Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery 0323851991, 9780323851992

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Woodhead Publishing Series in Biomaterials

EMERGING APPLICATIONS OF CARBON NANOTUBES IN DRUG AND GENE DELIVERY

Edited by

PRASHANT KESHARWANI Assistant Professor, Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

Woodhead Publishing is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2023 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-85199-2 For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Matthew Deans Acquisitions Editor: Sabrina Webber Editorial Project Manager: Joshua Mearns Production Project Manager: Prem Kumar Kaliamoorthi Cover Designer: Mark Rogers Typeset by TNQ Technologies

Contributors Mohammad A.S. Abourehab Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt Mustafa A. Alheety Department of Nursing, Al-Hadi University College, Baghdad, Iraq Duygu Beduk Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey Sanghamitra Chatterjee Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai, Maharashtra, India Pavan Kumar Chintamaneni Department of Pharmaceutics, GITAM School of Pharmacy, GITAM-Hyderabad Campus, Hyderabad, Telangana, India Rambabu Dandela Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India Mahdieh Darroudi Department of Physiology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, Iran Ceren Durmus Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey Lopamudra Giri Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India Israel González-Méndez Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, Mexico Kenguva Gowtham Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India Simge Balaban Hanoglu Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey

ix

x

Contributors

Duygu Harmanci Central Research Test and Analysis Laboratory Application and Research Center, Ege University, Bornova, Izmir, Turkey Gowtham Kenguva Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India Prashant Kesharwani Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India Renat R. Khaydrov Institute of Nuclear Physics, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan Majid Khazaei Department of Physiology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, Iran; Metabolic Syndrome Research Centre, Mashhad University of Medical Science, Mashhad, Iran Praveen T. Krishnamurthy Department of Pharmacology, JSS College of Pharmacy (JSS Academy of Higher Education & Research), Ooty, Tamil Nadu, India G. Kusuma Kumari Department of Pharmacology, JSS College of Pharmacy (JSS Academy of Higher Education & Research), Ooty, Tamil Nadu, India Javier Lara-Romero Facultad de Ingeniería Química, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México Ahmed R. Mahmood Department of Medical Laboratory Technology, Imam Ja’afar Al-Sadiq University, Kirkuk, Iraq Abdulwahhab H. Majeed Department of Chemistry, College of Science, Diyala University, Diyala, Iraq Leqaa A. Mohammed Department of Chemistry, College of Science, Diyala University, Diyala, Iraq Seyedeh Elnaz Nazari Department of Physiology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, Iran Ammu V.V. V. Ravi Kiran Department of Pharmacology, JSS College of Pharmacy (JSS Academy of Higher Education & Research), Ooty, Tamil Nadu, India Majid Rezayi Medical Toxicology Research Centre, Mashhad University of Medical Science, Mashhad, Iran; Metabolic Syndrome Research Centre, Mashhad University of Medical Science,

Contributors

xi

Mashhad, Iran; Department of Medical Biotechnology and Nanotechnology, School of Science, Mashhad University of Medical Science, Mashhad, Iran Ernesto Rivera Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, México Smruti Rekha Rout Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India Andrea Ruiu Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, México Amirhossein Sahebkar Applied Biomedical Research Center, Mashhad University of Medical Science, Mashhad, Iran; Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Kendra Sorroza-Martínez Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, Mexico Suna Timur Central Research Test and Analysis Laboratory Application and Research Center, Ege University, Bornova, Izmir, Turkey; Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey; Department of Biochemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey

CHAPTER 1

Background of carbon nanotubes for drug delivery systems Mahdieh Darroudi1, Seyedeh Elnaz Nazari1, Prashant Kesharwani2, Majid Rezayi3, 4, 5, Majid Khazaei1, 4 and Amirhossein Sahebkar6, 7, 8 1

Department of Physiology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, Iran; 2Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 3Medical Toxicology Research Centre, Mashhad University of Medical Science, Mashhad, Iran; 4Metabolic Syndrome Research Centre, Mashhad University of Medical Science, Mashhad, Iran; 5Department of Medical Biotechnology and Nanotechnology, School of Science, Mashhad University of Medical Science, Mashhad, Iran; 6Applied Biomedical Research Center, Mashhad University of Medical Science, Mashhad, Iran; 7Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 8Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

1.1 Introduction Biotechnology researchers have developed a keen interest toward nanotechnology and have been focusing on working with nanomaterials in recent decades [1,2]. Because of their unique properties, nanomaterials are particularly well-suited for biomedical applications. They are facile to synthesize, can be modified in size, contain tunable surface chemistry, provide large surface-to-volume ratios, and are generally biocompatible [3]. All of these features make nanomaterials promising for almost all aspects of biotechnology, overcoming the many shortcomings in existing conventional materials [4]. Following a pioneering study by Higuchi et al. on albumin nanoparticles, it was suggested that nanomedicine could be an effective tool to target tumors and cancer cells as the ability to avoid immune system clearance is enhanced [5]. Nanoparticles have shown to have positive results against coronary artery disease, and cancer cells, by effectively avoiding clearance from immune system clearance [6e9]. Nanoparticles can be used to deliver a large variety of pharmaceuticals in a way that is safer (through targeted nanomedicines by limiting the amount of drug delivered) and more effective [10]. Biological and medical nanomaterials have been used for years, including liposomes [11], carbon nanoparticles [12e17], dendrimers [18], ceramic nanoparticles [19], iron oxide nanoparticles [20], titanium dioxide nanoparticles [21], magnetic nanoparticles, polymer nanocomposites [18], silica and metal nanoparticles [22]. Additionally, many different types of nanomaterials have been Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00009-1

© 2023 Elsevier Ltd. All rights reserved.

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

proposed as means of drug/gene delivery that respond to external stimuli. In addition, drugs release from within these nanocarriers are triggered by changes in pH, redox potential, enzyme activation, thermal gradients, magnetic fields, light, and ultrasound [23]. Among the nanomaterials, carbon nanotubes (CNTs) have drawn tremendous interest in the biomedical field because of both their promising properties, including high drug loading capacity [24], high stability [25], needle-like structure, high surface area [25], biocompatibility [26], flexible interaction with cargo, considerable strength, outstanding mechanical and electrical properties [27], and the ability to deliver drugs to specific tissues [28]. Despite the advantages, it also has some disadvantages related to toxicity and low biodegradability [29,30]. Although CNTs exhibit some undesirable properties, they are still being utilized in medicine in innovative ways, such as in drug delivery systems, gene delivery, gene therapy, diagnostic applications, as well as biosensors and vaccine delivery [29]. CNTs have a variety of appealing properties in biomedical applications, as shown in Table 1.1. Though CNTs have several desirable biological properties, their biosafety is often an issue of concern, particularly in regards to their use and their biomedical applications. Therefore, a comprehensive assessment of the in vivo impact of CNTs is required before wide-scale commercial biomedical applications are undertaken. Studies on CNTs have been criticized for their inaccuracies and incompleteness, ranging from animal models that aren’t representative of human exposure routes to studies that lack even a thorough description of the impurities, chemistry, charge, and dimensions of the studied CNTs [37]. Graphene sheets are rolled seamlessly as a cylindrical tube to form single-wall carbon nanotubes (SWCNT), as well as multi-walled carbon nanotubes (MWCNTs), which are composed of layers of graphene sheets stacked on one another. Moreover, there are three main methods for the manufacture of CNTs: chemical vapor deposition (CVD), laser ablation, and arc discharge [38]. In the current chapter book, we summarize promising and not-so-promising studies showing the importance of CNTs in a variety of biomedical applications. Many research studies, particularly performed in year 2016e2022, are assessed with critical insight into what these studies conclude. Herein, the uptake of CNTs, the delivery of pharmaceutical agents using CNTs has been covered. We also discuss concerns raised about the toxicology of CNTs at the end of this study and outline what and where the field urgently needs to grow.

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Table 1.1 Various applications of carbon nanotubes. Application

Description

References

Diagnosing application

Bioimaging and biosensing

Carbon nanotube’s optical, electronic and mechanical properties make it a promising material for the production of electrochemical and optical biosensors and some other applications deriving from CNTs because of their high photostability and lack of quenching

[31,32]

Therapeutical application

Photothermal therapy Drug delivery

Tissue engineering

Lab-on-chipdevices

CNTs would produce heat by converting near-infrared radiation (NIR). The unique needle-like shape of carbon nanotubes with the ability to quickly penetrate cell membranes makes them ideal carriers of drugs/genes due to their high surface area, multifunctional surface chemistry, lack of immunogenicity, and high surface area. Carbon nanotubes can be used in tissue engineering because of their biocompatibility, stiffness, mimicking of natural tissue nanofibers, cell adhesion and proliferation stimulation, and ability to form 3D structures. Miniaturized systems such as lab-on-a-chip devices are used to examine drugs, grow cells, and model diseases using tiny volumes of fluid flowing in various channels. The CNTs would be used in LOC devices as membrane channels, sensors, and channels walls.

[33] [34]

[35]

[36]

1.2 Quantitative approaches A significant application of nanoparticles is the delivery of drugs; because of their large surface areas, nanoparticles are capable of delivering large quantities of drugs or other medical cargos [39,40]. Therefore, developing efficient drug delivery systems is vital to human health [41]. Theranostic with nanotechnology for cancer has emerged as a promising field that could integrate the

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treatment and diagnosis of cancer by combining nanotechnologies with therapeutic agents to enable targeted drug accumulation in cancer-specific cells without affecting normal cells [42]. Drug delivery procedures should be significantly improved to ensure sustainability, low disruptions, and precise and accurate controlled delivery of drugs [43]. Nanomaterial-based drug delivery systems have recently been studied, and a number of breakthroughs have been noted thereafter. There have been numerous studies on drug delivery systems in recent years [44,45]. The bibliometric is insufficient to assess a research area outputs; it should include other inputs such as literature reviews to discover the insight of publications trends [46]. This research aims to explore the research status done in this field of study from past to the current year by a bibliometric approach and qualitative literature review. To ensure the reliability of the analysis and input data for the software, scientometric studies utilizing recognized database such as Google Scholar, ISI Web of Science, and Scopus have been accessed. This study used the Scopus database for its extensive coverage and comprehensive content. From 1965 to 2021, bibliometric searches were conducted in Google Scholar, Scopus, PubMed, and Web of Science Core Collections (n ¼ 70,300). Data were obtained from the online version of the core collection in Web of Science on January 15, 2022 [47]. The “Carbon nanotube*” OR “CNT*” OR “SWCNT*” OR “MWCNT*” to identify all articles related to treatment from 1990 to 2022 that contain the keyword in the title list, and 189,358 publications were encountered. Also, articles using the keyword “Drug Delivery*” AND “Carbon nanotube*” OR “CNT*” OR “SWCNT*” OR “MWCNT*” to identify all articles from 1990 to 2022 that contain the keyword in the title which, 4748 publications met the selection criteria. Upon further screening, only 3137 publications were categorized through “Drug Delivery*” AND “Carbon Nanotube*” OR “CNT*” OR “MWCNT*” OR “SWCNT*” keywords that were utilized for further analysis. Moreover, more data for this study based on title search were derived from SCOPUS, and the time span was from 1985 to 7rd February 7, 2022. Also, bibliometric studies were carried out on the Google Scholar and PubMed databases, resulting in 70,300 publications and 1353 publications. This data collection with an initial title search “Drug Delivery*” in the “Title of Article” has been done on another well-known database Scopus [47]. There were almost 38,996 documents with the title of “Drug Delivery*”; and nearly 125,277 documents with the title of “Carbon Nanotube*” while the “CNT*” OR “MWCNT*” title search terms were used to retrieve the data. The search

Background of carbon nanotubes for drug delivery systems

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returned 3571 documents from SCOPUS database. After careful inspection, a total of 1846 publications were identified as suitable for subsequent analysis from SCOPUS database. The search returned 1230 review papers, 201 letters, and 149 conference papers (Fig. 1.1). Besides, Index Keywords capture an article’s content with greater depth and variety [46]. The total number of records is 1846, and the total number of articles is 1332 in the sample period from 2012 to 2022. There are 160 author contributions in 160 journals and 10 sub-divisions in the publications. 4637 words were identified as frequently used in this literature, and 1457 institutions contributed their articles from 62 countries (Fig. 1.2). In Fig. 1.2c (inset), documents are categorized according to type in a detailed classification. The articles with 52% and 1441 records represent a significant portion of the total. Review documents accounted for 36% of the total records, with 993 records. Total documents numbered 1332 in 7 categories accounted, due to their credibility and acceptance in scientific communities, articles are strongly preferred as the document type by the authors.

Record identified throughout database including: Google Scholar 70300, Scupos 125277, WOS 189358, PubMed 1353 searching n= 386288, 347292 Duplicates removed

38996 articles excluded on the basis of not including anticancer, drug delivery, or magnetic nanoparticles

3571 Selected Full-text articles assessed for eligibility

1846 Articles 11 Review papers 201 Letters 149 Conference papers

1846 Records included in the Systematic Review

Figure 1.1 Prisma flow diagram.

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

(a)

(b)

600 500

Book Chapter 6%

400

Conference Paper 3% Article Review Book Chapter

300 Review 36%

200

Article 52%

Conference Paper Editorial Conference Review

100 0

(c)

Figure 1.2 (a) The network of keywords, (b) clustering of keywords, and (c) categories of the subject area, and inset categories by article type.

Keywords are highly influential on the mechanism and effectiveness of document searches. Keywords serve as critical links among the range of available documents to be identified as information sources. In Fig. 1.2a, the top two keywords are carbon nanotubes and drug delivery, with 459 and 337 occurrences, respectively. In order to identify the document immediately and in a timely manner, it is obvious that the exact link words or phrases should be used. Based on the screening of the 25 top keywords, we found that the most popular keywords fall into two categories: the compound name and the application of the compound. Another effective keyword with more than 50 occurrences is carbon nanotube, walled

Background of carbon nanotubes for drug delivery systems

7

carbon, single-walled, multi-walled, molecular dynamic, delivery system, multi-walled carbon, drug release, wall carbon, and cancer therapy. These keywords will be helpful in identifying the document that best describes carbon nanotubes and their applications for future researchers. Fig. 1.3 displays the occurrences of the keywords. To understand the different phases of growth of the publication numbers, the collected data was organized chronologically. Fig. 1.3 depicts

(a) 16% 14% 12% 10% 8% 6% 4% 2% 0% 2013

2014

2015

2016

2017

2018

2019

2020

2021

2022

(b)

Figure 1.3 (a) Trend topic growth between 2012 and 2022, and (b) average article published per year time span 2012e22.

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the schematic representation of yearly increases in record numbers. Research on carbon nanotube and drug delivery shows a slow onset with few publications between 2012 and 2016. The trend was downward from 2016 to 2017. As exhibited in the trend topic growth, an increase was depicted in 2017 based on both keywords carbon nanotube, and drug delivery system, which there is a good consolidation with publication trends. By 2022, there will be an increasing trend in publications. The records showed growth between 2017 and 2020 from 9% to 14%. There were 135, 164, and 178 records published in 2019, 2020, and 2021, respectively, and a percentage of 10%, 12%, and 14% was recorded. New developments and application in drug delivery and carbon nanotubes related materials explains the increase in publications since 2017. Fig. 1.4 presents a ranking of the countries involved in carbon nanotubes research according to the number of documents they have produced. India, the United States, and China produce the most documents, with 576, 518, and 514. China had 6257 citations, while the United States had 3419. It is noteworthy that the China achieved a higher ratio of citations to documents than USA. Iran is ranked 4th overall, followed by the United Kingdom in 4th place, Italy in 6th place, and South Korea in 7th place. There has been a noticeable increase in research in Carbon nanotube usage in the drug delivery system as shown by the reported achievements. The majority of the contributions originate from China, India, and the United States. Several country collaboration clusters show the importance of group research contributions and the current trends. Chart 4C depicting various clustering patterns of Cluster 1 identifies it as the most productive. Furthermore, Fig. 1.4a presents a geographic map of the countries involved. This graph is drawn by the VOSviewer package (www.vosviewer.com), which is a tool for comprehensive science mapping analysis for quantitative research in bibliometrics [48,49]. Also, the red color intensity states the four highest number of related documents published in each country. From the data in the graph, Fig. 1.4a exhibited that between 50 countries, India, China, and the United States have specialized studies earlier than other countries with the most significant number of normalized strong collaboration links to other countries with the rate of 20% of publication. China, the United States, and Iran have the rate of 18.6%, 18.5%, and 12%, respectively, after India in the mentioned topic. A three-field plot (Sankey Diagram) listing the respective institute, authors, and keywords on the considered topic is shown in Fig. 1.4c. This figure shows the relationship among top institutes, top authors, and top authors’ keywords. The top five

Background of carbon nanotubes for drug delivery systems

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Figure 1.4 Publication of carbon nanotube in drug delivery system research papers: top countries and clusters (a and b), and (c) three-field plot of top-author, topcountries; and top authors’ keywords on the considered topic.

institutes in which these documents were published included “Ministry of Education China” (58 DOC), “Islamic Azad University” (53 Docs), “Chinese Academy of Sciences” (42 Docs), “CNRS center national de la Researche Scientifique” (35 Doc), and “Tehran University of Medical Sciences” (31 Docs). The top five authors also include Jain, N. K, Mehra, N. K., Kostarelos, K., Raissi, H., and Bianco, H., based on drug delivery and carbon nanotube keywords.

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

Figure 1.4 Cont'd

1.3 CNT morphology and structure The cylindrical sheet of graphene with carbon atoms bonded as sp2 hybridization is thought to resemble hollow fullerene tubes. The carbon nanotubes are composed of carbon sheets made from rolled graphite, which is a source of carbon allotropes as well as graphite and fullerenes [50]. In many fields, carbon nanotubes are used in the form of buckytubes, with their cylindrical shape and unique properties. In addition to their mechanical, thermal, optical, and electrical properties, they also possess the following characteristics [51]. High stiffness and robustness abound in nanotubes, which also permit reversible collapse and buckling. In hexagonal networks, the high axial Young’s modulus (degree of stiffness) results in tensile strengths of 150 GPa due to the high CeC bond rigidity [52]. CNTs are thus among the most rigid materials known while still being able to buckle (elastic deformation) when subjected to compression forces [53]. In addition to the rudimentary constituents, carbon assemblies form many different configurations and shapes [54]. Under high pressure, nanotubes can be assimilated, resulting in a strong, infinite-length wire by replacing several sp2 bonds with sp3. The uncovered CNTs were discovered in recent years by Irjima, who described multi-walled CNTs in carbon soot produced by the development of the C60 molecule in an arc evaporation method for the first time [54].

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1.4 Classification of CNTs Nanotubes may indeed be alienated into various classifications based upon the number of sheets of graphene existent in the CNTs. The following are therefore sub-divided into: 1.4.1 Single-walled CNTs (SWCNTs) Single-walled CNTs are formed by orienting a graphene sheet on a single wall. A catalyst is required for the synthesis of SWCNTs, resulting in a nonpure CNT without complexity with a readily twisted structure [55]. Sometimes, they appear as fluffy black powder or granular flakes with a metallic appearance [56]. A variety of SWCNTs are available, which can be trundled up as a seamless tube in multiple ways. When organized according to their chirality and diameter, SWCNTs may act more like clearly delineated semiconducting, metallic, or semi-metallic structures [57]. There is a new technique published in US patients for preparing arrays or tightly packed bundles of single-walled CNTs, with a diameter of less than 0.2m that would be helpful in meeting commercially feasible reaction requirements [51]. 1.4.2 Multi-walled CNTs (MWCNTs) These are the CNTs that contain multilayered graphene rolled over themselves have varying diameters ranging from 2 to 50 nm which in turn depends on the number of tubes [58]. Its synthesis is catalyst-free, and the MWCTs are highly complex and cannot be twisted easily [51]. These tubes have a distance of approximately 0.34 nm in between layers [59]. Their typical form appears in form of fluffy black and granular powders. There are MWCNTs of two different types based on the graphitic sheet arrangement pattern. As an example of this, we can look at the Russian-doll model where the graphitic sheets form concentrically aligned sheets, for example, a length of (0, 14) SWCNT surrounding a shorter length (0, 12) SWCNT. Graphite is rolled around itself in the second model, resembling a scroll of parchment or a rolled-up newspaper [60]. 1.4.3 Double-walled carbon nanotubes (DWCNTs) There is another type of CNT that is similar to SWCNTs which also has two concentric layers that enclose both the inner and outer tubes of these nanotubes. These nanotubes are of serious concern in the pharmaceutical industry [55].

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1.4.4 CNT types based on chirality Chirality is a crucial factor in determining the electrical properties of CNTs. Depending on the chirality, CNTs can be categorized as an armchair, zigzag, and chiral, as illustrated in Fig. 1.5 [60]. In the armchair, an arrangement of bonds in one of the chairs is perpendicular to the tube axis; in zigzag, there is an arrangement of bonds in which the tube has a V shape perpendicular to the tube axis. Chiral or helical configuration are both contrary to the above two types [57,61]. Conductivity and electrical characteristics of CNTs are both effective indicators of CNT chirality and may be used as a basis for nanoelectronic devices [62,63]. Based on their structural transformations, preparation technique, and solubility characteristics, CNTs are categorized into specific contexts, such as functionalized, surfactant-assisted, solvent dispersed, and biomolecular assisted nanotubes [64].

Armchair Zigzag

(a) Armchair

(b) Chiral

(c) SWCNTs

(d) MWCNTs

Figure 1.5 Type of carbon nanotubes.

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1.5 Drug loading on carbon nanotubes The term “drug loading” refers to the combination of drug and carrier, which delivers the active medication to the target cells or tissues. CNTs are widely used for this purpose, particularly for large-scale drug delivery because of their high surface-to-volume ratio and sphere-shaped shape [65]. Moreover, amphiphilic or hydrophilic polymers on CNT surfaces can increase their loading capacity [66,67]. In addition to this, the chemical functionalization of nanotubes’ surfaces could also enhance CNTs’ biocompatibility [68,69]. Modification of CNTs can be achieved through the covalent attachment of PEG layers on their surface, PAMAM dendrimers on their surface, amphiphilic deblock copolymers on their surface, or dispersing the modified materials in a matrix of hyaluronic acid. The mechanical strength of CNTs, like SWCNTs, enhances the properties of both polymeric and non-polymeric composites [70]. It is also noteworthy that these nanomaterials can also carry pharmaceutical agents by encapsulating them within hollow cavities [71], adsorbing them within the walls of CNTs, and binding them to their surfaces upon functionalization [72]. A drug delivery system involving encapsulation has more advantages because the drugs are released in a specific way within the targeted cells while preventing their degradation [73]. Table 1.2 shows how therapeutic drugs

Table 1.2 Mechanism of loading drug. Entry

Drug

1 2

Pregabalin Doxorubicin

3

Ifosfamide

4 5

Paclitaxel Doxorubicin

6

Cisplatin

7 8

Doxorubicin Ciprofloxacin

9

Doxorubicin

Type of carbon nanotube

Process of immobilization

References

SWCNT Armchair and zigzag CNT Armchair SWCNT f-MWCNT Covalent-fMWCNT Carboxyl-fMWCNTs MWCNT-FA PEG/GelChitMWCNT f-SWCNT

Encapsulation Adsorption

[74] [75]

Encapsulation

[76]

Encapsulation Adsorption and encapsulating of drug Encapsulation

[77] [78]

Encapsulation Grafting in nanocomposite matrix

[80] [81]

Encapsulation

[82]

[79]

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

would be attached to different types of carbon nanotubes. Various chemical and electrical methods are available for controlling the release of drugs from CNTs. CNTs can also be sealed with polypyrrole (PPy) film in order to prevent drug release [83]. Drug delivery systems could be made more selective by using homing devices, such as epidermal growth factor (EGF), or folic acid [84]. A wide range of biomedical applications may be possible for CNTs as a drug delivery carriers. Unlike encapsulation or “endohedral modification” [73], these CNTs serve to maintain the structural integrity of drugs, which helps to minimize their degradation and in turn maximize their release from within them under controlled conditions [85]. Due to hydrophobic and capillary forces, this approach is most useful for drugs with low surface tension. For instance, Fig. 1.6 shows how nanoparticles of gold are incorporated into the carbon nano bottles to prevent the uncontrolled release of the encapsulated drug (cisplatin) [86]. When the drugs are loaded by exohedral modification (as opposed to encapsulation), it becomes much more exposed. Tethering refers to covalent interactions with noncovalently attached drugs; as one of the bio-conjugation methods. A further step of oxidation is required in the tethering process before functional groups can be produced for conjugation. Additionally, a covalent bond may change the drug’s molecular structure, thereby altering its bioactivity and specificity [87]. This tethering approach is superior due to strong covalent bonds that produce a stable platform

Figure 1.6 Schematic illustration of cisplatin delivery system and possible internalization method of carbon nanotube including loading of the drug on the surface of carbon nanotube, then cisplatin carried inside, in vivo study of current nanocarrier and pH-responsive releasing drug leading to decreasing of tumor size.

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between nanocarriers and drugs rather than the non-covalent method, which ultimately causes the undesirable dissociation of therapeutic agents in biological fluids. CNTs are generally used to deliver drugs to target cells in the following way. Modified CNTs contain chemical receptors that attach to drug molecules. Drug molecules are transported by these chemical receptors inside nanotubes [88]. Depending on the method used, the received conjugate can be injected into the body through injection, oral administration, or directly upon contact with the targeted cell. Using the endocytosis pathway, the drug-loaded into CNT capsules can be internalized by the chemical receptors and eventually emitted by the cells [88]. Fig. 1.6 is a schematic illustration of an effective drug delivery system. 1.5.1 CNTs and cellular uptake It is crucial to understand how drugs are taken up by the projected cells to comprehend drug pharmacokinetics and pharmacodynamics. There must be a better understanding of biochemical pathways and drug permeation across cellular membranes. Drugs with high solubility are thought to be absorbed primarily through diffusion through the lipid bilayer. However, in the case of high molecular weight drugs, such mechanism is impaired, as low lipophilicity interferes in passing through the bilayer structure [89]. CNTs have remarkable properties that allow them to be absorbed by a wide variety of cells. CNTs can penetrate cell membranes efficiently because of their needlelike shape, which can be either good or bad depending on the intended application. Hence, CNTs are suitable for a range of biomedical applications, including the delivery of therapeutic agents and genes [90]. Although many studies have been conducted on cellular uptake, there are still many questions regarding cellular pathways initiated by CNTs and the delivery of therapeutic agents and genes [91]. Furthermore, not only is it essential to determine how cells take up CNTs, but if the CNTs will deliver drugs viably to the cells. Therefore, it is extremely important to carefully study CNT internalization. Several (not one) pathways, depending on the properties of the CNTs, have been elucidated for cellular uptake of CNTs [92]. Some comprehensive reviews show that there is no single mechanism for cellular uptake of CNTs. In particular, CNTs can be internalized via various mechanisms, including (1) direct penetration through the cell membrane or (2) passive and active uptake, which have also been called independent and dependent pathways, accordingly [93]. Below is a summary of the current literature on CNT entry into various cells, emphasizing dimensions, cell types, and CNT faces (Fig. 1.7).

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

Figure 1.7 Cellular uptake of carbon nanotube into endocytic vesicles.

The distribution of CNTs within cells can occur either passively through diffusion across the cell membrane lipid bilayer or through needle mechanisms. CNTs overcome such barriers due to their needle-like structure and high respect ratios [27]. CNTs can also be internalized by endocytosis, which can be classified into five groups: phagocytoses, pinocytosis, caveolin-mediated endocytosis, clathrin/clathrin-mediated endocytosis, and clathrin/caveolae independent. A phagocytic pathway is the process in which large particles (w1 mm) enter cells. It predominantly occurs within neutrophils, macrophages, and monocytes. Cellular uptake occurs primarily through receptor-mediated endocytosis, which involves clathrin-coated endocytic vacuoles. Caveolae gets invaded by nanomaterials 60 nm in diameter, which contains high levels of cholesterol and sphingolipids. The Caveolin-mediated endocytosis process is responsible for transporting vesicular material and entrapping viruses and bacteria. Caveolin and clathrin are known to play a role in cellular endocytosis and can facilitate the internalization of CNTs with a diameter of 100 nm. The macropinocytosis mechanism is thought to take up CNTs that are larger than 300 nm. While the entry of CNTs and their drug cargo may offer many advantages, each mechanism also has drawbacks that should be considered. There are several features that influence cellular uptakes, such as CNT types (MWCNTs or SWCNTs), dimension, surface charge, surface functionalization, degree of aggregation, functional group chemistry, cell type, and agglomeration [94].

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1.5.2 Size of CNTs The previous studies demonstrated that CNTs with a smaller diameter result in a higher cellular uptake [22,95]. A number of mechanisms are involved in the passage of short CNTs through cell membranes, but active endocytosis, particularly clathrin-mediated endocytosis, is generally the most important [96,97]. Indeed, the shorter the CNT, the greater the chance of passive internalization. In this regard, Zhang et al. evaluated cell uptake based on CNT particle size and compared CNT particle sizes of eight types, consisting of MWCNTs and SWCNTs, with macrophages (RAW264.7), ranging in sizes from 30 to 400 nm. Their results indicated that macrophages were able to take up CNTs in more significant quantities when their particle size increased after increasing cytotoxicity, with energy-dependent phagocytosis as the primary mechanism of cellular uptake [98]. 1.5.3 Degree of agglomeration and aggregation The degree of agglomeration and aggregation of CNTs may affect their internalization. A study by Song et al. found that higher concentrations of O-MWCNTs resulted in higher uptake within human epithelial cervical cancer cells (HeLa). However, these cells were not cytotoxic at concentrations of less than 150 mg/mL [99]. The amount of agglomeration and toxicity increased slightly when the concentration of O-MWCNTs was raised to 150 mg/mL. According to their findings, agglomeration assisted endocytosis of O-MWCNTs occurred due to their ability to interact effectively with cells. A regulated agglomeration of CNTs in some delivery systems is capable of facilitating the delivery of drugs/genes through their high uptake and low toxicity. A study conducted by Kuroda et al. demonstrated that aggregated CNTs enhances uptake in RAW264 cells [100]. This suggests that aggregation would be effective on uptake mechanisms. 1.5.4 Surface charge CNTs can be modified for their surface charge by altering the electrostatic interactions and dispersibility. Consequently, the surface charge would be relevant to the uptake of NTs by cells, as well as other biological processes [101]. The SWCNTs were functionalized by Budhathoki-Uprety et al. using polycarbodiimide polymers with carboxylic acids (COOH-CNT) or primary amines (NH2-CNT) attached to their side chains, which these

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complexes have surface charges of approximately 66.8 and 52.8 mV, respectively [102,103]. Cationic nanotubes could be more readily incorporated into HeLa cells than anionic nanotubes. However, serum proteins in the culture media adsorb CNTs in the cell culture media and influence cellular uptake of these CNTs. It is important to remember that the proteins adsorb to the nanomaterials that cells recognize, not the nanomaterial itself. That is referred to as the “protein corona effect” of nanomaterials, a layer of proteins adsorbed to a nanomaterial when exposed to body fluids [104]. CNTs can therefore be designed so that they can be taken up by cells. Consequently, CNTs can be designed to deliver drugs efficiently when their surface chemistry is taken into account. The reactivity of nanomaterials’ surfaces with cellular membranes contributes to their toxicity. It is the nonbiodegradability of CNTs that gives them their toxicological properties. The binding of blood proteins influences biological pathways of the CNTs, and cytotoxicity can therefore be decreased as a result [105]. According to Ge et al., blood proteins bind to the surface of SWCNTs, enabling changes to their cellular interactions. This ultimately reduces their cytotoxicity in two different human cell lines, including human umbilical vein endothelial and acute human monocyte leukemia (THP-1) cells. 1.5.5 Cell type Cellular uptake rates and mechanisms may vary by the cellular system [106]. The internalization of CNTs was evaluated in human lung cancer cells A549, human lung cancer cells Calu-6, human breast cells MCF-7, and mouse macrophages J774 by Summers et al. He in his work demonstrated that the highest CNT uptake occurred in the J774 cell line [101]. After exposure to CNTs for 24 h, A549 cells had a w40% lower uptake than J774 (which showed the highest uptake). There was no significant difference between the MCF-7 and Calu-6 cells, but both types comprised of about 30% J774 CNTs. Additionally, macrophages could take up SWCNTs more efficiently than fibroblasts. Although macrophages preferentially phagocytose particles with a diameter of over 500 nm, fibroblasts primarily endocytose particles with a diameter of 200 nm and less [107]. In addition, J774 cells can take up larger particles and also the aggregates of CNTs. By aggregating CNTs within phagocytic cells, nanoparticles are retained within them, which makes them ideal carriers for CNT transportation into tumor cells.

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CNTs and their cellular uptake have been analyzed using a wide range of techniques, including atomic force microscopy, transmission electron microscopy, dynamic light scattering, fluorescence microscopy, surfaceenhanced Raman scattering, and confocal Raman spectroscopy. The characterization and visualization of cellular uptake of CNTs are based on some characteristics of CNTs, such as optical characteristics [107e109]. The evolution of nanotechnology began with improvements in microscopy, and this here depicts is no lesser truth in terms of CNT cellular internationalization.

1.6 Drug delivery using carbon nanotubes In recent decades, biomedical researchers have focused significant attention on using CNTs for various purposes due to their high electrical, physicochemical, and mechanical properties and the high aspect ratio of CNTs. In addition, they have been used as nanocarriers for the delivery of several plasmid DNAs [110], proteins [111], peptides [112], siRNAs [113], and API [114]. 1.6.1 Antineoplastic APIs Worldwide, cancer ranks second behind cardiovascular diseases. Various unwanted toxic effects have been observed in the surrounding healthy tissues as a result of APIs therapy [115]. One of the most challenging aspects of therapeutic research remains drug delivery to cancerous cells [116]. This is because tumor cells express P-glycoprotein (P-gp), which will block the entry of therapeutic agents into the tissue. Therefore, most of the tumortargeted antineoplastic agents are unexpectedly destroyed before they can kill the targeted cells. For the treatment of cancer, innovative technologies are needed for delivering therapeutic agents [117]. Different nanomaterials have previously been utilized as drug delivery agents. A wide variety of antineoplastic agents can be loaded in CNTs, including doxorubicin, cisplatin, methotrexate, and camptothecin. As an example, cisplatin has been used to treat a variety of cancer-related disorders. Cisplatin stimulates cellular death by inhibiting DNA replication and cross-linking between DNA strands. Despite its anticancer effect, this drug produces many side effects, including ototoxic, nephrotoxic, and neurotoxic effects. The chloride ions in plasma can obliterate the therapeutic effect of drugs by causing an interference in their interaction with water. Due to this,

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CNTs are used as drug carriers to reduce side effects by preventing the deactivation of therapeutic agents. In one study, the synthesis of functionalized SWCNTs containing cisplatin to target prostate cancer cell lines PC3 and DU145 was reported. Cisplatin acts by penetrating deep within the cellular membrane and selectively accumulating within the cytoplasm of cancerous prostate cells based on findings of cellular uptake studies. Therefore, the encapsulation of cisplatin has proved to be very effective factor in stabilizing its administration to cancerous prostate cells [118]. It has also been extensively used for treating cancer with doxorubicin drug as an anticancer therapeutic agent. The chemical structure of doxorubicin has been shown in (Fig. 1.4b). The non-covalent complexation of MWCNTs and functionalized SWCNTs with polyethylene glycol (PEG) was also demonstrated by Hwang [119]. In an investigation, it was found that MWCNTs combined with doxorubicin work better against cancerous cells than doxorubicin alone. SWCNTs have been synthesized and modified with different types of polysaccharides as well as with doxorubicin and folic acid in recent years [120]. In some cases, the release of therapeutic agents from within these nanocarriers was reported to be pH dependent. In this regard, CNTs are functionalized on the surface to control the rate of drug release and loading efficiency. According to Fabbro et al. research [116], a novel method is essential for improving the construction and characterization of modified CNTs to allow them to be applied therapeutically. 1.6.2 Anti-inflammatory APIs Many studies have been conducted in order to enhance cellular uptake properties over the years. In addition to improving the molecule release profile, this also helps in reducing side effects [121]. Meanwhile, researchers have also been looking at CNTs as a means of delivering anti-inflammatory APIs. Zanella et al. (2007) investigated an anti-inflammatory drug known as nimesulide on both Si-doped capped and pristine SWCNTs using firstprincipal calculations (DFT). Based on DFT calculations, they reported the use of CNTs as potential carriers for aromatic residues and have also demonstrated improved physisorption with Si-doped SWCNTs due to their electronic properties [122]. Due to the inefficiency of conventional administration strategies, antiinflammatory delivery systems provide stable plasma levels of the therapeutic agent over a longer period of time, allowing for extended drug

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release profile. Additionally, osmotic pressure from the membrane surrounding the therapeutic agent causes the drug to be absorbed from the carrier. In one investigation, Madaenia et al. increased the hydrophilicity of MWCNT by adding cellulose acetate, which resulted in increased indomethacin release from the membrane [123]. One alternative drug release technique called “stimulated drug release” involves releasing therapeutic agents from a vehicle in response to various stimuli, such as temperature, pH, and other physiological conditions. Increasingly complex and intelligent systems can also be derived from electrical signals and through microsystems [124]. Electrical signals have stimulated dexamethasone release via SWCNTs-chitosan hydrogel films developed by Arti Vashist and colleagues. In this paper, the authors investigated whether electrostatic interactions would encompass indomethacin on functionalized SWCNTs. The CNTs are fully reversible once the charge is activated, and as a result, indomethacin is released due to electrostatic repulsion. Accordingly, SWCNTs can produce electrical signals that can control the release of therapeutic agents, improving the bioavailability of active molecules [125]. 1.6.3 Cardiovascular APIs Cardiovascular diseases are heart and blood vessel disorders that cause death in a large number of people worldwide [91]. In recent years, conventional medicine has become the preferred method of treating cardiovascular disease. However, in the treatment of atherosclerosis and other cardiovascular disorders, the use of anti-cardiovascular medications is confined by their failure to cross the endothelium layers effectively. Using rosiglitazone, for example, to treat atheroma through macrophage infiltration into atherosclerotic wounds, the drug acts as an agonist of the peroxisome proliferatoractivated receptor. The mechanism of action of this therapeutic agent may be viewed through its toxicity to normal healthy cardiovascular tissues, which can then result in the development of undesirable side effects such as fluid retention (fluid retention) and heart failure [93,94]. Another delivery system is the macromolecular and thiomersal [126] and utilization of silica particles within the delivery system [96] to deliver anti cardiovascular drugs. In cardiology, nanoparticles based on silica are used to deliver annexin V, which is crucial for achieving nanomaterials [127]. One study described how CNTs have progressed impacts on treating cardiovascular disease [128]. Additionally, Liu et al. investigated the feasibility of

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interactions between SWCNTs and nifedipine in order to further understand: (1) interaction between CNTs and nifedipine, (2) absorption of the therapeutic agent by the CNTs, and (3) the process of energy translocation. It is mainly used to treat angina pectoris, hypertension, and other cardiovascular disorders by antagonizing calcium channels. CNTs spontaneously encapsulate and adsorb nifedipine via van der Waals interactions by their internal cavity [129]. However, modified nanotubes are better at encapsulating therapeutic agents than pristine MWCNTs due to their hydrophilic nature [130]. The researchers used pristine and oxidized CNTs in the same study to investigate the loading mechanism of the therapeutic agent carvedilol. This drug works principally as an adrenoceptor/vasodilator antagonist for treating hypertension but may be beneficial to protecting the heart and nervous system as well. For the loading of carvedilol into the internal cavity of CNTs, the mentioned study used three different approaches, namely a solvent method, a wetness impregnation method, and a fusion method. Each approach contributes to the differences in loading capacity and carvedilol’s physical state. 1.6.4 Anti-infective APIs The emergence of strong microorganisms that are resistant to many broadspectrum antibiotics makes it even more challenging to treat infectious diseases in this modern age. In contrast, APIs that are used to treat infection tend to have poor physisorption by the cells because of their poor solubility and penetration [131]. Antibiotics remain a crucial part of modern medicine. Even so, the invention of new therapeutic agents is a long and costly process involving long-term laboratory studies and clinical trials. Consequently, currently available carbon-based nanomaterials like CNTs are used to deliver therapeutic agents in infected areas, that can in turn help overcome bacterial resistance, and enhance drug solubility [132,133]. Amphotericin B is a common agent used for treating fungal infections that is less soluble in water. It is not recommended to administer this drug parenterally because the chances of aggregation in the bloodstream is high. This issue has been addressed through several strategies, including formulation of amphotericin B with MWCNT: to increase its cellular uptake, enhance its therapeutic effect against different pathogens, enhance its dispersion in water, and reduce aggregation in the bloodstream after administering parenterally [134]. In addition to Candida albicans, Candida parapsilosis ATCC 90118, and Cryptococcus neoformans ATCC 90112, conjugation

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of Amphotericin B and MWCNT has been reported to have significant antifungal effects. Furthermore, API modified with MWCNTs showed no toxicity to mammalian cells when absorbed rapidly by cell membranes. MWCNTs can protect therapeutic agents, such as dapsone, from being metabolized by antibacterial agents. Thus, in the liver, the cytotoxicity is substantially reduced due to an increase in anti-mycobacterial activity [133]. 1.6.5 Gene therapy A new technology, gene therapy, was developed in the 1980s that allowed cells to deliver nucleic acids to treat genetic defects. Viruses and non-viral genetic systems require reliable and robust delivery systems to transport nucleic acid into their targeted cells. The transport of genes within the cell is also studied extensively with nanomaterials, such as carbon nanotubes, polymeric nanoparticles, and liposomes. Their immunogenicity, nucleic acid diameters, and suitability for scale-up processes make them excellent choices for nanoarchitecture [135]. It is generally recognized that cationic polyelectrolytes like dendrimers, protamine sulfate, and polylysine offer us an option for developing non-viral mechanisms of DNA delivery [136]. These systems enhance the ability of the cell to absorb DNA by endocytosis and safely transport it to the nucleus following non-covalent binding with the nucleic acid. There are several polycationic functionalized carbon nanotubes that have demonstrated the capacity to transport human DNA plasmids using polycationic carbon nanotubes. The findings demonstrate the transport of plasmid DNA around carbon nanotubes and the success of the therapeutic system to increase gene expression in marker genes. These studies confirm the feasibility and effectiveness of this approach [137]. 1.6.6 The delivery system using conjugated CNT-liposomes As an excellent vehicle for therapeutic agent delivery, CNTs are extensively explored due to their ease of transport by cellular membranes. CNTs bind to the covalent structure of liposomes and form conjugates with them [138]. Covalently attached liposomes are a reliable method for delivering therapeutic agents across cellular membranes. CNT can be loaded with a wide range of drugs which can be used to treat specific targets due to the cavity inside each CNT. CNTs modified in this way have opened up new possibilities in biological investigation. Various functionalized-CNT devices are reported to have demonstrated stable biological applications, including better cellular uptake of drugs and nucleic acid delivery [117,139,140].

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1.7 The safety profile of CNTs in terms of toxicology In modern medicine, the use of carbon nanotubes may provide a means for simultaneously addressing many of the difficulties associated with conventional healing methods [115,141]. One of the main issues that need further research for these nanomaterials is the toxicological profile they exhibit. Researchers at first emphasized the safety profile of CNTs and argued that CNTs have no apparent toxicity [142]. However, researchers have suggested that CNTs can produce toxic effects, especially when administered intravenously and pulmonarily [140,141]. The biomedical application of this method has been restricted by the inconsistency observed in different studies. It became apparent that a novel method should be developed for the synthesis of CNTs and that accurate methods should be used for characterization. There are many other strategies that have proven effective, though many have faced challenges. Polyethylene glycol (PEG) functionalization [81], diethylenetriamine pent acetic dianhydride surface modification [119], immunoglobulin surface modification [143], manipulation with genetic material [144], adhesion to blood proteins [145], conjugation with peptides [145], and vitamin E surface coating [146] are just a few. In summary, it is very difficult to atomize CNTs in their pure form in organic solvents or in aqueous solutions. Consequently, pure CNTs are more toxic than functionalized ones, which may to some extent alter the biological responses. CNTs must be functionalized in a way that loads small molecules to the surface of the nanotube in order to disperse in solvents [147].

1.8 Conclusion Due to the inherent physicochemical properties, high aspect ratio, electrical and mechanical properties, and their ability to be physisorbed to the cell membrane, CNTs have been brought to wide use in recent years for delivering therapeutic agents to the targeted tissues and cells. Because of CNTs’ good compatibility with all manifold functionalization, systemic toxicity is reduced, and therapeutic agents are delivered more efficiently. Furthermore, CNTs can be coated with a variety of substances that serve distinct purposes, including diagnostic agents that confirm the efficacy of drug delivery systems, stealth agents that evade the immune system, targeting agents that minimize side effects, and drug carriers that ensure therapeutic benefits. As compared to conventional methods, such as

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spraying therapeutic agents along the infected tissues, CNTs are also proving to be a promising treatment for various cancer types. In spite of this, the hydrophobicity of CNTs poses a significant challenge in their application. Because CNTs are nonbiodegradable and non-disposable, we are still unclear of their natural cycle after drug release in the cells. According to some studies, carbon nanotubes can deliver genes, drugs, and antigens across cell lines with little cellular toxicity. However, the potential toxicity and cellular uptake of CNTs in biological systems remain uncertain. Numerous in-vitro and in-vivo studies conducted by researchers have been inconclusive as additional evidence for different cell lines has been established. Various concerns were discussed in these reports concerning the size of CNTs, the multiple attachments of ligands, the accumulation of these nanoparticles in the body after administration, various surface properties and physiological properties of CNTs, and the possible occurrence of hypersensitivity reactions in patients injected with these nanoparticles. CNTs can be chemically functionalized using amphiphilic molecules such as phospholipids and PEGylated polymers. Therefore, synthesis method must be further investigated from a toxicological perspective in order to minimize the toxicity of carbon nanotubes in biological medium.

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[130] Y. Li, T. Wang, J. Wang, T. Jiang, G. Cheng, S. Wang, Functional and unmodified MWNTs for delivery of the water-insoluble drug Carvedilolda drug-loading mechanism, Appl. Surf. Sci. 257 (2011) 5663e5670, https://doi.org/10.1016/ j.apsusc.2011.01.071. [131] E. Imbuluzqueta, C. Gamazo, J. Ariza, M.J. Blanco-Prieto, Drug delivery systems for potential treatment of intracellular bacterial infections, Front. Biosci. 15 (2010) 397e417, https://doi.org/10.2741/3627. [132] I. Banerjee, M.P. Douaisi, D. Mondal, R.S. Kane, Light-activated nanotubeporphyrin conjugates as effective antiviral agents, Nanotechnology 23 (2012) 105101, https:// doi.org/10.1088/0957-4484/23/10/105101. [133] G.D. Vukovic, S.Z. Tomic, A.D. Marinkovic, V. Radmilovic, P.S. Uskokovic,  M. Colic, The response of peritoneal macrophages to dapsone covalently attached on the surface of carbon nanotubes, Carbon N. Y. 48 (2010) 3066e3078, https:// doi.org/10.1016/j.carbon.2010.04.043. [134] W. Wu, S. Wieckowski, G. Pastorin, M. Benincasa, C. Klumpp, J.-P. Briand, R. Gennaro, M. Prato, A. Bianco, Targeted delivery of amphotericin B to cells by using functionalized carbon nanotubes, Angew. Chem. 117 (2005) 6516e6520, https://doi.org/10.1002/ange.200501613. [135] I. Ojea-Jiménez, O. Tort, J. Lorenzo, V.F. Puntes, Engineered nonviral nanocarriers for intracellular gene delivery applications, Biomed. Mater. 7 (2012) 054106, https:// doi.org/10.1088/1748-6041/7/5/054106. [136] H. Xing, M. Lu, T. Yang, H. Liu, Y. Sun, X. Zhao, H. Xu, L. Yang, P. Ding, Structure-function relationships of nonviral gene vectors: lessons from antimicrobial polymers, Acta Biomater. 86 (2019) 15e40. [137] M. Zhang, P. He, L. Dai, Carbon nanotube biosensors, in: Carbon Nanomaterials, second ed. 3, Frontiers Media S. A, 2013, ISBN 9781466502420, pp. 187e216. [138] F. Karchemski, D. Zucker, Y. Barenholz, O. Regev, Carbon nanotubes-liposomes conjugate as a platform for drug delivery into cells, J. Contr. Release 160 (2012) 339e345, https://doi.org/10.1016/j.jconrel.2011.12.037. [139] X. Liu, D. Xu, C. Liao, Y. Fang, B. Guo, Development of a promising drug delivery for formononetin: cyclodextrin-modified single-walled carbon nanotubes, J. Drug Deliv. Sci. Technol. 43 (2018) 461e468, https://doi.org/10.1016/ j.jddst.2017.11.018. [140] A. Mazzaglia, A. Scala, G. Sortino, R. Zagami, Y. Zhu, M.T. Sciortino, R. Pennisi, M.M. Pizzo, G. Neri, G. Grassi, et al., Intracellular trafficking and therapeutic outcome of multiwalled carbon nanotubes modified with cyclodextrins and polyethylenimine, Colloids Surf. B Biointerfaces 163 (2018) 55e63, https://doi.org/ 10.1016/j.colsurfb.2017.12.028. [141] S. Prakash, M. Malhotra, W. Shao, C. Tomaro-Duchesneau, S. Abbasi, Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery carriers for cancer therapy, Adv. Drug Deliv. Rev. 63 (2011) 1340e1351, https://doi.org/10.1016/ J.ADDR.2011.06.013. [142] V. Amenta, K. Aschberger, Carbon nanotubes: potential medical applications and safety concerns, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 7 (2015) 371e386. [143] V.E. Kagan, N.V. Konduru, W. Feng, B.L. Allen, J. Conroy, Y. Volkov, I.I. Vlasova, N.A. Belikova, N. Yanamala, A. Kapralov, et al., Carbon nanotubes degraded by neutrophil myeloperoxidase induce less pulmonary inflammation, Nat. Nanotechnol. 5 (2010) 354e359, https://doi.org/10.1038/nnano.2010.44. [144] A.A. Shvedova, A.A. Kapralov, W.H. Feng, E.R. Kisin, A.R. Murray, R.R. Mercer, C.M. St Croix, M.A. Lang, S.C. Watkins, N.V. Konduru, et al., Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in

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myeloperoxidase-deficient mice, PLoS One 7 (2012) e30923, https://doi.org/ 10.1371/journal.pone.0030923. [145] C. Ge, J. Du, L. Zhao, L. Wang, Y. Liu, D. Li, Y. Yang, R. Zhou, Y. Zhao, Z. Chai, et al., Binding of blood proteins to carbon nanotubes reduces cytotoxicity, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 16968e16973, https://doi.org/10.1073/ pnas.1105270108. [146] J. Wang, P. Sun, Y. Bao, B. Dou, D. Song, Y. Li, Vitamin E renders protection to PC12 cells against oxidative damage and apoptosis induced by single-walled carbon nanotubes, Toxicol. Vitro 26 (2012) 32e41, https://doi.org/10.1016/ j.tiv.2011.10.004. [147] V. Mishra, P. Kesharwani, N.K. Jain, Biomedical applications and toxicological aspects of functionalized carbon nanotubes, Crit. Rev. Ther. Drug Carrier Syst. 35 (2018) 293e330, https://doi.org/10.1615/CRITREVTHERDRUGCARRIERSY ST.2018014419.

