://medicalfuturist.com/living-with-an-artificial-pancreas/)

necessary nutrients (Subramani et al. 2012). The pores in this device are big enough to allow glucose and insulin to pass through them, but small enough to hinder the passage of much larger immune-system molecules. These boxes can be implanted under the skin of diabetic patients. Though this can temporarily restore the delicate glucose control feedback loop of a body without any potent immunosuppressant, however, at the same time, this can lead to higher chances of infection. Scientists are also trying to create a new device called “nano-robot,” which would have insulin stored in the inner chambers, and glucose sensors on the surface to monitor/sense glucose levels. In the event of an increase in blood glucose level, the sensors on the surface will record it, and insulin will be released accordingly as per the signals. Yet, this kind of nano-robot is still only a theory (Arya et al. 2008; Martinac and Metelko 2005; Carino and Mathiowitz 1999). However, to deliver insulin experimentally, a smart insulin patch has been developed. “Smart” as it is called because it releases insulin as per the body’s need. It contains more than 100 micro-needles packed with insulin and glucose-sensing enzymes to monitor glucose levels (Fig. 6.8).

6.2.8

Nano Pumps

Nano pumps are powerful devices that have many possible applications in the field of medicine. Debiotech introduced for first-time insulin delivery through a nano pump. The pump injects insulin into the patient’s body at a constant rate resulting in

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Fig. 6.8 Smart insulin patch. Obtained with permission from (http://www.innovationtoronto.com/ 2015/06/smart-insulin-patch-could-replace-painful-injections-for-diabetes/ 2018)

a balanced amount of sugar in the blood. The pump can also administer small doses of the drug over a prolonged period, i.e., sustained-release (http://thefutureofthings. com/news/1286/insulin-nanopump-for-accuratedrug-delivery.html 2018). Nanopump depends on microfluidic MEMS (Micro-Electro-Mechanical System) technology that allows a tiny pump to be mounted on a disposable skin patch to provide continuous insulin infusion in the body (Fig. 6.9). Nanopump enables significant advancements in bioavailability, treatment, efficiency, and the quality of life of diabetic patients (https://www.medgadget.com/2007/04/debiotechs_insu lin_nanopump.html 2018).

6.2.9

Smart Cells

Smart artificial beta cells provide a new treatment option for diabetic patients. In Smart-cell technology, during hyperglycemia, glucose attacks the Smart cell by eating away its insulin-containing structure. This leads to the damage in the cell membrane leading to a break down of the protein matrix to release insulin and normalize blood glucose (Hampton 2018).

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Fig. 6.9 Nano-Insulin pump. Obtained with permission from (https://www.medgadget.com/2007/ 04/debiotechs_insulin_nanopump.html 2018)

6.3

Recent Advances of Nanotechnology in the Detection of Insulin and Blood Sugar Levels

Nanotechnology has emerged as a focal point in diabetes management and can rapidly be used to detect small amounts of insulin and blood sugar levels in the body. It also helps to assess the health of insulin-producing cells in the body. The various ways to detect insulin and blood sugar levels in the collection include:

6.3.1

Microphysiometer

The microphysiometer is built from multiwalled carbon nanotubes and it measures the insulin production at various intervals. These carbon tubes are similar to several flat sheets of carbon atoms stacked and rolled into tiny tubes to give the multiwalled appearance. It is used to detect and monitor the cell response to a variety of chemical substances, especially ligands for specific plasma membrane receptors (McConnell et al. 1992). The nanotubes have high electrical conductivity and the concentration of insulin in the chamber can be directly related to the current present at the electrode. It operates at a neutral pH which is characteristic of the living cells. The current detection method helps to measure insulin production at intervals by systematically collecting small samples and measuring their insulin levels. The sensor detects insulin levels continuously by measuring the transfer of

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Fig. 6.10 A schematic representation Microphysiometer with insulin detectors. Obtained with permission from (https://www.nextbigfuture.com/2008/04/microphysiometer-using-multiwall-car bon.html).

electrons, which are produced when insulin molecules oxidize in the presence of glucose. When the cells produce more insulin, the current in the sensor increases and vice versa, thus allowing the monitoring of insulin concentrations in real-time (Fig. 6.10).

6.3.2

Implantable Sensor

Implantable sensors have been used in medical research for measuring parameters such as pressure, force, torque, and temperature inside the human body. An implantable sensor has been developed for patients with diabetes, that is capable of longterm monitoring of tissue glucose concentrations by wireless telemetry (Gough et al. 2010). The use of poly(ethylene glycol) beads coated with fluorescent molecules to monitor blood sugar levels in diabetes is very useful using this method. The poly (ethylene glycol) beads are injected under the skin and stay in the interstitial fluid. When glucose in the interstitial fluid drops to dangerous levels, fluorescent molecules get displaced, and a glow is created. This glow is seen on a tattoo placed on the arm. This technique is designed to give diabetic patients an alternative to short-term glucose sensors or finger-sticking, and it also limits dangerous fluctuations in glucose level “glucose excursions” (Erin 2010). Sensor microchips are also being developed to continuously monitor key body parameters like blood

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glucose, temperature and pulse. In this method, the chip is implanted under the skin and it transmits a signal that could be monitored continuously (Arya et al. 2008).

6.4

Conclusion

The impact of nanotechnology on diabetes is increasing at a tremendous pace. Nanotechnology is useful in the detection of insulin and blood glucose levels (with the help of microphysiometer and implantable sensors) in the management of diabetes. It has facilitated significantly in advances for both self-regulated insulin delivery systems and glucose sensors and has proven beneficial in diabetes mellitus management and has also revolutionized insulin delivery through enhanced oral formulations and islet encapsulation. Insulin delivery through inhalable nanoparticles and developments of oral insulin, microspheres, artificial pancreas and nano pumps have improved the treatment options and quality of life of a person with diabetes. Applications of nanotechnology soon are going to be clinically very important, especially in the field of pharmaceutical drug designing and development. In conclusion, the next-generation nanoparticle-mediated insulin will improve the quality of life and life expectancy of diabetic patients. Acknowledgments The Department of Science and Technology, Government of India, Nano Mission, under Grant SR/NM/NB-1038/2016, supported this work.

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Recent Advances in the Emergence of Nanorobotics in Medicine Taha Umair Wani, Syed Naiem Raza, Nisar Ahmad Khan, and Faheem A. Sheikh

7.1

Introduction

Currently, the conventional route of drug delivery has some pitfalls which restrict their use in desired ways. The drug from such a dosage form (i.e., the traditional course of drug delivery) is released way before it reaches the actual site of action. This leads to unnecessary exposure of the drugs to other organs leading to side effects and toxicities. Ideally, a drug administered to a patient intended to address a particular disease is only required to act at some specific site/tissue/organ (e.g., in case of cancer, the chemotherapy needs to administrated at the tumor site only). However, due to numerous biological barriers (e.g., capillary endothelial, bloodbrain, cell membrane, blood-cerebrospinal fluid, etc.), the targeted delivery of drugs is seldom to be achieved regularly. In some instances, the target-specific delivery is acquired with the help of special techniques (e.g., intraventricular infusion and intracerebral injection for delivery of the drug to the brain) (Todo et al. 1991; Akdemir et al. 1995; Lee et al. 1995). But these approaches are quite invasive and fatal for such delicate organs such as the brain. Moreover, these barriers can be removed with the use of the nano-drug delivery system (Blanco et al. 2015). Recently, the introduction of nanotechnology has revolutionized the practices carried out while delivering essential drugs. Especially with the advent of nanomedicine that allows the site-specific drug targeting has coherently achieved. Nanocarrier systems are being extensively investigated for their possible roles for improvising the efficiency of drug delivery (Soppimath et al. 2001; Kumari et al. 2010; Rashid et al. 2018; Wani et al. 2014). Being small in size, these nanocarrier T. U. Wani · S. N. Raza · N. A. Khan Department of Pharmaceutical Sciences, School of Applied Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_7

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systems or nanoparticles can quickly enter cells, which is advantageous in specific cell-targeted therapy (Shang et al. 2014; Banerjee et al. 2016). By exploiting the nanomedicine, a drug can be delivered to the desired site of action without effecting the other organs of the body that are being exposed to the drug during delivery (Sahoo et al. 2017). This dramatically helps to reduce the toxicity and unnecessary wastage of medicines. This is particularly beneficial in the case of narrow therapeutic window drugs whose clinical use is obstructed due to their high dose-dependent toxicities (Karve et al. 2012). Besides, targeted delivery of drugs to cell organelles, e.g., mitochondria can also be achieved through nanoparticles (Durazo and Kompella 2012). Nanomedicine has improved our ability to detect the earlier indications of disease and diagnose its genetic susceptibility earlier and more efficiently (Baetke et al. 2015; Brigger et al. 2012; Fathi Karkan et al. 2017). Nanomedicine involves the use of nano-sized lipid and/or polymer-based carrier systems for the incorporation of expensive drugs. Other materials such as natural starch or inorganic materials, e.g., zinc, gold, iron, etc. are also used for this (Bobo et al. 2016). It is worth to mention that billions of dollars are being invested in the development of nano-drug delivery systems. In this course, a large number of nanocarriers systems, e.g., liposomes, niosomes, nanoparticles, nanospheres, nanotubes, nanoshells, and nanocapsules, are perfected. Among them are the nanorobots that have developed and evaluated for their potential applications for treating a vast number of diseases. The benefits of nanotechnology in medicine are tremendous compared to conventional drugs. Today, several nanoparticles are clinically approved for use in humans. In the next sections, we will be highlight some of the important application of nanotechnology in general and nanorobotics in particular.

7.1.1

Solubilization

It is a well-known fact that unless a drug is present in the solubilized form at the site of action, it would not be able to show its activity (Lipinski 2001). Intriguingly, a lot of drugs, especially the medications used to cure the cancers, are hydrophobic and water-insoluble in nature. To increase drug solubilization and give them to patient’s other organic solvents have frequently been used to solubilize these drugs. However, the biggest problem associated with these solvents is that these are the most toxic. Furthermore, the side effects experienced by patients are not due to the actual drug but are due to the toxicity from solvents used to solubilize the drugs. An alternative way to increase the solubility of drugs is to load these non-water-soluble drugs into nanoparticles (Kipp 2004). For example, one such formulation using nanoparticles is clinically approved anticancer drug (i.e., paclitaxel), which is commercially available in the US under the name of Abraxane. This formulation is a nanoparticle-based albumin-bound paclitaxel drug used for the treatment of several cancers such as breast, pancreatic and lung cancers.