CHAPTER 2

Properties, classification, synthesis, purification and characterization of carbon nanotubes Javier Lara-Romero Facultad de Ingeniería Química, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México

Carbon compounds are all around us; from DNA to CO2, more than a million, billion, or trillion molecules, organic and inorganic, are made of carbon. Elemental carbon has been known and used by men from ancient times. Graphite, diamond, and coal had been the familiar forms of carbon until the last 40 years, when new carbon structures were discovered. In 1996, the Nobel Prize in Chemistry was awarded to Robert F. Curl Jr., Sir Harold Kroto and Richard E. Smalley for the discovery of fullerenes, a new form of elemental carbon. Their experiments were conducted in 1985 [1]. A second Nobel Prize related to new carbon structures was awarded to Andre Geim and Konstantin Novoselov in 2010 for their groundbreaking experiments regarding the two-dimensional new carbon material, graphene. Another interesting carbon form are carbon nanotubes which are tubular structures created by rolling-up graphene sheets. They were first synthesized by Sumio Iijima in 1991 [2]. These discoveries are examples that carbon has surprised scientists in the last 35 years and more surprises are yet to come. This chapter will discuss the properties, classification, synthesis methods, purification, and characterization of carbon nanotubes.

2.1 Properties of carbon nanotubes The outstanding properties of carbon nanotubes (CNTs), which allow them to be incorporated in many applications, are directly associated to their chemical structure. It is then necessary to review the bonding of carbon atoms in these structures. First, in diamond, there are four sp3 hybrid orbitals in a tetrahedral geometry; each carbon is connected to four other Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00010-8

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carbons via s covalent bonds creating a tight tridimensional structure which makes diamond the hardest known material. There are no delocalized p electrons available in diamond which make it an insulating material. Second, in graphene, there are three sp2 hybrid orbitals in a planar geometry; each carbon is connected to three other carbons via s covalent bonds creating a planar hexagonal network. In graphite, hexagonal carbon sheets are held parallel to each other by weak van der Waals forces with a spacing of 0.34 nm. While the in-plane structure is as strong as diamond, the interaction among sheets creates a soft material where they can easily slide with respect to each other. Carbon nanotubes are hollow cylinders which are formed by rolling graphite sheets where the bonding is sp2. The circular curvature creates a distortion causing the three s bonds to be out of plane and the p orbitals to be more delocalized [3]. These facts provide CNTs some of the most exiting properties that a material can have such as mechanical strength, electrical and thermal conductivity and chemical, optical and electrical activities. 2.1.1 Mechanical properties The mechanical properties of any material are defined by its strength, elasticity, hardness, resilience and fatigue, among others. These properties depend on the type of interatomic bonds in the material. Since s bonding is perhaps the strongest type of bond found in nature and CNTs are composed of s bonds, theoretical predictions indicate that CNTs have high strength, flexibility, and resilience. Recent advances in instrumentation such as high-resolution transmission electron microscopy (HR-TEM) and atomic force microscopy (AFM) have allowed scientists to confirm these predictions. Knowing the Young’s modulus, tensile strength and the deformation under stress are the key parameter to establish a possible use of CNTs from the mechanical point of view. In this regard, both theoretical calculations and experimental observations indicate that carbon nanotubes exhibit higher Young’s modulus than steel and graphite. The reported Young’s modulus for CNTs is approximately 1200 GPa while the value for steel is in the order of 200 GPa. The response of CNTs to tensile strain is usually measured using AFM tips that pull the nanotube into the plastic deformation zone. Again, higher tensile strength values for CNTs compared to steel have been reported. In this case, value of approximately 150 GPa have been obtained for CNTs compared to 0.4 GPa for steel. In terms of deformation, it is important to observe that along the axial

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direction, CNTs show high strength and rigidity; however, in the transversal direction, they are easily deformed due to buckling. Due to these great mechanical properties, CNTs have found extensive application as reinforcement material in metal and polymer composites as well as in tribology as solid lubricants and lubricant additives dispersed in water and oil [4e6]. 2.1.2 Thermal properties Thermal properties explain the response of a material to the application of heat. Heat capacity and thermal conductivity define the thermal behavior of any given material. Graphite and diamond exhibit great heat capacity and thermal conductivity. Theoretical work has predicted that CNTs would have thermal conductivity greater than graphite and diamond. Thermal conductivity depends strongly on the structure of the CNTs. In general, low structural quality produces low thermal conductivity. A single nanotube has higher thermal conductivity than a CNT bundle and a CNT film due to poor energy transfer between the nanotubes. This can be explained in terms of the small contact areas among nanotubes in the bundle and film which reduces phonon transfer. Temperature deeply impact the thermal conductivity of CNTs. At low temperature, close to absolute cero, the increase of the thermal conductivity is linear. As the temperature is further increase, phonon modes control the thermal conductivity process until it reaches a maximum value at temperatures close to room temperature. Increasing the temperature even more tends to reduce the thermal conductivity. These thermal features of CNTs allow them to be considered as a thermal interface material where a continuous and good heat dissipation is needed [7e9]. 2.1.3 Electronic properties The remarkable electronic properties of CNTs offer a great number of novel nanoscale device applications. The electronic properties of CNTs are extremely sensitive to their geometrical structure. Small size and high symmetry allow unusual and unique quantum effects that defines the extraordinary electronic behavior. CNTs made of only one wall exhibit both metallic and semiconducting properties depending on their chirality without changing local bonding. Electronic properties depend on the tube radius, chirality, tube curvature, and the presence of defects. Depending on the combination of these parameters, the nanotube would show metallic or

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semiconducting characteristics. In particular, CNTs with semiconducting features have found applications for transistors, logic devices and memories [10e13].

2.2 Classification of carbon nanotubes CNTs are carbon allotropes which have a tubular structure formed by rolling graphene sheets. They can be classified depending on the number of sheets used to form the hollow cylinder structure. • Single-walled carbon nanotubes (SWCNTs). This type of CNT structure is formed by rolling up one single graphene sheet. Typical diameters are in the order of 1e5 nm and lengths vary between few nm to few cm. • Multi-walled carbon nanotubes (MWCNTs). This CNT structure is produced when two or more graphene sheets are rolled up to form a seamless tubular structure. They can be viewed as a group of coaxial or nested SWCNTs with an interlayer distance of approximately 0.34 nm, a value close to the interlayer distance of the graphene sheets in graphite. Typical diameters are in the order of 5e100 nm and lengths vary from 100 nm to few cm (Fig. 2.1). The folding direction of the graphene sheet defines the chirality of the CNT. In this regard, CNTs can be zigzag, armchair, and chiral. This is an important parameter that helps understand some of the outstanding properties mentioned before, in particular electronic properties which have a direct relationship with tube chirality (Fig. 2.2). Defects in the carbon skeleton creates different types of structures such as bent, helical, toroidal and branched just to mention a few. Doping

Figure 2.1 Types of carbon nanotubes.

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Figure 2.2 Different directions of folding a graphene sheet to form carbon nanotubes with different chiralities.

Figure 2.3 HR-TEM images of (a) toroidal carbon nanostructures and (b) N-doped CNTs with bamboo structures.

CNTs with heteroatoms (N, B, S, P) allows the formation of other type of structures. In particular, incorporating nitrogen to the hexagonal carbon network produces CNTs with bamboo structures which can be viewed as short capped nanotubes connected in a single tubular structure (Fig. 2.3).

2.3 Synthesis of carbon nanotubes The growing interest in using CNTs in various technological applications requires to develop methods that could satisfy the market while protecting the environment. It is important to recognize that three elements have to

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Figure 2.4 Elements needed to produce carbon nanotubes.

be properly combined in order to produce CNTs with the desired features: a carbon source, a catalyst and energy (heat) (Fig. 2.4). Many methods are available nowadays but they all are derived from the basic synthesis methods which are arc discharge, laser ablation, and chemical vapor deposition. A brief description of each of these methods will be given next. 2.3.1 Arc discharge This was the first method used to produce CNTs. At first, it was used to produce C60 fullerenes but it was noted that CNTs were also produced. In this method, the carbon source is a graphite rod electrode. Graphite rods are doped with metal particles used as catalyst such as Fe, Ni of Co. The set up of this synthesis method consists of a stainless-steel chamber where graphite electrodes are placed close to each other a high voltage power supply provides the temperature. In a typical synthesis, two graphite rods are set end to end separated by a short distance (w1 mm) inside the reaction chamber filled with an inert gas. A high current is applied to generate a high temperature (w3000
C) arc discharge between the electrodes. This vaporizes carbon atoms into a plasma and SWCNTs and/or MWCNTs are deposited on the surface of the cathode [14e17] (Fig. 2.5). 2.3.2 Laser ablation In this method, the carbon source is graphite which is heated by a laser beam in an inert atmosphere. The carbon species produced are transferred

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Figure 2.5 Schematic of the arc discharge synthesis method.

from the high temperature zone to a water-cooled conical collector by a flowing inert gas. Metallic particles are added to the graphite target in order to promote the CNTs growth. The fast condensation of the vaporized species mixed with the catalyst allowed the formation of the carbon walls until the carbon diffusion stops due to low temperature [18e22] (Fig. 2.6). 2.3.3 Chemical vapor deposition Chemical vapor deposition (CVD) is a very versatile and perhaps the most common method used to synthesize CNTs. Large scale production at relative low temperatures can be achieved by CVD. A great variety of

Figure 2.6 Schematics of the laser ablation synthesis method.

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carbon sources used in gas phase have been used. They include petroleumderived hydrocarbons like toluene, benzene, xylene or botanical compounds such as turpentine and neem oil. A conventional heating devise make this method economical and easy to implement. The catalyst (compounds containing Fe, Co, Ni, etc.) can be either mixed in the carbon source prior to be fed into the reaction zone or deposited on a substrate which is then placed inside the heating furnace. Ferrocene is the most effective catalyst reported in the literature. Controlling the temperature, type of catalyst, catalyst concentration, flow of reactants and resident time allows an excellent control of the desired features of the produced CNTs [23e27] (Fig. 2.7). The spray pyrolysis is a variation of the traditional CVD. In this method, the catalyst is mixed with the carbon source and the reactive mixture is atomized and introduce to the reaction zone in the form of a mist. The small drops containing the carbon source and the catalyst are thermally decomposed in a very efficient way [28e30] (Fig. 2.8).

Figure 2.7 Schematics of the chemical vapor deposition synthesis method.

Figure 2.8 Schematics of a spray pyrolysis synthesis method.

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2.4 Purification It is impossible to produce CNTs without impurities. Typically, these impurities consist of catalytic metal particles and amorphous carbon. Most of the applications require the removal of these impurities, so, different method to accomplish this have been developed over the years. Common removal techniques include plasma oxidation [31], oxidation in air [32], and hydrothermal treatments [33]. A wide spread method for this porpoise is the acid oxidation [34]. Since ferrocene is a popular catalyst, iron nanoparticles are impurities remaining after the synthesis. Along with the purification process, a functionalization procedure can be included in order to achieving their complete dispersion in water by forming carboxylic and hydroxyl groups on the CNTs’ surface. The complete purification and functionalization process consist of the following steps: First, 0.5 g of MWCNTs synthesized are dispersed in a ball flask by magnetic stirring followed by ultrasonic stirring in 100 mL of concentrated hydrochloric acid during 4 h, maintaining a constant temperature below the boiling point of hydrochloric acid. The MWCNTs are filtered and washed with deionized (DI) water. Next, MWCNTs from the previous step are brought to constant reflux for 8 h at 80
C with 100 mL of concentrated nitric acid in a 250 mL ball flask attached to a coolant with minimum water flow. The resulting MWCNTs are then filtered and washed with DI water. Finally, the MWCNTs of step 2 are refluxed with a 1:1 mixture of sulfuric acid and nitric acid with minimum water flow at 80
C. The MWCNTs are filtered and washed with DI water and subsequently dried in an oven at 60
C for 24 h. By performing the previous procedure, iron particles are successfully removed and CNTs are completely dispersed in water (Fig. 2.9). As an example of the effectiveness of this method, CNTs were characterized by infrared (IR) spectroscopy in each step of the purification and functionalization process to determine the functional groups that are formed in each step. The IR analysis is presented in two sections to have a better visualization of the signals detected. The first section comprises the wavelength range of 1000e2000 cm1 (Fig. 2.10a) and the second section is located between 2800 and 3800 cm1 (Fig. 2.10b). In both sections, it is observed that the synthesized and purified MWCNTs obtained by carrying out steps 1 and 2 of the acid treatments do not produce any signal related to the presence of an organic functional group. On the other hand, the functionalized MWCNTs obtained by

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Figure 2.9 Acid treatment method to purify and functionalized carbon nanotubes: (a) treatment with HCl, (b) treatment with HNO3, and (c) treatment with HNO3/H2SO4 mixture.

Figure 2.10 Sections of the IR spectrum of the as-synthesized, purified and functionalized MWCNTs. (a) Section 2.1 (from 1000 to 2000 cm1) (b) Section 2.2 (from 2800 to 3800 cm1).

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carrying out step 3 of the acid treatment generate signals that are associated with the formation of organic groups on the CNTs. In Section 2.1, signals are detected at the following wavelengths: 1240, 1330, 1402, and 1628 cm1, which correspond to the formation of carboxylic dimers that interact with each other by hydrogen bonds. In Section 2.2, signals located at 3220 and 3345 cm1 are detected, corresponding to the stretching of hydroxyl groups associated with carboxylic groups that interact by hydrogen bonds. The as-synthesized and purified MWCNTs (steps 1 and 2 of the acid treatment) do not have organic functional groups on their surface, so they cannot be dispersed in water. On the other hand, the MWCNTs processed with step 3 of the acid treatment, specifically their interaction with sulfuric acid, can form free carboxylic and hydroxyl groups that generate a certain degree of polarity in the CNTs to achieve their complete dispersion in water.

2.5 Characterization of carbon nanotubes This section is devoted to provide basic information of the different characterization techniques use to study CNTs. There is not a single technique that would give a complete characterization of CNTs. Technical information of CNTs include size (inner and outer diameters, number of walls, length), purity, crystallinity, thermal stability, and surface area, just to mention a few. A combination of microscopic, spectroscopic, and physicochemical tools is needed in order to have a complete picture of the structure, morphology and chemical features of CNTs. 2.5.1 Electron microscopy “A picture is worth a thousand words.” The use of electronic microscopes is essential in the nanotechnology world. There is no other way to identify the size and shape of CNTs but to use scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM). With SEM image at low magnification we can know if the synthesized CNTs are aligned, entangled or agglomerated. The length and, with the proper operation of the microscope, the outer diameter can be obtained. Energy dispersive X-ray spectroscopy (EDAX) allows the identification and quantification of elements in the CNT sample. SEM analysis has become a routine technique used to characterize the morphology and size of CNTs. Fig. 2.11 shows an example of SEM images of vertically aligned carbon nanotubes (VACNTs) and entangle CNTs produced by the thermal

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Figure 2.11 SEM images of (a) VACNTs grown on a quartz substrate and (b) entangled CNTs grown on a stainless steel substrate.

decomposition of a-pinene, a botanical hydrocarbon, and ferrocene by the spray pyrolysis method performed at 800
C on quartz and stainless steel substrates. HR-TEM images complement the information that one can get from SEM images since smaller dimensions can be resolve with a transmission microscope. More details of the wall’s alignment, inner and outer diameters, distribution of the catalytic particles, and the presence of amorphous carbon can be clearly determined with HR-TEM. The crystallinity of the CNTs can be established calculating the fast Fourier transform (FFT) of different tube zones. Sharp spots indicate high crystallinity. It the spots are diffuse (broad and blurred), the analyzed CNTs are less crystalline. This information has to be corroborated by other techniques such as Raman spectroscopy or X-ray diffraction [35]. As an example of the use of HR-TEM and the FFT analysis described above, Fig. 2.12 corresponds to the analysis of an amorphous carbon fiber and Fig. 2.13 shows the analysis of CNTs. In Fig. 2.12a, the image of tubular carbon structure is shown resembling a CNTs. However, the FFT analysis does not show any sharp spots that indicates that the analyzed fiber is amorphous (Fig. 2.12b). Fig. 2.12c is the line scan from which the plane distance can be estimated by measuring the distance that separates the sharp spots, which in this case cannot be measured. The same sequences of images for CNTs allows to determine the number of walls as well as the inner and outer diameters (Fig. 2.13a). The FFT analysis shows sharp spots which indicates that the analyzed CNTs are crystalline (Fig. 2.13b). The interlayer distance estimated from the line scan was w0.335 nm. Another use of HR-TEM is the identification of metal nanoparticles in the CNTs samples which may correspond to catalytic nanoparticles or to metallic particles deposited on CNTs. In dark field images, small metallic

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Figure 2.12 (a) HR-TEM image of amorphous carbon fiber, (b) FFT analysis, and (c) line scan of the FFT analysis.

Figure 2.13 (a) HR-TEM image of crystalline CNTs, (b) FFT analysis, and (c) line scan of the FFT analysis.

particles can be easily recognized by the large contrast difference between heavier and lighter elements. Their size and distribution can be estimated from these images. An example of this analysis is shown in Fig. 2.14. The images correspond to ceria nanoparticles deposited on MWCNTs by a microwave-assisted method. The lower magnification image (Fig. 2.14a), exhibits the excellent dispersion of ceria clusters on a single MWCNT. The higher magnification

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Figure 2.14 HR-TEM analysis of ceria nanoparticles deposited on MWCNTs: (a) and (b) high-angle annular dark field images at different magnifications, (c) EDS analysis of the nanoparticles, and (d) nanoparticle size distribution.

image (Fig. 2.14b) shows the tiny size of the clusters with a mean diameter less than 2 nm, and that their dispersion on the surface is highly homogeneous. The EDS analysis displayed in Fig. 2.14c reveal the presence of oxygen and cerium confirming the formation of ceria. The size histogram of the clusters synthesized by this method shows the closed size-dispersion, with an average diameter of 1.41 nm  0.40 nm (Fig. 2.14d). 2.5.2 Raman spectroscopy Raman spectroscopy has become a very important characterization technique to study carbon materials. In particular, Raman studies are essential in determining structural features of CNTs. The inelastic light scattering form CNTs which induces an increase or decrease of energy of the incident light due to absorption or emission of phonons present in CNTs is the basis of

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the Raman scattering process. Raman spectroscopy identifies the presence of SWCNTs and MWCNTs. Signals located at low frequencies (below 200 cm1) correspond to radial breathing mode (RBM). These signals are the Raman signature of SWCNTs and are absent in MWCNTs. There are three characteristic peaks of MWCNTs: the D band at w1340 cm1, which corresponds to the disorder-induced phonon mode (A1g), the G band at w1580 cm1 assigned to the Raman-allowed phonon mode (E2g) and the G0 band at w2660 cm1 assigned to the first overtone of the D mode. The origin of the D band is considered a measure of the disorder in the CNTs sample. The G band is a measure of the graphitization of the analyzed CNTs [36e39]. A typical Raman spectrum is shown in Fig. 2.15. It corresponds to the analysis of MWCNTs produced by spry pyrolysis of a turpentine/ferrocene mixture at 800
C. All the signals mention above are clearly identify. Changes in the intensities of these signals indicate changes in the structural features of CNTs. The D/Geband intensity ratio (ID/IG) is reported to increase with increasing structural disorder. The G0 /Geband intensity ratio (IG0 /IG) increases as the long-range order of C deposits is increased. These parameters have become an important indication of the

G'

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G

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2000

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Figure 2.15 Typical Raman spectrum of MWCNTs.

5000

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structural features found CNTs. As an example of how to integrate information from different characterization techniques, Raman spectroscopy and HR-TEM were used to analyze MWCNTs produced with three different catalysts: cobalt(II) phthalocyanine (CoPc), iron(II) phthalocyanine (FePc), and ferrocene (FecH). The temperature, injection flow, time and catalyst concentration where kept constant at 800
C, 5000 sccm, 30 min and 4.27 wt% of metal for the three catalyst respectively. Fig. 2.16aef show the HR-TEM images of MWCNTs obtained for each catalyst and their corresponding fast Fourier transform (FFT). Fig. 2.16a and c shows HR-TEM images of MWNTs produced using CoPc and FePc respectively. The main characteristic of these CNTs is their high disorder, due to the lack of alignment of the carbon layers throughout the entire nanotube structure. The corresponding FFT analysis (Fig. 2.16b and d) displays diffuse spots and the line scans (inset) revealing poor crystallinity of the formed MWCNTs with both catalysts, as indicated by the presence of wide peaks and low intensity of the central spots. It is important to notice that these catalysts formed bamboo-type structures which are characteristic of N-doped CNTs. Highly crystalline MWCNTs were obtained when using FecH as catalyst. The corresponding HR-TEM image and FFT analysis are shown in Fig. 2.16e and f. Clearly, the nanotubes exhibit noticeable crystallinity based on the fact that carbon layers are highly aligned and the presence of sharp spots in the FFT analysis and narrow spots in the line scan (inset). Fig. 2.17a shows the Raman spectra of the MWCNTs on a quartz substrate prepared with FecH, FePc, and CoPc as catalysts. The tendency of the band ratios is shown in Fig. 2.17b where it can be determined that the ID/IG value was the lowest and the IG’/IG value was the highest for MWCNTs produced with FecH. This indicates that the highest crystallinity as well as the highest carbon layer smoothness is achieved when using this catalyst. This is in excellent agreement with the HR-TEM results shown above. This example demonstrates that for the three catalysts tested, FecH is more active than FePc and CoPc toward the production of crystalline MWCNTs when using a-pinene as carbon source. It has been reported that, when using metal phtalocyanines (Fe, Co and Ni), N atoms are generated, dissolved into the catalytic nanoparticles and precipitate with carbon producing N-doped carbon nanotubes [37]. The incorporation of N atoms in the graphitic layers of the MWCNTs may be the source of the lower crystallinity when using FePc and CoPc as catalysts. FecH does not

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Figure 2.16 HR-TEM images and their corresponding Fast Fourier transform (FFT) of MWCNTs produced with different catalysts: (a) and (b) cobalt phtalocyanine, (c) and (d) iron phtalocyanine and (e) and (f) ferrocene.

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a D

G

b

2.0

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0.0 2.0 FePc

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ID/IG

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1.0 0.5

FecH 0.0 1000 1500 2000 2500 3000 -1

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Figure 2.17 (a) Raman spectra of MWCNTs obtained with CoPc, FePc and FecH catalysts, (b) Plots of ID/IG and IG’/IG for each catalyst tested. The solid lines are to guide the eye.

provide N atoms, hence producing more crystalline MWCNTs as revealed by HR-TEM and Raman analyses. 2.5.3 X-ray diffraction X-ray diffraction is the most commonly used technique to get information from crystalline materials. While it is not as frequently used to characterize CNTs as Raman spectroscopy, important information can be obtained from it. Values of the peak position and full width at half maximum (FWHM) of the graphite (002) signal located at 2q of w25.9 degrees provide insight information of the grown CNTs. It has been reported that the diffraction angle of the (002) signal decreases and the FWHM increases with increasing the curvature of the graphene layers. These changes depend on the interlayer spacing between layers of the CNTs and lattice distortion due to defective stacking of the graphene layers suggesting that a wellordered structure will exhibit a narrow FWHM [40, 41]. MWCNTs with the lowest diameter will have the highest curvature [6].

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Following the analysis of the previous example, XRD patterns from MWCNTs synthesized with the three catalysts mentioned before are shown in Fig. 2.18. The analysis shows a gradual up shift of the (002) peak position and an increase of the FWHM as the diameter of the MWCNTs increases. An increase of the d(002) spacing as the tube diameter decreases is also observed. The smaller FWHM was found for MWCNTs produced with FecH followed by those produced with FePc and CoPc. We relate these observations to the fact that more developed graphitic structures are produced when using FecH as catalyst. 2.5.4 Thermogravimetrical analysis The thermal stability and purity of CNTs can be measured by means of thermogravimetrical analysis (TGA). The initiation temperature (the temperature at which the MWCNTs start to decompose) as well as the residual mass, which is usually attributed to the metal catalyst used to produce the MWCNTs, can be obtained directly from the weight-loss curves. The

Figure 2.18 (a) XRD diffraction patterns of MWCNTs produced with different catalysts and (b) plot of the variation of position and FWHM of the (002) peak versus catalyst.

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oxidation temperature can be obtained from the derivative of the weight loss as a function of temperature from which. The oxidation temperature is often referred as the thermal stability of the analyzed material. The residual mass corresponds to the amount of catalyst used to produce the CNTs. Analyzing the samples produced with the catalyst mentioned in the previous sections, TGA analysis for the MWCNTs produced with FecH, FePc, and CoPc catalysts is shown in Fig. 2.19a. The initiation temperature (the temperature at which the MWCNTs start to decompose) as well as the residual mass, which is usually attributed to the metal catalyst used to produce the MWCNTs, can be obtained directly from the weight-loss curves [42e44]. Fig. 2.19b shows the derivative of the weight loss as a function of temperature from which the oxidation temperature can be obtained. The initiation temperature for MWCNTs produced using FecH as catalyst was 502
C, a greater value compared with 392 and 410
C obtained for the FePc and CoPc catalysts respectively. The oxidation temperature is often referred as the thermal stability of the material. This temperature was 635
C when using FecH, higher than 492 C and 460
C

a

b

-4.0

100 -3.5

90

-3.0

Derivative Weight (%/°C)

80 70

Weight (%)

FecH FePc CoPc

60 50 40 30 20

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FecH FePc CoPc

10

-2.5

0.0

0 200

400

600

Temperature (°C)

800

200

400

600

800

Temperature (°C)

Figure 2.19 Termogravimetrical analysis of MWCNTs formed with different catalysts: (a) TGA and (b) DTGA.

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obtained for FePc and CoPc respectively. The amount of metal measured after the complete decomposition of the MWCNTs was 3.7 wt% for FecH, 2.7 wt% for FePc and 5.5 wt% for CoPc. From these values and considering around 2 wt% of humidity content in each sample, the purity of MWCNTs was calculated to be 94.2%, 95.3%, and 92.5% when using FecH, FePc, and CoPc, respectively.

2.6 Conclusions The remarkable properties of CNTs are intimately associated to their structure. In order to identify possible applications, it is necessary to perform a complete and detail characterization of the produced carbon nanomaterials in order to establish a well-controlled production method.

References [1] H.W. Kroto, J.R. Heath, S.C. O’Brien, R.F. Curl, R.E. Smalley, C 60: buckminsterfullerene, Nature 318 (6042) (1985) 162e163. [2] S. Iijima, Helical microtubules of graphitic carbon, Nature 354 (6348) (1991) 56e58. [3] T.C. Dinadayalane, J. Leszczynski, Remarkable diversity of carbonecarbon bonds: structures and properties of fullerenes, carbon nanotubes, and graphene, Struct. Chem. 21 (6) (2010) 1155e1169. [4] J.P. Salvetat, J.M. Bonard, N.H. Thomson, A.J. Kulik, L. Forro, W. Benoit, L. Zuppiroli, Mechanical properties of carbon nanotubes, Appl. Phys. A 69 (3) (1999) 255e260. [5] J.P. Salvetat-Delmotte, A. Rubio, Mechanical properties of carbon nanotubes: a fiber digest for beginners, Carbon 40 (10) (2002) 1729e1734. [6] M.S. Dresselhaus, G. Dresselhaus, J.C. Charlier, E. Hernandez, Electronic, thermal and mechanical properties of carbon nanotubes, Philos. Trans. R. Soc. London, Ser. A: Math. Phys. Eng. Sci. 362 (1823) (2004) 2065e2098. [7] J. Hone, Carbon nanotubes: thermal properties, Dekker Encyclopedia Nanosci. Nanotechnol. 7 (2004) 603e610. [8] J. Hone, Phonons and thermal properties of carbon nanotubes, Carbon Nanotubes (2001) 273e286. [9] J. Hone, M.C. Llaguno, M.J. Biercuk, A.T. Johnson, B. Batlogg, Z. Benes, J.E. Fischer, Thermal properties of carbon nanotubes and nanotube-based materials, Appl. Phys. A 74 (3) (2002) 339e343. [10] J.P. Issi, L. Langer, J. Heremans, C.H. Olk, Electronic properties of carbon nanotubes: experimental results, Carbon 33 (7) (1995) 941e948. [11] J.E. Fischer, A.T. Johnson, Electronic properties of carbon nanotubes, Curr. Opin. Solid State Mater. Sci. 1 (4) (1999) 28e33. [12] Y.I. Prylutskyy, S.S. Durov, O.V. Ogloblya, E.V. Buzaneva, P. Scharff, Molecular dynamics simulation of mechanical, vibrational and electronic properties of carbon nanotubes, Comput. Mater. Sci. 17 (2e4) (2000) 352e355. [13] R. Heyd, A. Charlier, E. McRae, Uniaxial-stress effects on the electronic properties of carbon nanotubes, Phys. Rev. B 55 (11) (1997) 6820.

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[14] Z. Shi, Y. Lian, X. Zhou, Z. Gu, Y. Zhang, S. Iijima, S. Zhang, Mass-production of single-wall carbon nanotubes by arc discharge method, Carbon 37 (9) (1999) 1449e1453. [15] Y. Ando, X. Zhao, Synthesis of carbon nanotubes by arc-discharge method, N. Diamond Front. Carbon Technol. 16 (3) (2006) 123e138. [16] M. Keidar, Factors affecting synthesis of single wall carbon nanotubes in arc discharge, J. Phys. Appl. Phys. 40 (8) (2007) 2388. [17] N. Arora, N.N. Sharma, Arc discharge synthesis of carbon nanotubes: comprehensive review, Diam. Relat. Mater. 50 (2014) 135e150. [18] C.D. Scott, S. Arepalli, P. Nikolaev, R.E. Smalley, Growth mechanisms for single-wall carbon nanotubes in a laser-ablation process, Appl. Phys. A 72 (5) (2001) 573e580. [19] J. Chrzanowska, J. Hoffman, A. Małolepszy, M. Mazurkiewicz, T.A. Kowalewski, Z. Szymanski, L. Stobinski, Synthesis of carbon nanotubes by the laser ablation method: effect of laser wavelength, Physica Status Solidi (B) 252 (8) (2015) 1860e1867. [20] Y. Zhang, H. Gu, S. Iijima, Single-wall carbon nanotubes synthesized by laser ablation in a nitrogen atmosphere, Appl. Phys. Lett. 73 (26) (1998) 3827e3829. [21] M. Yudasaka, R. Yamada, N. Sensui, T. Wilkins, T. Ichihashi, S. Iijima, Mechanism of the effect of NiCo, Ni and Co catalysts on the yield of single-wall carbon nanotubes formed by pulsed Nd: YAG laser ablation, J. Phys. Chem. B 103 (30) (1999) 6224e6229. [22] H. Zhang, Y. Ding, C. Wu, Y. Chen, Y. Zhu, Y. He, S. Zhong, The effect of laser power on the formation of carbon nanotubes prepared in CO2 continuous wave laser ablation at room temperature, Phys. B Condens. Matter 325 (2003) 224e229. [23] L.C. Qin, CVD synthesis of carbon nanotubes, J. Mater. Sci. Lett. 16 (6) (1997) 457e459. [24] M.L. Terranova, V. Sessa, M. Rossi, The world of carbon nanotubes: an overview of CVD growth methodologies, Chem. Vap. Depos. 12 (6) (2006) 315e325. [25] A. Magrez, J.W. Seo, R. Smajda, M. Mionic, L. Forró, Catalytic CVD synthesis of carbon nanotubes: towards high yield and low temperature growth, Materials 3 (11) (2010) 4871e4891. [26] R. Purohit, K. Purohit, S. Rana, R.S. Rana, V. Patel, Carbon nanotubes and their growth methods, Procedia materials science 6 (2014) 716e728. [27] E. Couteau, K. Hernadi, J.W. Seo, L. Thien-Nga, C. Mikó, R. Gaal, L. Forro, CVD synthesis of high-purity multiwalled carbon nanotubes using CaCO3 catalyst support for large-scale production, Chem. Phys. Lett. 378 (1e2) (2003) 9e17. [28] S.R.C. Vivekchand, L.M. Cele, F.L. Deepak, A.R. Raju, A. Govindaraj, Carbon nanotubes by nebulized spray pyrolysis, Chem. Phys. Lett. 386 (4e6) (2004) 313e318. [29] G. Alonso-Nunez, J. Lara-Romero, F. Paraguay-Delgado, M. Sanchez-Castañeda, S. Jiménez-Sandoval, Temperature optimisation of CNT synthesis by spray pyrolysis of alpha-pinene as the carbon source, J. Exp. Nanosci. 5 (1) (2010) 52e60. [30] J. Lara-Romero, G. Alonso-Núñez, S. Jiménez-Sandoval, M. Avalos-Borja, Growth of multi-walled carbon nanotubes by nebulized spray pyrolysis of a natural precursor: alpha-pinene, J. Nanosci. Nanotechnol. 8 (12) (2008) 6509e6512. [31] A. Meyer-Plath, G. Orts-Gil, S. Petrov, F. Oleszak, H.E. Maneck, I. Dörfel, R. Mach, Plasma-thermal purification and annealing of carbon nanotubes, Carbon 50 (10) (2012) 3934e3942. [32] N. Dementev, S. Osswald, Y. Gogotsi, E. Borguet, Purification of carbon nanotubes by dynamic oxidation in air, J. Mater. Chem. 19 (42) (2009) 7904e7908. [33] G.S. Duesberg, S. Roth, P. Downes, A. Minett, R. Graupner, L. Ley, N. Nicoloso, Modification of single-walled carbon nanotubes by hydrothermal treatment, Chem. Mater. 15 (17) (2003) 3314e3319.

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[34] Y. Li, X. Zhang, J. Luo, W. Huang, J. Cheng, Z. Luo, H.J. Geise, Purification of CVD synthesized single-wall carbon nanotubes by different acid oxidation treatments, Nanotechnology 15 (11) (2004) 1645. [35] J.H. Lehman, M. Terrones, E. Mansfield, K.E. Hurst, V. Meunier, Evaluating the characteristics of multiwall carbon nanotubes, Carbon 49 (8) (2011) 2581e2602. [36] M.S. Dresselhaus, G. Dresselhaus, R. Saito, A. Jorio, Raman spectroscopy of carbon nanotubes, Phys. Rep. 409 (2) (2005) 47e99. [37] M.S. Dresselhaus, G. Dresselhaus, A. Jorio, A.G. Souza Filho, R. Saito, Raman spectroscopy on isolated single wall carbon nanotubes, Carbon 40 (12) (2002) 2043e2061. [38] M.S. Dresselhaus, A. Jorio, M. Hofmann, G. Dresselhaus, R. Saito, Perspectives on carbon nanotubes and graphene Raman spectroscopy, Nano Lett. 10 (3) (2010) 751e758. [39] S. Costa, E. Borowiak-Palen, M. Kruszynska, A. Bachmatiuk, R.J. Kalenczuk, Characterization of carbon nanotubes by Raman spectroscopy, Materials SciencePoland 26 (2) (2008) 433e441. [40] A. Cao, C. Xu, J. Liang, D. Wu, B. Wei, X-ray diffraction characterization on the alignment degree of carbon nanotubes, Chem. Phys. Lett. 344 (1e2) (2001) 13e17. [41] C. Müller, D. Golberg, A. Leonhardt, S. Hampel, B. Büchner, Growth studies, TEM and XRD investigations of iron-filled carbon nanotubes, Phys. Status Solidi 203 (6) (2006) 1064e1068. [42] L.S. Pang, J.D. Saxby, S.P. Chatfield, Thermogravimetric analysis of carbon nanotubes and nanoparticles, J. Phys. Chem. 97 (27) (1993) 6941e6942. [43] G.S. McKee, K.S. Vecchio, Thermogravimetric analysis of synthesis variation effects on CVD generated multiwalled carbon nanotubes, J. Phys. Chem. B 110 (3) (2006) 1179e1186. [44] D. Bom, R. Andrews, D. Jacques, J. Anthony, B. Chen, M.S. Meier, J.P. Selegue, Thermogravimetric analysis of the oxidation of multiwalled carbon nanotubes: evidence for the role of defect sites in carbon nanotube chemistry, Nano Lett. 2 (6) (2002) 615e619.

CHAPTER 3

Functionalization of carbon nanotube Mustafa A. Alheety1, Abdulwahhab H. Majeed2, 3 Leqaa A. Mohammed2 and Ahmed R. Mahmood 1 2

Department of Nursing, Al-Hadi University College, Baghdad, Iraq; Department of Chemistry, College of Science, Diyala University, Diyala, Iraq; 3Department of Medical Laboratory Technology, Imam Ja’afar Al-Sadiq University, Kirkuk, Iraq

3.1 General aspect 3.1.1 Synthesis of carbon nanotube The laboratory production of carbon nanotubes was started by Iijima in 1991 using the arc vacuum method. Since then, carbon nanotubes have become one of the most attractive materials, which researchers focused on in their study. During the last 3 decades, a number of different methods and techniques have been developed in which carbon nanotubes can be produced with high quantity and low production cost. Fig. 3.1 shows the most important of these methods.

Figure 3.1 The main methods of CNTs synthesis.

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00003-0

© 2023 Elsevier Ltd. All rights reserved.

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3.1.1.1 Electric arc discharge In the electric arc discharge of carbon, very high temperatures are used, during which carbon nanotubes can be produced, and fullerene is also produced. In fact, good quantities of fullerene were produced for the first time using the electric arc discharge method of carbon, using the KratschmereHuffman method [1]. The quality and quantity of the product prepared in this way depends mainly on the surrounding atmosphere and on the catalysts used in the preparation process. This method may be one of the simplest methods used to prepare carbon nanotubes with good quantities, but it requires several purification steps due to the formation of several forms of carbon compounds during the arcing process. In this technique, a continuous electric current is passed through two graphite electrodes (cathode and anode) which ignites an arc between the two electrodes in an inert gas atmosphere (usually argon, helium, or hydrogen) [2e6]. It was found that during the process of passing the constant electric current and forming the arc, the graphite in the anode began to gradually wear out, and a cigar-like sediment began to appear on the cathode. The outer covering of this sediment is gray and solid with a fine black inner core containing MWNTs, polyhedral particles, and amorphous carbons [6,7]. In the electric arc discharge of carbon method, there are two main different methods of producing carbon nanotubes, which are to produce carbon nanotubes using catalysts and without the use of catalysts. Experiments have shown that it is possible to produce multi-walled carbon nanotubes (MWCNTs) without the use of catalysts in the preparation, but the synthesis of single-walled nanotubes (SWNTs) does not occur except with the presence of cofactors. The catalysts used in the carbon arcdischarge technology to produce single-walled carbon nanotubes (SWCNTs) may be metallic such as Gd [8], Pd, Co, Ag, Fe, Ni, Pt, etc., or they may be mixed catalysts, where Co, Ni, and Fe that mixing with other elements to produce mixed catalysts such as CoeRu, CoePt [18], FeeNi, NieY, CoeCu, CoeNi, FeeNo, NieTi, NieCu, NieY, etc. Studies have shown that NieY mixtures with graphite can produce a high yield ( 90
.

carbon atoms to adopt a geometry that is not ideal for this type of hybridization. Indeed, in a perfect geometry sp2, there are three co-plane sp2 orbitals to form three sigma bonds and a p bond formed from the p orbital which is orthogonal to the sp2 orbitals. In addition, the p orbital is symmetrically distributed across the nucleus. This geometry is shown in Fig. 3.2. Plane graphene respects this geometry, but a deviation from it is inevitable when it is coiled to form a nanotube. As shown in Fig. 3.2, the sigma links are no longer quite co-plane and the p orbitals then become more delocalized on the outer side of the nanotube, i.e., on the convex surface, than on the inner side [42,43]. This deviation from the sp2 geometry, or re-hybridization [44], allows better orbital coverage with the reagents approaching the outer surface of the CNTs and promotes addition reactions [45]. Second, the curvature of the graphene sheet causes a misalignment of the p orbitals of neighboring carbon atoms, as shown in Fig. 3.3. This kind of misalignment in conjugate systems is known to cause great stress in the system and increase chemical reactivity. Of course, the magnitude of the deviation from co-planarity and the misalignment of the p orbitals vary with the helicity of the nanotube, causing a slight difference in reactivity [42]. Furthermore, the reactivity of CNTs also depends on their electrical type. For example, work has shown that the reactions of osmylation [46], addition of phenyldiazonium [47], and organolithians [48] are preferentially carried out on CNTs metallic. The greater reactivity of metals seems to be

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Figure 3.3 Illustration of the misalignment of pz orbitals (expressed as the angle 4) occurring in the structure of the carbon nanotube. Depending on the orientation of the carbon-carbon bond with respect to the axis of the nanotube, the p orbitals can be more or less misaligned.

related to the presence of electronic states at the Fermi level. To justify this, it is interesting to draw a parallel between CNTs and annulenes. According to Huckel’s rule [49], annulenes having [4n þ 2] conjugated p electrons (n being an integer) are aromatic. The diagram of molecular orbitals of [4 þ 2] annulene, benzene, is shown in Fig. 3.4. In this type of molecules, all the binding orbitals are filled while all the anti-binding orbitals are empty. The energy difference between the highest occupied energy orbital (HOMO) and the lowest vacant energy orbital (LUMO) is large, making it difficult to add or remove a p electron. The aromatic structures are, therefore, very stable. In contrast, annulenes having [4n] conjugated p electrons (n being an integer) are said to be anti-aromatic. They exhibit degenerate nonbinding orbitals, which are half-filled. These characteristics are shown in Fig. 3.4 illustrating the molecular orbitals of [43] annulene, cyclobutadiene. This configuration is however not very stable and in reality, a slight JahnTeller [50] type distortion takes place in order to remove the degeneration of the nonbinding orbitals. However, the energy difference between their

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Figure 3.4 Diagram of the molecular orbitals of cyclobutadiene (left) and benzene (right).

HOMO and their LUMO remains small. It is therefore much easier to add or extract an electron in this type of system. Therefore, the anti-aromatic structures are more reactive than the aromatic structures. The application of Huckel’s rule to CNTs cannot be formally done due to the unidimensionality and size of the structure. It is however possible to extrapolate it, as suggested by Joselevich [51]. Indeed, semiconductor CNTs have a completely filled valence band, a completely empty conduction band and a significant difference in energy between the two (see Fig. 3.5). They can therefore be associated with an aromatic system. On the other hand, metallic CNTs have electron states at the Fermi level (see Fig. 3.5) that could be associated with nonbinding orbitals. Unlike small organic molecules, the distortion of the backbone is negligible here given the rigidity of the graphene lattice. The energy difference between their HOMO and LUMO can therefore be considered as zero. These characteristics are associated with an anti-aromatic system. This simplistic reasoning therefore supports the fact that metallic CNTs are more reactive than semiconductors. However, we must be careful with such an analogy. The energy difference between the HOMO and LUMO orbitals is mainly

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Figure 3.5 Illustration of the energy of the highest occupied energy orbital (HOMO) and the lowest vacant energy orbital (LUMO) in the electronic structure of a semiconductor carbon nanotube (left) and metallic (right).

recognized to affect the kinetic stability of a structure [52]. The fact remains that all CNTs are extremely thermodynamically stable because of the very large delocalization of electrons in the system. On the other hand, there are two particularly reactive areas in CNTs, whether metallic or semiconductor: structural defects and the ends of the nanotube. Structural defects are usually formed during the growth of the nanotube [53]. Although these defects are difficult to quantify, it has been established that their density on the wall depends very much on the conditions of synthesis [54,55]. They consist of an irregularity in the graphene structure of the wall forming a gap or reconstructions involving five and seven carbon cycles associated with each other. These defects break the periodicity of the structure and cause deformation of the carbon network.

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The carbons that are part of these structural defects are more stressed than those that are part of the “honeycomb” structure of the graphene wall. They are therefore preferentially attacked during chemical reactions. Their importance in terms of the general reactivity of the CNTs is however limited. Indeed, their density varies greatly from one sample to another and can be negligible in some cases. For example, the density of defects on a sample of CVD-synthesized SWNTs was evaluated at only one defect per 4 mm [56]. They look like half fullerenes made up of an arrangement of pentagons. The deviation from co-planarity is much more pronounced there than in the rest of the wall. The stress on the carbon lattice is enormous, and the energy of re-hybridization atoms to sp3 configuration promotes chemical reactions at these sites [42,56]. 3.2.2 Classification of covalent functionalization on CNT It is possible to classify covalent functionalization reactions into two groups, namely mild reactions, affecting only the weak points of CNTs, and aggressive reactions, attacking the entire wall. The soft functionalization takes advantage of the greater reactivity of the ends of the nanotube and of the structural defects. They only affect these regions, leaving the rest of the wall intact. Oxidation of CNTs upon reflux in dilute nitric acid is one example [57]. As shown in Fig. 3.6, it leaves behind oxygen groups grafted onto carbons forming structural defects and ends of the body. The advantage of this type of functionalization is that it hardly disturbs the structure and properties of nanotubes. However, few groups are grafted to

Figure 3.6 Gentle oxidation of CNTs leading to the formation of carboxylic acids on the surface of carbon nanotubes. At reflux, dilute nitric acid attacks only structural defects and the ends of CNTs.

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the nanotube this which implies that the interactions between the CNT and its environment are little modified. On the other hand, aggressive reactions make it possible to functionalize not only the structural defects and the ends of the nanotube but also the rest of its wall [58,59]. Due to the high stability of CNTs, these reactions all go through a very reactive intermediate, for example, a radical [60e62], a carbene [63], a nitrene [64], or an azomethine ylide [65]. The grafts are either monovalent or bivalent, that is, say that they respectively form one or two bonds with the nanotubes. When a graft attaches to the wall of a CNT, it locally changes the “honeycomb” structure of the wall, creating a point of failure and disrupting the wave function of the SWNT. During soft functionalization, the number of defects does not increase, or almost, since the reaction mainly affects the defects already present on the wall. On the other hand, during aggressive reactions, the creation of defects is important. The periodicity of the network is then greatly reduced, which significantly modifies the optical and electronic properties of the CNTs. These modifications have been particularly studied for the grafting of phenyl units. The addition of a phenyl diazonium salt to CNTs is a popular functionalization reaction. Developed by Tour et al. [61] in 2001, it allows phenyl groups to be grafted onto the wall of nanotubes. Its popularity is related to its high yield and the fact that the phenyl chemistry is rich and well known. Thus, after having grafted phenyl groups onto the wall of the CNT, it is possible to modify them and couple them to a variety of molecules. This versatility provides control over the surface properties of the functionalized CNTs. The addition of the phenyl diazonium to the CNTs can be carried out in an aqueous medium [66] in an organic medium [67] or else without a solvent [68]. The latter method is usually used when a high graft density is desired and/or the reaction is over a large amount of CNT. The other two methods are however preferred when a lower grafting rate is desired. Indeed, good control of the graft density can be achieved by adjusting the concentration of phenyl diazonium and CNTs in the reaction mixture. In addition, it is likely that the use of individualized CNTs in an aqueous or organic suspension will result in more uniform grafting within the sample. The mechanism of this functionalization reaction, as proposed by Schmidt et al. [69], is shown in Fig. 3.7. First, phenyldiazonium breaks down to form a radical phenyl, either through the formation of an intermediate diazoanhydride (step A) or via electron transfer from an SWNT (step B). Second, the radical can add with an SWNT to form

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Figure 3.7 Mechanism of the reaction between phenyldiazonium and proposed CNTs.

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Figure 3.8 Illustration of the most stable configuration of two phenyl groups on the wall of the carbon nanotube. The attachment sites are made up of sp3 carbons and constitute points where no electron can be delocalized.

an SWNT-phenyl radical (step C). It should be noted that SWNT (metallic)dphenyl radicals are more easily formed than SWNT (semiconductor)dphenyl radicals, since they have electronic states near the Fermi level. Third, SWNT-phenyl radicals can reduce phenyldiazoniums, then regenerating phenyl radicals (step D) and at the same time stimulating step C. Finally, there are three termination processes, which are shown in steps E, F, and G in Fig. 3.7, respectively. A phenyl radical can couple with another phenyl radical, with an already oxidized SWNT, or with an SWNT-phenyl radical. This reaction makes it possible to obtain a grafting rate of up to 11% (11/100 wall carbons are linked to a graft). The group of X. Blase studied, from a theoretical point of view, the configuration of phenyl units on the wall of CNTs [70]. Blase showed that these must be paired to be stable at room temperature (Fig. 3.8). The para position appears to be the most stable, followed by the ortho position. Thus, a p bond between two CNT carbon atoms is transformed during the reaction into two CNT-phenyl sigma bonds. Attaching a graft to the wall therefore results in the formation of two sp3 carbons in the graphene structure, thereby breaking the delocalization of p electrons at these locations. 3.2.3 Non-covalent functionalization The covalent functionalization of carbon nanotubes often calls upon highly reactive species such as oxidants or reducing agents. This is why a nanotube

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grafting chemistry based on non-covalent interactions has developed significantly in recent years, either by adsorption of surfactants, by coiling of polymers, or by adsorption of compounds by p interaction. Very strong non-covalent interactions exist between nanotubes and anilines [71], amines [72], and pyrenes [73,74], thus increasing the solubility of nanotubes in organic solvents. This can be explained by the formation of donoracceptor complexes. This gentle functionalization of carbon nanotubes by formation of non-covalent bonds makes it possible to physisorb various chemical entities on the surface of the nanotubes without changing their properties. Amphiphilic molecules, surfactants, such as sodium dodecyl sulfate (SDS) or Triton-X 100 Fig. 3.9, bind to nanotubes by van der Waals interactions [75]. The carbon nanotube is then found inside a micelle, which allows it to be dispersing in an aqueous phase. A stronger interaction is created between the nanotube and the surfactant when the hydrophobic part of the amphiphilic molecule contains a group aromatic, due to the p-stacking type interactions that form with the walls of the nanotube. Several research works have focused on the functionalization of carbon nanotubes by conductive polymers and on the study of the physical and chemical characteristics of this new composite which has significant potential for technological applications in the field of transistors, diodes, etc. of optoelectronics. Baibarac et al. have proposed a functionalization of SWCNTs by electropolymerization of aniline in HCl [76]. Chemical treatment with NH4OH leads to an internal redox reaction between the polyaniline and the carbon nanotubes, causing the polymer chain to transition from a semi-oxidized state to a reduced state. Sahoo et al. reported functionalization of carbon nanotubes by polypyrrole by in situ chemical polymerization using iron chloride FeCl3.6H2O as oxidant [77]. First, the MWCNT carbon nanotubes are treated with a mixture of concentrated acids H2SO4/HNO3 (3:1) at 90
C for 10 min. The functionalization of the nanotubes is carried out by sonication in a mixture of methanol and acetonitrile containing pyrrole and FeCl3.6H2O. The ratio of Fe3þ/pyrrole is 2.3. FT-IR analyzes showed the existence of

Figure 3.9 Chemical structure of sodium dodecyl sulfate (A) and Triton X (B).

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Figure 3.10 Interaction between polypyrrole and MWCNT-COOH according to Sahoo et al. [73].

an interaction between COOH groups of nanotubes and NH groups of polypyrrole, as shown in Fig. 3.10. Salipira et al. reported functionalization of carbon nanotubes by a cyclodextrin polymer for the detection of pollutants in water [78]. Functionalization is carried out by mixing b-cyclodextrin and hexamethylene diisocyanate and heating to 70
C. The functionalization of carbon nanotubes by bcyclodextrin shows a remarkable capacity to absorb pnitrophenol and trichlorethylene. It is also possible to form supramolecular complexes by wrapping polymers around nanotubes [79]. Thus, the functionalization of singlewalled tubes with poly (metaphenylene vinyl) (PmPV) leads to an eightfold improvement in the conductivity of the composite compared with the polymer alone, without reducing its luminescence properties [80]. Attachment of SWCNTs to polymers containing polar chains, such as polyvinyl pyrrolidone (PVP) or polystyrene sulfonate (PSS), leads to stable solutions in the water of the SWCNT/polymer complex [81]. The Iijima team reports the use of aqueous suspension of CNTs stabilized by charged nanoparticles [82]. This approach is also used by Roth’s team, which uses SDS molecules adsorbed to the surface of CNTs to electrostatically interact with ammonium groups attached to the surface of a substrate [83]. 3.2.4 Endohedral functionalization The internal cavity of the carbon nanotubes provides a host space for the deposition of target systems. For example, gold and platinum nanoparticles

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are inserted by capillary action in the tubes by treating the SWCNTs with hydrochloric acid [80]. The incorporation of fullerenes such as C60 [81], or metallofullerenes such as SmC82 [82], are also impressive examples in the internal chemistry of carbon nanotubes. 3.2.5 Examples for functionalization of carbon nanotube Several methods are mostly used to produce carbon nanotubes, and these nanotubes can be produced with different structures depending on the type of method used for preparation [84]. Therefore, it is noted that in each method, carbon nanotubes can be produced with different electrical, thermal and mechanical properties. The extent of change in these properties can vary depending on the method of synthesis, the degree of purification, and other surface treatments performed on the product [85,86]. Carbon nanotubes can assemble together to form bundle-like structures, which affects their wonderful properties and causes poor solubility in both polar and non-polar medium. The reason for the weak solubility of carbon nanotubes is due to the high van der Waals attractions between the tubes [87,88]. During the past years, a different set of strategies have been studied to improve the solubility, surface interaction and dispersion of carbon nanotubes [88]. Chemical functionalization can lead to enhancing the usual poor dispersion in different solvents, improving their technological application, improving their interactions with other materials, and changing the curing behavior of some polymers [89,90]. Often, the carbon nanotube bundles are broken up by ultrasonication, followed by treatment with a chemical agent, which “wraps up” the carbon nanotubes. This process changes the surfaces of the carbon nanotubes, and prevents their reaggregation [28]. In general, the functionalization process for carbon nanotubes is described as covalent and non-covalent bonding, depending on the chemical process type. The covalent functionalization depends on the formation of a covalent chemical bond between the carbon nanotube wall and the added chemical agent. As for the non-covalent functionalization, it depends on the intermolecular interaction between the walls of the carbon nanotubes and the added chemical agent by the van der Waals forces [28,39]. Covalent functionalization is based on the formation of a covalent bond with the functional group present on the outer wall of the carbon nanotube (such as the double bond or defects on the surface of the wall). For

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example, these functional groups can interact with different oxygen groups (such as hydroxyl or carboxylate), so that these oxygen groups are attached to the surface of the outer wall of carbon nanotubes during the oxidative reaction process. Covalent bonding changes the hybridization of carbon atoms (sp2 to sp3) with simultaneous loss of the local conjugation of the double bond. This functionalization type requires very reactive chemical reagents, commonly with high functionalization yield [91,92]. Carbon nanotubes are functionalized with different chemical groups, such as carboxylic groups (eCOOH), Hydrogenation (eH), Nitro groups (eNO2), phenyl group (eAr), and substituted benzene (eAreR) [28,94,95]. Scheme 3.1 shows the some functionalization of carbon nanotubes.

Scheme 3.1 Functionalization of carbon nanotubes with carboxylic groups, nitro groups (eNO2), hydrogen (eH), phenyl groups (eAr), and acyl groups (eCOeR).

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Scheme 3.2 Ozonation of carbon nanotubes.

The oxidation process (Scheme 3.2) is an important process in any chemical reaction, so we note that the oxidation process varies according to the type of chemical reaction. The ozonation process is one of the important oxidation processes that can be performed on compounds with double or triple bonds. Carbon nanotubes can be oxidized by the ozonation method to produce carbon nanotubes and ozone, after which the reduction process prepares carbon nanotubes containing hydroxyl groups that are very important in many other modifications that can be made on carbon nanotubes [93,95,96]. On the other hand, the fluorination is very useful reaction, because the fluorine atoms in fluorinated carbon nanotubes can easily be replaced through nucleophilic substitution reactions. For example, the fluorine atom can be replaced by an alkyl group by using the Grignard or organolithium reagents [92,93], as shown in Scheme 3.3. Amines compounds have been successfully used as nucleophilic reagents, by their reaction with the double bond present in the outer wall of

Scheme 3.3 The fluorination and then alkylation of carbon nanotubes.

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carbon nanotubes. Moreover, by using bifunctional group compounds, such as diamines compounds, carbon nanotubes can be covalently bonded to each other using these compounds [92]. Scheme 3.4 shows the reaction of carbon nanotubes with mono- and diamine compounds. During the early years of carbon nanotube functionalization, the treatment with concentrated nitric acid or a mixture of concentrated sulfuric acid and nitric acid was found to be very important in cutting or shortening the carbon nanotubes. At that time, it was speculated that the final carbon and carbon at defect sites in carbon nanotubes had been converted into carboxylic acids. In fact, this was soon demonstrated by the Haddon group [97]. There is now good evidence that a number of different functional groups can be loaded through reactions with carboxylic groups on carbon nanotubes [93]. By treating these carboxyl groups with thionyl chloride (SOCl2), the carboxyl groups in the carbon nanotubes were converted to the corresponding acid chloride, which was later reacted with NH2e(CH2)11eSH forming a covalent bond to be the resulting amide derivative of the carbon nanotubes [98].

Scheme 3.4 Functionalization of carbon nanotubes with mono- and diamine compounds.

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Scheme 3.5 Functionalization of carboxylic carbon nanotubes.

On the other side, the acid chloride carbon nanotubes react with pphenylenediamine to form another amide derivative of carbon nanotubes, which was polymerized with aniline to form aniline derivative with carbon nanotubes [99,100], as shown in Scheme 3.5. On the other hand, the properties of some polymeric compounds can be improved by adding them to carbon nanotubes. Chitosan is loaded with chitosan MWCNTs using [2 þ 1] cycloaddition of nitrine to the carbon nanotube electron system followed by a concentric reaction with chitosan, as shown in Scheme 3.6. The effectiveness of the new material (composite) was verified by the dramatic improvement of the mechanical properties (the tensile strength of the compound was significantly increased to 81.3 MPa from 36.5 MPa of pure chitosan [101]. It has been noticed lately a large trend of researchers to develop antimicrobial agents to confront infectious diseases. A very useful agent as microbial flashes is the MWCNTs decorated with silver nanoparticles, which are highly effective as antibacterials that can be used in a wide range of medical applications and water disinfection to reduce the spread of infectious diseases. Recently, Hamouda et al. loaded silver nanoparticles onto the surface of MWCNTs via wet impregnation technique followed by heat treatment at 450
C in an inert atmosphere, as shown in Scheme 3.7. The

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Scheme 3.6 Synthesis of carbon nanotubes biofunctionalized with chitosan.

Scheme 3.7 Functionalization of carbon nanotubes with Ag nanoparticles.

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antibacterial activity of Ag-MWCNTs nanocomposites against different microbial strains (bacteria and fungi) was tested. The nanocomposite showed higher antimicrobial ability against Gram-negative and Grampositive bacteria. Biological studies indicated a large inhibition zone for each bacterial strain of up to 18.5 mm. Over the last 2 decades, numerous investigations have focused on coupling and nucleophilic addition reactions between polymers and modified nanotubes. For instance, functionalized SWCNTs with phenylalkyne groups by means of diazonium salt reactions; subsequently, these reacted with copolymers synthesized via free radical polymerization: benzyl chlorinated polystyrene-co-poly(pchloromethylstyrene) and polystyrene-co-poly(pchloromethylstyrene)-b-polystyrene. Under mild conditions, Pd was used as a catalyst to connect the alkyne-modified SWCNTs with the benzyl chloride groups, resulting in a grafting extent of 53 and 81 wt%, respectively. Similarly, another study including attached thiol-reactive moieties onto MWCNT sidewalls, which reacted with a thiol-terminated poly[N-(2hydroxypropyl)methacrylamide] (PHPMA) via a coupling reaction under mild conditions. This coupling approach is successful for the synthesis of polymer-modified MWCNTs soluble in aqueous media. Liu and coworkers used tert-butyllithium and ferrocene to perform a direct monolithiation onto oxidized MWCNTs resulting in p-chloromethylstyrene-terminated MWCNTs. Afterward, the modified CNTs were functionalized with polystyryllithium, and thermogravimetric analysis indicated a grafting extent of 80 wt%. Zhang and coworkers carried out a thiol-ene addition reaction between (3-mercaptopropyl)trimethoxysilane and vinyl-terminated lowdensity polyethylene (LDPE) and trimethoxysilane-functionalized MWCNTs under mild reaction conditions. This resulted in an efficient reaction with a high grafting degree (18 wt%). Novel method for functionalization of CNT has been made depending on organometallic point. The study reports that CeOeC can be linked on the surface of carbon nanotubes via a one-pot method with two steps. The former is the nucleophilic addition of an organolithium reactive to the aromatic rings of the CNTs leading to the formation of CNT carbanions. The other is the nucleophilic substitution with halogen or hydroxyl oxacylcopropanes, such as epichlorohydrin, and comprises the reaction between the nanotube carbanions and the carbon atom linked to the chlorine, with the subsequent removal of lithium chlorine. The epoxy group may be transformed into other functionalities via a ring-opening method, which allows anchoring to hydroxyl or amine-modified polymers. Following this

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Scheme 3.8 Synthesis of PPS-NH2 grafted to epoxy-functionalized SWCNTs.

methodology, an aminated polyphenylene sulfide (PPSeNH2) derivative was been grafted to epoxy-functionalized SWCNTs. A solution of n-butyllithium was added to an SWCNT suspension in toluene (Scheme 3.8). Then, epichlorohydrin was added and the mixture (SWCNT-EP) was stirred and centrifuged. PPS-NH2 was suspended in ethanol and both polymer and SWCNT-EP dispersions were mixed and refluxed at 140
C for 4 h under nitrogen atmosphere. The resulting product (SWCNT-EP/ PPS-NH2) was vacuum dried, and the degree of grafting was close to 25%. Wu and coworkers carried out the reaction between acyl-chloride-modified MWCNTs and polystyryllithium anions, and the grafting level was found to be quite high, close to 40%. Following a nucleophilic addition reaction, Blake and coworkers grafted chlorinated polypropylene (PP) to butyllithium-functionalized MWCNTs with an extent of 31 wt% [102]. The ATRP is a way of creating a carbonecarbon bond with a transition metal catalyst. It allows the polymerization of an extensive variety of monomers with different chemical functionalities and offers good control of molecular weight, molecular architecture, and polymer composition with a low polydispersity. However, it presents some drawbacks, including the high catalyst concentration required for the reaction. This catalyst usually comprises a copper halide and an amine-based ligand. The removal of the copper upon polymerization can be tedious and costly, thus limiting ATRP use at a large scale. It is an air-sensitive reaction, difficult to be carried out in aqueous media. This technique has been employed by different authors for grafting PEG, PMMA, and poly(polyethylene glycol methyl ether methacrylate), P(PEGMA), to CNTs [50e53]. The strategy for grafting polymers from the CNTs by ATRP includes four stages, as depicted in Fig. 3.4. First, acyl-chloride-functionalized MWCNTs (MWCNTs-COCl) are synthesized via reaction of thionyl chloride with

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carboxyl-acid-functionalized MWCNTs (MWNTs-COOH); then, these react with glycol, resulting in MWCNTs-OH. Subsequently, the hydroxyl-functionalized MWCNTs react with 2-bromo-2methylpropionyl bromide, leading to MWCNTs-Br, which are then grafted onto the monomer (i.e., MMA), to yield MWCNTs-g-PMMA. Grafting degrees in the range of 32%e82% were obtained depending on the CNT/polymer weight ratio. Another study described an in situ polymerization method to attach MMA chains to CNTs via former functionalization with alkyl bromide groups via a two-step approach. The functionalized CNTs acted as initiators for ATRP of MMA, resulting in a PMMA-SWCNT nanocomposite with a high glass transition temperature albeit insoluble in common organic solvents. Another study for ATRP of polystyrene (PS) and PMMA from MWCNTs. The quantity of PMMA covalently anchored was 70 wt%, whereas for PS, it increased from 18 to 34 wt% with raising the initiator content. Thus, it appears that the polymer molecular weight can be controlled via adjusting the initiator content. Furthermore, the newest study carried out the ATRP polymerization of PMMA with SWCNTs with a grafting degree of 17 wt% (Scheme 3.9). In another study, includes prepared of acid-treated CNTs and treated monomethyl PEG-NH2 with functionalized CNT. Then paclitaxel (PTX) as model drug physically loaded onto poly(ethylene glycol) (PEG)-graftsingle walled CNTs (PEG-g-SWNTs) or PEG-graft-multi-walled CNTs (PEG-g-MWNTs) for cancer therapeutics (Scheme 3.10).

Scheme 3.9 Schematic representation for grafting polymers from MWCNTs via atom transfer radical polymerization.

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Scheme 3.10 Preparation of PTX loaded PEG-g-CNTs.

Moreover, poly(acryloyl chloride) (PAC) was first reacted with carboxyl functionalized multi-walled carbon nanotube (oxidized MWCNT) to form oxidized MWCNT-g-PAC. Then amine-functionalized Fe3O4 magnetic nanoparticles (aminated MNP) were bonded to oxidized MWCNT-gPAC to form oxidized MWCNT-g-PAC-g-aminated MNP (Scheme 3.11). FT-IR, SEM images, SEMeEDX, and VSM were used for characterization. VSM measurements showed that both oxidized MWCNT-gPAC and oxidized MWCNT-g-PAC-gaminated MNP have a super paramagnetic nature. Thermal characterization indicated that grafted structures were thermally more stable than PAC and Tg values of the grafted structures were quite higher than that of PAC. A.C. conductivity and dielectric constant measurements also performed, and their frequency and temperature dependence were examined. Another studies on the functionalization of carbon nanotubes were listed in Schemes 3.11 and 3.12.

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Scheme 3.11 The binding reaction of oxidized MWCNT and aminated MNP to PAC.

Scheme 3.12 Fluorination and further functionalization of carbon nanotubes.

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Scheme 3.13 The synthesis of polyethylene glycol f-MWCNTs.

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[70] G. Schmidt, S. Gallon, S. Esnouf, J.P. Bourgoin, P. Chenevier, Mechanism of the coupling of diazonium to single-walled carbon nanotubes and its consequences, Chem. Eur. J. 15 (9) (2009) 2101e2110. [71] Y. Sun, S.R. Wilson, D.I. Schuster, High dissolution and strong light emission of carbon nanotubes in aromatic amine solvents, J. Am. Chem. Soc. 123 (22) (2001) 5348e5349. [72] J. Kong, H. Dai, Full and modulated chemical gating of individual carbon nanotubes by organic amine compounds, J. Phys. Chem. B 105 (15) (2001) 2890e2893. [73] M. Jönsson-Niedziolka, K. Szot, J. Rogalski, M. Opallo, Pyrene sulfonate functionalised single-walled carbon nanotubes for mediatorless dioxygen bioelectrocatalysis, Electrochem. Commun. 11 (5) (2009) 1042e1044. [74] C. Ehli, G.A. Rahman, N. Jux, D. Balbinot, D.M. Guldi, F. Paolucci, M. Prato, Interactions in single wall carbon nanotubes/pyrene/porphyrin nanohybrids, J. Am. Chem. Soc. 128 (34) (2006) 11222e11231. [75] R. Haddad, Fonctionnalisation de Nanotubes de Carbone pour le Développement de Bio-architectures Affines: Application aux Biocapteurs (Doctoral dissertation, Institut National Polytechnique de Grenoble-INPG), 2010. [76] N.G. Sahoo, Y.C. Jung, H.H. So, J.W. Cho, Polypyrrole coated carbon nanotubes: synthesis, characterization, and enhanced electrical properties, Synth. Met. 157 (8e9) (2007) 374e379. [77] K.L. Salipira, B.B. Mamba, R.W. Krause, T.J. Malefetse, S.H. Durbach, Carbon nanotubes and cyclodextrin polymers for removing organic pollutants from water, Environ. Chem. Lett. 5 (1) (2007) 13e17. [78] Z. Spitalsky, D. Tasis, K. Papagelis, C. Galiotis, Carbon nanotubeepolymer composites: chemistry, processing, mechanical and electrical properties, Prog. Polym. Sci. 35 (3) (2010) 357e401. [79] A. Star, J.F. Stoddart, D. Steuerman, M. Diehl, A. Boukai, E.W. Wong, J.R. Heath, Preparation and properties of polymer-wrapped single-walled carbon nanotubes, Angew. Chem. 113 (9) (2001) 1771e1775. [80] M.J. O’Connell, P. Boul, L.M. Ericson, C. Huffman, Y. Wang, E. Haroz, R.E. Smalley, Reversible water-solubilization of single-walled carbon nanotubes by polymer wrapping, Chem. Phys. Lett. 342 (3e4) (2001) 265e271. [81] J. Zhu, M. Yudasaka, M. Zhang, S. Iijima, Dispersing carbon nanotubes in water: a noncovalent and nonorganic way, J. Phys. Chem. B 108 (31) (2004) 11317e11320. [82] M. Burghard, G. Duesberg, G. Philipp, J. Muster, S. Roth, Controlled adsorption of carbon nanotubes on chemically modified electrode arrays, Adv. Mater. 10 (8) (1998) 584e588. [83] Q. Wang, B. Arash, A review on applications of carbon nanotubes and graphenes as nano-resonator sensors, Comput. Mater. Sci. 82 (2014) 350e360. [84] S.W. Lee, Mechanical properties of suspended individual carbon nanotube studied by atomic force microscope, Synth. Met. 216 (2016) 88e92. [85] L.D.S. Cividanes, E.A.N. Simonetti, J.I.S. de Oliveira, A.A. Serra, J. Carlos de Souza Barboza, G.P. Thim, The sonication effect on CNT-epoxy composites finally clarified, Polym. Compos. 38 (9) (2017) 1964e1973. [86] A.L. Alpatova, W. Shan, P. Babica, B.L. Upham, A.R. Rogensues, S.J. Masten, V.V. Tarabara, Single-walled carbon nanotubes dispersed in aqueous media via noncovalent functionalization: effect of dispersant on the stability, cytotoxicity, and epigenetic toxicity of nanotube suspensions, Water Res. 44 (2) (2010) 505e520. [87] M.U. Khan, V.G. Gomes, I.S. Altarawneh, Synthesizing polystyrene/carbon nanotube composites by emulsion polymerization with non-covalent and covalent functionalization, Carbon 48 (10) (2010) 2925e2933.

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[88] F.V. Ferreira, W. Francisco, B.R.C. de Menezes, L.D.S. Cividanes, A. dos Reis Coutinho, G.P. Thim, Carbon nanotube functionalized with dodecylamine for the effective dispersion in solvents, Appl. Surf. Sci. 357 (2015) 2154e2159. [89] W. Francisco, F.V. Ferreira, E.V. Ferreira, L.D.S. Cividanes, A.D.R. Coutinho, G.P. Thim, Functionalization of multi-walled carbon nanotube and mechanical property of epoxy-based nanocomposite, J. Aero. Technol. Manag. 7 (3) (2015) 289e293. [90] L. Meng, C. Fu, Q. Lu, Advanced technology for functionalization of carbon nanotubes, Prog. Nat. Sci. 19 (7) (2009) 801e810. [91] A. Jagusiak, J. Goclon, T. Panczyk, Adsorption of Evans blue and Congo red on carbon nanotubes and its influence on the fracture parameters of defective and functionalized carbon nanotubes studied using computational methods, Appl. Surf. Sci. 539 (2021) 148236. [92] H.C. Wu, X. Chang, L. Liu, F. Zhao, Y. Zhao, Chemistry of carbon nanotubes in biomedical applications, J. Mater. Chem. 20 (6) (2010) 1036e1052. [93] K. Balasubramanian, M. Burghard, Electrochemically functionalized carbon nanotubes for device applications, J. Mater. Chem. 18 (26) (2008) 3071e3083. [94] L. Cai, J.L. Bahr, Y. Yao, J.M. Tour, Ozonation of single-walled carbon nanotubes and their assemblies on rigid self-assembled monolayers, Chem. Mater. 14 (10) (2002) 4235e4241. [95] K. Tsvaygboym, Photochemical studies of single-walled carbon nanotube ozonides and alpha-azoxy ketones (Doctoral dissertation), 2007. [96] J. Chen, M.A. Hamon, H. Hu, Y. Chen, A.M. Rao, P.C. Eklund, R.C. Haddon, Solution properties of single-walled carbon nanotubes, Science 282 (5386) (1998) 95e98. [97] V.N. Khabashesku, Covalent functionalization of carbon nanotubes: synthesis, properties and applications of fluorinated derivatives, Russ. Chem. Rev. 80 (8) (2011) 705. [98] J. Wei, C. Zhang, Z. Du, H. Li, W. Zou, Modification of carbon nanotubes with 4mercaptobenzoic acid-doped polyaniline for quantum dot sensitized solar cells, J. Mater. Chem. C 2 (21) (2014) 4177e4185. [99] C. Oueiny, S. Berlioz, F.X. Perrin, Carbon nanotubeepolyaniline composites, Prog. Polym. Sci. 39 (4) (2014) 707e748. [100] S.K. Yadav, S.S. Mahapatra, M.K. Yadav, P.K. Dutta, Mechanically robust biocomposite films of chitosan grafted carbon nanotubes via the [2 þ 1] cycloaddition of nitrenes, RSC Adv. 3 (45) (2013) 23631e23637. [101] H.I. Hamouda, H.M. Abdel-Ghafar, M.H.H. Mahmoud, Multi-walled carbon nanotubes decorated with silver nanoparticles for antimicrobial applications, J. Environ. Chem. Eng. 9 (2) (2021) 105034. [102] A.M. Díez-Pascual, Chemical functionalization of carbon nanotubes with polymers: a brief overview, Macromolecules 1 (2) (2021) 64e83.

Further reading [1] T. Nozaki, K. Okazaki, Plasma process, Plasma Process. Polym. 5 (4) (2008). [2] M.E. Pekdemir, M. Coskun, Chemical bonding of Fe3O4 nanoparticles on the surface of poly (acryloyl chloride) functionalized multiwalled carbon nanotubes, Iran. J. Sci. Technol. Trans. A-Sci. 44 (4) (2020) 1001e1010.

CHAPTER 4

Methods for enhancing dispersibility of carbon nanotubes Ammu V.V. V. Ravi Kiran1, G. Kusuma Kumari1, Pavan Kumar Chintamaneni2, Praveen T. Krishnamurthy1 and Renat R. Khaydrov3 1

Department of Pharmacology, JSS College of Pharmacy (JSS Academy of Higher Education & Research), Ooty, Tamil Nadu, India; 2Department of Pharmaceutics, GITAM School of Pharmacy, GITAM-Hyderabad Campus, Hyderabad, Telangana, India; 3Institute of Nuclear Physics, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan

4.1 Introduction Carbon nanotubes (CNTs) are tubular sheets of graphene that contain either single or multiple layers. Based on number of layers, CNTs are classified into three types, i.e., single-walled (single layered), double-walled (double layered), and multi-walled (multiple layered) [1]. CNTs can be synthesized by three techniques, i.e., laser ablation, chemical vapor deposition (CVD), and electric arc discharge. These methods involve high temperature, pressure, and metal catalysts, contributing to their impurity and toxicities. Due to sp [2] hybridization of carbon atoms, CNTs possess exceptional electrical, optical, mechanical, and thermal properties [1,2]. Pristine CNTs possess smooth surface which is inert and incompatible with most of the organic and inorganic solvents which requires further modifications on their surface. Enhancing dispersibility of CNTs in many solvents by surface functionalizations or modifications using covalent and noncovalent strategies could promote its use as materials in many fields including nanomedicine [3]. Covalent functionalization involves sidewall and tips modifications with hydrophilic moieties such as eCOOH and eNH2, whereas noncovalent functionalization involves attachment of polymers or biomolecules. These modified CNTs are characterized by atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), optical absorption spectroscopy (OAS), attenuated total reflectance (ATR), and Raman spectroscopy [4,5]. In the present chapter, we discuss various methods for enhancing the dispersibility of CNTs in many solvents and their applications. Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00011-X

© 2023 Elsevier Ltd. All rights reserved.

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4.2 Methods for enhancing dispersibility of CNTs Pristine carbon nanotubes (pCNTs) exhibit greater van der Waals forces aggregate or entangled form, limiting their use as nanomaterials. Ultrasonication is the most affordable and easiest technique to debundle and disperse CNTs. Although simple, ultrasonication is limited due to rebundling on removal of sonication [6] (Fig. 4.1). Debundling of pCNTs using suitable solvents is one of the suitable strategies for desired applications [7]. For past decades, many solubilizations methods have been developed for uniform dispersion especially in biochemical and electrochemical platforms. Recently, surface functionalization methodologies such as covalent and noncovalent techniques is inexpensive resulted in greater dispersion in aqueous as well as nonaqueous solvents [3,8e10]. These techniques impart hydrophilic features as well as side chain moieties, aiding in attachment as well as decrease toxicity. Several factors/criteria such as structural variations, surface chemistry, surface-to-volume ratio, nanotube size, and purity are majorly responsible for the hydrophobicity, aggregation, and toxicity of pCNTs [11]. Various strategies involved in dispersing pCNTs are briefly discussed next. 4.2.1 Covalent modification of pCNTs Covalent modifications can be achieved by either direct sidewall functionalization or by defect group functionalization. Direct sidewall covalent modification is achieved by rehybridization of sp2 carbon into sp3 therefore forming covalency with attacking species. Cycloaddition of azomethine ylides/Prato reaction, radical additions, halogenation are the widely employed approaches in direct sidewall covalent modifications. Defect group functionalization involves generation of defects via oxidation to yield carboxylic moieties that could aid in attaching different functionalities such as peptides, proteins, or antibodies [2,11] (Fig. 4.2).

Figure 4.1 Dispersing of carbon nanotubes under ultrasonication.

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Figure 4.2 Different covalent modifications of pristine carbon nanotubes.

4.2.1.1 Oxidation of pCNTs Oxidation of pCNTs is the most employed to etch and exfoliate the surface of carbon nanotubes. These oxidation techniques are categorized into two types i.e., gas-phased oxidation and liquid-phase oxidation (Fig. 4.2). Early modification strategies involved gas-phase oxidation in air and oxidative plasmas. Gas-phase oxidation along with nitric acid vapor treatment demonstrated to be an efficient method for introducing oxygen moieties on sidewalls of pCNTs [12]. Liquid-phase oxidation is achieved by refluxing CNTs with strong acids such as H2SO4, HNO3, HClO4, and H2O2 to produce carboxylic, epoxide, and hydroxyl moieties [4,13,14]. Xing and coworkers demonstrated sonication-assisted oxidation of CNTs in sulfuric/ nitric acid mixture. The procedure exhibited promoted acidic etching along with increase in population density of surface carboxy groups. Based on the sono-chemical treatment time, structural damage of MWCNT surface was observed, thereby affecting electronic properties [15]. Sonication of CNTs in nitric acid followed by treating with H2O2 is reported as the most efficient method for oxidation of MWCNTs without damaging their

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carbon skeleton. Ziegler and coworkers reported oxidation of CNTs with piranha solution (H2SO4/H2O2). On increasing temperature, the treatment found to attack existing defect sites and use oxidized vacancies to generate short nanotubes. Increase in reaction time resulted in shorter CNTs as well as selective etching. In room temperature, the piranha treatment showed slow etch rates, less carbon loss with minimal sidewall damage [16]. Selective oxidation of CNTs with H2O2 at different heating temperature and monitored via UV-visible-NER spectroscopy. Oxidizing with H2O2 has helped in achieving controlled shortening and uncapping of CNTs (with 15% H2O2 at 100
C for 3 h). Chen and coworkers developed a two-step process for effective shortening and carboxylation of CNTs. The process includes dispersing CNTs in oleum followed by nitric acid treatment (as shortening agent) into the acid-intercalated CNT dispersion [17]. Oleum effectively intercalated individual CNTs, thereby enhancing nitric acid efficiency. Solubility of ultrashort carboxylated CNTs in organic solvents, superacid and water was estimated about 2 wt.%. Gromov and coworkers reported direct attachment of amino moieties of SWCNT [18]. Two approaches were employed for the synthesis of amino-derived CNTs from carboxylated CNTs. The initial approach involves Hofmann rearrangement of the amides and later approach is on Curtius rearrangement of carboxylic acid azide. Microwave chemistry is one of the fastest and more efficient method for oxidizing CNTs. Mitra and coworker treated CNTs sulfuric and nitric acid mixture with microwave radiation to induce carboxylic and sulfonate groups for 3 min. The oxidized tubes exhibited a great dispersibility in deionized water and ethanol [19]. 4.2.1.2 Esterification and amidation of pCNTs Oxidized CNTs are widely used as precursors for further fabrication and are transformed into carboxyesters either by esterification or amidation reaction. In most scenarios, carboxylic groups are converted into acyl chlorides using thionyl or oxalyl chlorides [4]. Further, these acyl chloride conjugated CNTs will be treated with suitable alcohol or amine (Fig. 4.2). These surface functionalizations of CNTs permit to fabricate with water soluble conjugates including peptides, proteins, polymers (or biopolymers), and nucleic acids thereby improving dispersibility [20,21] (Fig. 4.3). Mounting evidence have attempted to improve the aqueous dispersibility of CNTs by conjugating biopolymers or proteins for biological use [4]. In many reactions, acyl chloride modified CNTs were introduced into highly branched molecules such as poly(amidoamine) (PAMAM) dendrons,

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Figure 4.3 Theoretical conjugation of proteins to carboxylated CNTs using EDC in the presence or absence of sulfo-NHS. (Reprinted (adapted) with permission from Y. Gao, I. Kyratzis, Covalent immobilization of proteins on carbon nanotubes using the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideda critical assessment. Bioconjugate Chem. 19 (2008) 1945e1950, doi:10.1021/bc800051c. Copyright 2008 American Chemical Society.)

poly(benzylether) to form dendrimer-CNTs hybrid. Chen and coworkers synthesized modified CNTs with polypeptides using grafting-from approach [22]. Prato and coworkers confirmed the covalent functionalization of short oxidized CNTs with alkyl amines on sidewalls and tips of CNTs using scanning tunneling microscopy (STM) imaging. Lee and coworkers created an amino-modified MWCNTs by reacting ethylenediamine with acyl chloride modified MWCNTs. Further, these amino-modified MWCNTs were modified with ruthenium (II) dye, a well-known photosensitizer for dye-sensitized solar cells [23]. In another study, short oxidized CNTs were functionalized with pyrylium moieties which exhibited greater solubility due to debundling the aggregated CNTs. Covalent functionalization of CNTs with organic amines was achieved using carbodiimide coupling. In this procedure, oxidized CNTs were initially treated with N-hydroxy succinimide (NHS) or 1-hydroxy benzotriazole (HObt) in the presence of N, N0 - dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), forming CNTs with esters. Further, the esters were replaced by amino group forming amide bond. Several number of biomolecules such as enzymes, peptides, proteins, and antibodies were conjugated covalently to CNTs via EDC/NHS reaction, rendering them water-soluble [24,25]. For instance, streptavidin was attached to carboxylated CNTs via amide linkage

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using EDC/NHS coupling reaction, forming DNAzyme-biotin chains to nanotube. DNAzyme-CNT conjugates exhibited greater selectivity, high catalytic activity, and improved cellular therapeutics [24]. In similar way, Wei Wu and coworkers covalently conjugated succinate-based ester derivative of 10-hydroxycamptothecin, radiolabeled 99mTcO4 and 99mTc via cleavable ester linkage (Fig. 4.4). The resultant MWCNT-HCPT conjugates enhanced circulation time and improved drug accumulation in tumor region [26]. Yang and coworkers proposed conjugation of single stranded DNA (ssDNA) to MWCNTs. First, MWCNTs were oxidized and activated with EDC and reacted with amine groups of ssDNA. The conjugation of ssDNA on the surface of MWCNTs was confirmed by TEM imaging [27]. In another study, Yang and coworkers developed a two-step chemical method for protein linkage. Initially, the MWCNTs were oxidized using 3:1 conc. H2SO4/HNO3 and were reacted with EDC/NHS for both activation and linkage of mouse monoclonal IgG using amidation reaction. The covalent linkage between MWCNTs and antibody was confirmed via ATR-FTIR and X-ray photoelectron spectroscopy (XPS). Further surface confirmations were performed by atomic force microscopy (AFM) and validated [28]. 4.2.1.3 Halogenation of pCNTs Fluorinations of CNTs during synthesis (i.e., arc discharge or laser ablation) at 25-600
C and were characterized using IR (at 1225 cm1 for CeF bond stretching) TEM, SEM, and XPS [29]. Kelly and coworkers confirmed the pattern of halogenation i.e., 1,4-addition reaction via STM images and semiempirical calculations [30]. Moderate solubility (w1 mg/mL) was reported by fluorinated nanotubes in alcoholic solvents. Defunctionalization of fluorinated nanotubes was achieved via heat annealing or by using hydrazine in 2-propanol suspension [30,31] (Fig. 4.5). Fluorination reaction has been one of the suitable techniques to accomplish substitution reaction. Agents such as Grignard or organolithium reagents were used to replace fluorine atoms with alkyl groups [32] (Fig. 4.5). Chlorination or bromination of CNTs were accomplished via electrochemical method. In this method, electrochemical oxidation of inorganic salts aided in coupling of chlorine or bromine atoms on the CNT. The chlorinated or brominated CNTs were reported to be soluble in polar solvents [32]. Coleman and coworkers reported the iodination of CNTs using modified Hunsdiecker reaction. Oxidized CNTs were reacted with idosobenzene diacetate in the presence of elemental iodine and UV

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Figure 4.4 Synthesis route of the MWNTHCPT conjugates. (a) Thionyl chloride, reflux; (b) Boc-NH(CH2CH2O)2eCH2CH2NH2, triethylamine, anhydrous THF, reflux; (c) 4 M HCl in dioxane; (d) d-HCPT, EDC HCl, NHS, triethylamine, anhydrous DMF; (e) FITC, anhydrous DMF; (f) DTPA dianhydride, triethylamine, anhydrous DMSO; (g) stannous chloride, 99 mTcO. (Reprinted (adapted) with permission from W. Wu, et al., Covalently combining carbon nanotubes with anticancer agent: preparation and antitumor activity. ACS Nano 3 (2009) 2740e2750, doi:10.1021/nn9005686. Copyright 2009 American Chemical Society.)

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Figure 4.5 Reaction scheme for fluorination of nanotubes, defunctionalization, and further derivatization. (Reprinted (adapted) with permission from D. Tasis, N. Tagmatarchis, A. Bianco, M. Prato, Chemistry of carbon nanotubes. Chem. Rev. 106 (2006) 1105e1136, doi:10.1021/cr050569o. Copyright 2006 American Chemical Society.)

irradiation, to produce iodinated CNTs, which was confirmed by EDX and XPS analysis [33] (Fig. 4.6). 4.2.1.4 Cycloaddition of pCNTs Cycloaddition reactions such as 1,3-dipolar cycloaddition of azomethine ylides, DielseAlder reactions, [2 þ 1] cycloaddition reactions, etc. play

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Figure 4.6 Reaction scheme for the modified Hunsdiecker reaction on SWNTs. (Reprinted (adapted) with permission from K.S. Coleman, A.K. Chakraborty, S.R. Bailey, J. Sloan, M. Alexander, Iodination of single-walled carbon nanotubes. Chem. Mater. 19 (2007) 1076e1081, doi:10.1021/cm062730x. Copyright 2007 American Chemical Society.)

crucial role in functionalization of pCNTs. Cycloaddition reactions offer controlled functionalization i.e., without skeleton disruption as well as statistical distribution, which is advantageous over other functionalization methods [34e36]. Cycloaddition of pCNTs is classified into sections: (1) Direct cycloaddition (2) Post-cycloaddition. Direct cycloaddition Direct cycloaddition reaction of pCNTs can be accomplished by either 1,3-dipolar cycloaddition of azomethine ylides (or) DielseAlder [4 þ 2] cycloaddition (or) [2 þ 1] cycloaddition (or) [2 þ 2] cycloaddition. 1,3dipolar cycloaddition of azomethine ylides of CNTs is the well-known method which grasped interest of many scientists and researchers [34] (Fig. 4.7). In a study, SWCNTs were functionalized with azomethine ylides generated via N-(4-hydroxy) phenyl glycine and formaldehyde showed higher surface potential indicating their improve dispersibility. Further, the 1,3-dipolar cycloaddition reaction was used to conjugate a fluorescent peptide on to CNTs and were tracked in human 3T3 and mouse 3T6 fibroblasts [38]. Langa and colleagues first reported the DielseAlder reaction between SWCNTs and o-quinodimethane using 4,5-benzo-1,2-oxathiin-2-oxide (under microwave conditions). SEM imaging confirmed bumps of height 1e1.5 nm indicating the functionalization [39]. Similarly, electron-rich dienes such as antracenes and 2,3-dimethyl-1,3-butadiene to initiate DielseAlder cycloaddition. In a study, pristine MWCNTs were reacted for 24 h with bulk benzocyclobutene (BCB) at various temperatures (140e235
C) [34] (Fig. 4.8).

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Figure 4.7 Scheme of 1,3-dipolar cycloaddition. (Reprinted (adapted) with permission from N. Tagmatarchis, M. Prato, Functionalization of carbon nanotubes via 1,3-dipolar cycloadditions. J. Mater. Chem. 14 (2004) 437e439, doi:10.1039/B314039C. Copyright 2003 Royal Society of Chemistry.)

Post-cycloaddition Post-cycloaddition is performed on the surface of CNTs that is previously modified. In this reaction, the functionalized CNTs were reacted with Cu1-catalyzed azide-alkynes which were reported to have applications in biomedical science, organic synthesis, and material science [40]. The Cu1catalyzed cycloadditions played key role in synthesis of small molecules,

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Figure 4.8 Scheme of DielseAlder Adduct with various benzocyclobutenes with multiwalled carbon nanotube. (Reprinted (adapted) with permission from I. Kumar, S. Rana, J.W. Cho, Cycloaddition reactions: a controlled approach for carbon nanotube functionalization. Chem. Eur. J. 17 (2011) 11092e11101. Copyright 2007 American Chemical Society.)

biomacromolecules, and dendrimers as they have high yield, high regioselectivity, easy of reaction, and good reliability [40]. Polystyrene modified CNTs using Cu1-catalyzed Huisgen [3 þ 2] cycloaddition click chemistry by Adronov and colleagues [41]. In another example, azide terminated polystyrene was reacted to alkynated-SWCNTs using Cu1-catalysis using 1,2,3-triazoles. The reaction was reported to be efficient under favorable conditions, which could further process to other chemical modifications including atom-transfer radical polymerization (ATRP) [42]. Scientists or researchers grafted polymers using grafting to and grafting from approach using Cu1-catalyzed Huisgen cycloaddition reaction [42]. For instance, amphiphilic polymers were reacted with alkyl bromo functionalized CNTs for ATRP providing azide moieties for coupling reaction. Similarly, stimuli-sensitive/responsive polymers were also coated on CNTs to create nanosensors and nanoprobes. Azide-moiety containing copolymer (consisting N,N-dimethylacrylamide and N-isopropylacrylamide blocks) was coupled with alkyne functionalized MWCNTs [43] (Fig. 4.9). 4.2.1.5 Radical addition Classical molecular dynamics simulations proposed a model for functionalization of CNTs with the help of carbon radicals, thereby indicating the probability of reaction in real scenario. Radical additions could

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Figure 4.9 Scheme of functionalization of MWNTs with PDMA-PNIPAM. (i) DMA, AIBN, dioxane, 60
C; (ii) NIPAM, AIBN, dioxane, 60
C; (iii) TDI, toluene, 80
C; (iv) Propargyl alcohol, toluene, 100
C; (v) Sodium ascorbate, copper (II) sulfate pentahydrate, H2O. (Reprinted (adapted) with permission from C.-Y. Hong, C.-Y. Pan, Functionalized carbon nanotubes responsive to environmental stimuli. J. Mater. Chem. 18 (2008) 1831e1836, doi:10.1039/B713559A. Copyright 2008 John Wiley & Sons.)

be achieved by simple covalent sidewall modification via diazonium salts. Initially, electrochemical reduction was performed on small diameter CNTs with substituted aryl diazonium salts in organic solvents [44,45]. Electron transfer between CNTs and aryl diazonium salts triggered the formation of aryl radicals. Similarly, in another reaction water-soluble diazonium salts were reacted with metallic CNTs. This methodology provided highly surface functionalized with micelle coated CNTs. In a study, very rapid and eco-friendly method for radical addition functionalization of CNTs at room temperature with various aryldiazonium salts in presence of potassium carbonate and imidazolium-based ionic liquids [46] (Fig. 4.10). The characterization of CNTs confirmed heavy functionalizations, increase in debundling, and improved solubility of CNTs. The chemical functionalization of SWCNTs with diazonium salts (with electron withdrawing groups) created hybrid materials allowed identification of characteristics features. In another research, Billups and colleagues examined reductive lithiation of CNTs in liquid ammonia. The method yielded dispersible CNT-based salts, which could readily react with aryl -haldies, -sulfides, -disulfides to produce functionalized CNTs [45]. In these reactions, a single electron will be transferred to halide moiety thereby creating radical anion which dissociates carbon skeleton.

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Figure 4.10 Functionalization scheme of SWCNTs with aryldiazonium salts in the presence of ionic liquids: R ¼ F, Cl, Br, I, NO2, or CO2CH3.

4.2.2 Noncovalent modification of pCNTs One of the major disadvantage associated with CNTs is their poor solubility/dispersibility, in majority of commonly used solvents [47]. Covalent methods for functionalization lead to disruption of sp2 structure of CNTs, thereby altering the mechanical and electrical properties. Contrastingly, noncovalent functionalization retains the intrinsic properties of CNTs and impart new surface properties, thereby serving as an attractive strategy in improving the solubility/dispersibility of CNTs. In noncovalent functionalizations, CNTs were mechanically agitated with dispersants to debundle and stabilize CNTs in solvents [10,48]. This noncovalent surface functionalization can be achieved by polymer coating, polysaccharide coating and biomolecular coating [9]. 4.2.2.1 Polymer coating Polymers are effective dispersants due to their long chain structure, which allow them to wrap around CNTs and break the van der Waals connections between their walls. Polymer coating offers advantage of forming a thermodynamically stable coat on CNTs along with ease of removal of free polymer without disturbing the polymer wrapped CNTs. Polymer coating of CNTs can be achieved by several types of polymers such as conjugated polymer (CPs), aromatic polymers, non-aromatic polymers, block polymers, and pendent polymers [49]. The efficient dispersion of CNTs in organic solvents can be achieved by CPs. Due to their large p conjugated backbone, CPs attach with the sidewalls of CNTs via p-p interactions and forms stable warp. Further, the CNTs wrapped with CPs results in synergistic effects of optoelectrical properties of CPs and the mechanical, thermostable, and conductivity

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properties of CNTs. The optoelectrical and luminescent properties of CPs can be applicable for bioimaging [10,50]. Poly(m-phenylene-co-2,5dioctoxy-p-phenylenevinylene) (PmPV) was used for functionalizing SWCNTs where polymeric backbone of the CPs aligns with the chiral lattices of CNTs and angles which increase on p surface and result in improved dispersion [51e53]. Nish and his associates reported on SWNTs wrapped with p-conjugated polyfluorene (PFO) and its benzothiadiazole (PFO-BT) derivative for increasing dispersibility [54]. Pang and coworkers reported on the superiority of cis isomer of poly[(m-phenylenevinylene)alt-(p-phenylenevinylene) in dispersing SWCNTs that the trans isomer, this was attributed to the geometry of cis-isomer forming a cavity among the chains to wrap CNTs by interacting with the planar conjugated polymer backbone with the SWCNT [55]. Polymers such as polyimides (PIs) with aromatic systems in the main chain can also wrap CNTs. Here, it was observed that one dimensional aromatic polymer such as tetraphene, showed greater affinity toward CNTs that their analog like triphenylene or pyrene [57]. Sulfonic salt of PI (PIeSO Na) showed excellent dispersibility of SWCNTs in organic solvents for prolonged periods, and exerted good thermostability. Further reports suggest that solution of PI-SO Na in DMSO has gained concentration of SWCNTs up to 2e3 mg/mL [57,58]. Polybenzimidazoles (PBIs), another class of aromatic polymers, also showed an enhanced dispersibility of both SWCNTs and MWCNTs in organic solvents [58]. Several nonaromatic polymers such as polyvinyl pyrrolidine (PVP) and polyvinyl alcohol (PVA) were also reported to enhance the dispersion of CNTs by polymer wrapping mechanism [59,60]. Stable dispersions of MWCNTs in organic solvents with polymers such as polyisoprene, polybutadiene, polyethylene oxide, and poly methyl methacrylate were reported by Baskaran et al. [56], where the CH-p interaction and its importance in dispersion of CNTs were also mentioned (Fig. 4.11). Other non-aromatic polymers also include biopolymers like sugars, nucleic acids also aid in dispersion of CNTs which was covered in biomolecular coating heading. Block polymers majorly due to their amphiphilic nature help in effective dispersion of CNTs via micellar encapsulation mechanisms. Increase in dispersion of SWCNTs was reported by Kang, Taton, and their associates with micellar formation of polystyrene-b-poly (acrylic acid) (PS-PAA) in DMF solution, induced by water [61]. Other block polymer of polystyrene (PS), polyethylene oxide (PEO), was also reported to show an enhanced dispersion of CNTs [61e63]. A conjugated block copolymer of

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Figure 4.11 Illustration of the shear-induced polymer wrapping over MWNTs under melt stirring in a composite containing small amounts of MWNTs. (Reprinted (adapted) with permission from D. Baskaran, J.W. Mays, M.S.J.C.O.M. Bratcher, Noncovalent and nonspecific molecular interactions of polymers with multiwalled carbon nanotubes. 17 (2005) 3389e3397. Copyright 2005. American Chemical Society.)

PEOPANI was shown to improve the solubility of MWCNTs for several weeks in several solvents including water [64]. The straight chain polymers can improve the dispersion of CNTS in solvents; however, their interaction is weak and may not be stable for longer periods. In order to increase the stability of dispersion of CNTs with polymers, pendent moieties with strong affinity to CNTs can be incorporated into the polymer chains [64]. In a study by Petrov et al., pyrene moiety was used as pendent was shown to improve the dispersion of CNTs in solvents [65] and porphyrin as a pendent moiety also improved dispersion of CNTs [66], due to increased interactions with CNT surfaces [67]. Further, pyrene group was found to be efficient pendent moiety when compared to phenyl and naphthyl groups [68]. However, Bahun et al. reported that this dispersion ability of pendent polymers is chain length dependent and at higher polymer chain lengths a reduction in solubility was observed [69]. 4.2.2.2 Polysaccharide coating CNTs can also be functionalized with bioactive polysaccharides which can aid in improving the solubility and biocompatibility of CNTs, while preserving their intrinsic properties. Among the many biomolecules, carbohydrate could be an excellent choice for multivalent cell surface binding with minimal cytotoxicity and good solubility under in vivo. Moreover, studies reported that polysaccharides, due to their, high hydrophilicity enable increase dispersion/solubilization of CNTs via noncovalent wrapping. Polysaccharides, glycoproteins, glycolipids, and glycans are also

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important components of the cell membrane and extracellular matrix, with important functions in cellecell communication, celleprotein interactions, and antibody and hormone molecular recognition [70]. Shinkai and coworkers reported on SWNTs functionalized with Schizophyllan (SPG), with lactoside (Lac) appendages. Taking the advantage of dissociation of SPG in DMSO and its reconstitution in water, SPG-Lac/SWNTs were prepared by mixing, CNTs dispersed in water and SPG in DMSO. Further, the structure of SPG with pendent b-1,6-glycosides with 1,2-diols enables modifications for improving the dispersibility [71,72]. It is well known that b Cyclodextrins (b-CDs) form inclusion complexes with hydrophobic molecules, where the hydrophobic molecule sits in the hexagonal channel like structure of b-CDs, and the hydrophilic groups present on the outer surface of b-CDs make the complete complex soluble. Dodziuk and coworkers reported on complexes of SWCNTS with n-CD having 12 glucosidic units, which sowed increased solubility and can be used to assess the number of types of SWCNTs present in the bulk [8]. Ikeda and coworkers reported on the differential effects maltoheptaose, D-Glucose, a, b, and Y CD on the solubility enhancement of SWCTs, where they mixed SWCNTs with the above mentioned saccharides using “high-speed vibration milling technique” (HSVM) [73]. They observed that unmodified Y CD produced complexes with higher water solubility. Later Stoddart and group considered the ability of CDs with appropriate sizes to form complexes with fullerenes, wrapped starch helically around iodine with SWCNTs to improve their solubility. This technique has also an added advantage of separation of SWCNTs at higher temperatures or by mixing with glucosidases, which can be used for purification of SWCNTs prepared from starch complexes [8,73,74]. CNTs exhibiting photoluminescence were developed by Shinohara et al., using multivalent hybrid polysaccharide, poly(p-N-acryloylamidophenyl-a-glucopyranoside) (PAPa-Glc) having a Mn 71000. The random helical structure of the multivalent hybrid polysaccharide gets wrapped round the SWCNTs with hydrophobic interactions [75]. A series of polysaccharide modified CNTs were reported by Zhang X and his associates, where, used sodium alginate (ALG), Amylose (AMY) and Chitosan (CHI) for noncovalent functionalization of SWCNTS. It was observed that there is wettability of the scaffolds were increased in comparison to pure SWCNTs. Further it was notes that the contact angle was less with AMY modification than with ALG and CHI, demonstrating the effect of functional groups on the increase in solubility (Fig. 4.12). Polar and highly hydrophilic functional groups could

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Figure 4.12 Formation mechanism of A/SeC. (a) The driving force for the formation of primary A/SeC, including hydrophobic interaction, hydrogen bond interaction or their synergism. (bed) The driving force for the subsequent processdhydrogen bond interaction. (Reprinted (adapted) with permission from L. Meng, C. Fu, Q. Lu, Advanced technology for functionalization of carbon nanotubes. Prog. Nat. Sci. 19 (2009) 801e810, doi:10.1016/j.pnsc.2008.08.011. Copyright 2009. Elsevier Ltd.)