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Protection from Degradation

Protection of drugs from the harmful enzymes present in our body (e.g., in the gastrointestinal tract and bloodstream) can be accomplished by encapsulating them in nanoparticles. Generally, a significant portion of the drug, when administrated orally, breaks down and/or gets rapidly degraded in the acidic environment of the stomach. However, the use of nanoparticles provides a shield for the drugs in such conditions. It is noteworthy to mention that protein, peptide, DNA/RNA-based therapeutics, and vaccines are highly vulnerable to enzymes present in the blood or gastrointestinal tract. Such drugs can be loaded into nanoparticles to protect them from degradation (des et al. 2013; Marasini et al. 2014; Zhao et al. 2010; He et al. 2013). Patisiran is an example of siRNA (small interfering RNA) loaded lipid-based nanoparticle currently under Phase III clinical trials for the treatment of ATTR amyloidosis. siRNA represents a new class of drugs that work by interfering with RNA and silencing of genes. For instance, in the case of patisiran, the siRNA binds to the mRNA associated with the genes responsible for hereditary ATTR amyloidosis and terminates its expression (Adams et al. 2017; Garber 2015).

7.1.3

Targeting

Another essential advantage of the large surface area of nanocarrier systems than the actual drug molecules that allows exploring nanomedicine for further applications is the surface functionalization. Nanoparticle surfaces can be functionalized by targeting ligands that can recognize and bind to the overexpressed biomolecules or membrane receptors in diseased tissue or organ and preferentially accumulate there and release the drug in high concentrations. This is very beneficial in the case of cancer chemotherapy, where the drugs used for the treatment are so toxic that they not only harm tumor cells but can also destroy healthy cells (e.g., hair follicles which is very common in cancer patients receiving cancer drugs). Hence the targeted delivery of nanoparticles aims to reduce toxicity and increase therapeutic efficacy. A large number of drugs are being investigated for targeted delivery to several diseases, but to date, no drug has been clinically approved (Veiseh et al. 2010; Singh and Lillard 2009; Hans and Lowman 2002; Cho et al. 2008; Masood 2016; Prabaharan 2015; Jain and Stylianopoulos 2010).

7.1.4

Immune Evasion

Immune evasion is the phenomenon wherein a tumor avoids recognition by the host immune system. Nanotechnology inspires this natural strategy where the vital drug, when loaded in nanoparticles, remains escaped by phagocytic cells in the bloodstream. This can be achieved by surface modification of the nanoparticles by longchain polymers (e.g., poly(ethylene glycol) (Petros and DeSimone 2010; Moghimi and Szebeni 2003). The flexible and hydrated polymer chains are seen by the

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immune system as a hydration layer and are thus unable to recognize them as foreign particles. This increases the circulation time of the drug and hence the efficacy of the drug is also improved. Antineoplastic doxorubicin-loaded with poly(ethylene glycol) and coated liposomes (known by the trade name Doxil) has already been approved by US-FDA and is available for the treatment of HIV-related Kaposi’s sarcoma, multiple myeloma and ovarian cancer (Barenholz 2012, 2016). The PEGylated liposomal doxorubicin preferably accumulates in the skin that is beneficial in the treatment of Kaposi’s sarcoma. Moreover, it had been concluded that Doxil is less cardiotoxic than the pristine drug.

7.1.5

Imaging of the Nanoparticles

Fluorescent substances can be incorporated in the nanoparticles and can retain inside for longer periods. This can help in the location of disease sites such as in case of atherosclerosis, ischemia, tumors, inflammation. In addition to fluorescent dyes, a large number of imaging agents are being investigated (e.g., dendrimers, carbon nanoparticles, gold-coated nanoshells, quantum dots) for diagnosing the diseases. Recently, the Cornell dots, which are also known as C-dots developed by Hooi et al. at Cornell University USA, are designed to locate cancer cells in the body. These are silica-based imaging nanoparticles which mainly contain organic dye and are under clinical trials (Ow et al. 2005). It is noteworthy to highlight that inorganic nanoparticles or semiconductor nanoparticles (e.g., quantum dots) are more advantageous in producing high contrast images than the conventional dyes.

7.1.6

Multidrug Loading

Nowadays, nano-drug carriers are used for combinatorial drug delivery to improve the efficacy compared to the conventional method of delivering multiple drugs together. This approach of combinatorial drug therapy is particularly beneficial in the case of delivery of drugs with different physicochemical properties (e.g., hydrophilic and hydrophobic drugs) can be incorporated in the same nanoparticle. On the one side, the inner core of nanoparticle may encase the hydrophobic drug. On the other side, the outer shell of the nanoparticle may lay bare hydrophilic drug. Using the multi drug-loaded nanoparticles, resistant cancers can be cured (Shapira et al. 2011; Ma et al. 2009; Wang et al. 2017). In this regard, several nanoparticle formulations containing multiple drugs are under clinical trials (Stathopoulos et al. 2010; Shi et al. 2017). In addition to drugs, the permeation agents can also be incorporated into the nanoparticles that can breakdown the extracellular matrix and hence increase the permeability and diffusion of nanoparticles and/or drugs into the tissues (Liu et al. 2016; Kou et al. 2017).

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7.2

Some Unique Properties of Nanoparticles

7.2.1

Unique Electromagnetic Properties

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Nanoparticles show some unique optomagnetic, electronic and thermal properties that molecular or microscopic particles cannot explain, the reason being the large surface area to volume ratio and similar size range of nanoparticles as the wavelength of light. A clinically approved example of the same is the nanotherm for the treatment of glioblastoma. The principle of the working of nanotherm is based on the accumulation of magnetic nanoparticles into the tumors and applying alternating magnetic field increased the temperature of the nanoparticles is sufficient to damage the tumor cells (Bobo et al. 2016; Maier-Hauff et al. 2011; Marchal et al. 2015).

7.2.2

Distinct Processing by the Bodies Defense System

Nanoparticles are treated differently by diseased and healthy tissues. The tumor tissues differ in characteristics from the healthy tissues concerning their vasculature, pH, being hypoxic, etc. In this way, the distinct properties of diseased tissues can be utilized to attain high accumulation of nanoparticles there and low accumulation in healthy tissues. For example, the distinctive low blood flow and underdeveloped fenestrations in the tumor tissues are utilized for the deposition of nanoparticles. In healthy tissues, nanoparticles, after adhering to the walls of blood vessels, are immediately dislodged by the high blood flow, while as, the reduced blood flow in the tumor blood vessels is not enough to do so. Therefore, resulting in the high accumulation of nanoparticles at the tumor sites. A summary of the applications of nano-drug delivery systems is given in Fig. 7.1.

Fig. 7.1 A summary of the application of nano-drug delivery devices

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Nanorobots as Small and Efficient

These days, biomedical research is focused on constructing nano-robots that can be moved forward and backward with an external control system allowing to pour the drugs at a specific site, to cut any desired part of tissue (for biopsies), and to capture the pictures of complex and unreached areas of the stomach. The functioning of natural cellular components (e.g., proteins, nucleic acids and enzymes) is a complex, sophisticated and marvelous process. The biological world is the first place of inspiration for scientists to do manipulation at a small-scale following the principles of bionanomimetics (Ummat et al. 2005; Sharma and Mittal 2008). The bionanomimetics is biomimetics at the nanoscale level, where the inspiration for problem-solving or nanomachine designing is directly inspired by nature. In recent years, researchers have designed artificial structures, especially the nano/molecular machines driven by tiny motors that work on the principles of natural cellular processes and use energy by conversion of chemical energy (in the form of ATP) into mechanical strength (Mallouk and Sen 2009; Sengupta et al. 2012). In brief, the nanomachine components can be comprised of engines/motors transmission elements, joints, radars, ratchets, sensors, etc. In bionanomimetic, the nanodevices molecules such as proteins and nucleic acids are used as nanomachine components. Besides, molecular machines that use chemical (Kelly et al. 1999), thermal or solar (Koumura et al. 1999) energies to do work have been developed using different types of molecules (e.g., triptycene, rotaxane and catenane). Before proceeding further, it is essential to take a note on how this idea of miniaturizing started and know the basics of creating things on a small scale. Inspired by the structural and functional versatility of living cells, the first idea towards making of miniature machines was drawn by a physicist, Richard Feynman in 1959 when he talked about “there is plenty of room at the bottom” in his famous lecture (Feynman 2012). By this, he meant that there is unimaginable space for manipulating things at small-scale (as small as angstroms and beyond that). In his debate over the topic, he argued that why can’t we design tiny machines, and it is possible to exploit and control things at a miniature scale. To show how it could work, he explained the basic concept of going small and at first talked about little writing. He explained us how it was possible to write an entire encyclopedia on the head of a pin. He outlined the potential problems and solutions therein for the same. He asserted that if the pinhead were magnified to about 25,000 times, the area would equalize the field of the whole Encyclopaedia Brittanica pages. Hence, the text size, in this case, precisely requires to be reduced by 25,000 folds. The precise calculations done by Feynman are attractive as well as relevant in understanding the basics of small-scale manipulation. The calculations went the following way. De-magnifying smallest dot of halftone reproductions (of Encyclopaedia) by 25,000 times–shrinks to 80 Å (Angstrom) diameter dot. Wherein, 80 Å—is approximately equal to the diameter of 32 atoms, which is about 1000 atoms in the area covered by the dot. If such a dot can accommodate in the area of 1000 atoms, then it is easy to adjust the whole encyclopedia on the head of a pin. These calculations are quite inspiring for the futuristic implementation of the idea of manipulating objects

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Fig. 7.2 Lithographic and etching technique for manufacturing of nanochips of silicon as described by Richard Feynman

on a small scale, in continuation of this, concerning issues that he highlighted were the method of writing, the way to read that little writing, the need for better magnification microscopes. The next step was the development of miniature computers possessing tiny elements and wires inside and small structures with controllable movements. A possible way of manufacturing internal parts of such machines (e.g., chip-sets or transistors could be utilized for lithographic and etching techniques). A photoresist material can also be coated on the surface of an insulator (e.g., silicon dioxide) present as a layer over a conductor (silicon). The radiation directed on the surface of photoresist at intermittent places changes the properties of those portions which may harden or soften. Then etching would evaporate the part of photoresist material where the radiation was not incident. This is demonstrated in Fig. 7.2. Then uncovered silicon dioxide (i.e., the insulator) can be evaporated and the rest of the photoresist material can be dissolved using a chemical in two consecutive steps, which results in delicate structures of silicon dioxide over silicon layer. The silicon can then again be laid down in the gaps and the silicon over the top of the silicon dioxide is evaporated, as illustrated in Fig. 7.2. The silicon molds left in the gaps between the silicon dioxide act as excellent transistors that can be connected for proper working. Similarly, nanochips with desirable wires and condensers can also be fabricated using the same strategy. This process is demonstrated in Fig. 7.2.