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increase the surface energy and make the surfaces of the SWCNT scaffolds more hydrophilic. However, the combination of CHI/ALG when use used for surface functionalization of SWCNTs, showed grater hydrophilicity owing to the fact of increased hydrophilic groups [77]. 4.2.2.3 Biomolecular coating CNTs can be functionalized with biomolecules such as proteins, enzymes, nucleic acids etc., will enhance the hydrophilicity and thereby dispersibility of the CNTs. Biomolecular functionalization also enhances the biocompatibility of CNTs. Solubility and toxicity of CNTs has been addressed by the noncovalent interactions between biomolecules and CNTs. Nucleic acid functionalization RNA molecules and small double stranded DNA molecules were reported to enhance the aqueous solubility of SWCNTs, where the nucleic acid gets bound to SWCNTs via p stacking, exposing the Sugar phosphate groups to water, enabling the solubility. Single-stranded nucleic acid wrap around the CNTs with a periodic pitch, expose phosphate groups to water, and enhance solubility [78]. Single-stranded DNA sequences (>>100 base) with fully random sequence of bases was used for dispersing CNTs by Gigliotti and his coworkers. They observed that the ssDNA strand used formed a tight helix with distinct pitches around the CNTs. The distance between pitch remained same within a DNA-CNT complex, but changes between different DNA-CNT complexes [79]. Solid state mechanochemical reaction was used by Geckeler’s group to wrap DNA around both MWCNTs and SWCNTs. The CNTs were cut to shorter lengths and ere fully covered with DNA. For more than 90%, SWCNTs produced highly aqueous soluble products with 250 nm to 1 mm, whereas nearly 80% MWCNTs produced products of 500 nm to 3 mm. Both the products were found to be stable in aqueous solutions for more than 6 months [80]. Further, with molecular dynamics simulation, a DNA molecule could be spontaneously inserted into an SWCNT in a water solution. The van der Waals and hydrophobic forces were both crucial in the insertion process, with the former having a greater influence on how DNA entered the CNT. Further, experimental results also confirmed the encapsulation of CNTs with p labeled DNA molecules [81].

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Peptide functionalization The surface of carbon nanotubes can be functionalized with a variety of proteins to increase their solubility. The hydrophobic portion of the protein binds non-specifically to the sidewalls of SWCNTs, exposing the hydrophilic ends to the surface. As a result of the hydrophilic character of the surfaces, they become more water soluble [82]. Efficient dispersion of SWCNTs was reported by Dalton et al. [83], using peptides which fold into amphiphilic a-helix. They also demonstrated the p staking on the surface of SWCNTs interact with the phenylalanine (Phe) residues on the hydrophobic face of the peptide helix. Here, the hydroxyl groups and nitro groups present on the Phe residues affects the interactions between the peptide and SWNTs. Further, this dispersibility of individual SWCNTs increases with increase in the density of aromatic residue of hydrophobic face [83]. Kum Maxwell and their associates reported on suspension of protein-SWCNTs conjugate, where they different varieties of proteins like cytochrome C, BSA, HRP in deionsed water were sonicated with oxidized SWCNTs. The obtained suspension with SWCNTsProtein conjugate was found to be stable for over 15 days. However, in these conjugate, nonspecific bindings can happen as the conjugates get stacked near the hydrophobic sidewalls of SWCNTs and also near the hydrophobic parts of proteins, and increase of sizes can be observed [84]. Functionalization of biomolecules on the surface of CNTs also have several theragnostic applications in biomedical field as the biomolecular functionalized CNTs can get translocated through cell membrane and can even reach cell organelles.

4.3 Conclusion The above-mentioned processes can be practical strategies to make pCNTs highly stable dispersions for further applications. Based on the degree of functionalizations and nature of functional moieties, surface functionalized CNTs exhibited enhanced solubility in polar solvents. However, extensive level of knowledge and understanding is required to preserve the dispersive CNTs with an extensive level of characterizations and validations. Developing an inexpensive, green synthetic modifications to disperse pCNTs has to be the focus, which could revolutionize the area of nanotechnology.

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Declarations Funding details: This work was supported by the Department of Science and TechnologydIndo-Uzbek Joint Research Programme (INT/Uzbek/P-01), New Delhi.

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[79] B. Gigliotti, B. Sakizzie, D.S. Bethune, R.M. Shelby, J.N.J.N.L. Cha, Sequenceindependent helical wrapping of single-walled carbon nanotubes by long genomic DNA 6, 2006, pp. 159e164. [80] D. Nepal, J.-I. Sohn, W.K. Aicher, S. Lee, K.E.J.B. Geckeler, Supramolecular conjugates of carbon nanotubes and DNA by a solid-state reaction 6, 2005, pp. 2919e2922. [81] D. Cui, F. Tian, Y. Kong, I. Titushikin, H.J.N. Gao, Effects of single-walled carbon nanotubes on the polymerase chain reaction 15, 2003, p. 154. [82] K. Nagaraju, R. Reddy, N.J.J.O.A.B. Reddy, F. materials, A review on protein functionalized carbon nanotubes 13, 2015, pp. 301e312. [83] V.Z. Poenitzsch, et al., Effect of electron-donating and electron-withdrawing groups on peptide/single-walled carbon nanotube interactions 129, 2007, pp. 14724e14732. [84] M.C. Kum, K.A. Joshi, W. Chen, N.V. Myung, A.J.T. Mulchandani, Biomoleculescarbon nanotubes doped conducting polymer nanocomposites and their sensor application 74, 2007, pp. 370e375.

CHAPTER 5

Drug delivery aspects of carbon nanotubes Andrea Ruiu, Israel González-Méndez, Kendra Sorroza-Martínez and Ernesto Rivera Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, México

5.1 CNTs for drug delivery Drug delivery systems are defined as formulation, approaches and engineered technologies aiming to protect, transport, and release therapeutic agents into a targeted site [1e6]. These systems avoid the decomposition of the biologically active compounds in the main blood stream. These technologies lead to several benefits, such as an improved selectively to a specific site, increased efficiency and stability of the drug, less frequent dosing, better absorption and mechanism of action, together with the reductions in toxic metabolites. Up to the date, different technologies have been studied for this application: nanoshells, dendrimers, metallic nanoparticles, micelles, and nanocapsules can be mentioned among others. However, an interesting alternative to the above mentioned methods is related to CNTs [7e10]. CNTs are part of the family of carbon nanostructures including systems like nanodiamonds, nanocones, nanohorns, and graphene (Fig. 5.1). These structures have been widely studied and described since 1985, year of the discovery of fullerene derivatives [11e14]. These structures find application in different areas such as energy storage, magnetic force spectroscopy, microwave absorption, and lithium batteries [15e20]. Only recently, these derivatives have been investigated from the biological point of view, in particular, in drug delivery, being CNTs the most studied [21,22]. CNTs are microtubules constituted by graphitic carbon. Their discovery date is classically fixed in 1991, following the work of Iijima [23]. However, more in-depth literature studies [24] relocate their discovery to 1952, by the work of Radushkevich and Lukyanovich [25]. This latest work was reported in Russia, leading to a poor diffusion of the discovery to the worldwide scientific community. Nevertheless, other subsequent studies Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00008-X

© 2023 Elsevier Ltd. All rights reserved.

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Figure 5.1 Example of allotropic forms of carbon.

were inherent to the CNTs, starting from the work of Endo [26] in 1976 or the Abrahamson’s report [27] in 1979. In these cases, “enrolled graphite sheets” were observed, which would be later classified as CNTs. Briefly introduced the controversy of their history, CNTs are hollow tubular systems. These tubes are constituted by sp2 hybridized carbons, similar to graphene and fullerenes. Indeed, CNTs can be envisioned as rolled up graphene sheets. They are normally divided into two macrofamilies, meaning single-wall CNTs (SWCNTs) and multi-wall CNT (MWCNTs) [28]. SWCNTs have a diameter from 0.4 to 3 nm, with a nanometer scale length. They are normally self-assembled to form bundles, organized into a hexagonal crystalline system. SWCNTs normally present three different forms such as zigzag, chiral and armchair, according to their formation process (Fig. 5.2) [29e31]. MWCNTs can have an inner diameter, whose dimension differs from 0.4 nm to several nanometers, depending on the number of layers forming the structure. The external diameter can reach up to 30 nm. They normally both-end capped by a half-fullerene like system. MWCNTs are divided in two categories, depending on their structural composition. If they are constituted by concentric CNTs containing one into the other, they can be classified as Russian Doll model. Instead, if they present a single graphene layer wrapped around itself, they can be called Parchment model, forming a scroll-like system [32,33].

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Figure 5.2 Classification of CNT.

CNTs can be prepared through different techniques, mainly involving gas phase processes. Among others, three fabrication methods are being thoroughly studied and reported (Table 5.1). The most used process are the chemical vapor deposition (CVD) techniques, the laser ablation technique, and the carbon arc-discharge method. The latest techniques are the first ones used for the CNTs preparation, but they involve high temperatures (>800
C) or only small-scale accessibility, requiring long purification processes. For these reasons, the CVD method is nowadays the most used one [34]. The introduction of CNTs in the medical field took place at the beginning of 21st century. Their high surface area, impressive chemical Table 5.1 Preparation methods of CNT. Method

Laser ablation

Arc discharge

CVD

Yield Type of CNT Advantage

>75% SWCNT and MWCNT Room temperature and high purity process Lab-scale method

>75% SWCNT and MWCNT High quality tubes, simple and inexpensive method High temperature and purification needed

>75% Mainly MWCNT

Disadvantage

Simple, large-scale CNT production, low temperature MWCNT with defects

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stability, and electron-rich chemical structure, candidate CNTs for potential absorption and/or conjugation with biological active molecules. The use of CNTs offers several advantages: - Reduce side effects - Prove therapeutic effect even as drug carrier - Avoid adverse impact on the immune system - Application as diagnostic agents - Retaining capacity for different types of drugs The application of CNTs in the biological field has been tested in both pharmacy and medicine (Fig. 5.3). To pursue these applications, they are mainly used as carriers. The biological active entity, which can be a drug, a gene, or a sensor, is linked or absorbed to the CNTs (the method will be discussed later in this chapter) [7,33,35]. The resulting conjugate is introduced into the patient’s body through conventional (oral or injection) or unconventional (i.e., magnetic conjugated) ways. The conjugated is the adsorbed in the targeted organ or cell to deliver the bioactive molecule. CNTs find application in different biomedical fields, where most of them involve them as carriers for bioactive compounds. The most important applications are the following: - Cancer therapy - Infection therapy - Gene therapy by DNA delivery - Tissue regeneration - Neurodegenerative diseases therapy - Antioxidants - Biosensors - Drug extraction CNTs can be easily functionalized to be used as carriers. Thanks to its reduced dimensions, they can cross biological membranes and introduce the biological active compounds in the site of interest. In addition, the high aspect ratio of the nanostructure allows multiple attachment sites for the drugs, leading to a lower loading of the overall doses. CNTs have been loaded with anticancer drugs, such as epirubicin, doxorubicin, and cisplatin, showing higher cytotoxicity for the cancerogenic cells. Additionally, thanks to their strong absorbance in near-infrared region, CNTs are potential candidates for hyperthermia therapy, showing already promising results for pancreatic cancer [36]. CNTs have been also coupled with antiviral and antibacterial agents such as Pazufloxacin. Moreover, they have been used in vaccine delivery

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Figure 5.3 Applications of functionalized CNT in medicine.

procedures, thanks to the functionalization with different cell peptide epitopes, giving strong immune response [37e39]. The coupling of DNA probes to CNTs protects the organic bioactive derivative from potential enzymatic cleavage and other biological processes. Consequently, this gene therapy approach permits to obtain DNA-CNT complexes characterized by higher biostability and DNA delivery capability [40e42].

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The use of CNTs for tissue generation takes great advantages from the physical and mechanical properties of the nanostructures. CNTs are characterized by their biocompatibility, high biodegradation resistance, and ability to reinforce the mechanical strength of the recovering tissues and improved conductivity [43,44]. The use of organic compounds to trap oxygen-free radical has been studied for a long time. However, only recently the relation between these radicals and the disease has been proved and studied. For this reason, the development of novel radical traps in the biological field has exponentially increased in the last years. CNTs are the new entry in this family, and their scavenger abilities have been tested very recently. Acid CNTs are natural antioxidants which are proposed in useful biomedical applications for the prevention of chronic ailments, aging, and food preservation [45e47]. The high functionalization ability, which makes CNTs an excellent bioactive molecules carrier, enables the nanostructures as drug extractors. Thanks to the possibility of p-p stacking, CNTs have been tested as solid phase extractors. They have been found to be more efficient than classical silica base sorbent and macroporous resins, showing extracting ability in different media as biological fluids, drug preparations, environment, plants, and animal organs. Their efficiency has been verified for aromatic pesticides, phenolic derivatives, and different natural compounds. In addition, CNTs have been tested for the extraction of inorganic and organometallic compounds and are already applied in the preparation of analytical equipment as GC and LC columns [48,49].

5.2 Surface engineering of CNTs for drug delivery CNTs are characterized by a highly hydrophobic behavior, due to their fully conjugated aromatic structure. This kind of structures would potentially lead to high cytotoxicity or poor solubility in biological media, annihilating their application in the medical field. For this reason, to enable their application in biological fields, their surface functionalization is mandatory and can lead to enhanced dispersion, solubilization, biocompatibility, and reduced cytotoxicity [50]. This functionalization can be achieved through a covalent or a noncovalent attachment (Fig. 5.4). The covalent modification of CNTs is obtained by introducing different functional groups like halogens, carbenes, arynes, carbonyl, hydroxyl, and carboxylic acids among others. The introduction of these functional groups allows a direct linkage of the active molecules to the CNTs [51,52].

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Figure 5.4 Covalent and noncovalent functionalization of CNT.

The noncovalent modification can be achieved through simple reactions and conditions (sonication, centrifuge), avoiding time and energy demanding reactions and purification. This method relies on the use of surfactants and biological polymers, which can interact with CNTs by van der Waals forces, pep, and CHep interactions. 5.2.1 Noncovalent functionalization of CNTs The noncovalent functionalization of CNTs presents the main advantage to keep intact their structure, which maintains the impressive mechanical and physical properties (Fig. 5.5). This functionalization is mainly obtained following two different methods [50]: - Use of amphiphilic molecules able to interact with CNTs through hydrophobic units - Formation of acid-base salts, using the ionic interaction of CNTs. These functionalization methods can lead to the exfoliation of the typical rope-like supramolecular assembly of the CNTs as well. For example, reacting octadecylamine with acid SWCNTs, it is possible to obtain smaller assemblies with a diameter of a few nanometers, which are stable for over 10 days [53]. The interaction of the CNTs with hosting systems is possible through p-p interactions, functionalizing the system. An example of such functionalization is the use of pyrene and aromatic moieties, which can enhance the CNTs solubility if functionalized with hydrophilic groups. Higher molecular weight systems can be used to noncovalently functionalize CNTs [54e56]. In this case, the use of proteins or conventional polymers can be taken into account. The interactions between CNTs and the hosting system can be defined as van der Waals forces, between the hydrophobic part of the polymer and the surface of the nanostructure. In

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Figure 5.5 Characteristics of noncovalent and covalent CNT functionalization.

this case, the high molecular weight of the functionalizing system allows enrolling the CNTs, but it can take place through different mechanisms depending on the nature of the used polymer and/or protein. The wrapping can undergo through two different mechanisms: - Thermodynamic model - Helical model

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In the first case, the polymer interferes with the supramolecular CNTs organization with its hydrophobic part, disrupting the common rope-like assembly and wrapping the aromatic nanostructure. The hydrophilic part will be exposed to the water solution, to minimize the hydrophobic interface and subsequently increase the solubility of the functionalized CNTs. The polymer enrolls uniformly the CNTs, despite a random functionalization, increasing the polymer-nanostructure interactions and the thermodynamic stability of the functionalization. The second model instead allows a better explanation of the increased solubilization of the CNTs after functionalization, but at a molecular level. When rigid aromatic polymers (i.e., polyarylethynylene) are employed, these interact with the CNTs surface by forming a structure parallel to the nanostructure axis. In this case, the wrapping is forbidden, due to the rigidity of the polymers. On the contrary, by using more flexible polymers, a wrapping process can be observed through AFM studies [57,58]. A very interesting characteristic of such functionalization stands in its reversibility. Despite the high stability of the process due to the strong ongoing interaction between the CNTs and the functionalizing polymer, it is possible to reverse the process by changing the solvent or substituting the polymer with other typology of surfactant units. 5.2.2 Covalent functionalization of CNTs The covalent functionalization of CNTs can be divided in two categories. The first one concerns the ends or the defect sites of the CNTs, which allows the addition of amides and esters without interfering with the psystem. The second category mainly involves the sidewall of the CNTs, functionalizing the double bonds of the conjugated system. The covalent functionalization of the CNTs ends is normally started by multistep acid treatments. These oxidative processes are historically the first functionalization of CNTs, being used as well as purification step to eliminate the catalysts employed in their synthesis. Different kinds of gasphase and solution-phase methods are used for the CNTs oxidation, including H2 S þ O 2 , O3, H2SO4 þ KMnO4, HNO3 and H2SO4 þ HNO3. These oxidation methods introduce easily functional chemical groups such as COOH, OH, and C¼O (Fig. 5.6) [59e62]. The carboxylic group (COOH) is the most used functional group, thanks to the good control of the active site generation through the different oxidation methods (i.e., HNO3 treatment leads 1-3 wt% of COOH groups) [63].

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Figure 5.6 Examples of end-functionalization of CNT.

This kind of functionalization provides two main advantages: - The p-conjugation of the CNTs stands unaltered, allowing to maintain the mechanical and electronical properties of the nanostructure. - The chemical functionalization brings a huge impact on solubility of the CNTs and facilitates a precise manipulation of the active sites. The first reported functionalization of the CNTs was based on the end amidation of the structure. This was obtained by a first conversion of the CNTs through acid, followed by thionyl chloride reaction and concluded by a thermal treatment with octadecylamine for 3 days at 100
C [64,65]. The obtained system was then soluble in the most common organic solvents, allowing a first characterization of the soluble CNTs. This amine treatment was a breakthrough moment for the CNTs chemistry, leading to a direct functionalization by using glucosamine, amine-rich polymers, and proteins. The end functionalization has usually low effects on the physical and electronical properties of the CNTs, since it involves only side sites and defects, and does not modify the p-conjugation of the structure [66e69]. Due to the different chemistry of the carbon atoms at the end and in the sidewall of the CNTs, the latter needs a different treatment. The sidewall carbon bonds have a lower reactivity, needing high reactive reagents to allow their functionalization. Examples of these chemicals are carbine [35,70], fluorine [71], aryl radical [59,72], and azomethine ylides [73] (Fig. 5.7). The sidewall functionalization leads to a drastic modification of

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Figure 5.7 Examples of sidewall CNT functionalization.

the physical properties of the CNTs, due to the introduction of defects into the p-conjugation of the structure. Indeed, this modification brings a change in the hybridization of the carbon atoms, which change from sp2 to sp3. This conversion modifies the surface potential, altering the electrical properties of the systems, as well as leading to a distortion of the tubular shape itself. The solubility in organic or aqueous solution of sidewall functionalized CNTs is normally achieved by introducing different functional groups, such as long alkyl chains and nitrenes.

5.3 Recent applications of CNTs for drug delivery of non-anticancer drugs Nowadays, the delivery of cancer chemotherapeutic agents using CNTs has been the most studied strategy; however, CNTs stand out for their multiple

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properties as drug delivery systems (DDS). In this regard, CNTs are promising candidates as a potential carriers for the delivery of multiple types of drugs, such as antimicrobial, anti-inflammatory, antihypertensive, and antioxidant agents. These CNT-based DDS, despite being less frequently reported, have shown to successfully enhance the therapeutic efficacy and modulate the release profile of various non-anticancer drugs [74]. 5.3.1 CNTs for improving the antimicrobials treatments 5.3.1.1 CNTs for improving antimicrobials formulations An antimicrobial agent is a general term that is mainly concerned with antibiotics, antibacterials, antifungals, antivirals, and antiprotozoans. Antimicrobial agents are drugs, chemicals, or other substances that are capable of acting by two modes either kill (microbiocidal) or slow the growth of microbes (microbiostatic) [75]. These agents represent one of the most popular classes of non-anticancer agents assembled in CNTs. Amphotericin B (AMB) is one the most important examples of antimicrobials proved in CNTs. AMB is a polyene macrolide considered as one of the first-line agents to combat opportunistic systemic fungal infections in immunocompromised patients. Despite its broad activity spectrum, high effectiveness and relatively low cost, the use of AMB is, however, limited by numerous undesirable side effects, ranging from infusion reaction (an effect postulated to result from proinflammatory cytokine production) to nephrotoxicity [76]. One of the factors contributing to the toxicity of AMB lies in its natural tendency to aggregate due to its low water solubility. CNTs chemically derivatized with AMB were hence attempted, with the aim to improve the solubility of AMB and decrease its aggregation potential [74]. In order to improve the AMB biopharmaceutical properties, MWCNTeAMB and SWCNTeAMB conjugate that were designed with PEG linker were prepared with AMB loading of 25% and 10% w/w, respectively [77]. These conjugates were tested for their antifungal activities against a collection of reference and clinical fungal strains, in comparison to native AMB and a conventional colloidal dispersion AMB deoxycholate formulation. Both CNT conjugates displayed broad spectrum of antifungal activity that was considerable more potent than AMB alone. The MWCNT-based AMB conjugate, in particular, resulted to be active in several AMB-resistant fungi, and demonstrated a nonlytic mechanism of action, where the conjugate only permeabilized fungal membrane after extended period of incubation and induced membrane depolarization at slow kinetics.

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In this way, the work of Pruthi et al. reported another strategy where the AMB was physically adsorbed on mannosylated MWCNT to achieve specific delivery of the drug to macrophages for the potential treatment of leishmaniasis [78]. With around 0.66 mM of mannose present in every g of MWCNT, the mannosylated MWCNT possessed high affinity to bind lectins (Concanavalin A) and were able to be uptaken by J774 macrophages in sufficient amount. Nevertheless, AMB was adsorbed onto the mannosylated MWCNT with good entrapment efficiency of around 75% and demonstrated a sustained in vitro release profile. Other drug used as antimicrobials agent is pazufloxacin mesylate, an antibiotic belonging to the class of fluoroquinolones, was adsorbed onto MWCNT functionalized with ethylenediamine [79]. In vitro release suggested that the adsorption of pazufloxacin was reversible, and its release profile consisted of an initial rapid burst release followed by a period of sustained release. Interestingly, it was revealed that the total amount of pazufloxacin released from the amino-functionalized CNT was almost a fold higher at pH ¼ 5.7 than pH ¼ 7.0, possibly due to enhanced hydrophilicity of the protonated pazufloxacin in acidic conditions, which consequently diminished their hydrophobic pep interaction with the CNT carriers. The sensitivity of this DDS to pH may benefit the treatment of tumors, whose pH value is lower than that of the normal tissue. Besides, it could be also advantageous in the treatment of infections, where the intracellular environment of infected cells is likely to be more acidic [74]. Another family of antibiotic agents are the aminoglycosides, for example gentamicin that was incorporated into bullfrog collagen hydrogel doped with 1% w/w CNT [80]. In this context, CNTs served as an additive to enhance the physical stability of the hybrid hydrogel and to retard the release of gentamicin. The release modulation effect was attributed to the formation of an irregular CNT network in the hydrogel that impeded solvent diffusion, and to the presence of carboxylic functional groups on the CNTs that could interact with the amine groups of gentamicin. Thus, the presence of CNTs might improve drug loading by simple chemical attraction between CNTs and gentamicin, potentially resulting in differential drug loading in both control and CNT-laden hydrogels, thereby affecting the rate of drug release. In a recent work, Zomorodbakhsh et al. proposed the use of MWCNTs for improving the tuberculosis (TB) treatment [81]. TB has been recognized as one of the most fatal infectious diseases, which is caused by Mycobacterium tuberculosis (M.tb). Isoniazid (INH) is the most commonly utilized drug in the

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treatment of TB. Patients need to take 300 mg of INH daily for 6 months in combination with another anti-TB drug and tolerate several side effects of INH. On the other hand, the emergence of resistant strains of anti-TB antibiotics is one of the major problems in the treatment of this disease. So, with the aim of optimizing TB treatment in this work, it has been prepared a conjugated MWCNTs with INH by ester bonds and then a nanofluid was fabricated which was evaluated on M.tb. The new formulation MWCNTsINH had a significant antibacterial effect in low dosages, in comparison with INH alone. Furthermore, this nanodrug has more potential advantages, such as better penetration of the bacterial membrane, increased yields at the lower concentrations than the usual therapeutic doses and a decreased bacterial resistance toward the usual form of antibiotics. 5.3.1.2 CNTs as antimicrobials agents According to some recent reports, carbon-based nanomaterials such as fullerenes and CNTs, especially SWCNTs, show potent antimicrobial properties [82]. In this regard, Kang et al. provided the first report showing that SWCNTs had strong antimicrobial activity on Escherichia coli (E. coli) [83]. They demonstrated that SWCNTs could cause severe membrane damage and subsequent cell death (Fig. 5.8). In another study, the same author presented the first evidence that the size of the carbon nanotubes is an important factor that affects their antibacterial activity [39]. They

Figure 5.8 Proposed mechanism of antimicrobial activity of CNT.

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prepared SWCNTs and MWCNTs and investigated their antibacterial effect against E. coli. The results revealed that SWCNTs were significantly more toxic to bacteria than MWCNTs. The authors also reported that direct cell contact with CNTs influenced the cellular membrane integrity, metabolism processes and morphology of E. coli. According to the authors, SWCNTs could penetrate into the cell wall better than MWCNTs due to their smaller nanotube diameter. Furthermore, the superior surface area of SWCNTs initiated better interaction with the cell surface [39]. Then, Arias and Yang investigated the antimicrobial activities of SWCNTs and MWCNTs with different surface groups toward rod-shaped or round-shaped Gram-negative (Gram ve) and Gram-positive (Gram þve) bacteria [84]. According to their results, SWCNTs with surface groups eOH and eCOOH showed improved antimicrobial activity to both Gram þve and Gram ve bacteria, while MWCNT with the same surface groups did not exhibit any significant antimicrobial effect. The results showed that the formation of cell-CNT aggregates caused a damage to the cell wall of bacteria and consequently release of their DNA content. In a study by Yang et al., the effect of SWCNTs length on their antimicrobial activity was investigated [85]. Upon their findings, longer SWCNT exhibited stronger antimicrobial activity due to their improved aggregation with bacterial cells. Finally, Dong et al. investigated the antibacterial properties of SWCNT dispersed in different surfactant solutions (sodium holate, sodium dodecyl benzenesulfonate, and sodium dodecyl sulfate) against Salmonella enteric (S. enteric), E. coli, and Enterococcus faecium [86]. According to the results, SWCNTs exhibited antibacterial activity against both S. enterica and E. coli, which augments with the increase of nanotube concentrations. The combination of SWCNTs with surfactant solutions showed to be less toxic to 1321N1 human astrocytoma cells, so they can be employed in biomedical applications especially for drug-resistant and multidrug-resistant microorganisms. 5.3.2 CNTs for improving the anti-inflammatory therapy Anti-inflammatory agents can be broadly classified into two major classes: (1) the steroids-based molecules, specifically glucocorticoids and (2) the non-steroidal anti-inflammatory drugs (NSAID). Both of these antiinflammatory agents have been delivered with CNTs acting either as the main drug carrier or as adjuncts to assist or modify their release from another parent delivery systems [74].

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Using MWCNTs as nanoreservoirs, oxidized MWCNTs loaded with dexamethasone (Dex) phosphate, a steroidal anti-inflammatory drug, were sealed and incorporated into a film of polypyrrole via electropolymerization [87]. The use of CNTs and polypyrrole complemented each other by mitigating their weaknesses. Since polypyrrole is a conducting polymer, it is able to release drug in response to an electrical stimulus. However, it is limited with issues such as low drug loading and non-sustainable drug release per stimulation. On the other hand, while the interior of CNTs with open ends can be filled with drugs, drug leakage from CNTs could lead to uncontrollable drug release [74]. By combining both components, polypyrrole film could be used to seal the open ends of drug-laden CNTs to abate leakage. In exchange, drug-loaded CNTs could serve to raise the system’s overall drug loading capacity and to modify the release profile of the encapsulated drug. Indeed, Dex was successfully loaded within the inner cavity of oxidized MWCNTs and the polypyrrole coating was able to store the encapsulated Dex effectively, without any significant leakage in the absence of electrical stimulation. Drug loading was increased by almost a fold with the incorporation of CNTs nanoreservoirs. Thus, upon the application of electrical stimulation Dex was released out of the polypyrrole film via a de-doping process, where positive charges in the polymer backbone were neutralized to promote the dissociation of negatively charged Dex phosphate. The CNT-containing film was able to release Dex in a sustained and linear manner due to the extra drug stored in the nanoreservoirs. More importantly, Dex retained its pharmacological activity after loading and stimulated release, as demonstrated by measuring the amount of nitrogen oxide, an inflammatory product secreted by lipopolysaccharide-activated microglial cells. Such delivery system is useful in the development of implantable microelectrodes as neuronal prosthesis that can deliver anti-inflammatory agents to combat inflammation-induced neuronal loss and scar formation due to chronic application of microelectrodes. Transdermal delivery of NSAID is particularly useful in alleviating pain associated with arthritic conditions and musculoskeletal damage. While transdermal drug delivery causes less systemic toxicity compared to conventional routes such as oral administration, release of drugs from transdermal devices is often low and lacks precise control [74]. Capitalizing on the excellent electrical conductivity of CNTs, these have been used as an additive in electro-responsive gel systems to modulate and enhance the release of several NSAID at a relatively low voltage that is non-irritating to

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skin. Diclofenac sodium, for instance, has been incorporated into spherical gelatin/MWCNT hybrid microgels to form an electro-responsive DDS [88]. CNTs confer to the gelatin microgels greater thermostability and electrical conductivity. Interestingly, incorporation of CNTs of as high as 35% w/w did not affect drug loading and resulted in a higher rate and total amount of released drug. The authors reported a higher gel swelling arising from larger capillary networks formed at higher CNTs concentration. The release of diclofenac from the hybrid microgel was further increased by 20% with voltage application due to electrically induced shrinkage. In another study, the same drug, diclofenac sodium, was loaded into carboxymethyl guar gum and oxidized MWCNT hybrid polymer hydrogel at different CNT concentration (0.5%, 1.0%, and 3.0% w/w) [89]. It was depicted that 1.0% CNT w/w hybrid hydrogel possessed the highest drug load and the slowest rate of drug release, resulting from strong drugeCNT association and high matrix viscosity. Higher CNT concentration of 3% w/w, however, gave poorer gel performance, since CNTs were more likely to aggregate and reduce the available free surface area. It seems that there is a significant difference between the two studies related to the concentration of incorporated CNT and their eventual effects on the mechanical properties and the release profile of diclofenac from CNT-hybrid gel DDS. This shows an urgent need for standardized drug load studies in CNT. Ketoprofen is another example of NSAID that has been incorporated into an electro-sensitive transdermal DDS impregnated with CNTs. Specifically, a semi-interpenetrating polymer network composed of polyethylene oxide (PEO) and pentaerythritol triacrylate interspersed MWCNT (10% with respect to PEO) and ketoprofen were electrospun into fibers [90]. According with the design, the electrical conductivity of the fibers was increased with the addition of MWCNTs. In the absence of voltage application, in vitro skin permeation of ketoprofen was slightly lowered with the introduction of MWCNTs, blocking available space for diffusion. Under electrical stimulation, however, CNTs accelerated the effect of electric voltage, leading to higher drug release. The CNT-laden fiber also did not affect the viability of mouse L929 fibroblasts, suggesting the biocompatibility of such transdermal DDS. Other than transdermal delivery, prolonged and sustained release of NSAID can be also achieved with the use of membrane technology, where a drug-core is surrounded by layers of membranes that control drug release via diffusion [74]. Oxidized MWCNT containing semipermeable cellulose acetate polymer membrane was employed to coat an indomethacin osmotic

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pump tablet system, and the effect of CNT on the release characteristics of indomethacin was investigated [91]. At 0.01% w/w of CNT against cellulose acetate, a higher amount of indomethacin was released in a zeroorder release pattern, as result of increased membrane porosity and higher extent of water uptake, attributed to the presence of oxidized CNTs with hydrophilic groups. However, higher concentration of CNTs failed to form homogenous and porous membranes because of the aggregation and reduction in the effective surface area of CNTs. From the results of this study, we can affirm that it exists an optimum concentration of CNTs to be used as additive to modulate the drug release. Excessive amount of CNTs could be counterproductive, especially if they aggregate and form bundles with reduced surface area. Repetitive use of intra-articular corticosteroid injections are the way for treating synovial inflammation in advanced arthritis. However, short- and long-term use of corticosteroids usually triggers serious side effects (i.e., adrenal insufficiency, hyperglycemia, Cushing syndrome, osteoporosis, Charcot arthropathy, etc.). Recently, another study demonstrated that conjugation of a corticosteroid (triamcinolone) on polyethylene-glycol (PEG)-fabricated MWCNTs enhances intracellular drug delivery via increased lysosome transport and besides suppresses the expression of major pro-inflammatory cytokines (i.e., TNF-a, IL-1b, and IL-6) and matrix metalloproteinase-1 and -3 from fibroblast-like synoviocytes at a very low drug dose [92]. Therefore, low-dose triamcinolone conjugation with carbon nanotubes suggested a potential drug candidate for resolving side effects associated with conventional arthritis treatment. 5.3.3 CNTs for improving the antihypertensive therapy Treatment of hypertension typically involves oral administration of synergic mixtures of different antihypertensive drugs, which has various mechanisms of action. Examples of the different classes of antihypertensives include diuretics, angiotensin converting enzyme inhibitors, angiotensinogen receptor blockers, b-blockers, and calcium channel blockers. In addition to lowering blood pressure, some of these antihypertensives are also useful in the treatment and management of other cardiovascular diseases, such as angina, ischemic heart disease and heart failure. While these agents are typically orally administered, increased attention has been devoted to the development of novel DDS that can prolong the drug’s half-life and enable facile administration with reduced frequency to improve patient compliance [74].

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One way to help the patient to adhere the treatment is through the development of transdermal DDS, where drugs can be released from a reservoir system in a sustained and controlled topical manner. To address this strategy, Bhunia et al. reported the preparation of transdermal composite membrane consisting of PVA and carboxylated MWCNTs, loaded with diltiazem hydrochloride, a water soluble non-dihydropyridine calcium channel blocker [93]. Incorporation of CNTs increased the tensile modulus, ultimate tensile strength, percent elongation and viscosity of the composite membrane, resulting in low bursting tendency and sustained release of diltiazem when tested with a Franz flow diffusion cell. In addition, CNTs were also able to adsorb diltiazem and retard its release. Further increase of CNTs concentration past 1% w/w, led to poorer membrane mechanical properties and performance due to the formation of CNTs aggregates. Another way of achieving controlled and sustained release of highly water-soluble drugs is through microencapsulation. Metoprolol tartrate, a water-soluble antihypertensive belonging to the class of b-blocker, was chosen to be the model drug to formulate a sustained release polymeric microparticulate formulation with ethyl cellulose (EC) microsphere impregnated with MWCNTs via solvent evaporation technique with acetone [94]. The ability for metoprolol to be homogenously adsorbed onto CNTs minimized leaching of the drug from EC, leading to elevated drug loading and sustained drug release profile up to 24 h in CNT-laden EC microspheres, compared to plain EC microspheres. Finally, to bring these systems closer to the clinic, long-term safety evaluation is critical, especially for antihypertensives where chronic drug intakes are required. Candesartan cilexetil and diltiazem hydrochloride, belonging to the drug class of angiotensinogen receptor blockers and non-dihydropyridine calcium channel blockers, respectively, were noncovalently loaded successfully onto MWCNTs by nanoprecipitation [95]. The differences in hydrophobicity of the two drugs led to different release profiles, where hydrophobic candesartan cilexetil showed slow release of only 20% while highly water-soluble diltiazem hydrochloride was released rapidly to almost 90% within 15 h. Recently, data has been obtained on the effects of repeated systemic administrations of water-dispersible SWCNTs to spontaneously hypertensive rats with respect to constitutive NO-synthase (cNOS) [96]. As known, NO is an inhibitory transmitter in the cardiovascular system. It was found that the systemic (i.p., subcutaneous, and i.m.) introductions of SWCNTs

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for 2 weeks resulted in considerable elevations of the NOe 2 level (a marker of NO bioavailability) in the blood of experimental hypertensive animals. Thus, SWCNTs may be used in the future for antihypertensive therapy as a novel agent capable of activating cNOS, and thus increasing the NO production in central and peripheral elements of the cardiovascular system. 5.3.4 CNTs for antioxidants delivery One molecule with antioxidants properties is an entity with the ability to scavenge free radicals, consequently this class of compounds can reduce cellular oxidative stresses related to aging and a variety of human disease states, such as Alzheimer’s disease, Parkinson’s disease, cardiovascular diseases and diverse types of cancer. Co-delivery of antioxidants with CNTs is particularly relevant in mitigating some of the nanotoxicological concerns associated with SWCNTs, specifically antioxidant depletion and ROS generation [97,98]. Tocopheryl PEG succinate (TPGS) is a synthetic water-soluble precursor of the natural antioxidant a-tocopherol (Vitamin E), commonly used as surfactant to enhance the solubility of drugs in pharmaceutical industries. In a study by Yan et al., TPGS was found to be capable of dispersing both MWCNTs and SWCNTs better than the conventional non-ionic surfactant Triton X-100. TPGS formed amorphous coating on the CNTs surface after drying, indicating the potentiality of such system to be used as delivery system for Vitamin E [99]. Quercetin and rutin, belonging to the flavonoid class of antioxidants, were co-precipitated with SWCNTs with ultrasonic field and the antioxidant ability of the biohybrid conjugates were evaluated in a chemiluminescence assay with luminol and hydrogen peroxide [100]. According to the design, SWCNTs simple or hydroxylated and carboxylated show an intrinsic antioxidant activity. In increasing sequence SWCNTCOOH > SWCNT-OH > SWCNT lead to that proton donating is more efficient than electron donating in the mechanisms of free radicals scavenging. This phenomenon is contradictory to previous reports of oxidative damage induced by SWCNTs mentioned above. With the ability to scavenge free radicals, gallic acid is a polyphenolic antioxidant that has been used in food and cosmetics to prevent rancidity of fats and oils. Covalent attachment of gallic acid onto pristine MWCNTs by free radical grafting with hydrogen peroxide and ascorbic acid as biocompatible redox initiators pairs was conducted to form an antioxidanteCNT bioconjugate [101]. With a gallic acid loading of around 2 mg per g of

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CNT, the conjugate retained the antioxidant activity of gallic acid and showed significantly higher ability to scavenge 2,20 -diphenyl-1picrylhydrazyl, hydroxyl and peroxyl radicals than non-gallic acid loaded CNT control. The biocompatibility of the antioxidanteCNT conjugate was also assessed with hen’s egg test-chorioallantoic membrane (HETCAM). The conjugate was found to be non-irritant and noninflammatory as no significant hemorrhage, intravasal coagulation or lysis of blood vessels were observed on the membrane after applying the construct for 5 min and washing with normal saline. Interestingly, in addition to its ability to act as antioxidant, the CNTegallic acid conjugate also possessed good activity at inhibiting cholinesterase, which may be beneficial for Alzheimer treatment, where ROS damage is already contributing to the pathogenesis of the disease [74]. 5.3.5 CNTs for delivery of diverse drugs As a naturally occurring neutrotransmitter, acetylcholine plays an imperative role in the process of signal transmission between neurons, both in the central and peripheral nervous system. Deficiency of acetylcholine could potentially lead to the development of Alzheimer’s disease, a neurodegenerative disease affecting memory and cognition. Furthermore, other acetylcholine dependent diseases such as myasthenia gravis, depend on an external supply of this neurotransmitter. Since it is difficult for hydrophilic acetylcholine to cross the hydrophobic BBB, there is therefore a need for novel strategies to deliver acetylcholine across BBB and into brain effectively. SWCNTs noncovalently loaded with acetylcholine (around 200 mg/g of CNT) were able to penetrate BBB and effectively reverse learning and memory loss in kainic acid-induced Alzheimer’s mice model [102]. Theophylline is a xanthine derivative commonly employed for the treatment of respiratory diseases. Using it as a model drug, theophylline was incorporated into hybrid microsphere comprising alginate and CNT [103]. Incorporation of CNT conferred greater mechanical resistance and stability to the microspheres. In addition, introduction of CNT also enhanced drug loading, diminished drug leakage and slowed down the rate of theophylline release from the hybrid microspheres. Lastly, the CNT/alginate microspheres also lacked significant cytotoxicity on mouse fibroblasts L929, achieving similar cell viability as neat alginate microspheres.

5.4 Current status of CNTs toxicity CNTs possess remarkable physicochemical properties such as, ordered structure with high aspect ratio, ultralight weight, high mechanical strength,

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high electrical conductivity, high thermal conductivity, metallic, or semimetallic behavior and extreme penetrating capacity in the cell membrane, high drug load, pH-dependent use, and high surface area [104,105]. The combination of these characteristics makes CNT good candidates for biomedical applications ranging from imaging to therapy [106e111]. Along with the development of nanomaterials with potential for biomedical applications, the question of their potential toxicity has gained increasing attention. Generally, the harmful effects of nanomaterials arise from the combination of several factors, two of which are particularly important: (a) the large surface area and (b) the intrinsic toxicity of the surface. Unlike conventional particles with larger mean diameter, nanoparticles smaller than 100 nm can be potentially more toxic to the lungs (portal of entry), redistribute from their deposition site, escape normal phagocytic defenses, and modify the structure of proteins. Therefore, nanoparticles can activate inflammatory and immunologic responses and affect normal tissue function. CNT, in the context of toxicology, can be classified as ’nanoparticles’ due to their nanoscale dimensions, so unexpected toxicological effects can be induced when they come into contact with biological systems [104,112,113]. Studies have been performed on the effects of pristine CNT on different cell lines, such as human epidermal keratinocytes, human embryonic kidney cells (HEK293), human acute monocytic leukemia cell lines, human T cells, and alveolar macrophages [114e117]. Exposure to SWCNTs in keratinocytes was studied and oxidative stress and cellular toxicity were detected due to the presence of free radicals and peroxides, which caused the depletion of antioxidants and the loss of cell viability [118]. Also, inflammatory responses were reported in the same cell line caused by MWCNTs [119]. In recent years, it has been reported that various strategies can be used to modify the properties of pristine CNTs to achieve the best performance in biological systems [120]. 5.4.1 Modification of the CNTs surface A major drawback of CNTs, particularly relevant for their compatibility with biological systems, is their incomplete insolubility in all types of solvents. Modification of CNT surfaces has made nanotubes dispersible in physiologically relevant aqueous environments and this, in turn, changes their behavior under these conditions (Fig. 5.9). The biodistribution profiles determined for different types of CNT showed a strong dependence on the nature of the surface modification [121,122].

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Figure 5.9 Covalent and noncovalent modification of the CNT surface to eliminate toxicity.

5.4.1.1 Noncovalently functionalized CNTs This type of functionalization is particularly attractive due to its possibility of joining several groups, through weak interactions, without disturbing the p system of the graphene sheets and the intrinsic physical properties of CNT, such as near-infrared, fluorescence, and Raman scattering can be maintained [123,124]. This type of functionalization mainly involves surfactants because they are easy to obtain and inexpensive. Furthermore, CNTs can be decorated with macromolecules (nucleic acids or peptides) or coated with polymers [125,126]. Shim et al. [127] developed a platform of SWCNTs coated with biotin, a surfactant, Triton, and poly (ethylene glycol) (PEG) polymer for the specific binding of proteins, with the objective that this platform does not present toxicity. Also, Lui et al. [128] constructed noncovalently functionalized SWCNTs with phospholipids and PEG, and investigated SWCNT biodistribution by positron emission tomography (PET) and Raman spectroscopy. The effect of the PEG chain length (2000 or 5400) on targeting for cancer treatment using the RGD peptide was investigated. A significant accumulation of all lipid coated SWCNT types was observed in liver and spleen. Furthermore, no obvious toxicity or negative health effects (weight loss and fatigue) were observed in mice injected with SWCNT-PEG for control periods of up to several months.

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In another study, Mohammadi et al. [129] reported the design of noncovalently functionalized SWCNT with chitosan and its derivatives (palmitoyl chitosan and carboxymethyl chitosan) for the administration of doxorubicin (DOX). Among other studies, the cell viability of Npalmitoyl-chitosan-SWCNT (NPCS-SWCNT) was determined, because they were the ones that presented a high loading efficiency and an adequate release profile in cancer cell lines (HeLa cells). The nanotubes functionalized with the polymer did not show any toxic effect on the cells due to the functionalization with the polymer, while the SWCNT loaded with DOX caused significant death of the HeLa cells and the cell viability decreased to 64% after 72 h. 5.4.1.2 Covalently functionalized CNTs Various chemical strategies have been developed for the formation of a variety of chemical bonds on the surface of CNTs, either on the sidewalls or at the end. The covalent approach can be divided into two categories: functionalization by modifying the carboxylic acid groups attached to the surface on the nanotubes or via direct reactions on the sidewalls of the nanotubes [126,130,131]. A study by Sayes et al. [132] performed functionalization on the sidewalls of SWCNT with different functional groups, SWCNT-phenyl-SO3H, SWCNT-phenyl- (COOH)2, SWCNT dispersed in 1% Pluronic F108 and SWCNT-phenyl-SO3Na. The cytotoxicity of the functionalized SWCNT samples in cultures of human dermal fibroblasts (HDF) cells was determined. According to the results, covalently functionalized SWCNT were not cytotoxic to HDF cells, on the other hand, an SWCNT sample was dispersed in Pluronic F108 at 1% as control of noncovalent modification of SWCNT. Such mixture was cytotoxic since this coating is reversible because it is not covalently linked. On the same topic, MWCNT conjugated with poly (ethylene glycol) (PEGylated) were prepared and their intracellular behavior in mammalian cells has been tested. The PEGylated MWCNT were internalized in HeLa cells without damaging the plasma membrane and did not alter cell proliferation in the same cell line. The results of the studies showed that PEGfunctionalized MWCNT are biocompatible with the mammalian cells tested [133]. Due to its biocompatibility and good solubility under various physiological conditions, PEG has been extensively studied for the covalent functionalization of CNT to prolong the time in the bloodstream, reduce uptake by the reticuloendothelial system, and block nonspecific binding of serum proteins [111,127,128,134].

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The covalent and noncovalent functionalization of the sidewalls of MWCNT with ammonium (MWCNT-NHþ 3 ) and coated with Pluronic F127 (MWCNT: F127), respectively, were reported. Cell uptake studies in human lung carcinoma cell lines (A549) showed that MWCNT-NHþ 3 had higher cell uptake compared to MWCNT:F127. Cell cytotoxicity studies showed that MWCNT:F127 were more likely to elicit cytotoxic responses compared to MWCNT-NHþ 3 , which showed high cell viability even with exposure to the highest dose [135]. 5.4.2 Dimensions CNTs vary significantly in length and diameter depending on the synthesis method and post-synthesis treatments, CNT can exhibit diameters ranging from 1 to 100 nm and lengths from nanometers to microns. The nanoscale dimension of SWCNTs is in the range of 0.4e2.0 nm in diameter and an average length of 300e1000 nm, while MWCNTs have average diameters of 20e30 nm and average lengths of 500 at 2000 nm. Clean and processed CNT are typically shorter than native nanotubes due to the destructive conditions used in the purification [136]. SWCNTs have a greater tendency, compared to MWCNT, to bundle into fibers due to attracting van der Waals forces, analogous to the forces that join graphite sheets. Fibers typically contain many tens of nanotubes and can be considerably longer and wider than the nanotubes from which they are made [137]. This could have important toxicological consequences, given their structural similarity in size, shape, and cell persistence to carcinogenic needle-shaped asbestos fibers [138,139]. Wang et al. [140] investigated the impact of CNT diameter on biodistribution, using two series of MWCNT with different diameters, narrow (9.2 nm) and wide (39.5 nm). The trial was tracked by single-photon emission computed tomography/computed tomography (SPECT/CT) and by g-scintigraphy organ biodistribution study. Narrow MWCNT demonstrated an improved affinity for tissues, including non-reticular endothelial tissues, compared to wider MWCNT. Nagai et al. [141] also studied the influence of MWCNT diameter on cytotoxicity and carcinogenicity. MWCNT (diameter w 50 nm) with high crystallinity showed mesothelial cell membrane perforation and cytotoxicity in vitro and consequent inflammation and mesotheliomagenicity in vivo. In contrast, thick (diameter w 150 nm) or entangled (diameter w 2e20 nm) MWCNT were less toxic, inflammatory, and carcinogenic. Thin and thick MWCNT similarly affected macrophages.

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In another study, it was analyzed how the diameter of MWCNT influences pulmonary toxicity. Two short, highly pure MWCNT samples (length M3 and S4, the latter numbers like those of the control. These results indicated that, unlike SWCNTs, MWCNTs cause potent inflammation and DNA damage in vitro and in vivo, the extension is related to the thickness and length of CNTs. These effects are related to an increased risk of carcinogenic activity due to DNA damage and the inflammatory effect. Furthermore, these results indicate that short and thin MWCNTs do not cause DNA damage or inflammation, indicating that

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these MWCNTs can be safely used [148]. In addition to the previous study, similar studies have been carried out to investigate the relationship between CNTs and asbestos, and their influence on the development of respiratory diseases, including mesothelioma; some results showed that there is a carcinogenic risk of CNTs related to their length, although more studies are needed to verify this [149e152]. Other studies, on the contrary, did not find any relationship between the dimensions of the CNTs with the risk of the development of some pathogenesis [153,154]. 5.4.3 Purity Metals such as Cu, Ni, Fe, Pb, and Mo are used in the synthesis of CNTs, which function as catalysts to promote the growth of CNTs. After synthesis the residual metal can be encapsulated in a carbon layer, either amorphous soot or graphite layers, and the purification processes are not entirely successful in removing metals from CNTs. These metallized impurities can remain as high as 30% by weight in CNTs, while “high quality” CNTs with low content of metallic impurities occupy from 0.5% to 1% by weight of CNTs. Metallic impurities have been found to be one of the factors influencing CNTs cytotoxicity because these metallic impurities can significantly participate in redox reactions of important metabolic biomarkers and intermediates. It is important to consider the catalyst content when investigating CNTs toxicity [155e158]. Other impurities that can be found in CNTs are disordered carbons and polycyclic aromatic hydrocarbons (PAHs). Koyama et al. [159] studied the influence of impurities on the toxicological profile of MWCNTs. This study was conducted by controlling the remaining amount and types of impurities within the tubes by heat-treating the highly contaminated and newly developed nanotubes at 1800
C, 1800
C in argon atmosphere, and 2800
C. The obtained results show that the disordered carbons and PAH that grow in the newly developed nanotubes at 1800
C, give rise to acute toxicity, like that of black carbon. For nanotubes heat-treated at 1800
C in argon, the functional groups decompose as evolved gases, toxic PAH and disordered carbons evaporate and are easily transformed into crystalline carbons, but the iron particles diffuse from the inside to the outside and a small amount of iron is present on the outer surface of CNTs. Although the remaining iron content is low, metallic iron can be oxidized to release water-soluble ions that can serve as redox catalysts for the generation of reactive oxygen species. Extremely clean tubes, prepared with high-temperature heat treatment at 2800
C, have excellent biocompatibility in the assay.