7.2.4

Building Machines Atom by Atom

These days, the nanotechnologists are aiming towards the manufacturing of molecular machines, known to be assemblers, which will be working under programmed control and will be capable of assembling and/or building objects with precision at the atomic level (Drexler 1981). Such machines will be consisting of parts at a small scale, e.g., miniature engines, cables, motors, levers, etc. Furthermore, these machines would be capable of manufacturing machines at the nanoscale. This will be creating structures through atom-by-atom arrangements rather than in bulk would

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mean perfection in manufacturing. Nanomachine components built by directing the cellular machinery, e.g., DNA synthesis, to synthesize new proteins and molecules that can be assembled to construct nano-sized objects. These components may then be assembled through attractive forces between their complementary surfaces in-vitro. The naturally occurring example of this type of structure is a ribosome whose constituent proteins spontaneously join in solution to form ribosome (Traub and Nomura 1979). The argument is such when precise chemical syntheses of compounds are carried out successfully through such specific atomic rearrangements, then how does it make a difference to arrange atoms for building automated nanostructures. This will be advantageous in the sense that more accurate and perfect machines can be created since manufacturing is a matter of arranging only a few 100 or 1000 atoms. In this assembly, free sigma bonds (without steric effects) acts as rotary bearing, a series of sigma bonds as hinges, conformation changes as force for linear motion.

7.3

Challenges in Building Molecular Machines

7.3.1

Brownian Motion

Minute particles and molecules are constantly moving randomly in all directions in space, which is known for its Brownian motion. The unique movement of materials makes it challenging for such small size objects to move in one particular direction. Efforts have been made in this regard to covert such a chaotic motion into a one-directional motion, as will be discussed later in this chapter.

7.3.2

Resistance in Miniature Electric Circuits

Electric circuits of the size of nanometers have been anticipated by researchers to show considerable resistance to the flow of current. Although nanomachines are more extensive than simple atomic or molecular structures to efficiently display the effects of quantum mechanics but still small enough to avoid wholesome of those effects. It is the result of those quantum effects that pose a severe problem of resistance in miniature electrical circuits. One of the prominent effects shown by electric circuits of a small scale is the coulomb blockade, which is the blockade to the electric conductance as the electronic circuits become small.

7.3.3

Different Laws and Principles at an Atomic Level, e.g., Forces of Attraction (Viscosity and Van der Waals Force) and no Reasonable Gravity

Things on a small scale do not follow the same rules as the big objects. As gravity, weight and inertia are not significant for nano-sized objects, the laws like laws of

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kinematics, laws of gravitation are not obeyed. Still, instead, laws of quantum mechanics are followed. As we move down the scale the forces of attraction or repulsion dominate, e.g., Van der Waals forces, viscosity, electrostatic attraction or repulsion, etc.

7.3.4

Lubrication of Miniature Machines

Another seemingly important consideration about the working of miniature machines is the lubrication of their moving parts. But in reality, it may not produce a significant problem because the heat dissipated out would disappear instantaneously. Further, the effective viscosity of the lubricating fluid would be tremendous concerning the tiny machine parts.

7.3.5

Magnetic Properties

The property of a magnitism is determined by the direction of the individual magnetic domains. A magnetic area is a small part of the magnet, which is composed of particular atomic magnetic moments pointed in the same direction. Such magnetic moments of the atoms determine the amount of magnetization of a magnetic material. Since miniature machines are constructed from only a few numbers of atoms, therefore, the number of domains present would be minimal to display proper magnetism.

7.4

Manufacturing Nanostructures

7.4.1

Nanorobot Motors/Engines

A large number of molecular-level structures have been developed over recent years that have the potential to become building blocks of miniature machines. A typical nanorobot will consist of (1) body (2) motor or engine (3) a radar system. In 2005 James Tour at Rice University developed a nano car, which was a molecule consisting of an “H” shaped molecule acting as the chassis and four fullerene molecules attached to the four ends of the chassis (Shirai et al. 2005). The fullerenes are spherical soccer-ball-shaped molecules attached through single carbon-carbon single bonds to the chassis molecule. These carbon-carbon individual bonds can rotate at high temperatures due to which the fullerenes roll, and the car can move (back and forth randomly). Figure 7.3 shows the 3D and molecular structure of nano car described. In 2002 Rustem Ismagilov et al. of University Harvard demonstrated that small boats of few centimeter dimensions fitted with platinum strips moved on the surface of water and H2O2 autonomously (Ismagilov et al. 2002). The catalytic action of platinum broke H2O2 into water and oxygen. Oxygen came out in the form of

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Fig. 7.3 A nano car consisting of H shaped molecule attached at four ends to spherical soccer formed fullerene molecules acting as four wheels for the nano car developed by James Tour of Rice University (a) 3D structure of nano car (b) Molecular structure of nano car, Reprinted adapted with permission from (Shirai et al. 2005) Copyright # 2005, American Chemical Society

bubbles that pushed the boat forwards by recoil. In 2004 Paxton et al., for the first time, demonstrated a synthetic nano/micro-robot, which was a micron-sized variant of this self-propelling boat, which was a gold-platinum rod that propelled spontaneously when placed inside the water/H2O2 solution (Paxton et al. 2004). The propelling force, in this case, was not of recoiling type as in case of a boat. At the platinum, each H2O2 molecule breaks down to the water and an oxygen molecule releasing two protons and two electrons. The electrons move towards the gold end through the rod. At the gold end, each H2O2 reacts with an electron and a proton to generate two water molecules. Thus protons (hence positive charge) exceed at the platinum end than that at the gold end. Therefore, protons also move in the same direction but along the surface. During their motion along the surface, protons bind water molecules through ionic attractions and drag them also. This dragging force generates an opposite power to push the rod forward (Fig. 7.4a). But these nanorods move in random directions, as shown in Fig. 7.4b. The external magnetic field restricted these nanorods from moving in one direction, as shown in Fig. 7.4c. Later, several other bimetallic nano/micromotors have been demonstrated to move in the direction of a lighter anodic component, e.g., Pd-Au, Ni-Au, Ag-Au, Rh-Pt, Ag-Pt, and Si-Au, Zn-Pt (Wang et al. 2006; Zhou et al. 2017; Yoshizumi et al. 2013).

7.4.2

Unidirectional Motion: Ratchet System

As already mentioned, things at molecular and atomic levels are in absolute chaos all the time due to Brownian motion wherein things move in all possible directions randomly. Hence guiding a nanostructure to move in only one particular route is a challenging task. Biological systems also face the same kind of problems, and still, everything in the natural or cellular world goes on with absolute perfection. This idea has led scientists to design various systems that can bring order from chaos to restrict

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Fig. 7.4 Nanorods (a) Gold-Platinum nanorod that moves spontaneously over the surface of hydrogen peroxide solution with the help of catalytic reaction at the gold end. (b) Nanorods are containing nickel discs moving in random motion without application of external magnetic field (c) Nickel-containing nanorods moving in one direction on the implementation of the magnetic field

nanorobots to move in one direction (Hoffmann 2016). One such method is the ratchet-pawl system (Davis et al. 2010; Kay and Leigh 2015; Musser 1999). These systems are capable of converting the random forces they experience into a net positive force that is effective in one direction. Richard Feynman first described the ratchet system for miniature machines. The principle of how a ratchet converts a chaotic motion into an organized one-directional action resembles Maxwell’s Demon model (Mandal and Jarzynski 2012). Maxwell suggested that by merely gathering information about the molecules, specific processes can be performed without the utilization of external energy. He described it by a model where if the speeds of all the moving air molecules in a box are known, and somehow

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slow-moving molecules can be gathered at one end of the box and fast-moving molecules at the other end then one half of the box can be made hot while the other half cold. Ratchets work on the principle of utilizing Brownian motion forces in the direction of desired motion and blocking the effects of these forces in the opposite direction. A typical ratchet is a combination of a gear having asymmetric teeth, a pawl to push the teeth, and an arm to prevent a backward motion of the gear. As the forces of Brownian motion push the propeller, connected to an apparatus, forwards it rotates (equal to, e.g., one tooth of the gear), the arm then comes in place and jams the gear so that the Brownian motion forces are not able to rotate the propeller backward. In this way, only the forward thrust (i.e., one-directional motion) by the propeller is favored, and the machine is forced to move only in one direction. Such a ratchet system can move against all forces under ten piconewtons (almost 105 times the gravitational force) at the molecular level. Kelly et al. in 1997 designed a molecular pawl ratchet system consisting of triptycene (a “Y” shaped molecule) serving as gear having three blades (each blade is a benzene ring) (Kelly et al. 1999). A “G” shaped molecule, helicene (consisting of four benzene rings), served as a pawl and spring setup. From NMR spectroscopy, it was observed that the triptycene moves in both directions with equal frequency. This was found despite an asymmetry in the shape of helicene, i.e., it has a twist in it. This observation was in accordance with the analysis done by Feynman earlier. Feynman had stated that thermal energy would induce vibrations in the ratchet system itself, such that the spring attached to the pawl jiggles to engage and disengage the pawl from the gear. Thermal fluctuations would occasionally lift the pawl, and while disengaging the gear will slide back by a nick due to the skewed gear teeth. When the system is in thermal equilibrium, the forward thrust of the colliding molecules cancels with the backward movement due to the thermal vibrations of the pawl. The net result is that the rotation of the gear in any direction is prevented. Any shift in the equilibrium would allow the ratchet guided motion. A new type of engine known as Brownian motor has the potential to address this problem. Brownian motors utilize the concept of using thermal noise to activate other processes, e.g., chemical reactions to drive the ratchet. The biological world uses molecules that are kind of Brownian motors to carry out various operations like the movement of proteins, transcription processes, ionic pumps, etc. Such systems utilize a chemical reaction, light to produce an asymmetrical force that would drive the ratchet. Kelly et al. used a sort of similar approach to jamming the backward rotation (but not to drive the motor) wherein they utilized a chemical reaction to do so. Kelly et al. incorporated a hydroxyalkyl group to the pawl (i.e. helicene) and an amino acid to the triptycene, and in the presence of phosgene gas, the two reacted to prevent the backward rotation of the gear (Kelly et al. 2000). From the biological world, ionic pumps are the best examples of Brownian motors that carry charged substrates (ions) against the electrochemical potential gradient. These pumps let the unidirectional flow of electric current only. Typically, a pump may be a V-shaped protein bearing a channel inside. The mouth of the pump opens to inside when ATP binds to it, and the interior of the channel having high interaction with the ions attracts the ions and the ions travel through the channel.