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5.4.4 Route of administration Routes of administration of CNTs include oral route, intravenous injection, administration by inhalation, transdermal, subcutaneous, and intraperitoneal injection. Different research groups have discovered that CNTs toxicity is related to the route of administration. Studies have indicated that intravenous injection, oral and dermal administration of CNTs could lead to mild inflammation, while inhalation of CNTs results in severe inflammation. Passive diffusion and energy-dependent endocytosis are the two suggested methods for CNTs entry into living cells [157]. Several reports indicate possible negative effects of CNTs on dermal cells and their possible absorption through the skin barrier [160]. Zhang et al. [161] investigated the effect of exposing human epidermal keratinocytes to different concentrations of 6-aminohexanoic acid derivatized SWCNT (AHA-SWCNT). The results showed an increased release of interleukin-8 and -6 (IL-8 and IL-6) and a decrease in cell viability, suggesting a dose-dependent irritation response [162]. In another study, Mayard et al. investigated the effect of handling raw CNTs in a laboratory production facility and found around from 0.2 to 6 mg per hand of CNT deposits on gloves, indicating that dermal exposure can occur in unprotected body regions. Therefore, the use of protective clothing was suggested to minimize dermal exposure to CNTs. 5.4.5 Hemotoxicity Hemotoxicity encompasses the different ways in which the blood compatibility of new biomaterials is evaluated before they are marketed. The hemotoxic profile of a chemical can be considered as the identification of its possible adverse effects, resulting from its interaction with blood components including cells and proteins [112]. Since it is very likely that the circulatory system is the first entry of CNTs, it is essential to understand how the adsorption of blood proteins in CNTs alters behavior and produces cellular responses and other biological effects. Ge et al. [163] studied the interactions of SWCNTs with human serum proteins. Experimental and theoretical results demonstrated that there are interactions between SWCNTs and fibrinogen, immunoglobulin, albumin, transferrin, and ferritin. Selective binding of these proteins on the SWCNTs surface can affect cellular responses and result in different cytotoxicity. In another study, Salvador Morales et al. [164] used Western blot techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to elucidate the mechanism of complement activation and to

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analyze the interaction of complement and other plasma proteins with CNTs. The results demonstrated that SWCNTs and double-walled carbon nanotubes (DWCNTs) activate the human serum complement system through the classical pathway. DWCNTs also activate the alternate path, in comparison with SWCNTs that barely activate this path. Activation of complement by CNTs through the classical pathway will lead to the generation of inflammatory peptides. When these nanomaterials are introduced into a host, complement activation will result in the accumulation of neutrophils and the adherence of surrounding phagocytic cells. Furthermore, since these nanotubes are too large to be phagocytosed, it is possible for neutrophils to discharge degrading enzymes, causing tissue damage. There will probably be granuloma formation caused mainly by macrophage adherence. The cytotoxic effect of CNTs on erythrocytes has also been studied. In the study, surface-functionalized SWCNTs with acid groups (AFSWCNT) were used to examine the effect they had on mouse red blood cells. The results demonstrated dose- and time-dependent erythrocyte lysis. The confocal microscopy results indicated that AF-SWCNT could enter erythrocytes. Administration of AF-SWCNT intravenously resulted in transient anemia, as seen by a sharp drop in the red blood cell count accompanied by a significant drop in the blood hemoglobin level [165]. Although some of the studies mentioned above on the toxicity of CNT have indicated serious risks associated with exposure to nanotubes, they are not entirely clear since they depend on the evaluated dose, type of CNT (SW or MW), route of administration, CNTs synthesized by different methods, degree of purity, dimensions, surface modification, etc. Additional studies, such as tissue distribution, organ accumulation, the pathway of excretion, and any physiologic abnormalities associated with CNT administration, are needed using a variety of detection methodologies [166]. Another critical aspect when considering the interpretation of toxicity data is the possible interaction of CNT with the reagents used for well-described and used viability tests, which can give false results [167,168].

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CHAPTER 6

Gene cargo delivery aspects of carbon nanotubes Ammu V.V. V. Ravi Kiran1, G. Kusuma Kumari1, Praveen T. Krishnamurthy1 and Renat R. Khaydrov2 1

Department of Pharmacology, JSS College of Pharmacy (JSS Academy of Higher Education & Research), Ooty, Tamil Nadu, India; 2Institute of Nuclear Physics, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan

6.1 Introduction Advances in cell biology, as well as the completion of the Human Genome Project, have greatly improved our knowledge and understanding of genes and their pathological roles in recent years. Because current drug therapies are limited by their inability to target and silence genes, there has been a surge in interest in the use of nucleic acids in the treatment of diseases/ disorders [1,2]. Recently, biotechnological advancements have resulted in the development of novel therapeutically active gene interventions such as DNA plasmids, small interference RNA (siRNA), microRNA (miRNA), oligonucleotides (ODNs), and DNA/RNA aptamers that can manipulate pathological outcomes at the genetic level (i.e., either at post-transcriptional or translational level). Although these interventions may aid in the treatment of pathological conditions, their delivery has been a challenge for several decades [1]. There are several challenges that limit the use of nucleic acids as therapeutic agents in clinic, such as intravascular degradation, tissue penetration and cellular entry, endosomal degradation, immune-mediated toxicities, non-specificity, off-target side effects, and so on. Recently, viral and nonviral vectors were used in gene therapy to deliver nucleic acids. Viral vectors have been shown to be effective and efficient, but their clinical use is limited due to their immunogenicity, genetic load limit, and oncogenicity [1,3]. Nonviral vectors, on the other hand, such as solid-lipid nanoparticles, polymeric nanoparticles, carbon nanotubes (CNTs), gold nanoparticles, and so on, have demonstrated the ability to overcome viral vector restrictions and are considered inexpensive by many pharmaceutical companies [4,5]. CNTs are tubular, rolled graphene sheets that can be single or multilayered. CNTs are classified as single-walled (for single sheets) (SWCNTs), Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00006-6

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double-walled (for double sheets) (DWCNTs), or multi-walled (MWCNTs) based on the number of layers (multilayered). Because of their small size, high aspect ratios, high loading capacity, and ease of chemical functionalization, CNTs have piqued the interest of scientists and researchers in nanotechnology communities [6,7]. CNTs can be synthesized using three techniques: chemical vapor deposition (CVD), laser ablation, and electric arc discharge [8e11]. CNTs exhibit toxicity issues due to their pristine nature, such as inducing detrimental effects due to greater lengths, aggregation, the presence of impurities, and so on [12,13]. To overcome these constraints, CNTs are surface modified or functionalized (f-CNTs) with suitable biological or biocompatible agents, transforming them into biomimicking agents. There has been a growing body of evidence in recent years pointing to the successful use of CNTs in gene therapy. This chapter examines CNTs as nonviral vectors, their advantages, and therapeutic applications in a variety of diseases.

6.2 Functionalized CNTs as nonviral vectors Pristine CNTs (pCNTs) are insoluble and toxic in physiological systems due to their high lipophilicity and residual metal catalysts. Surface modifications of pCNTs with biocompatible agents such as biopolymers or biomolecules improve their miscibility (Fig. 6.1). Surface modification or functionalization of CNTs is divided into two types: exohedral modification (surface changes) and endohedral modification (internal changes) (filled internally) [14e16]. 6.2.1 Exohedral modification Surface functionalizations of various functional group moieties are used in the exohedral modification of CNTs. Exohedral modifications are classified into two types based on their functionalization, namely covalent and noncovalent functionalization [13,15,16]. 6.2.1.1 Covalent modification of CNTs CNTs can be covalently modified by either direct covalent sidewall functionalization or defect group functionalization. Direct covalent sidewall functionalization involves the initial rehybridization of carbon atoms from sp2 to sp3 configuration, which aids in covalent linking with various functional molecules. For rehybridization, methods such as halogenation, cycloaddition of azomethine ylides (or the Prato reaction), and others are

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Figure 6.1 Possible combinations of carbon-mediated nucleic acid delivery: types of CNT, surface modifications, and nucleic acid cargos. (Reprinted (adapted) with permission from K. Bates, K. Kostarelos, Carbon nanotubes as vectors for gene therapy: past achievements, present challenges and future goals. Adv. Drug Deliv. Rev. 65 (15) (December 2013) 2023e2033. Copyright 2013, Elsevier Ltd.)

used [17]. Surface defects are generated during defect group functionalization by oxidation (i.e., using strong acids) to generate carboxylic acid moieties, which aid in conjugating different targeting moieties via amidation or esterification (Fig. 6.2). Many biomolecules, such as peptides, proteins, antibodies, and nucleic acids, prefer amide functional moieties. The most significant limitation of covalent modification is the loss of optical and electrical properties, which prevents detection in biological systems [13,15]. 6.2.1.2 Non-covalent modification of CNTs CNT aggregation is one of the reasons for their poor aqueous solubility. Non-covalent functionalization of CNTs could be accomplished through direct adsorption of nucleic acids or wrapping with polymers (e.g., polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyethyleneimine

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Figure 6.2 Different possible functionalization for attaching nucleic acids in carbon nanotubesdcovalent, non-covalent and endohedral filling.

(PEI)) or biopolymers (e.g., Lipid conjugated PEG, DSPE-PEG) or surfactants (e.g., Triton-X, Sodium dodecyl sulfate) (Fig. 6.2). PEGylated moieties, primarily biopolymers, are a popular functionalizing agent for attaching various drugs and biomolecules because they have been shown to have the longest circulation time, the least reticuloendothelial system uptake, and excellent clearance [16,18]. CNTs are reported to disperse properly when surfactants are added, but they have lower solubility and are less preferred due to their toxic effects. 6.2.2 Endohedral modification Endohedral modification entails inserting compatible materials into the inner cavity of CNTs (Fig. 6.2). Endohedral filling could be accomplished in two ways: (a) in situ (i.e., during nanotube synthesis) or (b) ex-situ (i.e., by opening the tips of CNTs). Supercritical CO2 extraction, low concentrations of protein solutions, or nano-extraction and nano-condensation methods are examples of strategies [19e21].

6.3 Intracellular fate of CNTs: uptake and elimination mechanism CNTs have remained an intriguing gene vector for decades due to their ability to cross cell membranes, favorable lipophilicity, size, and nanoneedle effect [14,22]. CNT-based gene vectors, on the other hand, have the potential to cause cellular toxicity as well as recognition and clearance by the

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mononuclear phagocyte system (MPS) [23]. CNT-based gene vectors, on the other hand, have the potential to cause cellular toxicity as well as recognition and clearance by the mononuclear phagocyte system (MPS) [23]. Because pCNTs have longer lengths and toxic metal catalysts, they are highly susceptible to “frustrated phagocytosis” of immune cells, resulting in an increase in ROS outburst [24e26]. CNTs exhibited efficient entry into mammalian cells via dual-uptake mechanisms, i.e., either by nanoneedle effect or endocytosis (Fig. 6.3). Endocytosis traps CNTs in endosomal vesicles, which are then directed to lysosomal enzymes for further degradation. Endosomal entrapment is undesirable because endosomal enzymes degrade gene cargoes [27]. Many studies suggest that using cationic polymers like PEI could aid in avoiding endosomal entrapment (i.e., via Protonsponge effect) [28e30].

Figure 6.3 Pathways for the penetration of CNTs into the cell. (a) Nonreceptor mediated endocytosis: (1) membrane that surrounds the drug loaded functionalized CNTs, (2) internalization of drug loaded CNTs, and (3) release of drug; (b) receptor mediated endocytosis: (4) membrane surrounds the CNT-receptor conjugate by forming endosomes followed by internalization, (5) release of drug, and (6e8) regeneration of receptor; (c) endocytosis independent pathway: (9) direct penetration of drug loaded functionalized CNT and (10) release of the drug. (Reprinted (adapted) with permission from V. Rastogi, P. Yadav, S.S. Bhattacharya, A.K. Mishra, N. Verma, A. Verma, et al., Carbon nanotubes: an emerging drug carrier for targeting cancer cells. J. Drug Deliv. 24 (2014) 1e23, under the Creative Commons Attribution License, Copyright 2014.)

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CNTs, on the other hand, penetrate through the cellular pores in the nanoneedle effect. This nanoneedle effect can be broken down into three steps: (1) floating and landing on the cellular surface, (2) cellular penetration, and (3) sliding through the cellular membrane. Individual CNTs prefer the nanoneedle effect, whereas clustered CNTs enter via the endocytic mechanism. CNTs would either accumulate in various cell components (including the cytosol, mitochondria, endosomes, and nucleus) or be eliminateddvia exocytosis or enzymatic degradationdupon cellular entry [31,32]. Increasing evidence suggests that stress-induced macrophages and endothelial cells are primarily responsible for toxic CNT exocytosis [25,33]. Enzymatic degradation is one of the most important elimination mechanisms used by cells to eliminate exogenous CNTs. In one study, oxidized SWCNTs were degraded by enzymatic catalysis in an abiotic environment, with eCOOH moieties being proposed as a triggering factor for enzymatic degradation [25,34,35].

6.4 CNTs as an ideal gene cargo vector in various diseases Scientists have detailed some desired properties for an effective nano-based nonviral gene vector, including optimum gene cargo loading, cellular transport, intracellular off-loading of gene cargo, and less/no immunogenic reactions, over the course of more than three decades of research. Many studies have used functionalized CNTs as a nonviral gene vector to deliver nucleic acids such as plasmid DNA (pDNA), siRNA, miRNA, shRNA, ODNs, and DNA/RNA aptamers [7,17,32,36e42]. 6.4.1 CNTs for plasmid DNA delivery CNTs functionalized with ethylenediamine, polyethylenimine (PEI), polyethylenimine/poly(acrylic acid) (PEI/PAA), chitosan, and other polyethylenimines are preferred for pDNA delivery. Pantarotto et al. performed the first in vitro plasmid DNA transfection using CNTs [37,43]. According to the authors, surface area and charge density of CNTs are important factors to consider when synthesizing DNA-CNT complexes [37]. CNTs were also functionalized with agents such as luciferase and green fluorescent protein (GFP) to facilitate biological readouts. Fluorescein-labeled (FAM) dsRNA and surface modified folic acid-modified phospholipids (SWNTsdsDNA-FAM-PL-PEG-FA) demonstrated improved DNA transfection and tumor targeting in HeLa cell lines, for example [44]. In addition, ethylenediamine-functionalized SWCNT attached to the p53 gene

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(f-SWCNT-p53) increased cellular uptake and induced apoptosis in the MCF-7 breast cell line [45]. Another study used a nanoparticle-carbon nanotube (NP-CNT) hybrid to deliver pDNA. The NP-CNT hybrid was made with a polyacrylic acid (PAA) coating to which pDNA (encoding angiopoietin 1 and VEGF) was electrostatically attached. In an in vivo canine model, the developed nanohybrid improved reendothelialization of injured arteries [46]. Layer-by-layer functionalization with biopolymer is a novel strategy for reducing the size, toxicity, and protecting the hydrophobic surface of MWCNTs. Chi-Hsien Liu and colleagues tested the effectiveness of pDNA delivery using layer-by-layer modification (i.e., ovalbumin protein, polyethylenimine, and polysaccharide). The developed construct demonstrated improved DNA dispersibility and intracellular release [47]. X. Yang and colleagues created multi-functionalized SWNTs loaded with FAM labeled short ds DNA encapsulated in folic acid modified phospholipids for tumor targeting (SWNTs-dsDNA-FAM-PL-PEG-FA). According to the authors, the developed SWNTs-dsDNA-FAM-PL-PEGFA had good tumor cell targeting properties [44] (Fig. 6.4).

Figure 6.4 Preparation of functionalized SWNTs which has targeting function for tumor cells. (Reprinted (adapted) with permission from X. Yang, Z. Zhang, Z. Liu, Y. Ma, R. Yang, Y. Chen, Multi-functionalized single-walled carbon nanotubes as tumor cell targeting biological transporters. J. Nanoparticle Res. 10(5) (May 2008) 815e822. Copyright 2007, Springer Ltd.)

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6.4.2 RNA interference (RNAi) Since its discovery, RNAi has piqued the interest of scientists and researchers seeking to solve clinical problems. Despite the fact that various drugs and monoclonal antibodies (mAbs) have been successful in treating various diseases, many important targets remain difficult to inhibit using these strategies. For example, mAbs could only target proteins that are found on the cell surface [48e50]. In brief, Dicer, an endo-ribonuclease, recognizes dsRNA and cleaves it into small fragments of 19e23 base pairs in length. The fragmented dsRNA binds to the RNA-induced silencing complex (RISC), directing the guide strand to the complementary target mRNA. Exogenous dsRNA (siRNA) is introduced in the RNAi technique, and Argonaute 2, a cleavage enzyme within the RISC, degrades the target mRNA, preventing translation. Endogenous microRNAs (miRNAs) will also be pre-processed by Drosha, a nuclear RNase III, before being transported into the cytoplasm (via nuclear transport receptor complexes). Many researchers used CNTs as nano-vectors to deliver RNAi components. Wang and colleagues’ initial studies demonstrated successful delivery of cyclin A(2) siRNA using f-CNTs. Surface-modified CNTs have been shown to have a high transfection rate and to inhibit cell proliferation [33,51]. In one study, Al-Jamal and colleagues used cationic dendron f-MWCNTs to deliver two different siRNAs, siTOX, and siNEG, which induced cellular apoptosis in lung carcinoma cells [33]. To link siRNA, phospholipid-PEG (DSPE-PEG) was adsorbed on SWCNTs with a maleimide moiety in a proof-of-concept study. When the siRNA entered the cell, it was cleaved off and released from the nanotube conjugate, resulting in greater transfection than Lipofectamine [52]. A synergistic combination with siRNA-loaded CNTs has been reported to reduce the risk of various cancers [38,53]. Lei Wang et al. created and used hTERT siRNA-SWCNT to treat tumors in mice in conjunction with photothermal therapy. According to the authors’ findings, photothermal therapy (NIR 808 nm) combined with siRNA-loaded SWCNT could improve therapeutic efficacy and cytotoxicity (Fig. 6.5) [53]. Similar findings have been reported in various cancer cell lines, including lung, liver, breast, and ovarian, with a 40% mRNA knockdown and a 70% protein knockdown [38,39,51, 52,54e56]. Apart from their siRNA delivery in cancer, CNTs have also exploited in few other diseases including cardiac cells [56e59], T-cells [39], and skeletal myocytes [55]. Yi Li and colleagues created f-CNTs attached to siRNA

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Figure 6.5 Tumor-bearing nude mice model under SWNT-PEI/siRNA/NGR treatment: (a) After siRNA treatment without laser irradiation; (b) Image of dissected tumors after siRNA therapy; (c) In the process of treatment; (d) After treatment; (e) Image of dissected tumors in the group of SWNT-PEI/siRNA/NGR þ 808 nm NIR laser. Average tumor size in a PC-3 nude mouse model under treatment without (f) or with (g) Laser in vivo. The SWNT-PEI/siRNA/NGR þ laser group shows significant (P < .05) suppression of tumor growth compared with the other experimental groups with laser irradiation (n 1/4 5), and each SWNT-PEI þ laser group shows significant (*P < .05) suppression of tumor growth compared with SWNT-PEI group, respectively. (Reprinted from L. Wang, J. Shi, H. Zhang, H. Li, Y. Gao, Z. Wang, et al., Synergistic anticancer effect of RNAi and photothermal therapy mediated by functionalized single-walled carbon nanotubes. Biomaterials 34 (1) (January 2013) 262e274. Copyright (2012), with permission from Elsevier.)

(targeting Caspase3) in their study (F-CNT-siCas3). The developed construct was water soluble, biocompatible, and had a high transfection efficiency (up to 82%), which downregulated the Caspase3 gene miRNA in cardiomyocytes (Fig. 6.6) [57]. According to the authors, this study provided proof-of-concept for use in treating cardiovascular diseases. Another study used hexamethylenediamine (HMDA) and

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Figure 6.6 Flowchart illustrating the preparation of siRNA delivery vectors based on FCNTs: (a) SWCNTs and PL-PEG2000 were combined through hydrophilic and hydrophobic interactions. (b) SWCNT-PEG-SPDP was formed by mixing SWCNT-PEG and Sulfo-LC-SPDP under mild conditions. (c) siRNA was conjugated to SWCNTs through a cleavable disulfide bond. (d) In vitro gene silencing process initiated by F-CNT-siRNA. (e) In vivo myocardial infarction treatment based on injecting F-CNT-siRNA delivery vehicles into the MI region. (Reprinted (adapted) with permission from Y. Li, H. Yu, L. Zhao, Y. Zhu, R. Bai, Z. Jin, et al., Effects of carbon nanotube-mediated Caspase3 gene silencing on cardiomyocyte apoptosis and cardiac function during early acute myocardial infarction. Nanoscale 12 (42) (2020) 21599e21604. Copyright 2020, Royal Society of Chemistry.)

poly(diallyldimethylammonium)chloride to electrostatically complex ERK targeting siRNA with SWCNTs (PDDA). The nanotube construct successfully crossed the cell membrane and transfected siRNA into primary cardiomyocytes, effectively silencing the ERK gene [60]. Z.Liu and colleagues created a nonviral nanotube-based delivery system that was loaded with siRNA for silencing HIV-specific CD4 receptors on T cells or CXCR4/CCR5, preventing virus entry and reducing infection [39]. TRPC3 siRNA loaded with f-CNT knockdown Ca2þ-TRPC3 resulted in decreased insulin-mediated glucose uptake in adult skeletal muscle cells in another study [55]. Bcl-xL shRNA-attached PEIfunctionalized SWCNTs tagged with the 5TR1 aptamer were created, and they demonstrated improved shRNA transfection and antitumor efficacy (Fig. 6.7) [42].

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Figure 6.7 Schematic illustration of shRNA delivery using aptamer-conjugated polyoplexes. (Reprinted (adapted) with permission from S. Taghavi, A. HashemNia, F. Mosaffa, S. Askarian, K. Abnous, M. Ramezani, Preparation and evaluation of polyethylenimine-functionalized carbon nanotubes tagged with 5TR1 aptamer for targeted delivery of Bcl-xL shRNA into breast cancer cells. Colloids Surf. B Biointerfaces 140 (April 2016) 28e39. Copyright 2015, Elsevier Ltd.)

Using PEI-g-GNR, Haifend Dong and colleagues investigated the first miRNA delivery in HeLa cell lines. The PEI-g-GNR shielded the locked nucleic acid modification from nucleases and aided in probing, resulting in improved transfection into cancer cells [61]. 6.4.3 Oligonucleotides (ODNs) ODNs are biologically active gene components that regulate gene expression at the transcriptional or translational levels [62]. Early research revealed that covalently (eCOOH) and non-covalently (PEI, Chitosan) CNT are used to deliver ODNs [41,63,64]. Pan and colleagues used MWCNTs to deliver antisense c-myc ODNs that inhibited HL60 cell proliferation, induced apoptosis, and down-regulated the overexpressed c-myc [65]. Unmethylated CpG ODNs were used in another study to load MWCNTs conjugated with H3R6 polypeptide (MHR-CpG) for immune-based oncotherapy. The nanotube conjugate demonstrated

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enhanced immunogenicity in both humoral and cellular immune systems, improved expression of CD4þ, CD8þ, TNF-, and IL-6, and suppressed tumor growth [64]. Villa and colleagues created a functionalized SWCNT conjugated with oligonucleotide and RGD ligands for tumor targeting (Fig. 6.8). The SWCNT-oligonucleotide demonstrated good biodistribution and self-assembly of multifunctional SWCNTs [63]. Similarly, Zhao and colleagues conjugated f-SWCNT with CpG ODNs, which increased CpG uptake and boosted antitumor immunity in a brain cancer model [66]. 6.4.4 DNA/RNA aptamers Aptamers are single-stranded, long-sequenced ODNs with high affinity and selectivity for a specific target molecule. When compared to protein

Figure 6.8 Synthetic scheme for the production of targetable, oligonucleotidefunctionalized single-wall carbon nanotubes. [4] was used for biodistribution studies, whereas [6] was used to study tumor-cell specific binding. (Reprinted (adapted) with permission from C.H. Villa, M.R. McDevitt, F.E. Escorcia, D.A. Rey, M. Bergkvist, C.A. Batt, et al., Synthesis and biodistribution of oligonucleotide-functionalized, tumor-targetable carbon nanotubes. Nano Lett. 8 (12) (December 10, 2008) 4221e4228. Copyright 2015, ACS Publications.)

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delivery and therapeutics, aptamers are easier to synthesize, alter, and are smaller in size, mimicking the properties of antibodies in various diagnostic formats [17,67]. Jeroen van and colleagues covalently grafted aptamers onto the surface of MWCNTs-COOH to create a novel nano-delivery system for easy passive translocation into different cell types (MWCNT-Apt). The developed nanotube-based delivery system efficiently delivered aptamers into MCF-7 and Calu-6 cells [68]. In another study, PEGylated MWCNTs were loaded with a nanoultrasound contrast agent and an anti-PSMA aptamer, which demonstrated improved imaging guidance and accuracy in targeting prostate cancer cells [69].

6.5 Conclusion The ability to modulate target genes and elicit the desired therapeutic effect is the primary goal of translating gene therapy from bench to bedside. CNTs have a number of appealing properties that increase their potential as gene vectors, including a wide range of transport mechanisms for various nucleic acids. Several challenges, such as incorporating genetic loads into CNTs, penetrating cellular membranes, targeted delivery, controlled gene expression, and toxicity concerns, must be overcome in order to provide effective gene therapy in clinics, for which CNTs may be a viable nonviral vector. Before any further clinical use, a thorough pharmacological and toxicological aspects and route of administration are needed to be addressed, which could further improve treatment options and patient compliance.

Declarations Funding details: This work was supported by the Department of Science and TechnologydIndo-Uzbek Joint Research Programme (INT/Uzbek/P-01), New Delhi.

References [1] A.D. Miller, Human gene therapy comes of age, Nature 357 (6378) (June 1, 1992) 455e460. [2] K. Bates, K. Kostarelos, Carbon nanotubes as vectors for gene therapy: past achievements, present challenges and future goals, Adv. Drug Deliv. Rev. 65 (15) (December 2013) 2023e2033. [3] K.B. Kaufmann, H. Büning, A. Galy, A. Schambach, M. Grez, Gene therapy on the move, EMBO Mol. Med. 5 (11) (November 2013) 1642e1661.

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CHAPTER 7

Carbon nanotubes for anticancer therapy: new trends and innovations Israel González-Méndez, Kendra Sorroza-Martínez, Andrea Ruiu and Ernesto Rivera Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior Ciudad Universitaria, México City, Mexico

7.1 Introduction Under the denomination of “cancer” can be included different diseases (approximately 200) that vary in their clinical manifestations and in their response to therapeutic measures, but share common triggering mechanisms, such as exposure to chemical toxic substances, suppressor gene mutations, as well as hereditary genetic components [1]. Cancer is characterized by uncontrolled cell growth that destroys normal tissues and organs. Several mutations, uncontrolled growth, and division of cancer cells allow these cells to acquire properties such as self-sufficiency in growth signals, unlimited proliferation potential, and resistance to signals that stop proliferation or induce apoptosis in normal cells [1]. Tumors have evolved to utilize additional supports through interactions with surrounding stromal cells, promotion of angiogenesis, evasion of immune detection systems, and metastasis to other organs [2]. Throughout several decades, cancer has been one of the deadliest noncommunicable diseases, affecting at least 7.1 million people worldwide [3]. Normally, the treatment strategies are based on the use of diverse anticancer agents (chemotherapy), combining with radiation therapy to reduce the tumor size, and have the possibility of remove it by chirurgical intervention (in the case of solid tumors) [4]. Besides, gene therapies (RNA interference, oligonucleotides, aptamers) are being employed as a promising tool [5,6]. The routine above-mentioned strategies constantly fail due to various reasons such as poor solubility, unfavorable pharmacokinetics, nonspecificity that is responsible of off-target side-effects, sensibility of multidrug resistance mechanisms such as an efflux for P glycoprotein, and instability in the physiological environment [7e9]. Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00005-4

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Despite the inconveniences previously described, the interest in the implementation of combined therapy grows every year, which employs the integration of systems of diagnosis and treatment (theranostics) [10], or two or more forms of treatment, which has taken a greater boom as an innovate alternative [11e13]. In this regard, nanotechnology has emerged as powerful tool to improve conventional treatments. Nanotechnology is an applicable aspect of a broader area of nanoscience (nano means 10
9 m), which is one of the upcoming and highly challenging as well as a rewarding key research area in the modern scientific set-up [14]. It is the science of small particles having unique properties, which change by altering the size of the particles as well as the melting point, electronic and optical properties [15].

7.2 Advantages of nanotechnology for cancer therapy With the proposed advanced systems by nanotechnology based on the smart design of multifunctional nanomaterials for the co-delivery/coassembly of two or more therapeutic agents [10], combined therapy has been made possible due to the integration of multiple therapeutic modalities within a single system [16]. Nanomedicine proposes a multimodal synergistic therapy through the integration of multiple therapeutics in a single nanoplatform rather than simple mixing to reinforce the synergistic effects of individual agents [17]. The rapid development of nanotechnology strategies has allowed the possibility of assembling several types of therapeutic agents into one nanostructure through physical adsorption (internal or superficial) by supramolecular interactions or covalent bonds at the platforms [18]. In this way, it is possible to get multifunctional nanomaterials for achieving the “mission” of multimodal synergistic therapy. It is worth to point out that nanomaterials exhibit several prominent advantages over small molecules in biomedical applications [19]. • Nanomaterials are able to passively accumulate and preferentially remain at the tumor site via the so-called enhanced permeability and retention (EPR) effect [20e23]. • Design of nanosystems with diverse functional groups; the surface of these systems can be easily modified with proteins, peptides, and other biomolecules to reduce nonspecific uptake by the reticuloendothelial system (RES) and then specifically bind to overexpressed tumor cell receptors for enhanced accumulation [24e26].

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•

The large surface-area-to-volume ratio of nanomaterials efficiently entraps high payloads of drugs, genes, and other therapeutic molecules and protects them from enzymatic degradation in the complex physiological microenvironment [27]. • The functionalized nanomaterials are able to control the release of the loaded drug molecules via diverse internal and external stimuli (e.g., temperature, pH, GSH, light, etc.), which help to prevent premature drug leakage in healthy tissues and mitigate the potential side effects [28,29]. To meet all these criteria, the research studies have turned to use nanotechnology for designing high-performance nanomaterials through selective physical/chemical co-loading of two or more kinds of therapeutic agents for multimodal synergistic therapy, which may substantially improve the therapeutic effectiveness and efficaciously treat those malignant tumors that harbor resistance to monotherapy [17].

7.3 Nanotechnology systems for cancer therapy Nanotechnology proposes different nanostructures with application in areas as diverse from imaging to tissue engineering [30,31]. These systems include the use of vehicles for enzyme encapsulation [32], DNA transfection [33,34], biosensors, and drug delivery [35e37]. The nanostructure-based products employed in nanotechnology include nanoparticles (NPs), which can be classified as organic NPs and inorganic NPs. Liposomes, dendrimers, virus, solid lipid NPs, and polymeric NPs are organic NPs which have been utilized in cancer therapy and diagnosis for a long time. Gold, silver, silica, magnetic particles, ceramic particles, quantum dots, and carbon particles are inorganic NPs that are considered as promising vectors for cancer treatment [38]. Among the aforementioned systems, carbon nanotubes (CNTs) are platforms that stand out for their excellent physicochemical properties. CNTs were fully described in 1991 by Sumio Iijima as tube-shaped, wellordered, flat network, high aspect ratio allotropes of carbon having a diameter measurable on the nanometer scale [30]. In particular, the exploitation of their diverse properties and small dimensions through biologically inspired functionalization techniques has enabled a broad range of biomedical applications via interactions at the molecular and cellular level. CNTs can be categorized according to their structures as single-walled CNTs (SWCNTs), comprising one layer of cylindrical graphene with

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diameters ranging from 1 to 10 nm, and concentric multi-walled CNTs (MWCNTs) which contain several concentric graphene sheets with diameters of approximately 5e30 nm and lengths ranging from several nanometers to a few millimeters [39].

7.4 CNTs advantages as nanocarriers for cancer therapy In the design and development of nanosystems, CNTs have attracted a great deal of attention due to their unique structure. The chief properties of CNTs that make them a prominent tool over other nanocarriers are: greater stability, biocompatibility, non-immunogenicity, ease of size alteration, and high drug-loading proficiency [15]. Inner and outer surfaces of CNTs can be modified separately as required and a variety of functional groups can be generated on their surface for further conjugation with targeting ligands as well as drug molecules. The inner volume of CNTs can be filled with a variety of biomolecules and drugs with high entrapment efficiency [40]. CNTs have unique absorption in the near infrared region, and specific properties of fluorescence can be used for biological sensing [41e43]. They have been used also as mediators for photothermal therapy (PTT) and photodynamic therapy (PDT) to directly destroy cancer cells [38]. A variety of cells can uptake the CNTs, so that they can be also employed for intracellular delivery of biologically active molecules [42,44,45]. Therefore, CNTs have recently emerged as a new option due to their possible use in diagnosis and treatment of cancer, bioengineering and gene therapy.

7.5 CNTs as drug delivery systems for cancer therapy Regarding their application as drug delivery systems, CNTs have been also popularly employed as carriers for controlled and targeted drug delivery in order to improve the pharmacological activity of bioactive molecules and simultaneously diminish their undesirable systemic side effects [46]. CNTs possess an inner space (suitable for encapsulating diverse molecules) and are proven not to be degraded into the human body due to their chemical properties and mechanical strength [47]. Furthermore, according to the NPs general properties, CNTs have great superficial area and are very small (in the dominium of nanoscale), thus they have highly specific surface areas, related to their needle-like shapes, which enable them to adsorb onto or conjugate with various therapeutic molecules [38]. Therefore, CNTs are

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considered promising nanocarriers for the delivery of drugs, genes, and proteins. Given these properties, various chemotherapeutic agents have been used as therapeutic loads in CNTs to fight the cancer process. These promising strategies are mentioned below and it is possible to differentiate them since they are dependent on the mechanism of the selected chemotherapeutic drug (Fig. 7.1). 7.5.1 CNTs as nanocarriers of topoisomerase I or II inhibitors Topoisomerases are a group of enzymes that relieve the torsional strain of supercoiled double helical deoxyribonucleic acid (DNA) by making either single or double stranded nicks at the DNA phosphate backbone and allowing the DNA to be unwind, before eventually resealing the cleaved DNA. Topoisomerase I catalyzes a transient break of one strand of duplex DNA and allows the unbroken complementary strand to unwind through the enzyme-linked strand. After successful DNA relaxation, topoisomerase I also relegates the broken DNA [46]. Topoisomerase II modifies the number of turns of a supercoiled DNA molecule, making one segment of DNA pass through another, for which they require cutting the double helix. In this process, topoisomerase II can relieve the torsional stress of the structure in both negative and positive supercoils. So, inhibitors of topoisomerase II prevent the rejoining of the nicked strands, resulting in double strain breaks and consequently cell death [46].

Figure 7.1 Anticancer drugs attached to CNTs and their biological targets.

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In summary, topoisomerase inhibitors are chemotherapeutic agents that arrest DNA replication and subsequent apoptosis, thereby slowing the growth of cancer cells. Clinically used topoisomerase I inhibitors include irinotecan, topotecan, and camptothecin (CPT), whereas topoisomerase II inhibitors include etoposide (ETO) and teniposide. In addition, anthracyclines, including doxorubicin (DOX), epirubicin (EPI), and daunorubicin (DAU), represent a unique class of anticancer drugs with multiple poisoning mechanisms that have the capacity of inhibiting topoisomerase II [38]. CNT decorated with topoisomerase I inhibitors are a very promising tool according to a report of Tian and coworkers [48]. In this work, a CPTloaded multiwalled CNTs (MWCNTs) with tri-block copolymer through pep stacking interactions succeeded in improving antitumor activity. To enhance the solubility in water, the MWCNTs were coated with tri-block copolymer Pluronic P123. In this design, the polymer-coated MWCNTs could effectively form non-covalent supramolecular complexes with CPT. Indeed, in vitro cytotoxicity studies using HeLa cells showed that these complexes enhanced antitumor activity compared to free CPT. These results suggest that functionalized MWCNTs improve the activity of anticancer drugs. However, an additional in vivo study was required to confirm the antitumor activity of CPT. To increase the water solubility and antitumor activity, 10hydroxycamptothecin (HCPT), a congener of CPT, was employed. Wu and coworkers [49] developed an MWCNT-based drug delivery system, via a cleavable ester bond conjugated to HCPT. When a HCPT loading of 16% w/w was achieved, the conjugate remained stable in the absence of esterases in buffer solution, and readily released HCPT in fetal bovine serum after hydrolysis of ester linkages by esterases present in the serum. This conjugate HCPTeMWCNT in both in vitro and in vivo tests, showed superior antitumor activity to clinical HCPT formulations. Besides, based on in vivo single-photon emission-computed tomography and ex vivo gamma-scintillation counting analyses, these conjugates showed a long circulation time (w3.6 h) in the blood and high accumulation level (w3.6% injected dose per gram of tissue [ID/g]) in the tumor area. Other strategy was presented by Tripisciano et al. [50], who encapsulated the antineoplastic agent irinotecan, a more water-soluble semisynthetic analog of CPT, within MWCNTs. In this experiment, it was observed that a larger inner diameter tube exhibits higher filling amount of irinotecan than a smaller one, and a loading efficiency of 32% was achieved. Since the stability and hydrophilicity of irinotecan are increased under

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acidic conditions, rapid and complete release was observed (pH 6.0 vs. 7.0) in a mild acidic environment. Intriguingly, further decrease of pH to 5.0 seems to have no additional influence on the rate of drug release [46]. However, in vitro analysis of colorectal cancer cells using this complex was not conducted. With the strategy of targeted drug delivery systems as a bioactive molecules, Chen and collaborators [51] fabricated ETO-loaded epidermal growth factor (EGF)echitosan (CS)esingle-walled CNTs (SWCNTs; EGF/CS/SWCNTeCOOHs/ETO). The CS improved the water dispersion of native CNTs and served as a linker for conjugation with the EGF. EGF/CS/SWCNTeCOOHs showed only slight cytotoxicity and the loading capacity of ETO was w25%e27% (w/w). The death of human alveolar carcinoma epithelial cells induced by EGF/CS/SWCNTe COOHs/ETO was 2.7-fold higher than that of ETO alone. Their work revealed the potential of this new drug delivery system to enhance the efficacy of ETO. On the other hand, anthracyclines represent a unique class of anticancer drugs that exhibit multiple mechanisms of action including the inhibition of topoisomerase II. With a flat and aromatic tetracyclic ring structure, anthracyclines are able to intercalate between DNA base pairs and inhibit the synthesis of DNA. In addition, the hydroquinone moiety of anthracyclines can also be metabolized and generate iron-mediated free oxygen radicals that damage DNA and cell membranes. In spite of their high clinical effectiveness against many cancers, the use of anthracyclines is unfortunately plagued with dose limiting myelosuppression, alopecia, acute nausea and vomiting, vesicant effects, and most notably, cardiotoxicity [46]. More effective and safer ways of delivering anthracyclines are hence of significant research interest. Among the anthracyclines mentioned above, DOX stands out since its loading and release from CNTs can be manipulated by changing the pH. In this regard, Liu [52] and coworkers proposed a new drug delivery system using polyethylene glycol (PEG)-functionalized SWCNTs attached to DOX. Water-soluble SWCNTs functionalized with PEG exhibited an extremely high loading efficiency of w400%. Their SWCNTseDOX complexes showed good stability in normal physiological buffer as well as in the serum and acidic environments, which is an ideal property for in vivo drug carriers. Diameter-dependent binding and release of the drug in SWCNTs showed the potential for multiple choices of use in drug delivery. Using a similar strategy and validating the results of Liu, Ali-Boucetta and

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co-workers [53] formed MWCNTs complexes with an aromatic chromophore and DOX via pep stacking interactions. Their complexes showed enhanced cytotoxicity in human breast cancer MCF-7 cells. However, cell viability was not decreased by DOX-free MWCNT carriers. More importantly, the DOX-free carrier of MWCNTs dispersed with Pluronic F-127 alone did not depress cell viability, implying that the cytotoxicity effect observed was entirely attributable to improved DOX efficacy rather than any inherent toxicity of CNTs. In general, the therapeutic efficacy of anthracyclines can be enhanced by conjugation with a targeting agent such a folic acid (FA), estradiol (ES), dexamethasone or with antibody binding [38]. With this in mind, Zhang and collaborators developed a targeted delivery system using FA-linked SWCNTeDOX [54]. This system employed polysaccharides (sodium alginate and CS) to control the release of DOX, while FA was used to improve the targeting properties of CNTs. This system showed good stability under normal physiological pH (pH 7.4) and efficiently released DOX at low pH such as in the tumor environment and intracellular lysosomes. Employing a similar strategy, Li and coworkers prepared a new delivery system by conjugating iron NPs and FA with MWCNTs [55]. Using this design, they improved the targeting efficiency of their nanocarriers by means of an external magnetic field. The nanocarriers showed sufficient load capacity of DOX and long releasing time. Their dual targeting method showed six-fold higher delivery than that of free DOX into HeLa cells. They also developed a CNT-based magnetic dual-targeted nanocarrier for drug delivery [56]. Their magnetic MWCNTs were conjugated to the targeting ligand FA to load the anticancer drug DOX. Under the guidance of a magnetic field and ligand receptor interactions, a dualtargeted delivery of DOX into cancer cells was successfully performed. DOX was loaded into MWCNTs, released into the cytoplasm with high efficiency, and showed enhanced cytotoxicity against U87 human glioblastoma cells with minimum side effects toward cells at the control site. These results demonstrated the potential of using magnetic nanocarriers for targeted delivery of DOX in cancer treatment. Another chemotherapeutic molecule of the anthracycline family, DAU, has been loaded into SWCNTs functionalized with a sgc8c aptamer, which is a three-dimensional single-stranded DNA structure capable of targeting the leukemia biomarker protein tyrosine kinase-7. The modified SWCNTs achieved a high loading efficiency of 157% (w/w) and showed a similar pH-dependent release profile as DOX [57]. Using one equivalent DAU

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concentration, their system showed higher uptake and selectivity toward the target cell line (Molt-4) than the nontarget cell line (U266), compared to free DAU. From these results, it was observed that the new system showed potentiality to reduce cytotoxic effects of DAU by selective delivery and controllable release of this drug to tumor cells [57]. In the most recent studies, for example, Ozgen and coworkers developed DOX loaded-MWCNT covered with both glycoblock copolymers and FA for dual-targeting of glucose transporter protein (they selected GLUT5 since it is a membrane protein that transports fructose into the cell cytoplasm) and folate receptors (FR) present in breast cancer [58]. In this study, two types of glycoblock copolymers-DOX conjugates were produced, namely P7 (P(FruMA-b-MAEBA)-Py/DOX) and P8 (P(FruMA-b-MAEBA)-N3/DOX). Furthermore, they were attached to MWCNT-COOH (both covalently and non-covalently) and FA to form two distinct hybrid CNT complexes, namely CNT/P7/FA and CNT/P8/ FA. Then, these were assessed by MTT assay using MCF-7 and MDA-MB231 breast cancer cell lines. The results showed that DOX-loaded glycoblock copolymer-CNTs with or without FA coating was more potent toward both cell lines than free DOX. However, the maximum cellular internalization and cytotoxicity was observed for FA coated hybrid-CNTs, suggesting the effective role of FA in cellular uptake of DOX. The performance of CNT/P7/FA was higher for the MDA-MB-231 cell line due to altered expression levels of GLUT5 in MDA-MB-231 compared to MCF-7, indicating that the accumulation of the CNT/P7/FA in MDAMB-231 was associated with fructose transporters. Also, comparison of cellular uptake for FA-unmodified hybrid-CNTs and FA-modified hybridCNTs confirmed these results, highlighting the superior role of FA for improving cell penetration. 7.5.2 CNTs as nanocarriers of alkylating agents The most common alkylating agents used in the clinical practice are Platinum (Pt) based compounds, which constitute an effective class of anticancer agents for a wide array of malignancies [59], since they chelate DNA and form intrastrand adducts that affect key cellular processes, such as transcription and replication, and ultimately trigger apoptosis [60,61]. While highly effective, the use of Pt based drugs is unfortunately limited by severe dose limit nephrotoxicity, neurotoxicity, and myelosuppression, arising from premature aquation and nonspecific target interactions [62]. In this regard, Pt(IV)-conjugated CNTs were constructed to effectively deliver

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cis-platin (cis-dichlorodiammine platinum, CDDP) [38]. Dhar and coworkers developed amine-functionalized SWCNTs (SWCNTse phospholipidePEGeNH2) as a “longboat delivery system” for Pt-based CDDP [63]. A complex of folate and CDDP was linked to amide bonds of SWCNTs to comprise the “longboat,” which was internalized into cancer cells by endocytosis. Next, CDDP release and subsequent interactions between the nucleus and CDDP progressed. In another work, CDDP-embedded SWCNTs using a wet chemical approach was also evaluated [47]. The in vitro release profile of CDDPeSWCNTs was smooth until 72 h, and the maximum release percentage was 68%. They demonstrated the inhibition of prostate cancer cell (PC-3 and DU145) growth by CDDPeSWCNTs. Another Pt anticancer agent, carboplatin (cis-diammine(1,1cyclobutanedicarboxylate) Pt(II), CP) was conjugated to CNTs [64]. Thus, Hampel and collaborators reported the incorporation of CP into MWCNTs. For this, CP was filled using a wet chemical approach based on capillary force after opening the CNTs. CP incorporation into the CNTs was confirmed by electron energy loss spectroscopy and X-ray photoelectron spectroscopy. In an in vitro analysis, CPeMWCNTs inhibited the growth of urinary bladder cancer cells, whereas unfilled MWCNTs had a minimal effect on cancer cell growth. 7.5.3 CNTs as nanocarriers of antimicrotubule agents Microtubules are slender, cylindrical filaments found in the cytoskeletons of plant and animal cells. They are dynamic, ubiquitous protein polymers composed of the protein, tubulin, which oscillates between phases of elongation and shortening [65]. With their dynamic structures, microtubules can adopt different organizations depending on their cellular functions such as maintenance of cell structure, protein trafficking, chromosomal segregation, etc. [66]. During mitosis, microtubules assemble into mitotic spindles that distribute chromosomes to opposite poles of a dividing cell. Thus, as a result of their essential roles in cell division and mitosis, microtubules are pharmaceutically validated targets for anticancer chemotherapy [67]. Among antimicrotubules, paclitaxel (PTX) and docetaxel (DTX) have been widely studied [38]. For example, Arya reported that SWCNTs increase PTX activity against lung cancer by reactive-oxygen-speciesdependent synergy between CNTs and PTX [68]. They confirmed the

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efficacy on A549 and NCIeH460 cell lines. In a similar study, Zhang and coworkers described the synergism between CNTs and PTX on ovarian cancer [69]. They reported that PTX-conjugated SWCNTs sensitized human ovarian cancer OVCAR3 cells and resulted in higher cell death. In their study, SWCNTs showed two different functions as molecular carriers and chemosensitizers. Thus the co-exposure of SWCNTs and chemotherapeutic drugs might be a promising approach to improve cancer treatment. To increase the water solubility of PTX, Sobhani and his research group proposed hyperbranched poly(citric acid) (PCA)-functionalized MWCNTs bearing high hydrophilicity and the conjugation of PTX with PCAe MWCNTeg-PCA [70]. In cytotoxicity studies using A549 and SKOV3 cell lines, MWCNTeg-PCAePTX showed a higher cytotoxic effect than the free drug over a shorter incubation time. These results showed the potentiality of their system for cancer chemotherapy. In the same way, Liu reported chemically functionalized SWCNTs conjugated with PTX via branched PEG chains as a new prospect in tumor-targeted accumulation with low toxicity [71]. In in vivo experiments, the blood circulation time of PTXeSWCNTs was longer and PTX uptake into the tumor as PTXeSWCNTs was ten-fold higher than clinical Taxol in a murine 4T1 breast cancer model. These results showed that PTXeSWCNTs have high efficacy in suppressing tumor growth. Similarly to the findings for anticancer activity, in another work Lay and collaborators found that the growth of MCF-7 cancer cells and HeLa cells was suppressed by PEGylated SWCNTs and MWCNTs with PTX [72]. Another antimicrotubule drug is DTX which has lower side effects than PTX used for breast and lung cancer treatment. In this work, AsneGlye Arg (NGR) were linked SWCNTs [73]. DTXeNGReSWCNTs showed higher suppression of tumor growth than DTX in PC-3 in vitro culture and murine S180 mouse cancer model. The tumor volumes in mice were decreased considerably under near infrared (NIR) radiation compared to those in the control group. Based on these results, DTXeNGReSWCNT drug delivery systems are potential for effective cancer therapy with minimal side effects. 7.5.4 CNTs as nanocarriers of antimetabolites agents Antimetabolites embody a class of anticancer drugs that disrupts the metabolic pathway for the formation of nucleic acids in cancer cells. There are two groups of drugs that fall into this drug category, namely the antifolates and the purine/pyrimidine antagonists [46].

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The first design containing MTX in CNTs and fluorescent probe FITC was reported by Pastorin and coworkers [74]. The authors studied the uptake of the complex that was analyzed with human Jurkat T lymphocytes. It was shown that the construct accumulated in cytoplasm in a timeand dose-dependent manner. In a different work, Samori and coworkers reported the covalently linked MTX to MWCNTs with two different cleavable linkers, namely the tetrapeptide Gly-Leu-Phe-Gly (which can be selectively cleaved by proteases overexpressed in tumor cells), and the 6hydroxyhexanoic ester (which is an esterase-sensitive hydrophobic spacer commonly used in other prodrug conjugate synthesis) [75]. Interestingly, the group showed that the cytotoxic activity of the conjugates in MCF-7 cells was strongly dependent on the presence and type of linker. Higher cytotoxic activity was observed with the protease-sensitive tetrapeptide linker than with the 6-hydroxyhexanoic ester linker, where cell death was induced in 90% and 20% of MCF-7 cells, respectively, after 24 h of incubation. Aside from covalent conjugation, MTX is also amendable for physical adsorption onto CNTs. A strategy of non-covalently functionalized MWCNTs with 1,2-distearoyl-phosphatidylethanolamine-methoxy-polyethylene glycol conjugate-2000 (DSPE-mPEG 2000) and then entrapment of MTX has been attempted by Modi and collaborators [76]. The product, MWCNTemPEGeMTX, was found to release MTX faster in acidic medium of pH 5.8 than in neutral of pH 7.4. This design is a proposed pH stimuli sensitive drug delivery system based on the fact that cancer tissues tend to be acidic, so it was suggested that this formulation is able to deliver and release MTX to cancer cells more effectively compared to other normal tissues. Another recent strategy which consists of the physical loading of different chemotherapeutic agents, including MTX, on an array of MWCNTs with different surface functionalization, as a PEG, hyaluronic acid (HA), FA and ES [77] was described. It was observed that regardless surface functionalities, the loading of MTX was estimated to be around 30% for all samples. Release of MTX was measured at two different pH values, 7.4 and 5.5. MTX release was higher at neutral pH (60%e70%) compared to acidic pH (35%e45%) after 24 h of incubation. This observed release profile suggests that MTX may be released and acts more efficiently if its carriers are localized in cytoplasm as compared to acidic lysosomes. Uptake and cytotoxicity of the various functionalized CNTs loaded with MTX were performed in A549, HeLa and MCF-7 cells. MTX, being an

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antifolate, exerts its antitumor activity by interfering with the biosynthetic pathway of DNA. Therefore, MTX exhibited slightly higher cytotoxicity in A549 and MCF-7 cells when it was delivered specifically to nucleus, where DNA is housed, by ES-functionalized carrier, as compared to FAand HA-CNT constructs, which were mainly localized in acidic endocytic vesicles and lysosomes in cytoplasm. The second group of antimetabolite drugs are purines and pyrimidines, since they are components of DNA, RNA and coenzymes that are required for cell proliferation. Therefore, purine and pyridimine antagonists that mimic the structures of their corresponding physiological molecules are able to hinder the synthesis of these essential metabolites and are used for cancer therapy [46]. There are many purine and pyrimidine antimetabolites that have been approved for clinical use. Examples of purine inhibitors include mercaptopurine, thioguanine, and azathioprine, while examples of pyrimidine inhibitors encompass 5-fluorouracil (5-FU), cytarabine and gemcitabine (GEM). The basic action mechanisms of purine and pyrimidine antimetabolites are similar. These compounds diffuse into cells, typically with the aid of membrane transporters, and are converted to analogs of cellular nucleotides by enzymes of the purine or pyrimidine metabolic pathway. These metabolites then inhibit one or more enzymes that are essential for DNA synthesis, causing DNA damage and inducing apoptosis [78]. The use of GEM in CNT was one the first designs. In this approach, a magnetic CNT-based DDS of GEM by physically adsorbing GEM onto poly(acrylic acid)-grafted MWCNTs with surface deposited iron NPs was created [79,80]. This carrier could only achieve similar reduction in cell viability relative to the group treated with free GEM. Although no improvement in in vitro efficacy was demonstrated, the comparable cytotoxicity profiles suggested that the carrier preparation process was at least not deleterious for the activity of GEM. Additionally, it was shown that the construct was able to be internalized by human pancreatic cancer cell line BxPC-3 and be found close to nucleus. In order to achieve specific tumor targeting, in another report GEM was loaded physically onto FA-conjugated MWCNTs to target cancer cells with overexpressed surface FA receptors [81]. With an entrapment efficiency of around 80%, the release of GEM was sustained and higher in acidic lysosomal pH 5.0 compared to pH 7.4, due to the ionization of GEM which increases its hydrophilicity in acidic medium. Owing to FA functionalization, the construct also experienced increase in hydrophilicity,

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consequently leading to a lower degree of hemolysis induction as compared to free GEM. When tested on MCF-7 cells with MTT assay, the CNT construct was found to be slightly more cytotoxic than free GEM and in comparison with the same CNT construct without FA functionalization in a concentration-dependent manner. Finally, the most recent outstanding work in the use of antimetabolites agents was reported by Solhjoo [82]. In this work, they used 5-FU in oxidized MWCNTs that were subjected to non-covalent PEGylation (OCNT-PEG). This investigation focused on the design of the pHresponsive 5-FU/OCNT-PEG drug delivery system and the prediction of release conditions through MD studies. The authors concluded that the best conditions for drug loading and release were neutral (7.4) and acidic (5.0) pHs, respectively. Although no in vitro assays were performed in this work, and according to the design, if we considered the acidic pH in the tumor sites, these conditions are more efficient in drug delivery to cancerous cells.

7.6 CNTs for radiotherapy Radiation therapy is a practical treatment option that is widely used in combination with other cancer treatment methods, such as surgery and chemotherapy. Tumors and cancer cells are exposed to ionizing radiation, which damages the DNA of cells and makes it difficult for them to grow and proliferate. There are two ways to expose tumors to ionizing radiation. First, the tumor can be exposed to a beam of radiation using an external source; but this type of radiation therapy has several disadvantages, such as low specificity for cancer cells, damage to healthy cells, and a limitation in the dose and area of exposure. Second, the tumor may be exposed to a radioisotope that can be implanted within the tumor or administered routinely to emit ionizing radiation continuously over time [83e86]. To solve the low nonspecific activity of radiopharmaceuticals, advances in nanotechnology, materials science, biochemistry, and molecular biology have allowed the development of more specific targeted compounds to reduce damage to healthy cells [85]. As a described earlier, nanotechnology describes the study, design, and application of nanoscale particles and systems, and has been widely exploited in drug development, discovery, and delivery [87,88]. Nanomaterials can be used to develop more powerful, active and targeted radiopharmaceuticals with fewer side effects and off-target activity.

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Therefore, special attention has been paid to the combination of nuclear medicine and nanomaterials to revolutionize current methods of diagnosis, detection and treatment of cancer. Nanomaterials have the potential to charge, transport, and deliver large numbers of radioactive atoms using only one nanomaterial. In this sense, metallic, polymeric, mesoporous silica nanomaterials, lipid-based, CNTs, etc. have been used to formulate effective radiopharmaceuticals [89e91]. Recently, CNTs have attracted significant attention due to their unique physicochemical and structural properties, including easy surface functionalization, photoacoustic effects, high drug loading capacity through pp stacking interactions. CNTs have a large area on the surface and all carbon atoms provide binding sites for attaching many ligands and are mechanically robust. The last two CNT features provide the advantage of amplifying a specific function or the ability to multi-functionalize the platform. Therefore, CNTs could be appropriate as delivery platforms for diagnostic or therapeutic agents, with multiple copies of target agents, radionuclides, drugs or fluorescent agents binding [85,92]. A key property of CNTs that is widely used is rapid clearance from the blood compartment. For example, Ruggiero et al. [93] reported the design and construction of SWCNT platforms, chelate molecules of radiometal ions (acid 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic) [DOTA] and antibody (Ab) E4G10 directed against neovascular tumor, were attached to the surface. Ab E4G10 was specifically directed to the monomeric vascular endothelial cadherin epitope (Ve-cad) expressed in the angiogenic vessels of the tumor. The obtained product was radioactively labeled with 225Ac, an alpha-particle emitting radionuclide, to obtain SWCNT-([225Ac] DOTA) (E4G10), which was used for targeted radioimmunotherapy and was tested in the tumor vasculature in a human colon adenocarcinoma murine xenograft model (LS174T). The specific platform reduced tumor volume and improved median survival relative to controls. Incorporation of SWCNT scaffold in construction design allowed to amplify the specific activity without negatively affecting the immunoreactivity of the rest of the targeted antibody. In another work [94], an alternative was studied to avoid the use of chelating molecules, such as DOTA, and to eliminate the need of binding the macrocyclic chelator to the nanotube. Gd3þ ions were used to increase the internalization and stable retention of 225Ac3þ ions within the SWCNTs, thus reducing the exposure of the radioisotope to serum proteins, besides making the outer wall clusters available, so that targeted

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peptides or antibodies can bind and can be preserved therapeutic efficacy. Furthermore, encapsulation of gadolinium ions within CNTs has been shown to enhance the contrast of MRI images. According to the loading studies, Gd3þ ions occupied the potential binding sites of the radionuclide 225 Ac3þ and therefore the loading efficiency decreased. The 225Ac @ CNT and 225Ac @ GCNT were tested in human serum to evaluate the possibility of purifying the material and removing weakly bound 225Ac3þ ions that are not embedded within the Gd3þ ion clusters and encapsulated within the nanotube. Das et al.[95] designed a selective platform for the possible treatment of tumors that overexpress FR while allowing real-time monitoring of the response to treatment through multimodal imaging. They took advantage of the large surface area of MWCNTs to bind four different molecules; an anticancer drug (MTX), a tumor-targeting module (FA), a radionuclide (99mTechnetium, 99mTc), and a fluorochrome (Alexa-fluor, AF 488, 647). Specifically, MTX was conjugated to MWCNTs through an intracellularly hydrolysable ester linkage, but stable in serum to ensure minimal drug loss in the circulation. The rest of the molecules that decorate the surface of the MWCNT were coupled by amide bonds and the radionuclide was coupled by coordination with the negatively charged hydroxyl and carboxyl groups. In vitro stability release MTX studies were performed from MWCNTs at different pH values, including phosphate buffer saline (PBS) (pH 7.4) and simulated lysosomal fluid (SLF) (pH 4.5). The obtained results highlight that in PBS 85% after 48 h of incubation. These data suggest that the ester bond between MTX and MWCNT is more stable in serum, compared to the intracellular medium, reinforcing the expectation that the tetrafunctional conjugate will be stable in serum, but can become the active prodrug immediately after their internalization to target cells through chemoenzymatic intervention. Finally, in vitro cytotoxicity studies in FR (þ) carcinogenic cells determined that the MTX-FA-MWCNT platform presented greater activity than free MTX.

7.7 CNTs for nuclear medicine imaging In recent years, nuclear medicine imaging (NMI) has been intensively explored as an advanced modality for diagnostic imaging, consisting of three stages: (1) introduction of radionuclides into the body, (2) detection of the emitted gamma rays and (3) the generation of images that provide details on

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the radionuclide distribution and the physiological characterizations of organs and tissues. The most common imaging modalities in IMNs are single photon emission computed tomography (SPECT) and positron emission tomography (PET) [96]. SPECT detects gamma rays directly from the decay of radionuclides, uses radionuclides with longer physical half-lives and that are more aligned with the biological half-lives of the physiological processes of interest, radiotracers are more readily available. In addition, they can run studies with multiple tracers simultaneously using different radionuclides that examine different biological pathways in a single imaging session; and the systems are cheaper [97,98]. The SPECT examination has the ability to detect an early stage of diseases that we cannot detect with other techniques, although it is less sensitive than PET [99]. On the other hand, PET has its foundation when a positron released by a decaying radionuclide travels in the tissue until it has exhausted its kinetic energy. At this point, it will find its antiparticle, an electron, and both will mutually annihilate each other, completely converting their mass into g-rays that are the ones that will be registered by the detector [100]. The power of this technique lies on the wide range of available radiotracers that produce image contrast directly related to the underlying physiology, metabolic pathways, or molecular targets [101]. Although PET is generally more expensive, at both clinical and preclinical level, it has undoubtedly several advantages over SPECT, such as the ability to quantify images, higher sensitivity, and therefore lower concentrations of tracer and higher resolution [102]. The most common application of PET in clinical oncology has been the detection of tumors and the evaluation of the extent of the disease by PET combined with computed tomography (PET-CT); the most used radiotracer in PET is 18F-fluorodeoxyglucose (18F-FDG) [103,104]. For years, the field was dominated by commercial probes composed of small molecules labeled with nonmetallic radionuclides with very short radioactive half-lives. In addition, the probes have other limiting characteristics, such as problems in administration to people with diabetes, high production costs, low specificity that can lead to false positives, among others [105]. These limitations highlight the need to develop new imaging probes focusing on a multifaceted approach, relying on areas such as radiopharmaceutical chemistry, biology, and nanomedicine [106e108]. Nanocarrier-based imaging systems have received much attention to address the limitations of traditional probes NMI in cancer diagnosis and treatment, because with the use of these probes accumulation in tumors can

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be predicted, tumor efficacy determined and evaluate the responses to nanomedicine therapies through the obtained images [109e111]. Recently, CNTs have emerged as promising platforms for imaging applications in the diagnosis and treatment of the disease. Compared with quantum dots and traditional fluorescent dyes, CNTs have several important advantages, such as robust chemical inertness, easy functionalization, high resistance to photobleaching, biocompatibility, large surface area and high absorption crosssection of two photons, which are particularly beneficial for their use as theragnostic nanoplatforms [112]. Wang et al. [113] designed and developed a platform composed of magnetic and radiolabeled MWCNTs to be used as dual contrast agents for Magnetic Resonance Imaging (MRI) and SPECT. As a first step, the surface of the MWCNTs was functionalized with carboxylic acid groups (eCOOH). Then, the MWCNT hybrids were obtained with the superparamagnetic iron oxide NPs, to obtain SPION-MWCNT. Radiolabeling of the hybrids was carried out using a functionalized bisphosphonate (BP), namely dipycholamine-alendronate, as linker between SPION and the 99mTc radioisotope. Stability tests of the SPION-MWCNT hybrids were performed in PBS at 37 C for 24 h, the results demonstrated a high stability of the hybrids because most of the 99mTc was still bound to the hybrids after 24 h of incubation. The SPION-MWCNT hybrids allowed SPECT/CT imaging and gamma scintigraphy to quantitatively analyze their biodistribution in vivo, demonstrating the ability of radiolabeled CNT hybrids as dual contrast agents for MRI and SPECT for in vivo use. Similarly, in another work [114], the surface of MWCNT was decorated with superparamagnetic iron oxide NPs, but the strategy to attach NPs on the MWCNT surface was through “click chemistry” in order to optimize and control the modification of the surface and prevent NPs from aggregating. These hybrids were efficiently internalized by tumor cells without showing obvious toxicity effects, and they were also detected at the single-cell level by MRI. In another work [115], the specific targeting toward a specific subpopulation of breast cancer cells that exhibit self-renewal capacity, pluripotency, high tumorigenic potential, and resistance to therapy was investigated. This study was carried out with a platform composed of SWCNTs functionalized with CD44 monoclonal antibodies and radiolabeled with Gallium-67 (67Ga) in order to monitor in vivo biodistribution using noninvasive SPECT images. The pictures showed the accumulation of 67Ga-SWCNT-CD44 nanoconjugates at the breast cancer site, compared to nanoconjugate samples that were not labeled with CD44.

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7.8 CNTs on hyperthermia therapy Drug delivery and light-activated therapies are potential platforms using light sources ranging from 300 to 1000 nm (UV-vis and NIR) to produce heat energy for cancer treatment. There are three typical methods of lightbased therapy, PTT, PDT, and light-mediated chemotherapy. PTT is a photothermal agent-based therapy, in which nanomaterials (with or without a photosensitizing agent) can absorb light and convert optical energy into thermal energy to produce heat, leading to photoablation of cancer cells/tumors [116]. PDT uses nanomaterials that can produce reactive oxygen species (ROS) at specific wavelengths, leading to apoptotic and necrotic cell death [117]. Light-mediated chemotherapy is a method of treating cancer cells, in which chemotherapeutics are loaded into carriers and then released by the action of light from nanomaterials (Fig. 7.2). These light-based therapies are often used in combination with each other, such as PTT-PDT, chemo-PTT, and chemo-PDT, which show significantly better therapeutic efficacy than individual therapies [112]. Among light sources, NIR with wavelengths ranging from 700 to 1000 nm is considered to provide deeper tissue penetration and less photodamage and autofluorescence, which are effective for light-mediated therapies [112]. CNTs can easily penetrate cell membranes and have intrinsic NIR photothermal conversion properties, that is why they are used for light-mediated chemotherapy and PTT, either individually or in combination for the treatment of cancer [117,118]. Surface functionalized MWCNTs with NP gold have been used for thermal therapy of cancer cells [119]. The cytotoxicity tests of the MWCNT/AuNPs hybrids in the breast cancer cell line (MCF-7) with NIR

Figure 7.2 PTT and PDT strategies for death cancer cells using CNTs.

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irradiation showed cell death. In comparison, the tests in the cell line that did not contain the hybrids but were exposed to radiation did not present any toxicity, proving that the hyperthermia was caused by the presence of the hybrids. In another work [120], a system targeting cancer cell mitochondria was developed, composed of SWCNT decorated with pHsensitive polymers and PEG. To estimate the therapeutic efficacy of the platform, in vitro cytotoxicity assays were performed with MB49 cells; the platforms showed minimal toxicity against MB49 cells without NIR irradiation. After NIR laser irradiation, cell viability drastically decreased. The study of CNTs for applications in thermal therapy has been widely studied over time [121e123].

7.9 CNTs for gene delivery CNTs, after undergoing an oxidation process, can be functionalized in their carboxylic groups with proteins, peptides, DNA, oligonucleotides, sugar residues, etc. Nucleic acid functionalized CNTs, such as small interfering RNA (siRNA) or DNA, are promising prospects for application in gene therapy and interference. An SWCNT platform was developed to carry telomerase reverse transcriptase siRNA (TERT) to silence TERT expression and inhibit tumor cell proliferation and growth. In in vitro cytotoxicity assays with LLC cancer cell lines, TC-1 and 1H8, it was shown that the platform can penetrate tumor cells to induce tumor cell suppression, although more studies are needed to evaluate the distribution of the platform after systemic intravenous administration [124]. In a very similar way [125], complexes were developed between MWCNT and siRNA, and the biological activity was evaluated in vivo; the treatment led to a statistically significant reduction in tumor volume.

7.10 Considerations for in vitro viability assays in CNTs The classical assays to determine the cell viability are based on quantifying a colorimetric change that allows extrapolating the rate of cell proliferation or cytotoxicity in a previously selected cell lines (carcinogenic or noncarcinogenic cells). These techniques are mainly based on determining the mitochondrial functionality of treated cells, this is achieved using water-soluble salts with tetrazole ring (classical salts MTT, XTT, WST-1, and INT). These scaffolds are reduced to an insoluble derivative of

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formazan in aqueous medium of an intense color that is characteristic of the salt from which they come (they can adopt blue and red tones) [126,127]. The reduction reaction of tetrazole ring to formazan is produced by the action of mitochondrial enzyme succinate-dehydrogenase, although cytosolic reductases or reductases from other subcellular compartments may also intervene. The resulting reduced coenzymes (NADH and NADPH) will convert the tetrazoles to their corresponding formazan product insoluble in water (Fig. 7.3), which is extracted and quantified spectrophotometrically [128]. Thus, the untreated cells are used as a positive control (100% viability) and all the values of the experiment are correlated with this data set at previously determined times (commonly 24, 48 and 72 h). Using this technique, it is possible to obtain relatively reproducible results for commonly employed chemotherapeutics (such as DOX or 5FU) [127]. In general, assays based on tetrazole salts can be interfered by factors such as the solubility of the obtained formazan, the selected time to terminate the assay, the toxicity mechanism caused by the evaluated substance and finally the chemical structure that is being analyzed. In this way, CNTs as potential nanotherapeutics against cancer should be evaluated with caution, since in some reports they have shown strong cytotoxicity [129] while in other studies with similar structures they appear to be innocuous when the MTT assay is used to determine the cell viability [130,131]. This behavior was noticeable when CNTs were tested in cell line A-59 (human

Figure 7.3 Classical viability assay based on tetrazolium salts for anticancer drugs and CNTs.

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alveolar epithelial cell line) in which through the MTT assay they showed a strong cytotoxic effect (the reduction in cell viability reaches at least 60%) after 24 h of incubation. Unlikely, if the WST-1 salt is used for the same molecule, no cytotoxicity is observed, even the same result of null cytotoxicity is observed when it is evaluated in other cell lines such as endothelial macrophages of rat (NR8383: rat alveolar macrophage cell line) or ECV304 line (endothelial cells derived from umbilical cord) [130]. To corroborate this observation, complementary studies were performed that began with the determination of the integrity of cell membrane through the lactate dehydrogenase (LDH) assay [1]. This enzyme is freely located in the cytoplasm and on the membrane, if there is any damage the release of LDH will occur into the surrounding environment [130]. To determine this possible damage, an assay that detects the presence of LDH in the medium through a tetrazolium salt (INT) is used. This salt is reduced in an LDH-dependent reaction, however, after incubating CNTs in various cell lines (A549, ECV304) for 24 h or more and at different concentrations, no reduction in viability can be observed since changes in concentration or incubation time have no significant influence on cell viability. To verify the cytotoxicity generated by the CNTs in the MTT assay, more robust studies were carried out such as the determination of the mitochondrial membrane potential assisted by FACS and annexin-V/PI staining, however, they did not reveal any cellular damage due to necrosis or by apoptosis. In general, CNTs interact with some tetrazolium salts like MTT, but not with other water soluble tetrazolium salts like WST-1, INT or XTT. This phenomenon can be explained by analyzing the type of chemical substitutions of each tetrazolium salt; MTTdtwo methyl groups, WST-1 and INTdnitro group. The thiazolyl cycle in the MTT molecule is the only possible partner to interact with nanotubes. It appears that the insolubility of MTT-formazan is crucial to nanotube binding and alteration of assay, and not for some interference of MTT in enzyme reduction (Fig. 7.4). This finding highlights the emerging need to standardize biological tests for new nanomaterials that include CNTs in all their versions, with at least two or more independent assays systems for this new class of nanotherapeutics. Finally, it is important to highlight that the clustering phenomenon of CNTs with MTT-formazan crystals, which affects the congruence of biological evaluations, can be exploited as a potential mechanism to guide bioremediation and consequently the elimination of SWCNTs from tissues

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Figure 7.4 Interferences in tetrazolium salts assays for carbon nanotubes.

in which they accumulate. This interaction is a potential strategy for the elimination of these nanostructures [130].

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K.S. Lam, A smart and versatile theranostic nanomedicine platform based on nanoporphyrin, Nat. Commun. 5 (2014). S. Kunjachan, J. Ehling, G. Storm, F. Kiessling, T. Lammers, Noninvasive imaging of nanomedicines and nanotheranostics: principles, progress, and prospects, Chem. Rev. 19 (2015) 10907e10937. X. Li, X.N. Zhang, X.D. Li, J. Chang, Multimodality imaging in nanomedicine and nanotheranostics, Cancer Biol. Med. 13 (2016) 339e348. R. van der Meel, E. Sulheim, Y. Shi, F. Kiessling, W.J.M. Mulder, T. Lammers, Smart cancer nanomedicine, Nat. Nanotechnol. 14 (2019) 1007e1017. K.D. Patel, R.K. Singh, H.W. Kim, Carbon-based nanomaterials as an emerging platform for theranostics, Mater. Horiz. 6 (2019) 434e469. J.T.W. Wang, L. Cabana, M. Bourgognon, H. Kafa, A. Protti, K. Venner, A.M. Shah, J.K. Sosabowski, S.J. Mather, A. Roig, X. Ke, G. Van Tendeloo, R.T.M. De Rosales, G. Tobias, K.T. Al-Jamal, Magnetically decorated multiwalled carbon nanotubes as dual mri and spect contrast agents, Adv. Funct. Mater. 24 (2014) 1880e1894. G. Lamanna, A. Garofalo, G. Popa, C. Wilhelm, S. Bégin-Colin, D. Felder-Flesch, A. Bianco, F. Gazeau, C. Ménard-Moyon, Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations, Nanoscale 5 (2013) 4412e4421. A. Al Faraj, A.S. Shaik, B. Al Sayed, R. Halwani, I. Al Jammaz, Specific targeting and noninvasive imaging of breast cancer stem cells using single-walled carbon nanotubes as novel multimodality nanoprobes, Nanomedicine 11 (2016) 31e46. L. Zhang, D. Sheng, D. Wang, Y. Yao, K. Yang, Z. Wang, L. Deng, Y. Chen, Bioinspired multifunctional melanin-based nanoliposome for photoacoustic/magnetic resonance imaging-guided efficient photothermal ablation of cancer, Theranostics 8 (2018) 1591e1606. D. Wang, N. Zhang, X. Jing, Y. Zhang, Y. Xu, L. Meng, A tumormicroenvironment fully responsive nano-platform for MRI-guided photodynamic and photothermal synergistic therapy, J. Mater. Chem. B 8 (2020) 8271e8281. K. Khalid, X. Tan, H.F. Mohd Zaid, Y. Tao, C.L. Chew, D. Chu, M.K. Lam, Y. Ho, J.W. Lim, L.C. Wei, Advanced in developmental organic and inorganic nanomaterial: a review, Bioengineered 11 (2020) 328e355. F. Saghatchi, M. Mohseni-Dargah, S. Akbari-Birgani, S. Saghatchi, B. Kaboudin, Cancer therapy and imaging through functionalized carbon nanotubes decorated with magnetite and gold nanoparticles as a multimodal tool, Appl. Biochem. Biotechnol. 191 (2020) 1280e1293. M. Wang, L. Ruan, T. Zheng, D. Wang, M. Zhou, H. Lu, J. Gao, J. Chen, Y. Hu, A surface convertible nanoplatform with enhanced mitochondrial targeting for tumor photothermal therapy, Colloids Surf. B Biointerfaces 189 (2020). N.W.S. Kam, M. O’Connell, J.A. Wisdom, H. Dai, Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 11600e11605. S.Y. Hong, G. Tobias, K.T. Al-Jamal, B. Ballesteros, H. Ali-Boucetta, S. LozanoPerez, P.D. Nellist, R.B. Sim, C. Finucane, S.J. Mather, M.L.H. Green, K. Kostarelos, B.G. Davis, Filled and glycosylated carbon nanotubes for in vivo radioemitter localization and imaging, Nat. Mater. 9 (2010) 485e490. M. Zhou, M. Melancon, R.J. Stafford, J. Li, A.M. Nick, M. Tian, A.K. Sood, C. Li, Precision nanomedicine using dual PET and MR temperature imaging-guided photothermal therapy, J. Nucl. Med. 57 (2016) 1778e1783. Z. Zhang, X. Yang, Y. Zhang, B. Zeng, S. Wang, T. Zhu, R.B.S. Roden, Y. Chen, R. Yang, Delivery of telomerase reverse transcriptase small interfering RNA in

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complex with positively charged single-walled carbon nanotubes suppresses tumor growth, Clin. Cancer Res. 12 (2006) 4933e4939. J.E. Podesta, K.T. Al-Jamal, M.A. Herrero, B. Tian, H. Ali-Boucetta, V. Hegde, A. Bianco, M. Prato, K. Kostarelos, Antitumor activity and prolonged survival by carbon-nanotube-mediated therapeutic siRNA silencing in a human lung xenograft model, Small 5 (2009) 1176e1185. T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation an cytotoxicity assays, J. Immunol. Methods 65 (1983) 55e63. D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nogziger, M.J. Currens, D. Seniff, M.R. Boyd, Evaluation of a soluble tetrazoliun/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines, Cancer Res. 48 (1988) 4827e4833. H. Tominaga, M. Ishiyama, F. Ohseto, K. Sasamoto, T. Hamamoto, K. Suzuki, M. Watanabe, A water-soluble tetrazolium salt useful for colorimetric cell viability assay, Anal. Commun. 36 (1999) 47e50. A. Semisch, A. Hartwig, Copper ions interfere with the reduction of the watersoluble tetrazolium salt, Chem. Res. Toxicol. 27 (2014) 169e171. J.M. Wörle-Knirsch, K. Pulskamp, H.F. Krug, Oops they did it again! Carbon nanotubes hoax scientists in viability assays, Nano Lett. 6 (2006) 1261e1268. V. Stone, H. Johnston, R.P. Schins, Development of in vitro systems for nanotoxicology: methodological considerations, Crit. Rev. Toxicol. 39 (2009) 613e626.

Further reading [1] T. Decker, M.L. Lohmann-Matthes, A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity, J. Immunol. Methods 115 (1988) 61e69.

CHAPTER 8

Carbon nanotubes as nanovectors for targeted delivery of platinum based anticancer drugs Sanghamitra Chatterjee Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai, Maharashtra, India

8.1 Introduction “Magic Bullet” was the term coined a century ago by the Nobel laureate Paul Ehrlich who was an immunologist [1], but it has now transformed into reality due to the emergence of nanotechnology and in particular the advent of carbon nanotubes as nanovectors. Nanovectors, which refer to the targeted delivery vehicle aiding in the transport of nanoscale material, has garnered prominence in the past decades being a pluridisciplinary approach in the realm of cancer theranostics [2e4]. Presently, nanomedicine has transpired into a conspicuous branch of nanotechnology which focusses primarily on targeted delivery of anticancer drugs for diverse forms of cancer [5,6]. A pivotal component of nanomedicine is the research pertaining to carbon nanotubes, which could function as potent drug delivery systems by augmenting the efficacy, specificity, and safety of anticancer drugs [7e9]. Divergent physicochemical properties of carbon nanotubes such as high surface area and aspect ratio, ability to be effortlessly conjugated with varied therapeutics, elevated cellular uptake, improved selectivity and efficacy, preferential tumor accumulation, and alleviated side effects have made them multifaceted and effectual drug delivery nanocarriers for novel cancer therapies [10e12]. Consequently, the usage of carbon nanotubes as nanovectors is advancing phenomenally making it feasible to envisage promising applications in the pharmaceutical industry. This chapter outlines the carbon nanotube based nanocarriers which have been extensively used for the targeted delivery of platinum based anticancer drugs.

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00004-2

© 2023 Elsevier Ltd. All rights reserved.

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In the present decade, platinum-based anticancer drugs are substantially used for the treatment of a wide spectrum of human malignancies, and they have revolutionized cancer therapy. Platinum drugs are administered in chemotherapy procedures either as a single therapeutic agent or in combination with other cytotoxic agents [13e15]. Conventional chemotherapeutics have been implemented for past decades in the treatment of malignancies, but the other side of the coin revealed onset of acquired resistance, off-target effects, systemic toxicity, and frequent recurrences [16]. In comparison with traditional chemotherapy, targeted drug delivery exhibited inhibition of drug resistance and restraint of normal tissue damage. In consequence, there was an ardent necessity to explore alternative strategies to improve the pharmacokinetic profiles of platinum complexes. Combinatorial therapy employing multimodular nanoassembly was effectuated to achieve enhanced therapeutic potency and low systemic toxicity. Platinum based anticancer drugs were merged with carbon nanotubes and in comparison, to the spherical nanoparticles, the tubular structure of the carbon nanotubes had a precedence in drug delivery applications [17]. Platinum drug conjugate with the carbon nanotubes manifested advantages of efficient drug loading with zero premature leakage and releasing the payload to the targeted site in a controlled manner [18]. Additionally, the adverse side effects could be prominently diminished due to the promotion of preferential accumulation of the anticancer drug in the malignant cells [19]. The inclusion of diverse classes of carbon-based nanomaterials such as single-walled carbon nanotubes (SWNTs), multi-walled carbon nanotubes (MWNTs), graphene, fullerene, and carbon nanohorn are potential nanocarriers for biomedical applications. Nanocarrier-based targeted delivery of platinum anticancer complexes has been envisioned as a versatile platform combining novel approaches and concepts as exemplified below in this chapter. The objective of this chapter is to summarize the synergism of platinum based anticancer drugs with carbon nanotubes as nanovectors, their versatility in physicochemical properties, efficacy and therapeutic safety, establishing the most functional and viable conditions for their utilization in cancer treatment and identifying the perspectives followed by further improvement of the nanoassembly.

8.2 Platinum anticancer drugs The substantial contribution of platinum-based anticancer drugs in oncology have been discerned in the past decades. The serendipitous discovery of

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cisplatin exhibiting antitumor activity revolutionized cancer theranostics. Michele Peyrone was the first person who described cisplatin in 1845, and for a long time it was known as the Peyrone’s salt [20]. Subsequently, when Barnett Rosenberg was working with his colleagues in Michigan State University on the influence of electric field on bacteria, it was then they discovered that inhibition of bacterial growth can be accomplished utilizing the products obtained from the hydrolysis of the platinum electrode [21]. Cisplatin which is cis-(diammine) dichloroplatinum being one of the most potent products was found to impede the growth of malignant cells and is the first platinum-based complex approved by the US food and drug administration (FDA) [22]. It is an alkylating and antineoplastic agent notable for both systematic toxicity and anticancer activity. Cisplatin is believed to bind covalently to the DNA producing distortions to its double helix structure [23]. The mechanism of action of cisplatin has been considerably explored and the researchers now concur that cisplatin with the aid of copper transporter 1 enters cells via both active transport and passive diffusion thereafter losing its two chlorides and binding purines in the genomic DNA. This leads to structural distortion and subsequent inhibition of DNA replication and transcription which finally results into apoptosis as illustrated in Fig. 8.1 [24]. Cisplatin is found to be stable in the presence of benzyl alcohol, mannitol, parabens, and glucose [25]. It is a widely administered drug possessing therapeutic effect against manifold tumors such as bladder cancer, breast cancer, prostrate carcinoma, ovarian cancer, cervix carcinoma, lung cancer, head cancer, endometrial cancer, and neck cancer [26]. Cisplatin is also a potent drug in the therapy of solid tumors including liver, gastric, and brain carcinomas [27]. Despite its widespread usage, the administration of the

Figure 8.1 Illustration of mechanism of action and the resistance pathways of cisplatin.

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drug is mostly restricted due to acute toxic side effects such as hematological, ototoxicity, and emetogenicity along with the display of acquired resistance possessed by diverse cancers [28]. The second generation of classical platinum anticancer drugs in which most markedly carboplatin and the third generation including most notably oxaliplatin thus came into existence and were globally approved. Carboplatin with its alleviated toxicity profile due to the presence of bidentate dicarboxylate ligands which lessen the aquation rate 100 times making it viable for high dose chemotherapy. Carboplatin being different from cisplatin in pharmacokinetics and pharmacodynamics have antineoplastic efficacy against kidney, breast, prostrate, and bladder tumor cell lines [29]. Oxaliplatin has minimal cross-resistance to cisplatin or carboplatin and targets a broad spectrum of tumors due to reduced toxicity. Other next-generation cisplatin derivatives include nedaplatin, lobaplatin, and heptaplatin which were clinically approved all over the world. Table 8.1 summarizes the attributes pertaining to these platinum anticancer drugs [26]. The classical platinum drugs exhibited drug resistance due to enhanced DNA repair, increased drug detoxification by thiols, modified cell signaling pathway, and diminished drug uptake [16]. To circumvent these drawbacks, nonclassical platinum anticancer drugs were developed which defied the structure activity relationship. Picoplatin, a sterically hindered platinum drug, was developed in which methyl pyridine substitutes one amine ligand thereby resisting glutathione mediated inactivation [30]. Multinuclear platinum drugs possessing multiple platinum atoms per molecule were developed which led to drastic conformational changes to the DNA and were instrumental in overcoming the drug resistance to cisplatin. BBR3464 being one of the potent members of this class showcased unparalleled efficacy on cisplatin resistant cell lines by functionally preventing the DNA repair machinery [31]. Platinum intercalators are another category of nonclassical platinum drugs. These drugs insert themselves reversibly between the base pairs of DNA’s double helix which prevents the replication due to lengthening and unwinding of the strands of DNA [32]. 56MESS is an elite member of this category. Additionally, other platinum complexes are also being evaluated which include platinum prodrugs. These are denoted as the fourth generation of the platinum complexes and are constituted of chemically reductive platinum prodrugs and photosensitive platinum prodrugs [33]. Satraplatin with enhanced oral bioavailability is a potent member of chemically reductive prodrug class and is under clinical trials.

Table 8.1 Platinum complexes in current clinical use.

Complex

Cisplatin

Brand names/ synonyms

Year approved

Structure

Non-leaving ligands, A2 (number of ligands)

Leaving ligands, X2 (number of ligands)

Market status

Clinical use

Abiplatin Platinex Platiblastin Platosin Briplatin Randa Cisplamerck Platinol Carbomerck Paraplatin

1979

Ammine (2)

Chloride (2)

Worldwide

Metastatic testicular and ovarian tumors, advanced bladder cancer

1989

Ammine (2)

1, 1Cyclobutanedicarboxylate (1)

Worldwide

Advanced ovarian carcinoma

Oxaliplatin

Dacotin Eloxatin

2002

1, 2Cyclohexanediammine (1)

Oxalate (1)

Worldwide

Metastatic colorectal cancer

Nedaplatin

Aqupla

1996

Ammine (2)

Glycolate (1)

Japan

Small and non-small cell lung cancer, head and neck tumors, esophageal and bladder tumors, cervix carcinomas

Carboplatin

Continued

Table 8.1 Platinum complexes in current clinical use.dcont'd

Complex

Brand names/ synonyms

Year approved

Lobaplatin

Miboplatin

Heptaplatin (SKI2053R)

Sunpla Eptaplatin

Non-leaving ligands, A2 (number of ligands)

Leaving ligands, X2 (number of ligands)

Market status

2004

1,2-Cydobutanedimethanamine (1)

2-Hydroxy-propanoate (1)

China

2005

2-(l-methylethyl)-l,3dioxolane-4,5-dimeth anamine (1)

2-Dioatopropanoate (1)

South Korea

Structure

Clinical use

Breast, testicular, ovarian, small cell lung and gastric carcinomas, chronic myeloid leukemia Gastric cancer

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8.3 Mechanism of action of platinum drugs The cytotoxicity of the platinum anticancer drugs is governed primarily by three major factors which include accumulation of the drug in the cells, intracellular aquation along with binding to the cellular targets, and cellular recognition of damage induced by the platinum leading to the cell death. The accumulation of platinum drugs in the cells is a major contributor to cytotoxicity and with a decrease in drug accumulation it leads to acquired cellular resistance. Copper transporter 1 is considered as the principal gateway for the drug accumulation in tumor cells for cisplatin and carboplatin primarily [34]. Oxaliplatin is relatively less dependent on copper transporter 1 in comparison to cisplatin and carboplatin. A direct interrelation was established between decrease in copper transporter 1 expression and increase in acquired cisplatin resistance in ovarian cancer cell lines [35]. Other copper transporters and organic cation transporters have also been reported which regulate the accumulation of platinum anticancer drugs [36], thereby indicating that active, passive, and facilitated transport mechanisms control the concerned drug accumulation. The rate of aquation of the platinum anticancer drugs plays a key role in the pharmacology which is illustrated in Fig. 8.2 using cisplatin as the model drug. The process of aquation chemically activates the drug, which is a primary requirement for its binding to intracellular targets such as DNA, proteins, and RNA [37]. The rate of aquation is a pivotal factor as the increased rate can lead to severe systemic toxicity due to reaction of highly active platinum complexes with varied molecules in the blood. On the contrary, slower aquation rates reduces the toxicity and prolongs the plasma

Figure 8.2 Schematic illustration of cellular accumulation of cisplatin, its intracellular aquation, activation of cellular signaling pathways by platinum induced DNA damage and the resultant cell death.

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half-life but also decreases the antitumor activity of the drug [38]. Oxaliplatin demonstrates reduced rates of aquation and consequently are more stable in the aqueous media with reduced toxicity [39]. DNA being one of the significant cellular targets for anticancer effect influences the cytotoxicity of the platinum complexes. The destabilization, bending, and unwinding of the DNA duplex occur due to formation of the platinum DNA adduct [40]. Varied DNA adduct profiles are formed depending on the different leaving groups in the concerned platinum drugs. Therefore, oxaliplatin produces a contrasting adduct profile in comparison to cisplatin and consequently differ in their cytotoxicity [41]. The formation of the platinum DNA adduct enables the DNA damage recognition proteins to selectively identify the distorted DNA. The existence of diverse pathways has been documented which involve the signaling of DNA damage followed by arresting the cell cycle and triggering cell death through apoptosis [42]. Literature survey reveals that the platinum complexes react with not only the DNA but also several non-DNA cellular components which influence significantly the cytotoxicity profile of the platinum anticancer drugs. Cisplatin has been shown to bind with tubulin, C-terminal part of the molecular chaperone and in some instances, it has interacted with phosphatidylserin and other phospholipid components of the cellular membranes [43e45]. Platinum-based anticancer drugs have radically transformed the cancer treatment by forming intrastrand adducts with DNA thereby affecting the primary cellular processes and eventually triggering the apoptosis.

8.4 Limitations of platinum drug therapy The platinum anticancer drugs have a significant impact in medical oncology and continue to be used clinically for the management of diverse malignancies. However, a key concern has been posed in the medical field due to the dose limiting toxicities such as neurotoxicity, nephrotoxicity, and myelosuppression related with platinum drug therapy, development of drug resistance, partial antitumor response in patients, and tumor relapse which consequently adversely affect the quality of life of patients. As a consequence, sub-lethal doses of the drugs are administered which thereafter trigger acquired resistance [46]. Cisplatin is associated significantly with nephrotoxicity which is observed 10 days post administration leading to irreversible renal failure. It manifests reduction in serum magnesium and potassium levels, increased serum creatinine, and permanent reduction in

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glomerular filtration rate [47]. Histopathological changes follow thereafter with death of tubular cells and renal tissue damage [48]. Carboplatin has diminished effects on the renal system with less severe renal toxicity, but its continued therapy may lead to overt nephrotoxicity [49]. Among the platinum anticancer drugs in medical usage, oxaliplatin is the least nephrotoxic in nature [50]. Neurotoxicity is a prominent side effect of platinum drug therapy in which cisplatin is responsible for the damage of the dorsal root ganglion [51]. The adverse neurological effects are generally reversible but, in some cases, they are even long lasting [52]. Carboplatin does not exhibit neurotoxicity when administered in relevant dosage forms whereas treatment with oxaliplatin leads to sensory neuropathy which may lead to impairment of normal life [53]. Cisplatin is also associated with ototoxicity especially in less than 5 years old patients with minimal effects on adults or adolescents [54]. High doses are responsible to induce visual impairments caused due to retinal damage [55]. Carboplatin on the other hand exerts severe hematological side effects in comparison to cisplatin and oxaliplatin [56]. Emetogenicity is associated with cisplatin therapy which is also responsible for asthma or hives and anaphylactic shock [57,58]. In order to combat these adverse effects, concomitant clinical strategies have been adapted which include forced diuresis, pre-hydration, co-administration of other drugs, and varied other chemoprotectant approaches which have limited benefits [59]. Development of drug resistance is a notable concern associated with platinum therapy and modalities to circumvent this problem continue to be an unmet need for most of the malignancies. Hence, albeit platinum anticancer drugs gained immense significance in the realm of oncology, the adverse side effects associated with them necessitated the exploration of alternative modus operandi.

8.5 Carbon nanotubes as platinum drug carriers In the recent years, the nanocarrier-based drug delivery of platinum complexes has gained prominence due to their advantages related to reduced side effects, enhanced drug efficacy, and circumventing drug resistance caused due to cellular accumulation [60]. Drug delivery systems play a pivotal role in improving the pharmacological profile of bioactive molecules and selectively targets the specific malignant cells without adversely affecting the neighboring tissues. Presently used drug delivery systems are majorly polymers, liposomes, virus-based systems, dendrimers,

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cyclodextrins, nanoparticles, and nanotubes. Carbon nanotubes are versatile therapeutic nanocarriers for the targeted delivery of manifold chemotherapeutic agents including primarily platinum anticancer drugs. It has been the most highly researched material as a drug delivery system in the last decade. The unique physicochemical properties of carbon nanotubes such as ameliorated strength and conductivity, optical properties, biocompatibility, their ease of functionalization and most importantly high surface to volume ratios render them as novel platinum anticancer drug carriers [61]. The needle-like structure, high drug loading capacity, and stability along with flexible interactions with the cargo make them promising candidates in the biomedical field [62]. Carbon nanotubes are functionalized to enhance their solubility and biocompatibility which include noncovalent and covalent functionalization on the external walls of carbon nanotubes, defect functionalization on the sidewalls and at the open tips, and lastly encapsulation of the drugs inside the carbon nanotubes [8,17]. The platinum anticancer drugs can be loaded into the carbon nanotubes via chemical conjugation, chelation, and p-p stacking [63]. The morphology of the carbon nanotubes has a significant impact on the safety and efficacy of the anticancer drugs making them as one of the most interesting nanocarriers for many therapeutic and diagnostic applications [17]. The delivery of platinum-based anticancer drugs utilizing carbon nanotubes as nanocarriers can be effectuated via three different modes. Firstly, the anticancer drug can be loaded into the cavity of the carbon nanotubes. Secondly, the surface of the carbon nanotubes can be functionalized employing carboxylic acid or amine groups and thereafter the drug can be attached directly to the carbon nanotube surface. Lastly, the drug can be attached to the carbon nanotubes through the utilization of a chemical tether as depicted in Fig. 8.3 [12]. This chapter focuses on the conjugates of platinum-based anticancer drugs with carbon nanotubes as nanocarriers, their versatility, therapeutic safety, and efficiency, and identifies the outlook for further advancement in this regime. 8.5.1 Single-walled carbon nanotubes The carbon nanotubes predominantly exist in two forms as SWNTs and MWNTs which exhibit prominent advantages as nanocarriers for platinum anticancer drugs. Literature survey unveils that the Lippard group reported soluble SWNTs loaded with cisplatin prodrug wherein the cisplatin was released via selective intracellular reduction [64]. The carbon nanotubes

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Figure 8.3 The four different methods of attachment of platinum compound to carbon nanotubes (a) Coordination to surface amine groups through the formation of a peptide bond, (b) cisplatin encapsulation within the cavity, (c) carbon nanotubes holds the platinum to the surface through hydrophobic effects, and (d) the coordination of platinum compound to carbon nanotubes surface with carboxylate groups.

enhanced the efficacy by more than 100 times (Fig. 8.4a) [16]. Platinum (IV) prodrug was later designed using axial folate targeting ligand which was subsequently tethered to the carbon nanotube (Fig. 8.4b) leading to enhanced possibility of selectively targeting the malignant cell [65]. Dhar

Figure 8.4 Carbon nanotubes for platinum (IV) drug delivery (aec). Platinum (IV) prodrug of cisplatin was either conjugated on the surface of carbon nanotubes (a, b) or entrapped into them for drug delivery (c). Folate was introduced as a targeting ligand (b).

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et al. conducted an experiment with covalently interlinked platinum (IV) composite attached to COOH group on SWNTs surface where amineended chain was conjugated non-covalently to form “longboat” for the platinum anticancer drug delivery system [65]. Tripisciano et al. encapsulated cisplatin into SWNTs and consequently with the usage of DMF solvent approximately 21% w/w of cisplatin drug loading was attained. Varied characterization techniques were employed to ascertain the insertion of cisplatin into SWNTs [66]. It was patently observed in another investigation on prostate cancer cells that the high concentration of cisplatin and SWNTs solution brought about a reduced toxic effect in comparison to the same amount of free cisplatin. Bhirde et al. designed a drug conjugate for targeted delivery against squamous cancer cells where the oxidized SWNTs was functionalized with cisplatin followed by the conjugation with epidermal growth factor [63]. Same group of researchers also experimented on mice xenografts with targeted SWNTs and demonstrated selective accumulation leading to marked regression of tumor cell growth in comparison to the controls [67]. Yang et al. synthesized magnetic (Fe3O4) nanoparticle layers on the inner surface of the carbon nanotubes and cisplatin was incorporated into the pores of carbon nanotubes [68]. Cisplatin was also encapsulated into zigzag SWNTs, and its behavior in aqueous solutions was examined by the usage of Monte Carlo simulations [69]. The binding free energies and solvation free energies were calculated to demonstrate the stability of the conjugate after incorporation of cisplatin into the SWNTs. Guven et al. entrapped cisplatin into ultra-short biocompatible SWNTs followed by wrapping with a surfactant namely Pluronic-F108 [70]. It showcased enhanced cytotoxicity in comparison to free cisplatin after 24 h on two different cancer cell lines (MCF7 and MDAMB-231). Several researchers were encapsulating platinum anticancer drugs within SWNTs as a drug delivery system. Mejri et al. reported a theoretical investigation to demonstrate that the encapsulation of anticancer drug cisplatin in the SWNTs is favored based on molecular dynamics simulation [71]. The capacity of the SWNTs is substantial which eliminates the necessity to close their both ends due to hydrophobic interactions and high confinement effects. The group also demonstrated the release of the drug which was favored near the membrane cell due to the advantageous electrostatic interactions with the hydrophilic part of the cell thereby paving the way for natural drug nanocapsule. Carboplatin has also been encapsulated within SWNTs and its efficacy on the growth of malignant cells has been investigated [72]. The anticancer drug carboplatin retains its structure, and the growth of bladder cancer cells is

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efficaciously suppressed. Magnetic functionalization of carbon nanotubes in combination with platinum-based anticancer drugs had remarkable potential as nanocarriers in drug delivery. SWNTs have been modified and functionalized via Bingel reaction by Irannejad et al. followed by the chelation of platinum (IV) by dicarboxylate groups on the surface of SWNTs. Diverse analytical techniques such as energy dispersive X-ray spectroscopy, thermogravimetric analysis, Raman and Fourier transform infrared spectroscopy were employed to ascertain the presence of the platinum (II) complex on the side walls of SWNTs [73]. The cytotoxic evaluation of the designed drug delivery system was investigated on the cervical carcinoma cells, and it was found to be higher than the anticancer ability of parent drug cisplatin. A variety of platinum anticancer drugs have been encapsulated in the SWNTs which subsequently enter the malignant cells using “needle like penetration” followed by the delivery of the molecule into the cytoplasm. The efficacy of SWNTs as nanovectors has been extensively researched and it has been found to be one of the most versatile nanocarrier owing to the properties of high specific surface area, the ease of functionalization to enhance the drug delivery attributes and its ability to cross biological barriers. 8.5.2 Multi-walled carbon nanotubes MWNTs possess diverse intriguing features which render them as promising candidates for the platinum based anticancer drug delivery. They are composed of multiple wall layers which minimize the feasibility of leakage of the encapsulated drugs from the side walls. This is a crucial factor which affects the measurement of drug release from the nanotubes [74]. Additionally, in MWNTs, more substances could be viably encapsulated in a shorter span of time because of the larger diameter in comparison to the SWNTs which facilitates facile entry of materials. The cisplatin prodrug which is highly hydrophobic and inert was entrapped within the MWNTs via hydrophobic-hydrophobic interactions (Fig. 8.4c) which was then reduced leading to the formation of platinum (II) species from inert platinum (IV) prodrugs [16]. Hampel et al. made a breakthrough by utilizing wet chemical approach wherein the capillarity was used as the driving force for carboplatin to enter the inner cavity of the carbon nanotubes [72]. The encapsulation of carboplatin inside MWNTs was investigated employing electron energy loss spectroscopy, X-ray photoelectron spectroscopy and other techniques which indicated that temperature had a significant influence on the amount of drug loading. The encapsulated MWNTs inhibited the growth of urinary bladder malignant cells whereas vacant MWNTs had negligible influence on the growth of cancer cells.