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Secondly, the products of the hydrolysis tend to close the gate of the pump inside, to widen the pathway and to decrease the interaction between the ions and the channel interior. Tian et al. in 1980 (Teissie and Tsong 1980) and Astumian et al. in 1988 (Tsong and Astumian 1988; Liu et al. 1990) confirmed this through electric fielddriven ions against electrochemical gradient when an alternating electric field was applied to the ionic pump. A similar mechanism is followed by another protein, kinesin working as a transporter to transport proteins within a cell guided by microtubules. The electrical potential barrier between kinesin and the microtubules prevents its movement from one tubulin to the other. Hydrolysis of an ATP molecule helps the shape of the kinesin molecule to change that favors a forward jerk due to the force of Brownian motion. Next, the hydrolysis products of ATP change kinesin to its original shape blocking any possible backward motion. In this way, the protein propels forward with the help of the natural Brownian ratchet mechanism. A similar device was designed by Imre et al. that used random voltage changes to pump electrons irreversibly (Astumian and Derényi 2001).

7.5

Steering Nanorobots

Another approach to move nanorobots in the desired direction is through external control. It is known that many bacteria hold themselves still by aligning themselves along the magnetic field of the earth. A similar approach was adopted by Paxton et al. They devised their gold-platinum nanorods with nickel disks that served as miniature compasses and responded to an external magnetic field placed a few millimeters away. In the presence of a magnet, these nanorods would align in position and overcome the random forces of Brownian motion in all other directions except the power provided by the catalytic reaction of H2O2, along the length of the nanorod making them capable of moving in straight lines. The nanorods were also able to change their path when the direction of the magnetic was altered. These nanorods have been observed by Velegol et al. to follow straight runways similar to that of bacteria driven by chemotaxis (Hong et al. 2007). Bacteria leave a breadcrumb trail, as shown in Fig. 7.5, composed of discontinued straight runs increasing in length as the bacteria move closer to the target. A similar type of breadcrumb trail like the path was observed in the case of nanorods. These workers have also designed particles that move by phototaxis using light to furnish positive and negative ions that diffuse away at speeds that create a driving force for the particles. In 2017 Zhou et al. developed tadpole-shaped Si–Au micromotor activated by light (Zhou et al. 2017). The motor is sensitive to light, and its velocity can be controlled by varying the intensity of light. It was able to

Fig. 7.5 Breadcrumb trail

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move in deionized water autonomously and organic solvents using light as an energy source and not the chemical fuel. Like the Pt-Au nanorods, the direction of the motion of Si-Au micromotor can also be controlled through an external magnetic force when a nickel layer is introduced in it. Light-driven nano or micromotors work on two types of principles viz. (1) those propelled by photothermal effects and (2) those powered photoelectric effects. A photothermal result is a thermal gradient that is created when the motor is illuminated by near-infrared radiation that drives the nano or micro motor. In the case of motors guided by photoelectric effect where usually semiconductor materials are used, a chemical reaction, e.g., catalysis of H2O2 initiated by light, generates electrons and holes that drive the nano or micro motor. Besides chemical or light-driven nanomotors, other types have also been developed that use forces like acoustic, electric, fluidic, magnetic, or electromagnetic forces to drive. Many microorganisms and cells have been observed to follow distinct paths and change directions in response to changing fluid properties. The best example from the biological world is provided by the movement of sperm cells against the fluid flow during fertilization (Miki and Clapham 2013). A rheotaxis bimetallic micromotor (Au-Rh rods) driven by a chemical-acoustic force and directed to one-directional motion by tuning rheological properties of fluid has been developed recently by Ren et al. (2017). These workers used acoustofluidics, which deals with the use of ultrasonic waves to move microscopic objects in a microfluidic system to propel the micromotors alone (or in combination with the chemical fuel) using an acoustofluidic device. This device generates surface acoustic waves (SAW) that ultimately lead to the propulsion of the bimetallic rod near the surface. In the absence of SAW these micromotors exhibited random Brownian motion. As the SAW is switched on, the micro rods align themselves along the surface and the Rh ends of the micro rods face either direction due to which bidirectional motion of the rods is observed. The advantage of using acoustic propulsion is the velocity that it imparts to the micromotors compared to the rheotaxis by chemical propulsion (200 μm/s) (Wang et al. 2012) compared to 50 μm/s (Jang et al. 2016) respectively). Ren et al., in their model, combined the advantages of both types of propulsions, i.e., the unidirectional motion of chemical propulsion and higher speeds provided by acoustic propulsion. But still, the direction of the movement of the bimetallic micro rods depends on the combination of the metals, e.g., in H2O2, the action will be in the course of the anode (i.e., Au). In contrast, in an acoustic field, the rod will move in the direction of a lighter component (i.e., Ru). Therefore, in a hybrid type of area, they will make a mess between the two effects. A solution to this problem is to use a lighter anode, which will favor only positive rheotaxis.

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Types of Nanorobots

7.6.1

Actuating Nano Transducers

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Actuating nano transducers, developed by researchers at the University of Cambridge, are considered as the smallest nano engines (Ding et al. 2016). These nano engines are capable of producing forces 100 times more than any other motor. The motor consists of a cluster of gold nanoparticles held together by temperaturesensitive polymers. Upon heating to a specific temperature (32  C), the nanoparticles clump very tightly, and subsequent cooling results in water uptake and the explosion of the aggregated nanoparticles that convert Van der Waals energy into elastic energy (Fig. 7.6). The polymer between the nanoparticles acts like a spring that pushes the nanoparticles outwards like a tightly compressed spring. In this way, force is generated by heating the device (with the help of a laser). Materials, e.g., ionic polymer-metal composites that show substantial deformation with the application of low voltage and least resistance, are best suited for making artificial actuators and many other biomimetic small scale machines. These materials are used to make ion exchange membrane self-actuating machines, e.g., artificial muscles (Shahinpoor et al. 1998). Such materials show interesting properties suitable for such fabrication, e.g., the capacity of absorbing large amounts of water and other polar solvents resulting in deep metallization of the polymer. This results in large bending/deformation of the ion exchange membrane with the application of the electric field. Periodic deformations can be induced in such an actuator with the implementation of an AC voltage. This imparts full applications to the type of actuators like biomimetic sensors, catheter wires, and artificial muscles, etc. (Chung et al. 2006).

Fig. 7.6 Gold nanoparticles held together by a temperature-sensitive polymer. Upon heating to 32  C and subsequent cooling, the nanoparticles burst to generate large force

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Graphene Engine Based Nanorobots

Recently researchers have developed a nano engine from a material of excellent mechanical strength known as graphene. When chlorine and fluorine molecules are inserted into the two-dimensional lattice of graphene and a laser is fired, the sheet expands. When the laser is rapidly turned on and off, the graphene is pumped back and forth, similar to the working of an internal combustion engine.

7.6.3

Frictionless Nano-Rotor

Atomic vibrations in crystal lattices govern the thermal and electrical properties of the solids and are also crucial in determining their piezoelectric, pyroelectric and ferroelectric properties. Besides atomic displacements, desired molecular motion can be achieved by tuning the cavities of the crystal lattices. Action in molecular species can be induced by restricting them in small spaces at the nanoscale, e.g., in nanometric hexagonal holes, as demonstrated by Dirk Kühne et al. (2010). These researchers designed nanoporous lattices containing thermally activated supramolecular caged chiral trimers (called dynamers) that rotated inside the hole. Such rotating molecules can be used to drive nanorobots inside the body to specific sites. Figure 7.7(a and b) show trimers assembling inside nanometric hexagonal holes and fully assembled trimers nano rotor, respectively.

Fig. 7.7 Nanoporous lattices are containing thermally activated supramolecular caged chiral trimers. These trimers rotate spontaneously and continuously when confined in small cages (hexagonal holes). (a) Trimers assembling inside the hexagonal cage; blue and green trimers known as λ and δ rotator trimers respectively differ in the direction of their rotation (b) Fully assembled trimers rotating inside the hexagonal cage

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Nanorockets

Researchers have developed a nanoscale molecular rocket capable of moving at high speed and controlled remotely. These nanorockets are robust in design that use catalytic reactions to gain high-speed propulsion in liquids, e.g., H2O2. Typically, nanorockets are nanotubes that are filled with rocket fuel hydrazine. Since such types of fuel are toxic, researchers have developed nanorockets that are metal-coated known as nanojets (Sanchez et al. 2011a). Sanchez et al. developed the world's smallest nanojet engine that are nanotubes made from a combination of metal layers spontaneously rolled over an AlAs surface upon treatment with hydrofluoric acid, as shown in Fig. 7.8a. These nanotubes are capable of propulsion at high speeds in a solution containing H2O2 and a surfactant (Fig. 7.8b). Figure 7.8c shows different trajectories, followed by nanorockets in different concentrations of H2O2 (Sanchez et al. 2011b). Nano-rockets are built from the combination of nanoparticles and biomolecules, e.g., numerous catalytic molecules are conjugated with gold/chromium rocket bodies through DNA. The catalytic reaction by the catalytic molecules releases the large number of oxygen bubbles that force the movement of the nanorocket in the forward direction. When UV light is shined on the rocket, the DNA linkages break, causing the break and the catalytic engines to detach, which helps in changing the direction of the rocket. Such devices are promising for use in the targeted delivery of the drugs

7.6.5

DNA Nanomachines

Since DNA can be stretched to an enormous length, it can be used to knit/construct miniature machines (nanorobots) using a technique known as DNA origami, with greater flexibility that can carry out specific functions, cellular and in-vivo, e.g., to measure the activity of a protein. DNA based nanomachines have been demonstrated to precisely monitor the cellular process, e.g., entry of cargo into a cell, viral infections, acidification of tumor cells or functioning of lysosomes. These nanomachines work as pH sensors inside living cells that can measure pH changes associated with or necessary for cellular processes. For example, lysosomes function at precise pH values, a slight variation of which results in dysfunctional lysosomes leading to a number of diseases. A DNA nanomachine can help in accurately detecting any difference in lysosomal pH and, therefore, the severity of the lysosomal dysfunction. A demonstration of how a DNA nanomachine changes its conformation with the change in pH is shown in Fig. 7.9a. Such machines can be precisely loaded at specific locations inside cells to measure the activity of proteins and not just their movement inside cells that simple fluorescence probes cannot do (Modi et al. 2009). These workers have also been successful in demonstrating the simultaneous working of two DNA nanomachines inside a cell precisely (Modi et al. 2013). These workers have been able to track and map two different pathways, the furin, and transferrin endocytic pathway in the same cell, simultaneously with the technology they call SympHony (Fig. 7.9b). With a technology like this, it is hence