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Figure 8.5 MWNTs with a molecule of therapeutic cisplatin (Red), gold nanoparticles (yellow) blocking MWNTs tip to prevent the release of cisplatin from the narrow passage MWNTs.

Encapsulation of anticancer drugs inside carbon nanotubes provide protection from external deactivating agents but conversely, the encapsulated drugs remain exposed to the environment due to the open ends of the nanotube which eventually might lead to the uncontrolled release of the drug even before reaching the desired target. Li et al. capped the ends of the MWNTs to design a “carbon nanotube bottle” which enhanced the specificity of the anticancer therapy. They modified MWNTs with 1octadecanethiol gold nanoparticles followed by successful loading of cisplatin into it which facilitated the effective delivery of the drug from the “carbon nanotube bottle” as depicted in Fig. 8.5 [74]. In another study, Wu et al. performed an investigation wherein oxaliplatin was embedded into the inner cavity of polyethylene glycolated MWNTs and MWNT-COOH by employing nano-extraction method [75]. The cytotoxicity was assessed in colorectal human malignant cells, and it was observed that in comparison to free oxaliplatin, polyethylene glycolated MWNTs, and MWNTCOOH exhibited reduced effects of toxicity. MWNTs were also investigated to determine their influence on cellular uptake, cellular function, and their capability to sensitize malignant cells such as human prostrate and bladder cancer cells in comparison to the conventional drug therapy using cisplatin and carboplatin [76]. Yang et al. synthesized magnetic MWNTs functionalized with poly(acrylic acid), which did not indicate systemic toxicity [77], thereby manifesting the versatility of magnetic carbon nanotubes in cancer therapy. Carboplatin has been integrated to functionalized MWNTs leading to high levels of toxicity in diverse malignant cells [78]. Multiple functionalization of MWNTs has been performed utilizing carboxyl or amino groups

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followed by the incorporation of anticancer drug carboplatin [29]. The functionalization was validated by high performance liquid chromatography and Fourier transform infrared spectroscopy. Inductively coupled plasma mass spectrometry was employed to assess the amount of platinum ions liberated in the body fluid. The biological studies indicated that the aminated MWNTs with carboplatin in comparison to the oxidized MWNTs encapsulated with carboplatin induced the cell death rapidly by inducing autophagy. Thus, the developed hybrid drug could be used in the treatment of breast cancers, perhaps with reduced side effects on the normal cells. Oxidated MWNTs have been functionalized with hyaluronate and conjugated with carboplatin to act as a novel drug nanovector. The developed nanocarrier demonstrated therapeutic potential and was used satisfactorily for the in vitro treatment of a lung cancer model [79]. Hybrid nanomaterials with fascinating thermal, magnetic, biological and optical properties have been designed by a combination of the magnetic nanoparticles with carbon nanotubes. Mehdipoor et al. deposited iron oxide nanoparticles onto the surface of MWNTs and the obtained hybrid nanomaterial was conjugated with the anticancer drug cisplatin [80]. It manifested high functionality, biocompatibility and water solubility with enhanced drug loading and the ability to cross cell membranes effortlessly. The group investigated the potency of the nanocarrier in targeting the drug cisplatin toward the malignant cells. Adeli et al. synthesized a hybrid nanomaterial for the anticancer drug delivery comprising of MWNTs and linear dendritic copolymers conjugated to cisplatin [81]. MWNTs was functionalized and solubilized by noncovalent interactions with polycitric acidepolyethylene glycolepolycitric acid linear dendritic copolymers. The developed hybrid nanomaterial-based drug delivery system was subjected to endocytosis and released cisplatin into malignant cells to affirm its efficacy. MWNTs have been proven to be more efficient as nanocarriers because increased drug loading and high protection can be attained due to their higher diameter range. It is conveniently feasible to attach another drug covalently to the surface of MWNTs consequently creating a double drug delivery system and imitating a bivalent therapy which is prominently used in the biomedical field. 8.5.3 Carbon nanohorns Carbon nanohorns with remarkable properties such as high purity, biodegradation routes, low toxicity, and drug loading capacity are considered as promising nanovectors for platinum anticancer drugs. It has

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several drawbacks associated such as low dispersibility in polar solvents, low reactivity, and high hydrophobicity, which can be overcome by the chemical functionalization of the carbon nanohorns [82]. The experimentalists report that the varied functionalization approaches lead to the opening of the nanowindows on the surface of the carbon nanohorns which would facilitate the drug release through this route. Ajima et al. encapsulated cisplatin into single walled carbon nanohorns which resulted into amelioration of in vitro anticancer efficacy by 4e6 times and enhanced tumor suppression in comparison to intact cisplatin [83]. Oxidized single walled carbon nanohorns with incorporated cisplatin are thus a versatile drug delivery system. It has been also investigated that the insertion of polar functional groups on the oxidized carbon nanohorns cause an improvement in the biocompatibility of the platinum anticancer drug conjugate due to the increased number of hydrogen bonds being formed with the solvent [82]. The chemical modification of the structural holes in the single-walled carbon nanohorns altered the loading and release of cisplatin drug [84]. Additionally, the modified single-walled carbon nanohorns with encapsulated cisplatin demonstrated ameliorated in vitro anticancer cavity and exhibited a significant tumor suppression in vivo [85]. 8.5.4 Graphene and fullerene Graphene and fullerene have emerged as potent nanocarriers for platinum based anticancer drugs and showcasing remarkable outcome in the field of therapeutic nanomedicine. The high aqueous solubility along with surface functionalization of graphene render it as a versatile nanocarrier for drug delivery [86]. The silver nanocomposite loaded cisplatin modified with a coating of graphene was synthesized for the augmentation of apoptosis and autophagy in the human cervical malignant cell [87]. The quantum dots manifested enhanced cytotoxicity, cellular uptake and apoptosis against the HeLa cells. Nanoscale graphene oxide has also garnered considerable attention as a nanocarrier in the realm of drug delivery. It exhibits high loading efficiency due to the presence of abundant oxygen containing groups and its sp2 aromatic structure. Tian et al. designed a polyethylene glycol functionalized nanoscale graphene oxide for the delivery of platinum anticancer drug, cisplatin [88]. The drug of interest cisplatin was attached onto the functionalized nanoscale graphene oxide by the covalent reaction between platinum (II) and the carboxylic group. The nanohybrid material was characterized by transmission electron microscope imaging, atomic

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force microscope imaging, Raman spectroscopy and Fourier transform infrared spectroscopy. The nanocarrier was nontoxic in nature and released the drug cisplatin in a sustained profile for 72 h. The designed nanovector manifested phenomenal cytotoxicity to oral adenosquamous carcinoma CAL-27 cells and human breast cancer MCF-7 cells by in vitro assays. Fullerenes demonstrate antioxidant properties owing to their high activity as free radical acceptors. They do not display any toxicity in both in vivo and in vitro conditions at low physiological concentration. Fullerene was used to synthesize nano complexes for anticancer drug delivery and these conjugates were investigated employing diverse characterization techniques [89]. Comprehensively, the use of carbon nanotubes as nanovectors for drugs and biomolecules is a noteworthy development in the arena of therapeutic nanomedicine. Carbon nanotubes-based drug delivery systems are associated with several challenges but are still substantially promising over the existent conventional technologies.

8.6 Conclusions The realm of nanotechnology offers an incredible wide spectrum of innovative nanovectors with augmented adaptability and versatility for both therapy and diagnosis of cancer. Platinum anticancer compounds have certainly made a profound influence on cancer management, but their medical usage had its share of limitations. Nanovector based delivery of platinum-based drugs has come to the fore during the last decade as an expedient alternative. The synergism between the platinum drug and the nanocarrier has a pivotal role in the novel designs of the drug transport and delivery systems extending the regime of anticancer activity. The significant advantage of using nanocarriers is that they can be multifunctional, containing not only the platinum drug but also targeting diverse molecules such as antibodies, cell-penetrating peptides, overexpressed receptors, and aptamers. This chapter outlines a comprehensive overview of carbon nanotubes being utilized as nanovectors for platinum anticancer drugs which paves the way for the next generation of nanomedicine. Carbon nanotubes are the emerging viable option as nanocarrier for platinum anticancer drugs which exhibit enhanced toxicity, poor stability, and reduced retention. It is discerned that the platinum drugs when conjugated with the carbon nanotubes as nanocarriers manifest improved pharmacokinetics and biodistribution, increased loading capacity, enhanced

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cytotoxic influence against malignant cells in vivo and in vitro in comparison to the drug therapy alone. Despite the numerous remarkable features of carbon nanotubes as nanocarriers, there still prevails a few concerns pertaining to their clinical applications. The most noteworthy being the toxicity of carbon nanotubes which remain contentious till date. The toxicity investigations were comparatively short course conducted in vivo which lack clarity on long term toxicity effects. Another pressing concern related to the applications of carbon nanotubes as nanovectors is their inability to release drugs controllably and adequately. Additionally, since the carbon nanotubes are inherent heterogenous, it thus becomes challenging to ensure the consistency and batch to batch uniformity of the fabricated platinum drug conjugate. There is a dire necessity to address these shortcomings which would then lead to the advancement of carbon nanotubes as commercially viable nanocarriers. Therefore, efforts need to be focused on revamping the targeted delivery of platinum anticancer drugs with appropriate carbon nanotubes as nanovectors. Further developments in this arena can be brought about by designing platinum anticancer drugs which are better than cisplatin in terms of alleviated side effects, enhanced tumor targeting specificity and demonstrating higher efficacy in cisplatin resistant malignant cells. Carbon nanotubes which are used as nanocarriers must exhibit reproducibility, precision, surface functionality, augmented drug loading and negligible batch to batch variability. Researchers might gain profound understanding of the complexity of tumor heterogeneity and design the nanocarrier drug delivery platform correspondingly. The recent developments in targeted drug delivery have strikingly piqued the interest in new methodologies and techniques for future advancements of anticancer drugs. The unification of nanotechnology in pharmacology has patently revolutionized the drug delivery system permitting the emergence of novel treatments with ameliorated specificity. It has conspicuously allowed to step over manifold milestones toward the development of the “magic bullet,” albeit immense work remains to be executed.

Acknowledgments S. Chatterjee is thankful to the Department of Science and Technology (DST), Govt. of India for the DST INSPIRE Faculty Award [IFA14-CH-155] and research funding. Institute of Chemical Technology (ICT) is acknowledged for providing the congenial environment.

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CHAPTER 9

Biomimetic carbon nanotubes for neurological disease therapeutic Lopamudra Giri1, Smruti Rekha Rout1, Kenguva Gowtham1, Mohammad A.S. Abourehab2, 3, Prashant Kesharwani4, 5 and Rambabu Dandela1 1

Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India; 2Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; 3Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt; 4Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 5University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India

9.1 Introduction The discovery of nanotechnology (NT) has become increasingly relevant in biomedicine, resulting in the development of a novel and unique area of research. Nanomaterials have innumerable applications in biotechnology which include drug delivery and targeting, diagnosis of disease, implants, imaging, etc. [1e6]. Because most biological processes are carried on at the nanoscale range, nanomaterials fit suitably with the biomedical field. Carbon nanotubes (CNTs), liposomes, and metal nanoparticles are common materials utilized to build NT products. NT methods can be used to manage a variety of drug features, such as solubility, environmentally induced controlled release, or very specific site-targeted delivery [7,8]. CNTs are considered to possess outstanding qualities due to their distinct structure and topological arrangement of carbon atoms. CNTs are tubular structures with a lattice-like structure made up of linked carbon atoms in a periodic hexagonal network. Rolling up a single graphite sheet and closing the ends of the tube with fullerene-like end caps, that can be used to view the formation of the tubular structure of CNT (Fig. 9.1) [9e17]. Hence, this NT has the potential to revolutionize human health by providing a wide range of diagnostic options and treatment applications. Several efforts are carried away to convert these scientific breakthroughs into clinical practice, which can welcome a new era in the field of biomedicine [18]. CNTs are considered as fourth allotropic type of carbon discovered by Iijima in 1991. Iijima revealed that multi-walled CNTs (MWCNTs) which are graphite tubes that are tubular in shape. The tubes were made up of at Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00001-7

© 2023 Elsevier Ltd. All rights reserved.

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Figure 9.1 Carbon nanotube structure defined as a roll-up vector. (a) Graphene lattice and (b) carbon nanotube [9].

least two layers, or may be more, with an outer diameter ranging from 3 to 30 nm [19e23]. CNTs can be compared to a rolled-up sheet of graphene in terms of structure. Single-walled carbon nanotubes (SWCNTs) and MWCNTs are the two primary forms of CNTs. The architectures of MWCNTs can be described using two different models. The Russian doll model is made out of concentric cylinders of graphite sheets. A single sheet of graphite is folded in around itself in the parchment model, resembling a parchment scroll or rolled newspaper [24e26]. The number of concentric nanotubes determines the diameter of an MWCNT. SWCNTs have an exterior diameter of less than 2 nm in most cases. SWCNTs are sometimes referred to as a sole big molecule; however, MWCNTs are a mesoscale graphite scheme with width ranging from 2 to 200 nm. MWCNTs are classified into two categories, each of which is characterized by a distinct model (Fig. 9.2). CNTs have been extensively studied and utilize for a variety of applications like gene delivery, biosensors, drug carriers, and tissue engineering [27,28]. Among all the allotropes of carbon, CNTs can be considered as the most researched one. CNTs have the grabbed a lot of interest due to its capability to change bio-medical research because of their various properties such as chemical, electrical, thermal, mechanical, and structural capabilities. CNTs can have functionalization moieties

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Figure 9.2 Representation of CNTs distinguishes using (a) single-walled carbon nanotube, (b) double-walled carbon nanotube, and (c) multi-walled carbon nanotube having different structures [111].

added to them so that they bind with cell external receptors that guide their uptake. These receptor-facilitated directing technologies can make it easier to load specific cells, reducing the number of medicines required for disease treatment. Furthermore, these treatments can aid in the reduction of systemic toxicity and inflammation [2,29e32]. In a number of literatures, in vivo and in vitro synthesis methods of CNTs have been published. The major barrier of using CNTs widely is toxicity issue, and this issue can be addressed by tuning its synthesis method. There are several methods of synthesis of CNTs but the most common method of synthesis are Chemical Vapor Deposition method (CVD) [33], Electric Arc Discharge method (EAD), Laser Deposition Method (LD). In CVD process, the vaporized reactants such as hydrocarbon gases react chemically to generate a nanosized product that is then placed on a substrate such as zeolite, silicon plate etc. The Laser Ablation method is a Physical Vapor Deposition technique where a laser source vaporizes a solid graphite target [34]. Besides the issue of toxicology, there are several other obstacles associated with CNTs such as biodistribution of aggregated nanoparticles, which are controlled by different physicochemical parameters such as aggregation, size, surface solubility, shape, capability, chemical arrangement, and efficient functionalization. One of the greatest vital limitations of CNTs is their inability to liquefy in aqueous conditions. To address this issue, scientists have started tuning the surface of CNT that improves water solubility and biocompatibility. There are several previous research work carried out that has shown CNTs are

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water soluble are considerably more biocompatible with human bodily fluids and do not cause any hazardous side effects [35,36]. CNTs have a lot of unique features that have been discovered. Several papers have mentioned possible usage of CNTs in recent decades, as well as remarkably depicted promising applications which can replace the traditional method of treating various diseases. Nowadays, CNTs are used in a wide range of industries, including medical, nanotechnology, manufacturing, peripheral hardware, and electronics. Several research works have been conducted that validate the efficiency of CNTs. As the use of CNTs as a basic material for biomedical applications grows, the impact of CNT on living cells attracts more and more interest. Lovat et al. used CNTs that have been shown by to be an excellent surface for cellular development and to promote neural signal transmission. Furthermore, they have demonstrated that single-walled CNT (SWCNT) can directly trigger brain circuit activity by exploring the coupling model among hippocampal cells and SWCNT, indicating SWCNT as a viable material [37]. In the area of biosensors, CNTs proposed as a sensing part for detecting and monitoring a variety of ailments, including diabetes and bacterial infection. A group of researchers used electrochemical detecting of immune complexes to identify salmonella, lowering discovery time, and simplifying sample preparation compared to conventional approaches [38]. CNTs have remarkable cell transfection properties, the utilization of CNTs as nanomaterial has got a lot of attention in recent years. Non-covalent boundaries allow pharmaceuticals like Doxorubicin (DOX) to be loaded onto the poly (ethylene glycol) (PEG) surface which is coupled with SWCNTs, and the increased permeability and retention (EPR) effect allows them to reach tumorous tissue. A pH-dependent release would also allow administration near to the tumor tissues. To load medicines on CNTs, in a study, there is use of mesoporous silica covering. In the acidic atmosphere, DOX was easily liberated (acidic) observed in tumor cells, but remained bound to CNT at alkaline and neutral pH, which prevents toxicity in healthy physique regions [39,40]. Using the photothermal characteristics of CNTs in combination with NIR laser stimulation was thought to be a better technique to treat cancer directly. In an animal study, intra-tumoral administration of MWCNT suspension followed by short laser excitation resulted in tumor death and a higher survival rate. In a separate study, Wang et al. found that injecting SWCNT with anti-CTLA-4 generated an immune response and boosted cytotoxic activity when combined with Photothermal Therapy (PTT), consequential in the complete eradication of residual metastases

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[41,42]. In several studies it was also demonstrated that embedding CNT in hydrogels considerably improved their mechanical and electrical characteristics, and nanocomposites can overcome toxicity and can be employed for biosensing, tissue engineering, and drug administration deprived of the risk of CNTs leakage into cells [43]. Among all the wide variety of applications mentioned, CNTs are mostly used because of their unique physicochemical features and can efficiently govern nervous system behavior. CNT is applied to numerous neuropathological condition treatments in vivo and vitro, as an innate therapeutic medicines, extending their biomedical uses. A wide range of diseases impact a substantial percentage of the world’s population, and neurological diseases are one of the primary causes among them. Neurological illnesses are a major public health concern around the world. NT has emerged as an intriguing and hopeful new technique for treating neurological diseases, with the potential approach to prepare drug, that target central nervous system (CNS) fundamentally [44]. NT engineering of materials can address several problems related to neurological disorder and enable molecular-level multimodal therapeutic targeting. Therapies for CNS illnesses must overcome obstacles such as the bloode brain barrier, the CNS’s complex cellular structure [45]. Currently used therapy techniques, however, have not proven very effective in treating many of neurology related illnesses. A number of pharmaceutically active chemicals are being incapable to spread to the intended sites of action in the living cells, which is one of the major causes of their failure. CNS extracellular and intracellular barriers obstruct the transport of medicines from the bloodstream to the site of action, the CNS. The use of nanoscale drug delivery devices is one possible way to get over these obstacles. These nanosized drug carriers provide a number of advantages in drug delivery systems, including drug loading ability, lower toxic level, and improved therapeutic impact [46]. Nanoparticles does have the ability to modify the treatment of neurological illnesses such as stroke, tumor, Parkinson’s and Alzheimer’s. Multiple studies have indicated that nanostructures can be used to effectively treat CNS disorders in the case of neurodegeneration [47]. Neurological disorders affecting a large number of populations around the world and lead to severe conditions (Fig. 9.3). Among the various nanomaterial, CNTs improve the structural properties of a variety of materials and generated in large quantities due to their morphological and physicochemical qualities. CNTs are used as a delivery method for the diagnosis of CNS because of their structural features, including enhanced

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Figure 9.3 Schematic representation of various neurological related conditions.

bioavailability in biological solvents due to functionalization, capacity to be easily modified with bioactive compounds, large specific surface area, and neural arrangements biocompatibility. To treat Alzheimer’s disease, Zhang et al. employed SWNT that had been treated with acetylcholine. By precisely controlling the doses and ensuring that SWCNTs preferentially enter lysosomes, rather than mitochondria, the target organelles for SWCNT cytotoxicity, SWCNTs were satisfactorily used to supply acetylcholine into the brain for the diagnosis of experimental model Alzheimer’s disease with a medium safety range [48]. Brain tumors are difficult to treat, despite several therapeutic improvements. Low permeability of antitumor medication compounds across the BBB barrier opened up novel opportunities for CNT-based methods. In an investigation, a medication delivery system based on CNTs significantly improved the effects of CpG oligodeoxynucleotide immunotherapy in the healing and act as potential anti-glioma [49]. Hence, from the above-mentioned examples we can conclude, CNT-based therapy could be beneficial for a variety of neurodegenerative diseases.

9.2 The purpose of using carbon nanotubes (CNTs) in neuronal tissue CNTs is widely studied and the most recent advancements in CNT-based nanotechnologies emphasis on carrier characteristics, processing and cell

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internalization, mechanism and cellular uptake in neuronal tissue. The scientific community is putting up several efforts to learn more about how carbon nanoparticles interact with the CNS. These nanomaterials have high hopes since they show extraordinary interaction with neural cells in vitro, making them ideal contenders for building new diagnostic systems and treatments for brain disorders including neuronal or glial malignancies [50,51]. CNTs are proving to be an appealing therapy option for neural diseases and nerve tissue injury. CNTs show morphological similarities to neurites, and short CNT bundles have dimensions similar to branched extensions of neuron cells, expanding the potential for investigating, mending, and stimulating neural networks [52,53]. CNTs are a form of carbon allotrope that has been researched for its better physical qualities and ability to interface with neurons and neurological circuits. The importance of CNTs in neuroregeneration is discussed in Table 9.1. CNTs are favorable substrates for primary neuron attachment and development. They also promote differentiation of stem cells into neurons, the appearance of action potentials in undeveloped neurons, and the implementation of development potentials in immature neurons, all of which accelerate the propagation of dendritic back currents in isolated neurons. The finding shows that they could be used to treat neurodegenerative diseases and spinal cord injuries in the future. In addition, CNTs are used to monitor brain activity in spinal cord sections, individual neurons and ganglia. In comparison to state-of-the-art equipment, microelectrodes coated with CNTs demonstrate improved sensitivity when recording neural activity. Finally, CNTs can transport functional chemicals into neurons [61e63].

9.3 Application of CNTs toward prevention of neurological disease 9.3.1 CNTs for neurodegeneration CNTs have been projected as hopeful contenders for neurological utilizations due to their proper nanostructure and dimension, which are similar to neurites, ion channels, and elements of the neuronal cytoskeleton. Electrical interfaces for neural modeling, increasing neuronal survival, growth, and function, are now among the neurological applications of CNTs. CNTs can lead to formation of neuronal electrical characteristics and stimulate neurite extension [64,65]. CNTs play an important role in the stem cells differentiation, such as human embryonic stem cells, which have

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Table 9.1 Importance of CNTs in neuroregeneration. S. No.

Types of CNTs

Applications

References

1.

SWCNTs, MWCNTs

[54]

2.

CNTs coated with bioactive molecule 4-hydroxynonal Varying the charge carried by functionalized CNTs. (MWCNTs coated with polyethelenediamine) Polyethyleneimine functionalized SWCNTs Functionalized SWCNTs conjugated with CpG

Promising candidate for tissue engineering Improve the practicality of using nanotubes as nerve cell growth platforms Can be used to control expansion and branching pattern of neural processes Shown to increase neurite branching and outgrowth Antitumor therapy is improved when drugs are delivered effectively Improves neuronal tolerance to ischemia injury and proved to useful treatment for neurodegenerative disorders Used for treatment of Alzheimer’s disease by precisely controlling the dosage Enhance axon regrowth into the lesion cavity and hindlimb functional recovery Supply electrical stimulation which induces neuronal signaling

[57]

4.

5. 6.

7.

Amine modified SWCNTs

8.

SWCNTs carrying acetylcholine

9.

SWCNTs functionalized with polyethylene glycol

10.

SWCNTs-neuron hybrid system

[55]

[56]

[49]

[58]

[48]

[59]

[60]

a lot of scope in regenerative medicine domains including cell replacement therapy, tissue or organ transplantation. Mesenchymal stem cells are attractive candidates for central nervous system repair, which allows them to quickly develop into neuronal cells [66]. Because of advances in nanomedicine using nanotechnology such as CNTs, researchers can improve healing procedures for neurological intervention will have a big impact on neuroscience field. In a study, the human bone marrow mesenchymal stem cells produced for neural

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differentiation on carboxylated MWCNT films put onto a collagen-coated polystyrene substrate. It was discovered that cells grown on this substrate proliferated at a slower rate than cells grown on a control substrate, possibly due to the non-organized structure of CNTs on the scaffold. Furthermore, when cells were cultivated on this substrate, carboxylate MWCNTs were shown to enhance the expression of numerous neural-associated genes while inhibiting bone-associated genes. CNTs can aid in the adsorption of increased neural growth factors, allowing for long-term neuronal differentiation to occur [67]. According to a study, CNTs have been found to have electrical conductivity capacity and high mechanical capabilities, and their morphological traits are alike to neurons. Furthermore, the size and construction of CNTs are similar to some brain constituents such ion channels, signaling proteins, and cytoskeleton components. CNTs’ likeness to these neural elements may have an additional benefit in terms of improving neuronal connection at the molecular level, and so improving regulation of neuronal physiological activity and information processing [53,68]. The substrate appropriateness of CNTs with neural stem cells was detected in a study based on cell viability and the development of neuronal processes. The compatibility of CNT substrates with neural stem cells has been established, allowing for the delivery of appropriate CNTs to wounded CNS sites and subsequent growth of neural stem cells into neurons [69]. CNTs have been shown to be useful in the treatment of neurodegenerative disease. In a study, PEG-modified SWNTs were successfully employed for neuroregeneration. A reduction in the capacity of injured tissue and a rise in the quantity of neurofilaments in the part nearby the location of injury, proved the effectiveness of the CNT based nanotechnology. This research work showed that CNT-based substrates can help injured neurons and brain tissues regenerate over time, opening up new possibilities in the field of neuroregeneration [59,70]. 9.3.2 CNTs for neuroprotection Several ways for attaching numerous types of nanoscale substance to CNTs have been published, allowing CNTs to combine their distinctive capabilities with the creative properties of novel nanoparticles in a single distinct structure [71]. A biocompatible bacterial magnetic nanosubstance from Magnetospirillum sp. AMB-1 were covalently attached to form functionalized SWNTs (f-SWNTs) in a study. Bacterial magnetic nanoparticles (BMPs) operate as a highly effective intrinsic peroxidase for the self-

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degradation of BMP-decorated SWNTs, and this f-SWNT-BMP hybrid has been revealed to demonstrate highly synergetic peroxidase-like activity. Because of the constant lipid membrane around the magnetic core, the BMPs from Magnetospirillum sp. AMB-1 employed in this experiment are can be successfully conjugated with other biomolecules, scatter well in aqueous solutions and naturally biocompatible. The lipid membrane contains a high percentage of phosphatidyl-ethanolamine, which delivers amine-binding sites, enabling BMPs simple to distribute and couple to other biomolecules including antibodies, enzymes, and polypeptides. Furthermore, BMP-SWNT hybrids have also been discovered to suppress the development ofdamyloid (A) fibrils, which are a crucial component of Alzheimer’s disease. These hybrids may pave the path for a new neurodegenerative disease therapy or inhibition method. Hence, in this therapy, BMP-SWNTs hybrids show neuroprotective benefits against amyloid (A) fibrillation-induced neurotoxicity human neuroblastoma cells [72]. In a study, the existence of CNTs was studied in numerous brain locations after intranasal delivery, with the goal of identifying the precise cell types engaged in CNT uptake. In this research work, it was observed if the two types of CNTs had neuroprotective properties, by giving them to mice with initial diabetic encephalopathy and looked at how nerve growth factor metabolism was impacted, as well as the effects of CNTs on glial phenotypes and neuronal. The goal of this study was to observe the way how two intranasally given MWCNTs, distributed in the brain parenchyma, which were distinct from each other. Among the CNTs the first one can considered as non-electroconductive and the second was electroconductive. CNTs have been linked to cellular and molecular consequences in specific areas of the brain, which could be part of a CNT-induced neuroprotective mechanism in a brain region associated in the progression and onset of certain neurodegenerative illness [73]. 9.3.3 CNTs for drug delivery across the bloodebrain barrier CNS illnesses, particularly neurodegenerative disorders, are a serious public health concern that researchers must address. Different therapy options have been used over the last few decades, but their therapeutic success is insufficient and only shows partial symptom abatement. The bloodebrain barrier (BBB) guards the CNS from toxic substances and is one of the most difficult barriers to overcome when delivering drugs into the CNS various diseases such as stroke, Alzheimer’s disease, tumors, Parkinson’s disease,

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epilepsy, Huntington’s disease etc. [74]. The BBB is made up of a single layer of polarized endothelial cells joined by complicated tight junctions, with cells like neurons, astrocytes, and pericytes controlling its activity. The BBB’s intricacy, as well as the occurrence of large amounts of efflux carriage proteins including P-glycoproteins and Multidrug Resistant Protein-1 and metabolic enzyme expression, all limit drug entrance into the brain [75]. Nanotechnology-based medication delivery is a potential strategy in the arena of neuro diseases, as nanomaterials have been shown to be successful in crossing the BBB, delivering pharmaceuticals to the target spot within the CNS [76]. The development of nanotechnology has resulted in the emergence of various nanocarriers such as liposomes, polymeric nanoparticles, micelles, and solid lipid nanoparticles, with diameters ranging from 1 to 100 nm and have established themselves as suitable nanocarriers for the treatment of numerous neurological illnesses. However, nanotechnological methods have been used to create a variety of sophisticated nano systems, including dendrimers, nano emulsions, nanogels, and nanotubes, and have proved to be greater potential than previous delivery systems [77,78]. Effective drug delivery to the CNS across the BBB requires the use of an appropriate nanocarrier technology. Nanocarriers’ surface area, size, shape, and surface charge, all have a significant influence on their ability to pass through CNS barriers. The nanocarriers utilized in CNS drug administration must be biocompatible, biodegradable, nontoxic, site-specific, and cost-effective, with optimal size, surface area, and surface charge. Basically, nano-system is used to cross BBB must touch the field of safety and efficacy which can be useful to target drugs to specific locations [79,80]. CNTs have a number of unique features that make it a suitable tool for drug delivery vehicles. CNTs’ cylindrical shape provides a significant advantage in transmembrane penetration, allowing intracellular CNTs to effectively traverse the BBB. CNTs can function as an excellent nanomaterial in as it can act as an electrical conductor and have good photothermal properties, as well as the ability to absorb optical intensity and photo luminesce. CNTs have a very high drug loading efficiency due to their wide aspect ratio. Furthermore, CNTs’ ultrahigh exterior area ratio permits functionalization with various chemical moieties [81,82]. The remarkable strength of particular cell or tissue targeting, therapy, and imaging demonstrated by the functionalization of CNTs with various material. In a study, a multifunctional oxidized single-wall carbon nanohorns is used as medications delivery tool that may improve methotrexate’s

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antitumor efficacy. The nanoparticle was loaded with medicine and covered with solubility enhancer DSPEePEGeNH2 to lengthen blood circulation half-life with the assistance of covalent grafting of transferrin. This SWCNTs combination has a very good antitumor effectiveness [83]. Although NT has been linked to the production of nano formulations of numerous neuroprotective, there is need to be aware about their side effects. Because of their potential to nanoengineer drug carriers to cross the BBB, target specific cells, diffuse inside brain tissue, or signaling systems for drug delivery, the future of NT in CNS delivery of drug is highly hopeful and offers up new paths in the treatment of neurological illnesses [84]. The relative impermeability of the BBB is caused by tight connections created by endothelial cells in brain capillaries, and some tiny and big therapeutic substances are unable to pass through it. Several nanotherapeutics have been extensively researched and used as a viable and promising method of medication delivery into the brain. CNTs have been proven to be capable of penetrating the BBB in a number of investigations. Many researchers developed a unique chemical compound of CNTs to carry medications to the brain with high therapeutic efficacy [85,86]. CNTs are well-received in the field of medication delivery and are gaining a lot of interest for their ability to carry drugs into the brain. 9.3.4 The use of CNTs for functional neurosurgery At the cellular level, neurosurgery is beginning to engage with the nervous system. Neurosurgeons will be able to inspect, manipulate, repair, and control the nervous system using various methods. Various technologies, as well as breakthroughs in molecular biology and genetics, are the requirements in the field of neurosurgery. Until molecular and genetic targeted alternatives bring treatments to severe diseases, surgery is more likely to become minimally invasive. Using of nanotechnology for neurosurgery can further enhance the therapeutic property of certain drugs. There are various nanoimaging applications pertinent to neurosurgery, and have undergone various preclinical and clinical trials. The use of nanoparticles has not only proved to be delivering agent but also can act as a better contrast agent for MRI scanning in brain tumor patients. Nanoimaging techniques such as quantum dots are rapidly expanding our understanding of neuronal function, such as the activities that take place in the axonal growth cone during axonal regeneration, and also identifying malignant cells in the central nervous system for both treatment and localization of

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chemotherapeutic drugs. Quantum dots and other fluorescence approaches, on the other hand, are most likely to have the most potential for nano neuroimaging [87]. At the cellular and subcellular level, neurosurgery is interacting with the nervous system. Neurosurgeons will be able to inspect, manipulate, repair, and control the nervous system using nanotechnology [88,89]. Among all the nanotechnology, CNTs have drawn great attention because of their impressive properties such as mechanical and electrical. A CNT with many walls has a 1000-fold higher electrical current density than a copper nanotube. An ideal electrode should be thinner, lighter, nonmetallic, stable, and radiolucent for liquid sterilization when used in conjunction with CT, MRI, or X-ray, especially in the field of neurosurgery. In this study, the usefulness of a novel electrode made from MWCNTs is investigated. The sheets belong to nanotube which were soft and well-suited and the newly designed electrodes had an impedance of 5 kU or less, which was alike to that of ordinary metallic electrodes. Intraoperative CT scans, MRI scans, and cerebral angiography can all be used in conjunction with these electrodes. The electrodes did not interfere with rays, just like traditional silver electrodes, since they did not contain any artifacts that would distort the images and hence, they also did not harm the skin. MWCNTs were used to make a very thin electrode. This electrode accurately measured brain waves and evoked potentials without altering images obtained. The MWCNT electrode is a low-cost surgical tool with a lot of potential [90]. Hence, different nanotechnology along with CNTs use in biomedical research has changed the way people approach several industries and medical professions. Because of a unique chemical arrangement, it has a wide range of applications. This material can be used in neurosurgery for therapeutic purposes, such as delivering controlled drugs or genes in the treatment of brain tumors [91]. 9.3.5 The use of CNTs in the treatment of ischemic stroke The global health burden of neurological illnesses is enormous. When blood flowing to the tissue of is suddenly cut off, a stroke develops. Numbness in the arms, face, and legs, as well as aphasia, are all symptoms. The indications will vary depending on the damaged part, and the patient’s recovery could range from complete to severe neurological impairments and death. Ischemic stroke, also known as cerebral ischemia, happens when flow of blood to the brain is disrupted due to a blockage in the arteries. A clot or a plunger can produce this occlusion. As discussed earlier, the brain is

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the important part in humans, and BBB is shielded, that prevents hazardous substances from entering through the peripheral blood circulation which resulted in prevention of drugs from reaching the damaged part of the brain, making CNS illnesses extremely treatment very challenging [92]. Stroke is a brain disease that is the fifth leading reason of mortality and the leading source of permanent disability worldwide. Ischemic stroke accounts for 80% of all strokes, while hemorrhagic stroke accounts for 20% [93]. NTs has appeared as a viable method of enhancing delivery of medications to the brain, particularly the ischemic region. CNTs have a lot of potential for use for treatment and recognition of ischemic stroke. CNTs can act as an attractive carrier for biological utilizations such as the diagnosis and dealing with neurological illnesses, especially ischemic stroke. CNTs have been studied for ischemic stroke and prospective applications because of their antioxidant activity, ability to reduce BBB paracellular tightness, intrinsic photoluminescence, ability to transport oligonucleotides, ability to traverse the BBB, and ability to drive cell differentiation. As a result of their unique physicochemical features and advancements in functionalization processes, they now have a wide range of uses [94]. Hence, CNTs have been employed in a number of biosensing applications and as well as biomedical field, which includes an immunosensor for detecting T-2 mycotoxins in Swine meat [95] and a biosensor for detecting 17b-estradiol in humans [96]. In a study, to develop an enzyme-free electrochemical sensor for uric acid, a clinically relevant molecule used in pregnancy-induced hypertension diagnosis known as carboxylic acid-functionalized MWCNTs, which is modified via ultrasonication with cyclodextrin. This nanocomposite material act as an electrochemical sensor for uric acid detection in blood and urine [97]. MWCNTs were also discovered to significantly decreased blood pressure in the mice who were given the treatment when stereo tactically administered to the brain. In brain cells, MWCNTs increase acetylation and nuclear translocation of nuclear factor-kB [98]. Vertically aligned MWCNTs (VA-MWCNTs) were successfully synthesized using a CVD technique after tuning preparation parameters such as temperature, time, substrate position in the catalyst, reactor, and gas flow rates. In the experiment for the first time, dexamethasone (DEX) was loaded and released from PEGylated VA-MWCNTs. These findings suggest that VA-MWCNTs play an important character in the extended and rapid release of DEX, which could result in to a more active treatment for ischemic stroke. The Functionalized CNTs having cytotoxicity effects in cells was very minimal. According to the results, functionalized

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VA-MWCNTs have the ability to transport DEX to the sick region over an extended length of time [99]. In another study, a functionalized nanoelectrode could be used to detect biomarkers in neurological illness especially ischemic stroke in the future. The bathochromic and hyperchromic alterations that occur when super aligned VA-MWCNTs are acylated and PEGylated should improve the nanoelectrode’s sensitivity to biomarkers and reduce opsonization of CNT-based nanocarriers for targeted drug delivery. Hence, we can conclude that effectively manufactured and functionalized CNTs can be used to construct nanoelectrodes for illness diagnostics and potential utilizations in the dealing with a variety of neurological conditions [100]. Carbon-nanotubes-doped sericin scaffold (CNTs-SS) which is used for stroke healing has programmable shape memory, photoluminescence, and the ability to transport bone marrow mesenchymal stem cells into brain regions while simultaneously encouraging neuron growth. This research combines substances characterizations, cell-biological information, mathematical modeling, molecular and genetic data, imaging methods and computational 3D reconstruction, 3D printing and casting, and stroke animal models to promote scaffold into stroke cavities for treating an acute ischemic stroke in vivo. As a result, our CNTsSS introduces an innovative class of shape-memory constituents with therapeutic effect [101]. Functionalized with amine MWCNTs are promising building blocks for transporting Nerve Growth Factor (NGF) and delivering constant concentrations and effects of this neurotrophin. The MWCNTs-NGF combination appears to be an excellent nanocarrier for neuroprotective applications since it suppresses OGD-induced cytotoxicity while also increasing the activity of key enzymes involved in the metabolic breakdown of superoxide and hydrogen peroxide superoxide radicals. In the meantime, rather than using artificial media that mimic biological fluids, more in vivo investigations should be conducted to estimate the efficacy of the MWCNTs-NGF complex inside a living organism. Aminefunctionalized MWCNTs are potential basic components for transporting nerve growth factor and delivering sustained concentrations and effects of neurotrophin. The MWCNTs-NGF combination appears to be an excellent nanocarrier for neuroprotective applications since it suppresses oxygen glucose deprivation-induced cytotoxicity while also increasing the activity of crucial enzymes involved in metabolic breakdown to form radicals of superoxide radicals and hydrogen peroxide [102]. Active endocytosis and passive nanopenetration are the two most common processes by which functionalized CNTs are absorbed into cells. As a result of

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the activation of oxidants and toxic enzymes, cells experience oxidative stress during CNT internalization. Reactive oxygen species (ROS) and free radicals are formed by oxidative stress. Free radicals in excess promote lipid peroxidation, protein breakdown, and DNA oxidation. DNA damage, apoptosis, amino acid oxidation, and enzyme inhibition are all possible effects of ROS in cells [103].

9.4 Cytotoxicity and immunogenicity of CNTs CNTs are a type of carbon nanomaterial that has been extensively explored. CNTs’ unique physical properties and chemical characteristics that make them ideal contenders for a variety of applications, including gene therapy, medication delivery, and electrical properties. CNTs’ toxicity, on other hand, has been a serious issue to use in biomedical field and tissue engineering. Impurities, length, aggregation, and size are also several factors of CNTs that all contribute to toxicity. MWCNTs with no functional group have poor toxicity, solubility, and dispersibility which resulted in key concern for the potential applications in tissue engineering and other biomedical fields. The interactions of CNTs with life systems are exceedingly complex and unpredictable. The biological properties, behavior, and performance of CNTs must all be thoroughly studied. Based on their composition, production technique, surface-area-to-volume ratio, concentration, size, shape, functional groups, extent of oxidation, and useful doses, CNTs are known to have varying levels of toxicity. CNTs can also cause damage to the DNA and cell membrane because of high hydrophobicity. Protein synthesis, apoptosis, mitochondrial activity, necrosis, alterations, intramolecular metabolic and oxidative stress, are pathways which can increase CNT toxicity [104,105]. Many in vitro and in vitro research-related toxicology of CNTs have been conducted; however, they are incongruent due to differences in kind of synthesis technique, functionalization, and the amount of CNTs. Furthermore, the cytotoxicity results were influenced by the kind of cell viable indicator dye used. AlamarBlue, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, neutral blue, and Coomassie Brilliant Blue are some of the most used indicator dyes [106]. CNTs have been examined for their toxicological effects, with SWCNTs and MWCNTs which are acid purified, showing a considerable impact on amount of toxicity. Commercial CNTs produced more ROS, which raised oxidative stress and decreased mitochondrial membrane potential. Acid-purified SWCNTs, on

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the other hand, have a lower toxicity effect due to the absence of metal impurities [107]. MWCNTs and SWCNTs’ fibrous surfaces revealed different mechanisms in the plasma membrane. MWCNTs were harmful because they induced abnormal phagocytosis and damaged the plasma membrane, whereas SWCNTs caused oxidative harm to the cells. Hence, SWCNTs and MWCNTs have varied levels of toxicity depending on their various features [108]. Functionalization can change the surface morphology of CNTs by introducing multiple functional groups. CNTs’ solubility, dispersibility, and agglomeration are all improved by functionalization. Covalent bonding or noncovalent binding can be used for functionalization. Functionalization allows different groups to be conjugated to CNTs, which aids in cellular processing, cell receptor binding, and elimination. As a result, the utilization of functional group in combination improves CNTs’ specific affinity to biomolecules of interest [109]. CNTs have proven to be an effective nanostructure in the construction of nanosized structures such as nanoplatforms that can carry antigens and promote the synthesis of T lymphocytes, which are key immune cells in the initiation and maintenance of immunological responses. T cells are commonly targeted for therapies that require the immune system to be stimulated, such as immunization. Nanocarrier-nanoplatform systems can boost a molecule’s immunogenicity [110]. Hence, CNTs have number of applications which can treat neurological disorders (Fig. 9.4).

Figure 9.4 CNT is used to treat a variety of neuropathological conditions in vivo and in vitro, broadening its biomedical applications.

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9.5 Clinical status of CNTs and future outlooks CNTs are investigated the most among nanomaterials for potential uses in detection and treatment disorders related to brain. CNTs have strong compatibility with brain tissues and have neuroprotective benefits against stroke-induced neurodegeneration. CNTs not only carry drugs and nucleic acids, and can enable applications related to neuroregeneration but also has better compatible with tissue of brain. Despite of these hopeful results, there are still some worries concerning about their toxicity which highly dependent on the location where CNTs are administered, their aggregation state, and the presence of metal impurities in the substance. CNTs are great tool for biomedical applications but few obstacles must be overcome. The first is concerned with safety, and it necessitates the use of CNT of extremely high purity in order to restrict the discharge of harmful ions while operating in any biological environment. This occurs in significant difficulty because high purity CNT samples are rarely produced in large quantities, necessitating a trade-off between quality and quantity. The other major obstacles include, which are not limited to the biomedical field, are more formulation-related. One of the most important challenge is the ability to obtain good CNT dispersion in solvents, particularly water. CNTs are difficult to separate in a solution due to their high hydrophobicity. As previously mentioned, this can be accomplished through functionalization of CNTs in various ways. CNTs rarely cause toxicity in brain tissues, according to the studies, owing to the high dispersibility and of the nanoparticles used. To date, the most significant impediment to the use of CNTs in the treatment of CNS diseases has been the removal of catalyst metal nanoparticles, which demands the use of many techniques that block the cost-effective manufacture of high purity material on a big scale. It’s important to look at how connecting molecules interrelate with enzymes and how this affects the arrangement and structure of enzymes on CNTs. Nanoscale materials’ interfacial properties, mobility can confine effects can impart sole properties to biocatalyst systems, allowing for the development of a class of innovative in biocatalysts that differ from old immobilized enzymes in terms of preparation, application potential and catalytic efficiency. Events and new mechanisms in field of CNTs may emerge in the future. The area of CNTs role in field neurology plays a great role in curing number of disease. This field’s popularity is constantly expanding, which will certainly lead to more fascinating breakthroughs in the near future.

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Acknowledgments Rambabu Dandela thanks DST-SERB for Ramanujan fellowship (SB/S2/RJN-075/2016), Core research grant (CRG/2018/000782) and ICT-IOC start-up grant. The authors acknowledge ICT-IOC Bhubaneswar for providing necessary support.

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Biomimetic carbon nanotubes for neurological disease therapeutic

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property and neuro-differentiation-promoting activity for individualized brain repair of severe ischemic stroke, Bioact. Mater. 6 (7) (2021) 1988e1999. P. Hassanzadeh, E. Arbabi, F. Atyabi, R. Dinarvand, Nerve growth factor-carbon nanotube complex exerts prolonged protective effects in an in vitro model of ischemic stroke, Life Sci. 179 (2017) 15e22. M. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (1) (2007) 44e84. N. Mehra, K. Jain, N.J.-D.D. Today, Undefined Pharmaceutical and Biomedical Applications of Surface Engineered Carbon Nanotubes, Elsevier, 2015. E.E.-I. journal, and S. Vardharajula, S. Ali, P. Tiwari, Undefined Functionalized Carbon Nanotubes: Biomedical Applications, 2012, ncbi.nlm.nih.gov. H.F. Cui, S.K. Vashist, K. Al-Rubeaan, J.H.T. Luong, F.S. Sheu, Interfacing carbon nanotubes with living mammalian cells and cytotoxicity issues, Chem. Res. Toxicol. 23 (7) (2010) 1131e1147. O. Vittorio, V. Raffa, A. Cuschieri, Influence of purity and surface oxidation on cytotoxicity of multiwalled carbon nanotubes with human neuroblastoma cells, Nanomed. Nanotechnol. Biol. Med. 5 (4) (2009) 424e431. M.L. Di Giorgio, S.D. Bucchianico, A.M. Ragnelli, P. Aimola, S. Santucci, A. Poma, Effects of single and multi walled carbon nanotubes on macrophages: cyto and genotoxicity and electron microscopy, Mutat. Res. Toxicol. Environ. Mutagen. 722 (1) (2011) 20e31. S.L. Montes-Fonseca, E. Orrantia-Borunda, A. Aguilar-Elguezabal, C. González Horta, P. Talamás-Rohana, B. Sánchez-Ramírez, Cytotoxicity of functionalized carbon nanotubes in J774A macrophages, Nanomed. Nanotechnol. Biol. Med. 8 (6) (2012) 853e859. H.A.F.M. Hassan, L. Smyth, N. Rubio, K. Ratnasothy, J.T.W. Wang, S.S. Bansal, H.D. Summers, S.S. Diebold, G. Lombardi, K.T. Al-Jamal, Carbon nanotubes’ surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo, J. Contr. Release 225 (2016) 205e216. W.S. Zhao, K. Fu, D.W. Wang, M. Li, G. Wang, W.Y. Yin, Mini-Review: modeling and performance analysis of nanocarbon interconnects, Appl. Sci. 9 (11) (2019) 2174.

CHAPTER 10

Theranostic applications of functionalized carbon nanotubes Duygu Harmanci1, Simge Balaban Hanoglu2, Duygu Beduk2, Ceren Durmus2 and Suna Timur1, 2, 3 1

Central Research Test and Analysis Laboratory Application and Research Center, Ege University, Bornova, Izmir, Turkey; 2Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey; 3Department of Biochemistry, Faculty of Science, Ege University, Bornova, Izmir, Turkey

10.1 Introduction Theranostics, as a term, consists of a combination of diagnostic and treatment approaches [1]. Classically, the process from diagnosis to treatment involves the use of therapeutic agents after diagnosis. Sometimes it can also be done as a diagnosis with the use of therapeutics by reverse engineering, or a combination of both conditions. Theranostics is an emerging field that offers a unified and personalized opportunity for definitive diagnosis and targeted therapy. Nanotechnology is one of the best platforms to obtain this integration. It can be defined as the processing of materials that are one billionth of a meter in size at the atomic and molecular level. The physical, chemical, biological, and morphological properties of these materials offer a unique advantage over materials of larger dimensions [2]. Carbon nanotubes (CNTs), one of the most extraordinary nanomaterials in nanotechnology, are small, lightweight, mechanically durable, carbon-based nanomaterials made of graphite with electrical and thermal features [3]. CNTs, first mentioned in the early 1950s, were finally defined in 1991 [4]. While their primary applications are in areas such as electronics, optics, and plastics, in recent years there has been increasing evidence of their use in the field of nanomedicine [5]. Thanks mainly to their surface area, chemical stability and ability to interact with biological molecules, they have made significant breakthroughs in the field of nanomedicine [6]. Despite their various properties, CNTs are particularly functionalized to adapt to physiological conditions, which is crucial for potential bio-applications. This functionalization can occur in a covalent or noncovalent pathway. In this way, hydrophobic carbon nanotubes can become water-soluble and degradable after some chemical treatments. Similarly, when used in the field Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00002-9

© 2023 Elsevier Ltd. All rights reserved.

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of nanomedicine, they can become more specialized for drugs or genes [7]. CNT functionalization also affects the clearance times of CNTs from the body [6]. Therefore, there is an increasing interest in the use of functionalized carbon nanotubes (fCNTs) in theranostic applications. Hence, this chapter focuses on functionalization methodologies and their potential applications as theranostic platforms based on current developments in various bio-related fields. We also discuss advantages of these materials depending on their types and functionalities and future directions, highlighting recent findings that critically demonstrate their biological safety.

10.2 Carbon nanotubes (CNTs) CNTs belong to a class of carbon similar in shape to a combination of graphene and fullerenes. Single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNTs) are the two types of CNT that can be generally categorized by their wall structure [8]. In SWCNTs, a single graphene sheet is folded into a cylindrical closed structure, while MWCNTs have multiple concentric tubes [9]. Sumio Iijima, a Japanese scientist, found the first CNT-related fullerene multilayer tubes with outer diameters ranging from 3 to 30 nm in 1991, which appeared in the form of rolled graphene in a cylindrical shape. Similar to graphene, CNTs have a hexagonal configuration with a nanometer-sized diameter and macrometer-sized length [8] (Fig. 10.1).