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Fig. 7.8 Nanorockets: (a) Schematic images of nanojets (developed by Sanchez et al.), which are nanotubes made up of consecutive layers of InGaAs, GaAs, Cr and Pt. These metals are initially put layer by layer on the AlAs surface and after the application of hydrofluoric acid, these metal layers roll into tubes. (b) Helix like trajectories followed by the nanojets of Sanchez et al. after immersed in a solution containing H2O2 and a surfactant. Reprinted adapted with permission from (Sanchez et al. 2011a). Copyright # 2011, John Wiley and Sons (c) Catalytic microjet engines developed by Sanchez et al. showing different trajectories in different concentrations of H2O2 (0.25%, 0.5%, 1%, and 2%). Reprinted adapted with permission from (Sanchez et al. 2011b) Copyright# 2011, American Chemical Society (d) Construction of self-propelling multi-layered Pt nanoparticlefunctionalized polymeric tubular nanorockets by Zhiguang et al. using template-based nanoporous layer-by-layer assembly. (e) Timeline of motion of nanorockets of Zhiguang et al. in H2O2 solution at 0, 0.5 and 1 sec. Reprinted adapted with permission (Wu et al. 2013) Copyright # 2013, John Wiley and Sons

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Fig. 7.9 DNA nanomachines. (a) A DNA nanomachine is known as I-switch by Modi et al., showing the opening and closing of its gates (based on measurement of fluorescence resonance energy transfer) at high and low pH, respectively. (b) Two DNA nanomachines developed by Modi et al. IFu and ITf programmed to map two different pathways (furin and transferrin pathways respectively) simultaneously in the same cell showing conformational changes with the change in pH

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possible to track pathways of cellular processes like those of lysosomal storage diseases. Another approach of cancer treatment using DNA nanorobots is to deliver coagulating payloads to the tumor vasculature (Bradley 2018). Li et al. have recently developed a DNA nanorobot that has addressed the problem of thrombin delivery to tumor vasculature. The specially designed DNA nanostructure shields the protein, thrombin until it reaches the tumor site where its opening is prompted upon the interaction of an aptamer attached to the DNA nanorobot with tumor marker, nucleolin (Li et al. 2018). The aptamer interaction with nucleolin guides the DNA nanorobot to the tumor site as well as triggers its opening. The thrombin protein then coagulates the blood vessels perfusing tumor cells, which results in infarction and necrosis of the tumor.

7.6.6

Nano Swimmers

The idea of how a nano or micron-sized robot would move by swimming in a fluid was first given by Purcell in his article “Life at Low Reynolds Number” (Purcell 1977). The motion of microorganisms is significantly affected by fluids of low Reynolds number (Lauga and Powers 2009). In his article, he explained the principles based on which organisms swim in liquids. Scallops are known for their swimming underwater by a reciprocal motion in which they open their shells slowly and close them fast, thus pushing themselves forward (Cheng et al. 1996; Jo et al. 2016). But such a move cannot be attained in liquids having low Reynolds number. He stated a scallop theorem which states that to swim in a low Reynold number, i.e., high viscosity fluid, a reciprocal motion is not sufficient for an object to float. At small Reynolds number, closing and opening movements at any speed would produce the same amount of displacement forward and backward, leaving the swimmer at its original position without any progress. Since the mass of microorganisms is very small, even a regular liquid behaves like a low Reynold number fluid. Hence organisms cannot swim with these kind of movements and instead adopt some different ways to swim. Purcell described different types of swimming patterns that microorganisms adopt to swim.

7.6.6.1 Two Arm Swimming The two arms are attached to two ends of the main body through two hinges like two flagella connected to two terminals of a bacterium. The arms act like the rudders at the ends of a boat that let the object go through a sequence of deformations, after which the purpose finds itself at a new position. A similar type of nano swimmers (Fig. 7.10) was developed by Li et al. comprising of two magnetic arms that moved under the effect of an oscillating magnetic field (Li et al. 2017). In such an oscillating magnetic field, the nano swimmer tries to align the magnetic momentum of its Ni arms along the axis of the magnetic field each time the magnetic field is altered. This results in the generation of forceful strokes by the magnetic arms that are powerful

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Fig. 7.10 Freestyle nano swimmers; Freestyle two-arm magnetic nano swimmer designed by Li et al., moving under the effect of an oscillating magnetic field. Reprinted with permission from (Li et al. 2017). Copyright # 2017, American Chemical Society

Fig. 7.11 Schematic representation of nano swimmer that propels through undulating motion

enough to propel the nano swimmers forwards (about 12 times its body length per second). Jang et al. have developed nano swimmers that are capable of swimming by planar undulating “S” shaped motion upon the application of the magnetic field (Jang et al. 2015). The undulating movement of the nano swimmer, as demonstrated by these workers, is shown in Fig. 7.11.

7.6.6.2 A Rotating Flagellum or a Corkscrew A helical flagellum is attached to a rotating hook-like structure that rotates around the flagellum. The motion couples with the translational motion of the helical flagellum, which drives the organism forwards. An example of this kind of action is provided by E. coli, as reported by Howard Berg in his paper (Berg and Anderson 1973)

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7.6.6.3 A Flexible Oar or Cilia In a low Reynold number fluid, a rigid oar would reciprocate motion of the object in forwarding and backward directions, and hence net displacement in any direction is zero. But a flexible oar would bend differently during forwarding and backward strokes that would produce a different amount of movement in either direction. 7.6.6.4 A Two Cell Toroidal Movement In this kind of motion by microorganisms, there may be two cells attached to each other that roll over each other surface and gain motion. Many workers have still been able to develop microswimmers with a scallop like movement successfully in non-Newtonian fluids and body fluids do not behave like water. They are non-Newtonian (shear thinning or shear-thickening) in nature. In such fluids, a scallop like movement can produce different amounts of thrust forwards and backward, leading to change in either direction depending on the liquid. Qui et al. recently have developed microswimmers or micro-scallops made of silicon-based organic polymer that use magnetic actuators as motors guided by an external magnetic field (Qiu et al. 2014). Figure 7.12(a and b) shows the movement of a scallop by opening and closing of its shells and the scallop like microswimmer developed by Qui et al. respectively. Micro swimmers consist of two shells or wings connected through a hinge to which micromagnets are attached. With the application of the external magnetic field, the micromagnets tend to align with the magnetic field. Thus, scallop closes and with the decrease of the magnetic field, the shells reopen to their original position due to the restoring force of the hinge. The question is that how can merely opening and closing of shells move the microswimmer in a particular direction. Since our body fluids are non-Newtonian fluids (shear-thinning fluids), moving the shells at different rates during opening and closing would change the viscosity of the fluid accordingly. At high speed, the high shear rate would decrease the thickness more than that at a low shear rate. Hence a fast closing followed by the slow opening of shells in a shear-thinning fluid would produce a net force in the forward direction. A similar kind of fluttering of the shells would produce a net opposite effect in a shear thickening fluid.

Fig. 7.12 Schematic representation of scallops (a) Reciprocal motion shown by scallop as described by Purcell. (b) 3D illustration of micro scallop developed by Qui et al. consisting of silicon-based polymeric shells and magnetic actuators

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Fig. 7.13 Helical nano propellers developed by various workers (a) Nano propellers moving within the complex network of hyaluronic acid polymer chains showing larger helices being hindered to a greater extent while the smaller ones moving freely Reprinted with permission from (Schamel et al. 2014) 2009, American Chemical Society (b) SEM image of a single helical nano propeller developed by Ambarish et al. Reprinted with permission from (Ghosh and Fischer 2009) 2014, American Chemical Society

Researchers also have developed magnetic nanopropeller, which has a corkscrew-like helical structure that rotates with the application of a rotating magnetic field. Due to its helical structure, the translational motion couples with the rotational motion and it moves forward. Figure 7.13(a and b) shows helical nano propellers synthesized by various workers.

7.7

Applications of Nanorobots in Medicine

Researchers today are interested in developing nanorobots that can sense and detect disease sites, accurately deliver drugs at target sites, do surgeries autonomously and much more. Although their development is in the preliminary phase, a large number of target-specific nanorobots have been tested. The applications of nanorobots seem tremendous some of them which are discussed as under

7.7.1

Early Diagnosis

Diseases are diagnosed through several techniques. One of the commonly used methods to detect a disease site is through imaging by using fluorescent substances. Nanoparticles loaded with fluorescent dyes are one of the promising devices to identify disease sites, e.g., tumors. For the purpose-specific biomarkers or ligands are attached to optical or fluorescent nanoparticles, which are then guided to specific disease sites, e.g., imaging of cell malignancy by silica nanoparticles containing fluorescent dye. Besides, semiconductor devices, e.g., quantum dots, because of their excellent photochemical properties, are being extensively investigated for use in imaging. Quantum dots are also beneficial in studying cell signaling among cancer cells

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(Gu et al. 2005), even the presence of oncogenes and orientation of DNA (Crut et al. 2005). Presently researchers are focusing on the use of nanorobots that can autonomously detect disease sites. These devices are micron or nano-sized electro-mechanical systems (MEMS or NEMS) based on micro/nanosensors, actuators, transducers and other electronic components. Since such devices contain biomarkers, e.g., antibodies, therefore, these are known as Bio-MEMS (Requicha 2003). The antibodies can accurately identify other biomolecules or biological systems, e.g., a specific virus (Zheng et al. 2005; Otto et al. 2003). Alternatively, Bio-MEMS consist of micro/nanowires fitted in microfluidic devices that can sense a biomolecular change and transfer the information through the electronic network. Bio-MEMS are already being used for several therapeutic purposes like monitoring glucose level in diabetes, in cochlear implants, pacemakers, etc. (Bhansali and Vasudev 2012; Lin et al. 2000; Miao et al. 2006; Nisar et al. 2008). Biosensors containing specific cells can be used to detect chemical or biochemical transformations, e.g., chick myocardial cells used in biosensors to detect verapamil, epinephrine, and tetrodotoxin (Wee et al. 2005; Hede and Huilgol 2006).

7.7.2

Targeted Drug Delivery

Nanorobots can be used to deliver drugs to specific disease sites anywhere in the body. Many hurdles in the treatment of diseases can be overcome by the use of autonomous nanorobots, e.g., cancer. Modern approaches of medication, e.g., chemotherapy and radiotherapy, are ineffective against various tumors due to the presence of certain hardcore zones (e.g., hypoxic zones). Due to rapid cell division, high oxygen consumption leads to hypoxic conditions in deep areas of tumors. Nanorobots can recognize these areas and preferably release the drug there. These nanorobots are equipped with miniature sensors that are sensitive to the oxygen concentration and can sense the hypoxic zones of a tumor.

7.7.3

Surgery

Nanorobots can be used to physically repair cells, organelles, or other cell constituents and cut pieces of tissues inside the body, e.g., in case of biopsies. These molecular machines contain high accuracy transducers that can provide precise information and mapping about the areas of the target in cancer surgeries (Cavalcanti et al. 2007).

7.7.4

Repairing

Nanorobots can be programmed for repairing DNA is a complex biological molecule that contains vast information about the structure and functioning of a living being. Certain defects in DNA (even very minute) can cause serious problems. Such errors

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are almost impossible to repair by standard methods. Nanorobots being very tiny autonomous structures and having sensors and repair system in them, can easily access DNA, grab it, scan and fix the defects.

7.7.5

Antibacterial

Certain bacteria are resistant to antimicrobial agents or antiseptics, e.g., Clostridium tetani, an anaerobic bacterium that is enormously found dwelling on rusty surfaces. A grave injury with such an object infects the more deep tissues where the bacterium grows comfortably. The bacterium produces numerous spores and secretes dangerous exotoxins. However, the bacterium cannot resist high temperature and its toxins are denatured at 180  F. Nanorobots that have specific markers on their surfaces to guide them to the infectious sites can be used in such cases. These nanorobots equipped with retractable heating elements can raise the temperature to about 302  F. When these nanorobots enter the spores of the bacterium, they increase the heat to high degrees resulting in the destruction and elimination of toxins and spores.

7.8

Conclusion

In conclusion, the fabrication of nanorobotics can be considered as a promising technology that has great potential to revolutionize the future of medicine and change the way of treating diseases. The advent of nanotechnology since the famous lecture of the physicist Richard Feynman has inspired almost every branch of science to think of every possible manipulation at a miniature scale. Nanomedicine has already been working on developing drug delivery devices at the nanoscale that provided high efficiency in delivering drugs to target sites. Now nanotechnology is being exploited to create miniature machines of the order of nanometers that move autonomously inside the body. The research so far has provided convincing and outstanding results of the precision and efficiency of nanorobots in independently targeting specific disease sites for drug delivery or eradicating certain pathogens. Treating diseases or pathological conditions with nanorobots would make it possible to avoid various invasive procedures that are routine in many cases, e.g., brain infusions, cancer surgeries, biopsies, radiotherapy, etc. and make the drug therapy patient compliant. However, a large number of nanorobots have been developed. This technology is still in its developing stage and would require more investment until it becomes clinically approved. Acknowledgments The Department of Science and Technology, Government of India, Nano Mission, supported this work under Grant SR/NM/NB-1038/2016.

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Composites of Ceramic and Polymeric Nanofibers for Photocatalytic Degradation of Dairy Effluent Muzafar A. Kanjwal and Faheem A. Sheikh

8.1

Introduction

It is surprising to note that effluents produced by industries relying on dairy businesses also contribute to waste, which is enormous in terms of volume. Furthermore, its nature is more polluting one if compared with other industrial effluents. These industries use and later on releases 2–3-folds more water than the milk which they use for processing, washing and cleaning (Ramasamy and Abbasi 2000; Prazeres et al. 2012). These wash waters contain 1–3% of milk components consisting of proteins (casein and lactalbumin) and sugars (lactose), lipids, sterols, vitamins (A, D and E), etc. (Vourch et al. 2008). In addition to these organic components, the dairy waste contains other used chemicals (e.g., acids, alkali and detergents, etc.) (Fernandez et al. 2010). These acidic and alkaline wastes require neutralization before further biological treatment. These wastewaters from the dairy company may contain the spoilage causing microbes (e.g., species of lactobacillus, streptococcus, bacillus, proteus, micrococcus, clostridium, coliform, alcaligenes, klebsiella, enterobacter, penicillium and geotrichum), which may or may not be pathogenic and may be released while washing. Ideally, if the industrial effluent discharges microbial load, the sterilization may be required. If somehow, the microbial population from dairy waste is completely sterilized before flushing it into water bodies, that will be most desired strategy, however, that process is bit expensive. Nevertheless, the organic constituent (which can still remain after sterilization) contains a lot of nutrients for the growth of algal blooms causing eutrophication to the water bodies in which they are discharged, thereby causing the death of aquatic M. A. Kanjwal Mechanical Engineering, The Hong Kong Polytechnic University, Hung Hom, Hong Kong F. A. Sheikh (*) Department of Nanotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India e-mail: [email protected] # The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2020 F. A. Sheikh (ed.), Application of Nanotechnology in Biomedical Sciences, https://doi.org/10.1007/978-981-15-5622-7_8

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animals and disturbing the ecological niche (Luo and Ding 2011). However, the brighter part is these wastes tend to degrade at a faster rate and can enervate the dissolved oxygen; thus, results in depleted oxygen content in water and causes foul odor. Since the organic part in the wastewater from the dairy industry can be degraded biologically and, on these products, microbial flora can grow on these nutrients. Therefore, traditional approaches used from the basics of the industrial microbiological processes are used. These include the use of oxidation ponds (Lansing and Martin 2006), coagulation/flocculation (Sengil and Ozacar 2006), anaerobic/aerobic sludge digestion (Demirel et al. 2005), and membrane separation techniques (Balannec et al. 2005). In this vein, it still needs much to be addressed, for instance, pH picture during the treatment, control on the type of microbes and temperature. Thus, it may be necessary to use holding tanks to tackle changes in weather and microbial activity. Owing to the requirement of less space, the physiochemical methods to treat dairy effluents are considered to be much useful to treat high organic contents; however, the only drawback is to remove the chemical products from residual sludge, and this may add to the cost burden. In order to overcome these issues, membrane separation (Fernandez et al. 2010; Vourch et al. 2005; Akoum et al. 2004), improvised with different technologies (Scott and Smith 1997; Sarkar et al. 2006), can be used to get reusable water from dairy effluents. It is pertinent to mention that biological treatment systems like activated-sludge aeration tanks and trickling-filter rocks are generally used to treat dairy effluents. Still, they represent quite a few disturbing facts, e.g., requiring ample space, specific technical staff, construction/maintenance of large structures and to bear with susceptible environmental conditions (e.g., temperature, pH variation, fluctuations in organic content concerning batches run in industries, etc.). In recent years, a technique called an advanced oxidation process had been continuously used. Primarily, it works on the generation of highly reactive species, i.e., hydroxyl radicals promoting the oxidation of organic components in the industrial effluents. In the classes of the advanced oxidation process, the photocatalysis using UV light for excitation of electrons is considered a promising strategy. Considering its ability to remove altogether and keep no accumulation of leftover pollutants, this technique is referred to as a green purification system; this provides faster oxidation for the effluents (Kanjwal et al. 2015a, 2016a, b; Kanjwal and Leung 2018). Selection of nanomaterials plays a crucial role while applying them for photocatalysis prompted applications (e.g., splitting of water for hydrogen generation), organic degradation, gas sensing, solar cell devices, laser diodes, photodetectors, catalysts in enhancing the reaction, nano-coating for energy-harvesting devices and conversion of CO2 (Chunyan et al. 2011; Obregón and Colón 2014; Zhang et al. 2014a, b). Among the plethora of nanomaterials, titanium dioxide (TiO2) has many applications because of its wide bandgap, different crystal structure (i.e., anatase, rutile and brookite having bandgap in the range of 3.2, 3.0 and 3.25 eV, respectively), excellent binding energy and photocatalytic properties (Kanjwal et al. 2015b; Nakata et al. 2013; Sheikh et al. 2016). The TiO2 in the form of nanoparticles, nanorods, nanorings, nanoflowers, nanocircles, nanobelts, nanofibers, etc. are easy to prepare, highly dispersed in aqueous media, photostable, low-priced,

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non-cytotoxic (Kanjwal et al. 2015b; Xia et al. 2014; Wu et al. 2014; Rioult et al. 2014). It is pertinent to mention that due to enlarged surface to volume ratios of the TiO2 nanomaterial (allowing them for tuneable requests), moreover, they represent extraordinary features in comparison to its bulk forms (Zanetti et al. 2014; Yu et al. 2013; Strini et al. 2013; Smith et al. 2013). These nanostructures are tested to remove various environmental pollutants, owing to their photocatalytic properties (Ali et al. 2014; Dalt et al. 2013; Liu et al. 2013; Manzanares et al. 2014). Nonetheless, these reactions occur in the presence of UV light at higher rates, which is only 5% present in the solar light; therefore, the use of additional expenditure (i.e., UV light) is unavoidable. Fundamentally, visible-light-driven catalytic activity is not as much due to a lesser electron transfer rate and higher electron-pair recombination. In this disposition, nanomaterials capable of performing photocatalysis reaction under visible-light-driven needs to be fabricated. In this regard, TiO2 is conventionally fabricated into different shapes and morphologies, including nanofibers, nanoparticles, nanotubes, core-shells, nanocubes and nanorods (Zhang et al. 2014a; Zhou et al. 2014; Wan et al. 2014; Sobolev and Koltunov 2014; Park et al. 2014; Miranda et al. 2014). In order to improvise the catalytic performance of TiO2 nanomaterials, so as the reaction will occur under solar light, thereby elevating quantum effectiveness for degradation of dairy effluents, hybrid nanocomposite is necessary to create (Reddy et al. 2015; Kanjwal et al. 2010a). In the pursuit of this, our research group created composite TiO2 nanofibers of CdO and GeO to improve the catalytic properties. In our other related work, we were successfully able to formulate the membranes using polyurethane nanofiber mats encasing the ZnO and NiO photodegradation of dairy effluents. It is noteworthy to mention that composites consisting of 2–3 phases; can considerably expedite the charge transfer and separation. Therefore, this resulted in an excellent photocatalytic outcome, using those nanofibers. It has been recommended that the use of nanomaterial (since the materials are reusable) to treat large amounts of effluents is not feasible because of the complicated and costly separation process involved in it. This may require nanofiltration and/or centrifugation at a large scale, and that is practically impossible. Therefore, the effort is to produce a nanofiber membrane with some excellent mechanical properties to hold pressure while filtration and will necessarily contain nanomaterial, which is photocatalytic in action. One the one hand, the urgency of nanofiltration will be solved. On the other hand, the simultaneous photocatalysis will occur along with the filtration of significant size pollutants that will happen.

8.2

Materials Used for Fabrication of Nanofibers

To fabricate the nanofibers incorporating nanoparticles helpful for filtration and photocatalysis following reagents were used in those studies. Tetrahydrofuran, N, N-dimethylformamide, silver nitrate and ethylene blue dehydrate were purchased from Showa Chemicals Ltd., Japan, titanium (IV) isopropoxide and cadmium acetate were obtained from Sigma Aldrich, USA. Polyvinyl alcohol, MW 65,000 g/mol was

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obtained from Dong Yang Chem. Co., South Korea. Polyurethane, MW 40000, Bayer, Germany. The diary effluent (unknown composition) used to test photocatalytic activity was a generous gift from Purmil Ltd. 197–3 Daeri-ro, Shinpyong-meum Imsil-gun, Cheollabukdo, South Korea.

8.3

Characterization of Fabricated Nanostructures

The morphology of the nanofibers after electrospinning was investigated using scanning electron microscope JEOL JSM-5900 Ltd., Japan. Before loading samples in the SEM machine, the nanofibers were pasted on aluminum stub using doublesided carbon tape and coated with a layer of gold by sputter coating. Later on, the samples were viewed under different accelerating voltages and magnifications to get clear images at the various location of the individual sample. In addition to view the morphology of the fibers, the elemental detection was performed using the same SEM machine equipped with EDS assembly. The crystal structure and phase character of nanofibers were detected by X-ray diffractometer purchased from Rigaku Co., Japan with CuKα (λ ¼ 1.54056 Å radiation over a range of 2θ angles from 10 to 90. To verify nanostructured phase in the nanofiber samples, the transmission electron microscope (JEM- 2200FS) fitted with a 200 kV field emission gun and EDS assembly was used. The samples were prepared by weighing 10 mg of sample dispersed in 200 μl ethanol and flushed on 600 mesh copper TEM grid. Furthermore, the surface nature was characterized by X-ray photoelectron spectroscopy analysis (XPS, AXIS-NOVA, Kratos Analytical, U.K). The conditions were set by fixing base pressure of 6.5  109 Torr, resolution (pass energy) of 20 eV and scan step of 0.05 eV/step. All the spectroscopic data obtained were analyzed using Origin 8 and plotted into graphs.

8.4

Photocatalytic Experiments

The photocatalytic property to degrade the dairy effluents using fabricated nanofibers was evaluated using a custom-designed glass reactor having a capacity of 1 liter, height 23 cm, and radius of 7.5 cm (Kanjwal et al. 2010b). The reactor was able to shelter UV lamp over its top and sunlight emitting lamp (2000 W), and the whole system was placed in a clean bench and was covered by aluminum foil all over. In the case of ceramic samples, 50 mg nanofiber as photocatalyst was tested against 500 ml of dairy effluent. For the polymeric samples (i.e., made up of polyurethane containing Ag-TiO2), the fiber mats were cut into 5 cm  5 cm fiber mats, which equal to 200 mg. Before providing the light source to the reactor, a pre-weighed amount of sample, the reactor was thoroughly mixed using magnetic stirring until the nanofiber got completed submerged in the dairy effluent. After the start of the reaction at different time intervals, the 2 ml of the sample was withdrawn from the reaction mixture. These samples were centrifuged to decant to supernatant for measurements in UV spectrometer corresponding wavelength (204 nm).

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Water Photosplitting

The ability of the fabricated nanofiber to perform photosplitting was tested by measuring the rate of hydrogen produced. The experiments were carried using a water-filled gas burette system, which was connected to the round-bottom flask. In total, 75 ml aqueous 0.5 M Na2SO3 was used as reaction media in which 50 mg of nanofiber catalyst was added. The reaction was allowed to move forward by agitating the mixture while steering at 600 rpm. During this reaction, the hydrogen gas evolved was noted by observing the displacement of the water level after every 60s. It was hypothesized that the gases produced are composed of oxygen and hydrogen with a mole (a consequent volume ratio) of 1:2, so the volume of hydrogen was calculated accordingly. Moreover, the control experiment was repeated by keeping a similar condition and without the presence of any catalyst. It is noteworthy to mention that no gas was evolved without the addition of a catalyst. The same experiments were repeated using methanol instead of the inorganic scavengers using methanol: water (1:1). Electrospinning is a perfect method to fabricate not only the nanofibers from polymeric solution but also the ceramic nanofibers using a metallic processor blended with the polymer. This technique uses a high voltage power to convert polymer droplets into a jet, which eventually solidifies during traveling to the depositor. The resultant fibers are continuously deposited on collectors resulting in micron-sized nanofibers mats, which can easily be removed from the collector (Kanjwal et al. 2015a, 2016a, b; Kanjwal and Leung 2018; Ofori et al. 2015). In the case of fabricating, the polymeric and ceramic nanofibers used as a photocatalyst in this study were spun using the electrospinning unit (CPS-60 K02V1, Chungpa EMT Co., Republic of Korea). This machine is capable of producing voltages up to 60 kV and works in an open environment. The solutions used to form nanofibers were loaded in a 5 ml plastic syringe, and the needle was replaced with 500 μl micropipette tips. The plunger from the syringe was removed, and the syringe containing solution was fitted on the burette clamp with 45 down-tilted facing towards the collector with keeping the working distance of 15 cm. The copper wire originating from the positive electrode (anode) was inserted into the solution, and a negative electrode (cathode) was attached to a metallic collector. The electrospinning was turned on, and the fibers were permitted to get deposited on a metallic plate, which was previously covered with an aluminum foil to produce the nanofibers. The electrospinning was allowed to continue with a voltage of 20 kV and 5 h. Figure 8.1a and b show scanning electron micrograph of the as-spun Ti(Iso)/ PVAc/GeIsp and Ti(Iso)/PVAc/CdAc nanofibers. One can observe that the nanofibers are non-defective and bead-free after the event of electrospinning. In the figure, captioned as c and d are the results of the same nanofibers after the heating of the samples at 500  C. An important observation from the nanofiber sample is drawn that after the high-temperature heat, the nanofiber diameter is reduced; this probably is accounted for due to the elimination of the polymer constituent from the nanofibers, therefore, indicating the removal of PVAc polymer. In detail, the

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Fig. 8.1 Scanning electron microscope images of the as-spun TiIspr/GeIsp (a), TiIspr/CdAc (b), TiO2-GeO2 heated at 500  C (c) and TiO2-CdO heated at 500  C (d), obtained with permission from (Kanjwal and Leung 2018)

nanofiber diameter of the as-spun nanofibers was 273 nm, and for the same nanofiber samples after high-temperature treatment, it decreased to 256 nm. Overall, the nanofibers still were able to maintain their orginal one-dimensional structure. Likewise, a specific preparation method of TiO2-CdO nanofibers in our previous study is reported (Kanjwal et al. 2010a). The internal contents of the nanofibers were observed using the transmission electron microscope, and the results are displayed in Fig. 8.2. The low and high magnification images of TiO2-GeO2 are in total agreement with the SEM images of this sample. A clear picture of the atomic planes in the high-resolution TEM image indicates good crystallinity (Fig. 8.2b), the crystal planes are parallel and are having a similar distance. The SAED in the upper left inset of (Fig. 8.2a) supports the highresolution TEM analyses. Moreover, the inset in Fig. 8.2b representative of fast Fourier transformation (FFT), which visibly confirms the good crystallinity following the SAED designs. The spectra of the TiO2 nanofibers loaded with GeO2 nanoparticle for the XRD pattern are shown in Fig. 8.3. The model revealed the existence of multiple crystalline phases with the presence of well-founded diffraction peaks at 2θ values of 25.24 , 38.12 , 48.00 , 53.86 , 54.95 , 62.71 , 70.10 , and 75.11 corresponding to planes of 101, 112, 200, 105, 211, 204, 220 and 215, respectively. The diffraction

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Fig. 8.2 Transmission electron microscopy results of the TiO2-GeO2 nanofibers after calcination at 500  C, a low magnification image of two adjacent nanofibers (a) a high magnification image of the same nanofibers indicating the presence of GeO2 nanoparticles embedded in TiO2 nanofibers (b), the inset images show the high-resolution transmission micrographs. Obtained with permission from (Kanjwal et al. 2015b)

Fig. 8.3 XRD pattern of TiO2 nanofibers loaded with GeO2 nanoparticle after calcination at 500  C. Obtained with permission from (Kanjwal and Leung 2018)

pattern is completely matching with JCPDS card no 21–1272; this indicates the presence of anatase titanium dioxide. Besides, clear GeO2 peaks were noticed according to JCPDS card no 36–1463 at 2θ values of 20.02 , 25.91 , 37.00 , and 68.58 , which corresponds to crystal planes of 100, 101, 102, and 301, respectively. The catalytic degradation of dairy effluents due to the ability of calcined nanofiber are presented in Fig. 8.4. In this result, the tests conducted in triplicates and data are plotted as a mean average. In the case of TiO2 nanofibers modified with CdO nanoparticles, we can observe the 75% removal of effluent at the first run after the

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Fig. 8.4 Removal percentage of the pollutants in the dairy effluent using TiO2 nanofiber loaded with CdO nanoparticles denoted as TiO2-CdO (a) and TiO2 nanofiber loaded with GeO2 nanoparticles indicated as TiO2-GeO2 (b). Obtained with permission from (Kanjwal and Leung 2018)

Fig. 8.5 The water photosplitting characters of the nanofibers in the presence of CH3OH (a), under inorganic scavengers Na2SO3 (b) obtained with permission from (Kanjwal and Leung 2018)

interval of 3 h (Fig. 8.4a). However, in the second run of the same catalyst, we can observe the 70% efficiency at 6 h. Furthermore, on the third run, the photocatalytic ability got reduced to 66% after 9 h. Similar passion is shown by another counterpart catalyst (i.e., TiO2 nanofibers modified by GEO2 nanoparticles). For instance, the catalytic activity reduced from 65%, 59% and 56% during the first, second, and third runs, respectively. Figure 8.5 shows the hydrogen generation using photosplitting of water in the presence of nanofibers under the UV light. In both cases, we can observe an unusual amount of hydrogen generation using CH3OH and Na2SO3 due to the addition of nanofibers as a catalyst. In detail, after 1 h, 150 ml of hydrogen is generated with the TiO2 nanofibers containing CdO nanoparticles, which is represented as 2.5 ml/min. Moreover, the rate of hydrogen production is considered outstanding and is calculated as 2999 ml/g.h (Fig. 8.5a). Almost a similar trend is shown by TiO2 nanofibers containing the GeO2 nanoparticles. For instance, 130 ml of hydrogen gas is evolved

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after 1 h, representing the release of 2.16 ml/min, recalculated as 2591 ml/g.h, which again is an excellent rate. In these experiments, Na2SO3 is used as a scavenger; however, CH3OH was also tested as some reports indicate it as another competent electron-denoting compound. Results displayed in Fig. 8.7b indicates that 75 ml hydrogen is generated in 0.5 h using TiO2 nanofibers incorporated with CdO nanoparticles, which is represented as 2.5 ml/min and recalculated as 2999 ml/g.h. Likewise, counterpart nanofibers incorporated with GeO2 nanoparticles indicated 65 ml of hydrogen produced after 0.5 h, which represents the release of 2.16 ml/min. Meanwhile, the real rate of hydrogen production is calculated as 2591 ml/g.h.

8.4.2

Synthesis of Ag-TiO2 Nanoparticles and Ag-TiO2 Nanofibers

200 mg of AgNO3 was dissolved in 800 mg of DMF to form a solution to be mixed with sol-gel. The sol used this study is prepared by dissolving 4 g of PVAc polymer into DMF, giving it final concentration of 14 wt%, after mixing well, 4 g of Ti(Iso) was added in a dropwise manner. During the mixing of Ti(Iso), small units of acetic acid were added so as the solution will not precipitate. To this, the heterogeneous mixture previously prepared AgNO3 solution was added, and the whole solution was allowed to mix again for 10 min. The resultant solution was placed in glass Petri plates and oven-dried at 80  C for 12 h and ground well. These dried powders were calcinated at 600  C for 1 h to obtain nanoparticles of an average diameter of 200 nm. For the preparation of Ag-TiO2 nanofibers, the solution used to prepare the nanoparticles was used to fabricate the nanofibers by electrospinning technique, as described earlier of this chapter. Initially, the solutions were prepared by dissolving 10 wt% of polyurethane by dissolving its pellets in THF and DMF (1:1) following the protocol, as mentioned in our previous study (Yousef et al. 2012; Sheikh et al. 2010; Sheikh et al. 2011a). From the literature, we know TiO2 and Ag nanoparticles are efficient photocatalytic and have good bandgap properties. Therefore, the ideal would be if these nanoparticles are incorporated into a polymeric solution to form membranes and these nanofibers will serve as the purpose of catalysis and will do the filtration at the same time. However, fabrication of those nanofibers is not an easy task because metallic precursor and polymer need a hydrolyzed and polycondensation in a universal solvent, which difficult to find. To prevent this, our group developed a strategy in which we were able to do colloidal electrospinning to form nanofibers (Barakat et al. 2010, 2011; Sheikh et al. 2011b). Taking advantage of previous experiences, a 10% polyurethane solution prepared by dissolving in THF and DMF (1:1) was added with Ag-TiO2 nanoparticles. This mixture was allowed to disperse well by vigorous stirring for 1 h and occasional bath sonication. Furthermore, these solutions were spun using voltage power supply CPS-60 K02V1, Chungpa EMT Co., Republic of Korea, as mentioned earlier in this chapter. However, the parameters for electrospinning were maintained as 20 kV current supply, 15 cm tip to collector distance, round metallic collector (diameter 12 cm) covered with

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Fig. 8.6 SEM morphology of the unmodified polyurethane nanofibers (a), SEM morphology of the polyurethane nanofibers incorporated with small ceramic AgTiO2 nanofibers (b), SEM morphology of the polyurethane nanofibers incorporated with small ceramic AgTiO2 nanoparticles (c), diameter frequency distribution for three nanofiber combinations (d) and EDS analysis of the nanofibers (e), obtained with permission (Kanjwal et al. 2015b)

aluminum foil and total 5 h as a collection time of nanofibers after the start of electrospinning. Results indicate that pure polyurethane nanofibers were smooth and continuous, with an average diameter of 550 nm (Fig. 8.6a). As the Ag-TiO2 nanomaterials infused into nanofibers, the width reduced to 30  15 nm, as shown in Fig. 8.6d. Moreover, the EDS data indicated the presence of TiO2 (20%) and Ag (2.5%) nanomaterials inside the polyurethane nanofibers (Fig. 8.6e). From the TEM analysis (Fig. 8.7) of all three nanofiber combinations, it is palpable that the surfaces of the fibers are smooth, these results are in complete agreement SEM analysis. After observing these results, a clear indication of the noncrystalline of polyurethane nanofibers appears in grey. Besides, Ag-TiO2 nanoparticles could be viewed by standard TEM encircled in red color (Fig. 8.7c). The next mage in this panel, i.e., Fig. 8.7d shows the elemental mapping from nanofibers containing Ag-TiO2 ceramic nanofibers. The line EDX TEM along a randomly chosen line is indicated in Fig. 8.7e. All these nanofibers have maintained the typical fibrous structure inside the core of nanofibers. Here it is noteworthy to mention that these small fibers resided inside the main polyurethane nanofibers and lasted the vigorous mixing during sol-gel formation and abnormally high voltages after its creation. Moreover, total metal weight percentages of 20.85 and 2.23 were determined for Ti and Ag, which approximately very close to the concentration used in the original precursors used to make the solution.

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Fig. 8.7 TEM images of pure nanofibers (a), nanofibers incorporated with AgTiO2 nanofibers (b), nanofibers incorporated with AgTiO2 nanoparticles (c), mapping of the nanofiber with AgTiO2 nanofibers (d), and line EDX TEM analysis in the inset of the previous image (e). The remaining panels represent the distribution of nanomaterial incorporated along with the selected line. Obtained with permission from (Kanjwal et al. 2015b)

The UV visible measurements from 200 to 800 nm three nanofiber combinations are shown in Fig. 8.8. The spectra of pristine polyurethane nanofibers shown in Fig. 8.8a indicate no absorption peak at 400 nm. After the incorporation of Ag-TiO2 nanoparticle (Fig. 8.8b) and nanofibers (Fig. 8.8b), the absorption curve was shifted in the 425 nm spectral region. This reveals the presence of nanomaterials in polyurethane nanofibers by exhibiting the typical surface plasmon absorption maxima (420 nm). The polyurethane nanofibers being noncrystalline represented a broad peak at 2θ value of 20 , in all the combinations (Fig. 8.9), XRD results. However, the other modified counterparts containing a load of TiO2 and Ag nanoparticles and nanofibers show other peaks at 2θ values of 24, 28 40 and 47 , in accordance with CPDS card no 04–0783 and the 111, 200, 220 and 311 are the correspondings to the crystal plane. The peaks at 2θ values of 24, 34, 47, 56 and 69 are corresponding to crystal planes of 004, 105, 217, 301 and 413, according to JCPDS card no 21–1272. Figure 8.10 shows the SEM results reflecting the changes that occurred on the morphology of nanofibers after using them for photocatalysis for degradation of dairy effluent. From the SEM imaged (Fig. 8.10), it is evident that nanofibers

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Fig. 8.8 UV spectra of the nanofibers membranes. Spectra of pure polyurethane nanofiber (a), spectra of nanofiber incorporated with Ag-TiO2 (b), spectra of nanofibers incorporated with ceramic Ag-TiO2 nanofibers (c). Obtained with permission from (Kanjwal et al. 2015b)

Fig. 8.9 XRD spectra of pure polyurethane nanofiber (a), spectra of nanofiber incorporated with Ag-TiO2 (b), spectra of nanofibers incorporated with ceramic Ag-TiO2 nanofibers (c). Obtained with permission from (Kanjwal et al. 2015b)

retained the fibrous shape after using them for more than 2 h. It is noteworthy to mention that samples were taken for SEM analysis after the second run, dried in an oven at 80  C for 24 h and viewed under SEM. These results indicate the mechanical integrity of nanofiber webs after going through the harsh conditions, therefore, are proved to be a better alternative to the existing filtration devices.

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Fig. 8.10 The SEM results of the nanofibers after using them in the catalytic reaction. Pristine polyurethane nanofibers (a), incorporated with AgTiO2 nanofiber (b) and incorporated with AgTiO2 nanoparticles (c). Obtained with permission from (Kanjwal et al. 2015b)

Fig. 8.11 Photocatalytic degradation using polyurethane nanofibers containing nanofibers and nanoparticles for two runs. Obtained with permission from (Kanjwal et al. 2015b)

The property of polyurethane nanofibers (with AgTiO2 nanofiber and nanoparticles) to show photocatalytic degradation of dairy effluent is shown in Fig. 8.11. In the case of pristine polyurethane nanofiber, the less than 10% degradation of dairy effluent occurred within 2 h. Surprisingly, the polyurethane nanofibers modified with AgTiO2 nanoparticles showed 50% of deterioration to diary effluent after 5 min and this continued increase 75% till 2 h. While in the case of nanofibers modified with AgTiO2 nanofibers, 70% of degradation occurred till 5 min and 95% of degradation occurred after 2 h. Moreover, the pH values of 4.47 remain the same before and after degradation.

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Conclusion

In conclusion, the first part of this chapter, electrospinning, was used to prepare nanofibers from TiIspr/GeIsp/PVAc and TiIspr/CdAc/PVAc. Post-electrospinning, these as-spun fibers were calcined to remove the sacrificial polymer and acetate from metallic precursor to obtaining ceramic fibers of TiO2-CdO and TiO2-GeO2. On the other hand, in the second part of this study, the polyurethane nanofibers were incorporated with ceramic nanofibers and nanoparticles of AgTiO2 using colloidal electrospinning. Both the nanofibers, which are in the form of powder as well as in the form of membranes, were tested for morphological, stereoscopic, water photosplitting and photocatalytic properties. Overall, both the nanofiber combinations resulted in having excellent photocatalytic activities. Acknowledgments The financial support for this work was given by the Danish Strategic Research Council (DSF-10-93456, FENAMI Project).

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