Figure 10.1 Structure of graphene and CNT types.

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CNTs and graphene materials have been selected as platforms for the development of novel adsorbents with enhanced or more functions due to their non-corrosive properties, customizable surface chemistry, large surface area, and the presence of oxygen-containing functional groups on the surface [10,11]. CNTs offer unique physicochemical properties such as a large surface area, high encapsulation ability, lightweight, biocompatible functionalization ability, low toxicity, and non-immunogenicity [12]. They can be considered as the best candidates for functionalization as they have a hexagonally closed cylindrical shape with strained sp2 hybridized carbons. Functionalized CNTs (fCNT) such as solvent dispersed, surfactant coated and conjugated CNTs which may contain drugs, monoclonal antibodies and ligands can be categorized based on their synthesis methods, structural modifications and solubility properties [9]. 10.2.1 Functionalized carbon nanotubes (fCNTs) Functionalization is the attachment of specific functional groups to the side chains or ends of CNT [13]. These materials could be functionalized via chemical bonding (covalent bonding), physical adsorption (noncovalent bonding) and electrostatic forces, resulting in increased hydrophilicity and a complete change in their biocompatibility profile (Fig. 10.2) [12,13]. Covalent chemistry is based on the establishment of a chemical bond between the CNT wall and the functionalizing agent [14]. Aromatic chemicals, surfactants and polymers can be used for this approach [15]. On the other hand, noncovalent interactions do not give damage to the graphene sheet structure, while covalent interactions establish additional chemical bonds in the main structure. In recent years, amphiphilic molecules have been used

Figure 10.2 Functionalization techniques used to obtain fCNTs.

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as an alternative way for noncovalent surface modifications. In particular, Van der Waals forces are the most common type of noncovalent interaction between CNT walls and the functional groups. The noncovalent functionalization of CNTs has attracted an interest as it allows the application of chemical modifications without changing the electrical network. Low molecular weight surfactants, amphiphilic polymers or organic molecules, polymers, and biomolecules such as DNA, siRNA, proteins, and peptides can be effectively loaded onto the surfaces of CNTs using the noncovalent strategy [9,14]. Although the sidewall system of the nanotubes is not altered, the noncovalent functionalization technique offers several advantages over covalent methods. However, in the case of either loading or binding of pharmaceuticals to the structure as well as functional groups could be more stable and efficient by using covalent functionalization. CNTs agglomerate due to their apparent hydrophobic nature which makes it difficult for them to disperse in solvents or viscous polymer melts. This could be achieved by exposing CNTs to a harsh environment, such as concentrated sulfuric acid or nitric acid, which creates high instability and breaks the hexagonal structure within the surface architecture forming reactive regions on the surface. Polymers and biopolymers can be bound to these reactive surface surfactants via p-p stacking. This pi-electron extension improves their solubility, the conductivity of the material and reduces the toxicity of CNTs, allowing them to be used in a wider range of applications [16]. Commonly used chemical approaches to functionalize CNTs and graphene for environmental applications are exclusively chemical techniques such as chemical oxidation, deposition, and advanced methods such as electrochemical, sol-gel, microemulsion, and hydrothermal methods [17]. For the nanomaterial synthesis, these approaches are typically relatively selective and simple compared to other techniques. They involve precursors of chemical reducing agents or photo reducing agents and a supporting, stabilizing, or additive agent to achieve the desired functional reactions. They can be combined with functional groups of carbon nanomaterials to obtain a variety of functionalities [11]. Determination of the exact functionalization position and mechanism for functionalized nanotubes are described as major challenges. The functionalization types can be divided into two categories: End defect and sidewall. End defect functionalization involves oxidation at the “endpoints,” while sidewall functionalization includes covalent binding of surfactants, proteins, and peptides on the surface of CNTs [9]. This type of

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functionalization requires highly reactive reagents with a high yield of functionalization [14]. CNTs can be efficiently modified to improve their solubility, biocompatibility, encapsulation tendency, and multimodal drug delivery and imaging, as they offer many advantages, such as enhanced cellular uptake and biological environment stability [9,18]. Not only it is capable of improving aqueous solubility, but it also has the potential to decrease toxic effects, improve biological compatibility and provide the ability to load pharmaceutical compounds, various biomolecules such as genes and proteins into fCNTs’ structure for obtaining effective delivery systems [13,18] (Fig. 10.3). These techniques provide enhanced diagnostic and therapeutic delivery capabilities in biomedical and pharmaceutical research and development. In addition, CNTs have the potential to be used as a new delivery mechanism for drugs such as peptides and gene therapies. These can be converted into smart nanoscale vehicles that are biocompatible, nonimmunogenic, and photoluminescent for drug delivery with or without diagnostic agent functionality with surface modification [12]. When all these are taken together, the importance and necessity of functionalization

Figure 10.3 Functionalizing agents of CNTs.

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for the applications of CNTs is obvious. In addition, the functionalization affects the CNT properties discussed next. 10.2.2 Properties of CNTs CNTs have unique mechanical, thermal, and electrical properties due to the presence of a pure sp2 hybridization network. Therefore, each property can result in different specifications according to different functionalization methods. The extent of these properties is highly dependent on the synthesis method, purification treatment, and further surface treatments. 10.2.2.1 Structural properties CNT structures can be defined as a hollow cylindrical structure framed by single or multiple layers of graphene sheets and a hemispherical fullerene structure at more than one closed-end. The curling of a graphene sheet gives rise to multiple allotropes of carbon [18,19]. The cylindrical structure contains sp2 hybridized carbon atoms [19], and they have a significant aspect ratio of over 1000 as their length is in the micrometer range. CNTs can have three different orientations depending on the atomic structure of the carbon bonds such as zigzag, armchair, and chiral arrangements [20]. Aforementioned before these materials are divided into two types as SWNTs and MWNTs depending on the amount of outer layer or walls. Based on the direction of rolling of the graphite sheets and the tube diameter, both SWCNTs and MWCNTs can have metallic or semiconducting properties [20]. SWCNTs are formed by a single graphene sheet wound tubularly around itself to form a cylinder, while MWCNTs are formed by two or more concentric tubes consisting of graphene sheets coaxially wound around each other with a spacing of 0.34 nm [21,22]. The different types of CNTs differ not only structurally, but also in their dimensions. Recently, it was discovered that a coaxial structure with two concentric graphene cylinders has better thermal and chemical stability than SWNTs [9]. Carbon-based nanoparticles are porous and stable, have a large specific surface area and high adsorption capacity [11]. Nanotubes can fuse under high-pressure, exchanging many sp2 bonds for sp3 bonds, so that strong, infinitely long cables can be created by high-pressure nanotube linkage [13]. Functional groups are always present on the surfaces of carbonous nanomaterials and they play a crucial role in the adsorption process.

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10.2.2.2 Mechanical properties CNTs can be considered the most flexible, strongest, and stiffest natural materials because of their covalent CeC bonding and flawless hexagonal network design [18]. The cylindrical graphitic structure gives them excellent mechanical features [19]. These mechanical properties of CNTs are mostly due to the covalent bonds between the carbon atoms [22]. The elastic modulus, commonly known as Young’s modulus, is used to determine the elasticity of a material. Due to the high CC bond stiffness of the hexagonal network, CNTs have an axial Young’s modulus of elasticity of nearly 1 TPa, making them one of the stiffest materials known, yet allowing elastic deformation when compressive forces are applied [18]. Many applications, such as probe tips for scanning microscopy, are made possible by the high modulus values [13]. The strength is achieved through sp2 covalent bonds between single and distinct carbon atoms [18]. However, CNTs are not nearly as strong under compression. Under bending load, they tend to buckle due to their hollow structure and high aspect ratio [13]. Mechanical properties such as strength, stiffness and impact resistance, as well as structural damping, can be improved by various surface functionalization [8]. 10.2.2.3 Thermal properties CNTs are of great importance not only because of their structural and mechanical properties but also due to their thermal properties. Nanotubes are believed to act as excellent thermal conductors along the tubes. They exhibit an important feature known as ballistic conduction, which allows CNTs to transfer heat more efficiently [13]. CNTs have temperature stability of up to 2800
C in a vacuum and about 750
C in air and they also show superconductivity below 20 K (about 253
C) thanks to the strong Table 10.1 General features of CNT types. Properties

SWCNTs

MWCNTs

Ref

Diameter (nm)

0.6e3.0

[22]

Young’s modulus (GPa) Tensile strength (GPa) Thermal conductivity (W/(mK) Density (g/cm3) Electrical conductivity (S/cm) Electrical resistivity (Um)

1500e2500 10e52 6000 1.33ee1.94 102e106 1  106

1.3e4.0 (internal) 2.0e50 (external) 200e950 10e60 2000 2.1e2.6 103e105 8.0e20  10-6

[20] [20] [20] [23] [24] [25]

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CeeC bonds in the plane of graphene. These bonds provide stiffness and quality against axial stresses. As a result, the adaptability to non-axial stresses is quite high. At low temperatures, the specific heat differs between SWNTs such as graphene and graphite. CNTs have a thermal property that is essentially isotropic and significantly anisotropic. It is known that even in bulk samples, the thermal conductivity of nanotubes is high and it is affected by ambient conditions [18]. The thermal conductivity of SWCNTs and MWCNTs is approximately 6000 W/mK and 3000 W/mK at room temperature, respectively [20]. The thermal characteristics of the tube are strongly affected by crystallographic defects. Such defects cause phonon scattering, which increases the phonon relaxation rate. Thus, both the mean free path and thermal conductivity of nanotube structures are reduced. 10.2.2.4 Electronic properties Carbon has three hybridization forms: sp3, sp2, and sp, which coordinate with four atoms having bond angles of 109.5
, three atoms having 120
, and two atoms having 180
. Strong s-bonds between carbon atoms are formed by three electrons remaining in sp2 orbitals, while weak Van der Waals bonds between planes are formed by orbitals. A network of electronic bonds is formed when the p orbitals of neighboring atoms overlap in a particular plane, giving graphite its high electrical conductivity [26]. One important feature that reflects the electrical characteristics of CNTs is called as chirality. The degree of chirality determines the distinctive electrical, conductive, and metallic properties [9]. The chiral vector is formed when the centers of two hexagons are joined together via vector additions. The electrical properties of nanotubes are affected differently by each chiral configuration which forms the basis of a rolling graphene sheet into a seamless cylinder [18,27,28]. CNTs have unusual electrical characteristics. Depending on their shape, they can be semiconducting or metallic. Although these materials are incredibly durable and inert structures, their electrical characteristics are exceedingly vulnerable to charge transfer and chemical doping effects caused by numerous substances [29]. CNTs establish a covalently cross-linked structure in the matrix of functionalized nanocarbon-based composites. In comparison to CNTs, graphene’s large specific surface area can considerably increase conductivity. At low filler loadings, graphene has a reduced percolation threshold and better conductivity, resulting in cost-effective filler and improved composite processability. Due to the extremely increased CNT dispersion, CNT functionalization has a considerable impact on the electrical

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263

conductivity of nanocomposites, resulting in the creation of conducting networks. However, functionalization must be done to avoid introducing a high number of heterogeneous atoms into the network, which act as a barrier to electrons and reduce electrical conductivity. Surface functionalization can provide a homogenous dispersion due to stronger contacts. Moreover, the CNT functionalization can increase the number of CNT sites that interact with the analyte, increase charge mobility and interchain in polymer chains [14]. 10.2.3 Potential applications CNTs have a wide range of applications due to their advantageous properties, which include biocompatibility, fast electron transfer kinetics, ultralightweight, chemical inertness, high tensile strength, a wide range of antibacterial and antifungal properties, acting as protein carriers, and containing exposed functional groups [30]. They can be used as strong fibers, reactants, and molecular switches, among other things. Due to the hydrophobic behavior of CNTs, they can be functionalized to have unique properties that make them suitable for a wide range of biomedical applications [19]. Drugs with low solubility, rapid deactivation and restricted bioavailability can all be addressed by using fCNTs, which are commonly utilized as drug carriers. However, one of the biggest drawbacks is the possibility of dissociation in biological fluids. Despite these characteristics, CNT is the most effective drug carrier for enhanced cell penetration and better-targeted drug delivery [19]. They might be conjugated with a vast variety of bioactive compounds. In biological applications, these molecular conformations have successfully offered newer routes [9]. UV stabilizing activity of CNTs is advantageous when they are exposed to UV radiation and oxygen. CNTs also have outstanding antioxidant properties such as high infrared optical absorption and photoluminescence, and echogenic characteristics, which are important for protecting polymeric materials from heat and photodegradation and tracking nanotubes in the biological environment [19,30]. They also have semi and metallic conductive qualities, making them a good fit for a variety of applications like clinical diagnostics, food safety, and environmental monitoring. The advancement of the bioelectronic system has opened up new possibilities for stabilization of DNA and proteins on the surface of CNTs. Their capacity to incorporate compounds such as proteins and oligosaccharides makes them an important carrier for the transport of active compounds such as medicines and related

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compounds. Recently, they have been utilized as electromechanical actuators, as well as electronic and optical biosensors for recognizing bioactive substances including DNA, protein, cells, and microbes due to their superior electrical characteristics. CNTs can be used in sensor manufacturing for detecting various pathogens and helping the diagnostic process of cancer treatment. Overall, fCNTs and their uses have recently sparked a lot of scientific research and have become a hot topic for new studies. Many studies based on CNTs have been conducted, and remarkable advancements have proven the superiority of CNT application in flexible electronics [31]. Several fields of science, medicine, and engineering have identified the potential of CNTs for practical uses. They have proven to be effective in a wide range of applications. Due to their unique structure, CNTs offer exceptional electrical, mechanical, optical, and thermal capabilities and have developed applications in a variety of sectors such as highperformance materials, nanomedicine, energy storage, environment, electronics, sensors, and molecular devices with nanotechnology being a major focus of much of their work. As attractive prospects for nextgeneration electronic materials, they have outstanding mechanical flexibility, high carrier mobility, current-carrying capability, an ultrathin body for effective electrostatic control and solution-processability for low-cost manufacturing [31e33]. Here, we mainly focus on the applications of carbon nanotubes in biomedicine in more detail.

10.3 Carbon nanotubes as theranostics The word theranostics was first coined by John Funkhouser in 1988 to describe an approach that combines diagnosis and treatment for individual patients [34]. Fields of study such as omics sciences, including proteomics and genomics, and bioinformatics, are molecular theranostics that contains genetic information and are used to develop diagnosis and treatment [35e37]. The use of theranostics provides images for diagnosis and response to treatment on the one hand, and on the other hand, it is possible to use the same agent for targeted therapy. Theranostics increases the effectiveness and choice of drugs for patients by eliminating unnecessary treatments and also develops targeted drugs and treatments for personalized medicine. The structures created by the use of nanomaterials in theranostics are nanotheranostics [38]. The ability of these agents to target the desired region, be synthesized with the desired properties, and have low side effects has increased their use in therapy. The theranostic strategies of carbon

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nanotubes used as nano-theranostic agents in the field of theranostics are discussed in the following sections. 10.3.1 Theranostics applications of CNTs As mentioned in the previous sections CNTs have been used for many theranostic applications due to their unique physical, chemical and biological properties that can be adequately functionalized, nanoscale size, large surface area, biocompatibility, electrical and thermal conductivity, good electrical transfer kinetics, flexibility, chemical inertness, lightweight, antimicrobial properties, and large production potential [39e41]. These unique properties have given them many applications, such as in cancer, infectious diseases, and neurodegenerative disorders, in gene therapy and areas such as sensing, drug delivery, imaging, and therapy (Fig. 10.4). In the present chapters, all these applications and related studies on CNTs are briefly discussed. 10.3.1.1 Cancer Cancer is one of the major diseases characterized by uncontrolled cell proliferation, which usually originates from a single cell as a result of the accumulation of mutations. It can affect surrounding cells and organs [42]. According to International Agency for Research on Cancer (IARC), about 19.3 million people had cancer and 9.96 million people had died from it in

Figure 10.4 Theranostic applications.

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2020 [43]. Similarly, 30.2 million people were expected to develop cancer and 16.3 million people expected to die from it by the year 2040 [44]. Certainly, the most important step in cancer treatment is the diagnosis. Survival or survival time is depending on the earlier diagnosis. Nowadays, laboratory tests, imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, and X-ray are widely used to diagnose cancer. After determining the cancer cells, if it is possible, the tumor is removed by surgery. Current techniques such as chemotherapy, radiotherapy, and their combination have been used to treat cancer [45]. The main purpose of the treatment methods is to prevent the recurrence of the tumor and metastasis. Although imaging tests such as MRI and PET have adequate sensitivity, these tests are expensive and require complex equipment [45]. In addition, the drugs used in treatment can destroy not only the cancer cells but also all other cells, including healthy cells [46]. In this point, new studies have gained importance to determine and treat cancer without damaging healthy cells and tissues using conventional methods. In recent years, nanotechnology, especially the promising CNTs, has started to be used for cancer diagnosis and treatment in all fields. Since their discovery, CNTs have been remarkable materials for sensing systems with their electrical, mechanical, and electrochemical properties as well as their sensitivity to biomolecules. The surface-to-volume ratio of CNTs is high, therefore CNTs enable rapid detection of biological samples even at low concentrations. Moreover, they are widely used for the development of biosensors due to their hollow tubular shape, large surface area, fast electron transfer kinetics, low potential for redox reactions, low surface contamination, good stability, and long lifetime [47]. CNTs have been successfully used in cancer research as functional molecules in biosensor systems for diagnosis and analysis, as drug delivery systems, and as active agents in tumor imaging systems [30]. Researchers have been developed hundreds of sensors for cancer diagnosis and treatment as recently as 2020. The many sensor studies and clinical study are summarized in Table 10.2. In this clinical study, it was reported that a nanosensor containing functionalized gold nanoparticles (GNPs) and CNTs was developed to analyze breath samples for the detection of gastric cancer. It was designed with 11 different organic ligand GNPs and SWCNTs coated with four different organic coatings to enable the sensor to work rapidly and reversibly against typical compounds found in breath samples. In the study, breath, saliva, cancer and para cancer tissue samples were collected from patients with gastric cancer. The sensor studies were confirmed with

Table 10.2 Cancer studies related to CNTs. CNT type

Therapeutic applications

Model/Cell line

Results

Ref.

• The sensor platform (SWCNT-GU modified DMEJ) was designed to identify the SOX-17 sequence. • Linear range and LOD were determined as 1 aM- 100 fM and 1 aM, respectively. • RMWCNTs surfaces were functionalized to FMWCNTs via acid digestion process and cytotoxic and genetic responses were compared with the artificial bone implanted material HA • Damaged cell profile changed CNT and HA dose-dependent. • CNTs and HAs induced the apoptosis in vitro

[48]

SWCNT

Gastric cancer

1.0 g SWCNT for modification of electrode surface

NA

RMWCNT and FMWCNT

Liver cancer

1.0e100 mg/mL for cell viability; 50 mg/mL to determine the apoptotic marker genes mRNA expressions

HepG2

[49]

267

Continued

Theranostic applications of functionalized carbon nanotubes

CNT treatment/dose

268

Table 10.2 Cancer studies related to CNTs.dcont'd Therapeutic applications

CNT treatment/dose

Model/Cell line

Results

Ref.

• MWCNTs was used for the detection of MUC1, functionalized with dopamine as signal generating probes in the sensing system. • Linear range and LOD were determined as 0.05 e940 U/mL - 100 fM and 0.01 U/mL, and MUC-1 spiked human serum samples were analyzed. • PPy@MWCNTs were used as a new sonosensitizer and multistep US irradiation of the composite was investigated in vitro as well as tumor irradiation. • MWCNT was bound Q for lower toxicity effect and with Pm drug.

[50]

MWCNT

Breast cancer

2.0 mg/mL

NA

MWCNT

Melanoma

5.0e250 mg/mL for temperature increment and cell viability studies; 100 mg/mL for intracellular ROS generation

Mouse malignant melanoma cell line C540 (B16/F10) and 15 male Balb/c mice (4-wk-old, weight of 20 g)

MWCNT

Breast and pancreatic cancer

3.125e50 mg/mL MWCNTs, 2.5e40 mg/mL Pm; 0.25e4.0 mg/mL Q for antitumoral studies;

MDA-MB-231 (breast cells) and PANC-1 (pancreatic cells)

[51]

[52]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

CNT type

25 mg/mL MWCNTs/ 20 mg/mL Pm/2.0 mg/mL Q for cytotoxicity and ROS studies.

• MWCNT-Pm-Q nanosystem caused an increase in antitumor effect and intracellular ROS level in pancreatic cells.

Brain tumors and Hodgkin lymphoma

NA

NA

MWCNT

Prostate cancer

100 mg MWCNT

NA

MWCNT

Colon cancer

12 mg GEM per kg body weight of rat for

HT-29 cell line and SpragueeDawley rats

• Adsorption and encapsulation of Lomustine, a chemotherapy drug, was investigated density functional theory calculations. • An electrochemical immunosensor was designed (COOHMWCNTs/PANI/ AuNP) for the detection of PSA. • Linear range and LOD were calculated as 1.66 ag/mL-1.3 ng/mL and 0.5 pg/mL. • Cytotoxicity and antitumor effects of

[53]

[54]

[55]

269

Continued

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SWCNT

270

Table 10.2 Cancer studies related to CNTs.dcont'd Therapeutic applications

CNT treatment/dose

Model/Cell line

pharmacokinetic study; 2.5, 5.0, 7.5, 10 mM for cytotoxicity studies; 100 mg/mL MWCNTs concentration for pharmacokinetic and biodistribution study

SWCNT

Pancreatic cancer

0, 1, 10, 25, 50, 100, 200, 400 mg/mL SWNT-CY7IGF-1Ra for cytotoxicity and photothermal studies; 50 mg/mL for cell-targeting capabilities; 300 mg/mL, 200 mL for NIR fluorescence and microscopic imaging

Results

Ref.

GEM loaded hyaluronic acid conjugated PEGylated MWCNTs were compared with free GEM. • It was found that GEM/ hyaluronic acid - PEG -MWCNTs displayed less hemolytic toxicity, reduced tumor volume and increased survival than free GEM. Human pancreatic cancer cell line BXPC-3; PANC1; ASPC-1 SW1990 BALB/c male mice

• CNTs labeled with antiIGF-1R antibody and CY7 as imaging agent was used to imaging of IGFR that is overexpressed by pancreatic cancer cells. • It was found that SWNT-CY7-IGF-1Ra can specifically accumulate, visualized in tumor and increased

[56]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

CNT type

survival of mice with laser irradiation exerts. Breast cancer

100 mL, 5.0 mge25 mg/ mL SWNT-GC solution for laser treatment

Murine tumor cell line 4T1 and mouse DC cell line DC 2.4; Female BALB/c mice aged 6 e8 weeks

MWCNT

All cancer types

NA

MCF-10A, MCF-7, MDA-MB23, MDA-MB468 HEK293 & ACHN, A-2780 & OVCAR-3, PC-3, KATO-III MC4L2 & 4T1

CNT

A375 and MCF7

• To increase antitumor efficacy of checkpoint inhibitors, CTLA-4 antibodies, SWCNT was modified by GC and was valued the treatment affection with photothermal therapy of metastatic mammary tumors. • Prolonged survival of the treated animal was found. • Lipidomics based EIS sensor was fabricated by CNTs on secretomes to detect lepidic contents of the secretomes of cancerous samples. • It had been confirmed that there was a higher lipid content in cancer secretomes.

[20]

[57]

[58]

271

Continued

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SWCNT

272

Table 10.2 Cancer studies related to CNTs.dcont'd

NCNTA

Therapeutic applications

CNT treatment/dose

All cancer types

0.1e150 mg/mL functionalized-CNT

H2O2 secreted from live cancer cells

Model/Cell line

Results

Ref.

• Carboxylic acid functionalized-CNT was conjugated with RGD, and CPT was encapsulated in the conjugate for targeted delivery. • Encapsulated CNTs were used to treat avb3expressing A375 cells and it was found increased the anticancer effect. MCF-7 and MBA-MD231

• Flexible nanohybrid microelectrode based on CF was modified with NCNTA and AuNP. • Sensor platform that had been mechanical flexibility and biocompatibility properties, was used to evaluating the sensitivity of different cancer cells

[59]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

CNT type

to chemotherapy or radiotherapy treatments. 2.0 g for carboxyl functionalized MWCNT

HepG2

MWCNT

10 mg MWCNT for GdN @ CQDs-MWCNT synthesis

A549, H522, MDA-MB231, MCF-10A cell lines and mice

• MWCNT was coated with PEG after functionalization with the carboxyl group. • PEG-MWCNT was loaded with DOX for tumor-specific drug release and was determined to exhibit enhanced inhibitory activity against HepG2 cells compared to free DOX. • MWCNT was coated with GdN@CQDs and DOX-EGFR antibody. • It has been reported that the prepared nanocomposites exhibit pH and temperaturesensitive drug release behaviors and can destroy tumors through chemo/

[60]

[61]

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MWCNT

Table 10.2 Cancer studies related to CNTs.dcont'd Therapeutic applications

CNT treatment/dose

Model/Cell line

Results

Ref.

SWCNT

Gastric cancer

AGS and L929

• After SWCNT was coated with PEI and PEG, AS1411 aptamer was conjugated on the surface. • Bcl-xL was attached to the SWCNT-PEI-PET conjugate due to its potent anti-apoptotic activity properties, and subsequently, a vehicle with intercalation of DOX was obtained. • This study demonstrated that the presence of nucleolin on the surfaces of AGS cells, but not on normal L929 cells and had tumoricidal efficacy of DOX and Bcl-xL shRNA.

[62]

AuNP, Gold Nanoparticles; CF, Carbon Fiber; COOH-MWCNT, Modified with Carboxylated Carbon Nanotubes; CPT, Topoisomerase I Inhibitor Camptothecin; DMEJ, Dimicroelectroted Junction; Dox, Doxorubicin; EIS, Electrical Impedance Spectroscopy; FMWCNT, Functionalized Carbon Nanotubes; GC, Glycated Chitosan; GCE, Glassy Carbon Electrode; GdN@CQDs, Gadolinium Containing Nanoparticles Magnetofluorescent Carbon Quantum Dots; GEM, Gemcitabine; GU, Gold Urchin; HA, Hydroxyapatite; HA, Hyaluronic Acid; HPV16, Human Papillomavirus; IGFR, Insulin-like Growth Factor Receptor; IGF-R1, IGF Type-1 Receptor; LOD, Limit of Detection; MUC1, Mucin-1; NA, Non-available; NCNTA, Nitrogen-doped CNT Arrays; PANI, Polyaniline; PEG, Polyethylene Glycol; PEI, Polyethyleneimine; Pm, Pemetrexed disodium; PSA, Prostate-Specific Antigen; PPy@MWCNT, Polypyrrole-coated Multi-Walled Carbon Nanotubes Composite; Q, Quercetin; RGD, Cyclic Arginyl Glycyl Aspartic Acid; RMWCNT, Raw Multi-Walled Carbon Nanotubes; ROS, Reactive oxygen species; SOX-17, SRY-box Containing Gene-17.

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

photothermal synergistic therapeutic effect.

274

CNT type

Theranostic applications of functionalized carbon nanotubes

275

the supporting gas chromatography (GC-MS), ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS), and RNA sequencing determination methods. The clinical trial of the sensor system, which they named Na-Nose, was completed in 2020 [63e66]. fCNTs have the potential to be used in delivery systems for drugs and biomolecules because they can pass through a biological barrier and be excreted via the kidneys and/or feces. Moreover, while the toxicity of CNTs is a controversial issue in vitro and in vivo, fCNTs have been shown to have low toxicity and are not immunogenic. This subject is discussed in detail under the title of Toxicity and Biosafety Considerations of CNTs. Another important feature that enables their use as drug delivery systems is that they can be modified with covalent and noncovalent interactions or used as drug delivery tubes. In particular, the noncovalent interaction (ionic bonding, hydrogen bonding, hydrophobic, or p- p interaction) not only damages the structure but also enables the controlled release of the drug [67]. In recent years, chemotherapeutic agents, herbal agents have been included in delivery systems prepared with CNTs. In the study conducted by Badea et al. [52], MWCNT was functionalized with quercetin and pemetrexed disodium. Quercetin is a fruit-derived flavonoid with low toxicity, antioxidant and anticancer activity, trigger programmed cell death by reacting with harmful reactive superoxide anions, while pemetrexed disodium is an anticancer antifolate agent. According to the study, the MWCNT-Pm-Q combination showed synergetic effects on pancreatic cancer cells and exhibited better stability and superior therapeutic efficacy compared to single-agent nanosystems. CNTs, especially SWCNTs, have been used for imaging due to their physical properties. Semiconductor SWCNTs exhibit a narrow bandgap that enables fluorescence emission in the near-infrared (NIR) region, including the NIR-I region (700e1100 nm) and the NIR-II region (1100e1400 nm). Moreover, CNTs convert the absorbed NIR-I radiation into thermal energy, which gives thermotherapeutic properties [68]. They are a good Raman probe because they exhibit strong resonance scattering. CNTs can be used as photoacoustic (PA) imaging contrast agents since they are among the darkest materials. Because of these properties, CNTs with various modifications have been used for imaging techniques such as magnetic resonance (MR) imaging, positron emission tomography (PET), and single-photon emission computed tomography (SPECT) [21]. Many studies have been used for imaging and therapeutic purposes in many different types of cancer, some of which are summarized in Table 10.2. In addition, devices are being developed to be used for

276

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

cancer imaging with CNTs. In a clinical study, the use of a CNT X-ray based stationary breast tomosynthesis device was compared with the results of traditional mammography. According to the results obtained, it was reported that image blur was largely eliminated and spatial resolution was improved [69,70]. 10.3.1.2 Infectious diseases Infectious agents are defined as viruses, bacteria, fungi, protozoa, and parasites and are transmitted from person to person through the air, body fluids, food, physical interaction, and vector organisms [71]. These frequently cause infectious disease and diagnosis, prevention, and treatment of them have always been difficult. Often, with the emergence of a resistant pathogen, all treatment methods have become ineffective and this condition leads to the rapid spread of the disease. Antimicrobial, antifungal and antiviral resistance are increasing day by day due to societal and technological changes provide the search for new ways to treat diseases. Therefore, these diseases are the area that is constantly open to research. Similarly, innovative studies are urgently needed for infectious disease [72]. CNTs have become very prominent nanomaterials in the search for a way to combat infectious diseases due to their unique properties. They have their place in studies of the infectious disease COVID-9 caused by the SARSCoV-2 virus. Especially considering the spreading speed of COVID-19, rapid diagnosis has become mandatory. In this context, it has been focused on rapid diagnosis kits. CNTs have also gained significance in sensor design, which is one of the point-of-care (POC) diagnostic platforms, due to their properties. In the study conducted by Shao et al. [73], an SWCNT-based field-effect transistor sensor was developed for the detection of spike and nucleocapsid antigens of SARS-CoV-2, and the sensor performance was evaluated using clinical nasopharyngeal specimens. SARSCoV-2 antibodies were bound to the carboxylic acid groups of SWCNTs via EDC/NHS crosslinkers by exploiting the simple conjugation property of CNTs. The sensor was reported to respond with LOD sensitivity for 0.55 fg/mL and 0.016 fg/mL for spike and nucleocapsid antigen in less than 5 minutes in the presence of SARS-CoV-2 antigens. In addition to sensing studies, theoretical studies have been performed with the idea that CNTs could be a good drug delivery system for COVID-19 considering their drug carrier properties. In one study [74], the interaction of remdesivir, an important drug for the treatment of COVID-19, with carboxylfunctionalized CNTs and S-, Al-, and Si-doped CNTs was theoretically

Theranostic applications of functionalized carbon nanotubes

277

calculated and it was reported that Si-doped CNTs are the best drug delivery system. CNTs can also be used to create detection systems or drug delivery systems for infectious diseases such as tuberculosis, leishmaniasis, and influenza, in addition to COVID 19. Furthermore, CNTs can be used not only to manage infectious diseases in humans but also to detect and treat infections in living things in nature, such as fish [75e77] and plants [78,79]. There are many studies conducted for the detection and drug delivery system for many infectious diseases, some of which are summarized in Table 10.3. 10.3.1.3 Neurodegenerative diseases Neurodegenerative diseases result from the degeneration of selected neurons in the central nervous system, and the most common forms include Parkinson and Alzheimer diseases [85]. There is a global need for effective treatment and diagnostic method for neurodegenerative diseases, for which carbon-based nanomaterials have recently been used. CNTs, which are promising nanomaterials, are gaining importance for diagnosis and treatment in the field of neuroscience due to their unique chemical and physical properties, ability to cross the bloodebrain barrier, and ability to effectively transport to the target. For example, taking Levodopa is necessary to treat Parkinson disease, but too much causes some movement disorders. Therefore, it is important to adjust the dose. To determine levodopa, Ji et al. developed an electrochemical sensor platform integrated into a smartphone [86]. Current changes on the chitosan, gold nanoparticle and SWCNTs-modified electrode surface were measured by differential pulse amperometry and integrated into the phone for real-time measurement. It was found that the sensor can detect concentrations as low as 0.5 mM in human serum and does not interfere with other substances in serum. In another study, Jiao et al. performed electrochemical measurements using dopamine (DA) modified indium tin oxide [87] electrodes with Pd-Pt alloy nanoparticles (Pd@Pt) with decorated polyoxometalate (PMo9V3) [88]. Studies in human serum also revealed LOD 1.25  108 M and a detection range of 2.50  108 to 1.78  104 M. 10.3.1.4 Others In addition to the broad use of CNTs in areas such as cancer, neurodegenerative diseases and infectious diseases, they are also used in topics such as tissue regeneration and engineering [87,89e92]. Carbon nanotubes are preferred among other nanomaterials and polymers for tissue regeneration

278

Table 10.3 Infectious diseases studies related to CNTs. Therapeutic applications

CNT treatment/dose

Model/Cell line

Carboxyl functional group and S-, Al- and Sidoped CNT

SARSCoV-2

NA

NA

SWCNT

SARSCoV-2

0.02 mg/mL SWCNT for electrode design

NA

SWCNT

SARSCoV-2

0.2 mg of SWCNT for the synthesis of ssDNA-SWCNTs

NA

Results

Ref.

• Interaction of simple CNT and functionalized CNT such as carboxylic group and S-, Al-, and Si-doped CNT with Remdesivir drug was calculated with density functional theory. • Si-doped CNT was determined as the best drug delivery system. • SWCNT-based FET sensor was designed with anti-SARSCoV-2 spike protein antibody and anti-nucleocapsid protein antibody for determination of SARS-CoV-2 antigens from nasopharyngeal samples. • LOD values were determined 0.55 fg/mL for S antigen and 0.016 fg/mL for N antigen. • ss-DNA-SWCNT-based optical sensing sensor was designed with ACE2 for the detection of SARS-CoV-2 spike protein.

[74]

[73]

[80]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

CNT type

• It was found that the sensor, when exposed to 35 mg/L SARS-CoV-2 viruses, showed 73% fluorescence turn-on efficiency within 5 s. SARSCoV-2

NA

NA

MWCNT

Malaria

NA

NA

CNT

Tuberculosis

NA

NA

• For the FET sensor designed, CNT channels were created and the reverse sequence of the RNA-dependent RNA polymerase gene of SARSCoV-2 was immobilized on these channels. • LOD was obtained as 10 fM. • Gold disc microelectrode was used as electrode and electrode surface was functionalized with nanostructured gold and MWCNs for determination of anti-PvMSP119 in serum human samples. • While the LOD of ELISA is 15 ng/mL, the LOD of the immunosensor was determined as 0.6 ng mL.

[81]

[82]

[83]

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Theranostic applications of functionalized carbon nanotubes

CNT

280

Table 10.3 Infectious diseases studies related to CNTs.dcont'd Therapeutic applications

CNT treatment/dose

Model/Cell line

Results

Ref.

• DNA biosensor was designed for the detection of the specific IS6110 DNA sequence of mycobacterium tuberculosis. • CNT was functionalized with PAN and the obtaining nanohybrid structure was used to generate the electrochemical signal. • Clinical applications were carried out with sputum samples. SWCNT and MWCNT

Cutaneous leishmaniasis

Cisplatin- SWCNT 0.028 e14.4 mM; cisplatin-MWCNT 0.021e11.2 mM; SWCNT, 0.034e17.6 mM; MWCNT, 0.028e14.4 mM; free cisplatin, 0.031e16 mM

Promastigotes of the reference strain

• Cisplatin (a drug for leishmaniasis) is bound to and encapsulated in the surface of both SWCNT and MWCNT. • It was found that considerable anti-leishmanial activity of cisplatin-MWCNT.

Anti-PvMSP119, Plasmodium Vivax Antibodies; FET, Field-Effect Transistor; NA, Non-available; PAN, Polyaniline; ssDNA, Single-Stranded DNA.

[84]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

CNT type

Theranostic applications of functionalized carbon nanotubes

281

because they are biocompatible, can be functionalized with biomolecules, and are difficult to biodegrade. With these properties, CNT-based materials have the potential to increase the strength and conductivity of the bone scaffold by integrating them into the patient’s body [93]. To investigate the mechanical properties of CNTs, Moura et al. fabricated biomimetic scaffolds with polymer nanofibers reinforced with CNT nanofibers in one study. According to the results, the fabricated polymer-CNT composites have the potential for tissue engineering of the knee meniscus [94]. In another tissue engineering study using CNTs, the fabrication of 3D-printed scaffolds with CNT-functionalized single-stranded DNA (ssDNA) using MC3T3 pre-osteoblast cells was reported by Liu et al. [95] According to the results of this study, CNT was found to increase electrical stimulation and thus conductivity by enabling cell stimulation, and ssDNA reduced the toxicity of CNTs. For more therapeutic studies, you can see Table 10.4

10.4 Drug and gene delivery Today, effective drug treatment is expected to target the damaged area and be used in the right dose without harming healthy cells and without side effects [99]. On the other hand, we often encounter problems like irreversible side effects, damage, low or high dose, drug resistance [100]. The development of drug delivery systems that can overcome all these problems is an important area of research. The functionalization of carbon nanotubes has also increased their ability to interact with biological molecules, thanks to the different surface properties they have acquired. In this context, approaches such as their use as drug delivery systems have also been proposed. The first of these approaches consists of creating a porous structure to hold the active components in a CNT network, while the second consists of functionally binding the component to the outer walls of the CNTs. The third and final approach is to use CNTs as catheters [101]. Studies have shown that there are serious differences between the use of these approaches and direct drug delivery [102e104]. Some of these differences include targeted delivery of the drug, facilitating the entry of the drug into the cell, increasing the absorption of the drug, and most importantly, reducing the toxic effects of drugs [105]. Stable binding of active molecules to cationically fCNTs is possible [106]. Compared to SWNTs, MWNTs have a larger diameter and are more commonly used in drug delivery systems due to their size, although they have limitations, especially in terms of optical properties. FCNTs have all the properties that a good delivery

282

CNT type

DWCNT

CNT SWCNT

CNT CNT

Therapeutic applications

CNT treatment/ dose

Cytokines IL-1b and TNF- a Neural regeneration L-DOPA

1.0 mg HOOC-PheDWCNTs 10 mg of SWCNTs 0.5 g of pristine SWCNTs

Tissue engineering Bone tissue engineering

0.05% and 0.10% CNT 0.5 mg/mL CNT

Model/Cell line

Results

Ref.

NA

• Simultaneous detection of 2 cytokines.

[96]

NSCs

[97]

MSC

• CNT-PLGA microspheres were found to increase the expression of neuronal biomarkers. • Analytical parameters for L-DOPA were obtained with the GCp/SWCNT-COOH@Nd2O3-SiO2 electrochemical sensor with 0.70 mmol L1 LOD and 2.30 mmol L1 LOQ. • Mechanical properties are increased with CNTs.

MC3T3 preosteoblast cells

• The sDNA@CNT nanocomplex increased the conductivity of the cells. • Cellular osteogenic differentiation was observed with sDNA@CNT.

NA

IL-1ß, Interleukin-1b; L-DOPA, L-3,4-dihydroxyphenylalanine; MSC, Mesenchymal stem cells; NA, Non-available; NSCs, Neural stem cells; TNF- a, Tumor Necrosis Factor a.

[98]

[94] [95]

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

Table 10.4 Therapeutic studies related to CNTs.

Theranostic applications of functionalized carbon nanotubes

283

system should have, such as carrying one or more active agents, motif recognition, compatibility with optical imaging, and targeted delivery [107]. Moreover, both hydrophilic and hydrophobic molecules can bind to CNTs [108]. Another reason for using CNTs as drug delivery systems is to prevent multidrug resistance. Drug-loaded fCNTs can be microencapsulated with poly-L-lysine alginate to avoid drug release problems. Thus, they have a new membrane so that they are protected from the external environment, become safe, and reach their destination [109]. With the ability to deliver a specific drug to a specific region in a controlled manner, fCNTs are an important candidate for reliable and effective therapy [6]. Especially in hereditary diseases, it is possible to increase the patient’s quality of life for a certain period by replacing a damaged or missing gene, but the most important problem here is the passage of DNA through the cell membrane [110]. At this point, it is possible to deliver the DNA into the cell via CNTs to replace the gene. CNTs facilitate this transition. In particular, ammonium-functionalized CNTs have proven successful in gene therapy [111]. Instead of sending DNA into a cell alone, it is more beneficial and reliable to send it by binding to SWNTs. With their surface area and electrical properties, they offer the natural application of the electroporation technique often used in delivering a DNA fragment to cells under in vitro conditions [112].

10.5 The importance of theranostics for personalized medicine The term of “personalized medicine” has been referred to as the personbased model, taking into account the individual’s own genetic and metabolic structures, for any medical procedure in the biomedical field [113]. Today, personalized medicine applications are becoming more and more common. This not only increases the patient’s quality of life but also reduces the burden on the healthcare system and prevents unnecessary treatment costs. The individual diagnosis is also important for the individual treatment. In both cases, it is possible to benefit from nanotechnology. In line with the combined approach understood in theranostics, CNTs are nanomaterials well suited for targeted therapy approaches in personalized medicine [114]. They can be used as hybrid systems for both diagnosis and treatment. CNTs made more specific by functionalization become stable and can remain in circulation for an extended time without degrading. This makes them useful for applications in personalized medicine. One of the

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Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

biggest problems is the toxicity of CNT used. Similar to other nanomaterials, carbon nanotubes have negative effects on human and environmental health. Studies in this area are continuing. 10.5.1 Toxicity and biosafety considerations of CNTs There are several conditions for approval of any material for health use. The most important of these conditions is the biosafety of that material. However, for theranostic use, detailed information should be obtained about the pathway of the material in question in the body, its diagnostic or therapeutic properties, its toxic or side effects, and its preparation. Theranostic use of any material should consider its suitability, preparation conditions, formulation and/or modifications, route of administration, biocompatibility, biodegradation, toxic effects, and metabolites [115]. There are many publications related to CNT toxicity [116e119]. The most important fact known today is that the findings obtained from in vitro and in vivo animal studies with non-functionalized/raw CNTs were always toxic [117]. CNTs are functionalized to reduce or even eliminate this toxic effect of CNTs. Thus, the toxic effect of non-functionalized CNTs in vitro and in vivo varies depending on the surface functionalization and the geometry of the CNTs. Although in vitro studies on non-functionalized CNTs emphasize the toxic effect, this is mainly related to the fact that these CNTs do not dissolve and aggregate in the culture medium due to their hydrophobic nature [120]. In vivo studies have reported inflammation, fibrosis and lung discomfort associated with the direct use of nonfunctionalized CNTs. Studies have shown that inhalation exposure of non-functionalized CNTs leads to outcomes such as inflammation, pulmonary fibrosis, mutations, neuronal damage in the brain, neurodegenerative diseases, hepatotoxicity in the liver and skin thickening, i.e., skin fibrosis [119,121]. On the other hand, fCNTs had either no or dosedependent toxic effects compared to non-functionalized ones both in vitro and in vivo [122]. Studies have shown that CNT-induced toxicity may have many causes. These are morphological and chemical properties, physical state, purity, route of administration, covalent, or noncovalent functionalization [120,123]. Since studies on the toxic effects of CNTs depending on their size are not mutually supportive, this issue is not yet fully resolved. CNTs exert their known toxicity via oxidative or nonoxidative pathways [122]. Therefore, the systemic effects of CNTs need to be investigated. If they cannot be removed from the body, they

Theranostic applications of functionalized carbon nanotubes

285

accumulate and cause an inflammatory effect [118]. Although more parameters and variables need to be evaluated in the long term to evaluate toxicity and side effects, the findings from cell and animal experiments will not translate directly to the three-dimensional organism. In addition, studies have shown that some of the tests frequently used to assess cell viability in CNT toxicology studies yield erroneous findings [124e127]. These tests are MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl))-2Htetrazolium-5-carboxanilide), and WST-1 (2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium). Firstly, in 2006, Wörle-Knirsch et al. [124] showed that CNTs cause erroneous results by binding MTT formazan crystals. In other studies, conducted following this research, it has been shown that the products of carbon black or magnesium-based alloys interact with MTT or XTT [125e127]. Finally, the WST-1 test with manganese or manganese-containing alloys has also proven to give inconsistent results [128]. Apart from these, the use of luminescence-based tests is required. Considering all this there is a great need for further clinical research in this field.

10.6 Pros and cons of CNTs CNTs are widely used in the field of theranostics due to their physical and electromechanical properties. Among their advantages is that they can be easily obtained in small sizes from abundant raw materials. Fluorescent dyes used for theranostic applications such as chemotherapy and gene therapy are not resistant to photobleaching, but CNTs have resistance to photobleaching. This property makes it possible to use them as Raman probes and fluorescent imaginary markers. Other advantages of CNTs can be listed as follows: modifiability with molecules such as antibodies, drugs; excitability in the NIR-I region; low phototoxicity; stability for fluorescence emission; short photoluminescence lifetime [129]. Although CNTs have many advantageous properties, also have many disadvantages. CNTs have the disadvantage of dispersion and not soluble in water; in addition, their toxicity is still a controversial issue. Some synthesis methods, such as arcdischarge, require high temperatures and are costly. However, recent studies have proposed to functionalize them in order to solve their dispersion problem and ensure solubility in biological systems and reduce their toxicity. However, this modification step has created an additional

286

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

Table 10.5 Pros and cons for CNTs. Pros

Cons

• Small size, lightweight, and modifiable surface • Resistance to photobleaching • Excitability in the NIR-I range • Low phototoxicity • Stability for fluorescence emission • Short photoluminescence lifetime • Wide range of use in theranostic applications

• • • •

Not soluble in water. Toxicity High production costs Requires surface functionalization for lower toxicity and to ensure solubility

cost problem [13,130]. All these pros and cons of CNTs are summarized in Table 10.5.

10.7 Conclusions and future perspectives In this chapter, CNTs have been extensively evaluated as a theranostic agent. The theranostics applications of them have been addressed in different pathological conditions, drug and gene delivery, and toxicity. Moreover, the importance of the functionalization of CNTs has been highlighted. In the last decade, CNTs have been actively explored for potential theranostic applications. In this context, various properties of them, such as the high conductivity, high drug loading capacity, and large internal surface area, have contributed to their widespread use as therapeutic nanomaterials. Functionalization and chemical modification of nanotubes has improved various therapeutic properties, providing new opportunities for using nanotubes as a drug delivery system. On the other hand, these engineered nanotubes have a significant level of toxicity. Various properties of CNTs, such as the homogeneity of the chemicals contained in the nanotubes, the metals used, and the sensitivity to the various gases released, must be carefully monitored. Meanwhile, the need to reduce the toxicity of nanoparticles cannot be ignored. Proactive measures must be taken to ensure the safe use of nanotechnology. There is a need to develop simple, affordable and non-costly functionalization and characterization methods to support the biodegradation of CNTs and make them more biocompatible

Theranostic applications of functionalized carbon nanotubes

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and nontoxic. In short, CNTs still have a long way to go before they become necessary material of the future. For the use of them to increase in the future, we need to make them safer and biocompatible for theranostic applications. However, due to the rapid progress and constant attention, we have a high level of confidence. We believe that CNTs will continue to surpass and enrich our lives in the future.

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[117] C.P. Firme, P.R. Bandaru, Toxicity issues in the application of carbon nanotubes to biological systems, Nanomed. Nanotechnol. Biol. Med. 6 (2010) 245e256. [118] J. Muller, F. Huaux, N. Moreau, P. Misson, J.-F. Heilier, M. Delos, et al., Respiratory toxicity of multi-wall carbon nanotubes, Toxicol. Appl. Pharmacol. 207 (2005) 221e231. [119] P.P. Simeonova, Update on carbon nanotube toxicity, Nanomedicine 4 (2009) 373e375. [120] J.M. Gernand, E.A. Casman, A meta-analysis of carbon nanotube pulmonary toxicity studiesdhow physical dimensions and impurities affect the toxicity of carbon nanotubes, Risk Anal. 34 (2014) 583e597. [121] H.J. Johnston, G.R. Hutchison, F.M. Christensen, S. Peters, S. Hankin, K. Aschberger, et al., A critical review of the biological mechanisms underlying the in vivo and in vitro toxicity of carbon nanotubes: the contribution of physicochemical characteristics, Nanotoxicology 4 (2010) 207e246. [122] A.A. Shvedova, E.R. Kisin, D. Porter, P. Schulte, V.E. Kagan, B. Fadeel, et al., Mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: two faces of Janus? Pharmacol. Ther. 121 (2009) 192e204. [123] M.I. Sajid, U. Jamshaid, T. Jamshaid, N. Zafar, H. Fessi, A. Elaissari, Carbon nanotubes from synthesis to in vivo biomedical applications, Int. J. Pharm. 501 (2016) 278e299. [124] J. Wörle-Knirsch, K. Pulskamp, H. Krug, Oops they did it again! Carbon nanotubes hoax scientists in viability assays, Nano Lett. 6 (2006) 1261e1268. [125] N.A. Monteiro-Riviere, A.O. Inman, L.W. Zhang, Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line, Toxicol. Appl. Pharmacol. 234 (2009) 222e235. [126] J. Fischer, M.H. Prosenc, M. Wolff, N. Hort, R. Willumeit, F. Feyerabend, Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays, Acta Biomater. 6 (2010) 1813e1823. [127] A. Almutary, B.J.S. Sanderson, The MTT and crystal violet assays:potential confounders in nanoparticle toxicity testing, Int. J. Toxicol. 35 (2016) 454e462. [128] E. Scarcello, A. Lambremont, R. Vanbever, P.J. Jacques, D. Lison, Mind your assays: misleading cytotoxicity with the WST-1 assay in the presence of manganese, PLoS One 15 (2020) e0231634. [129] N. Panwar, A.M. Soehartono, K.K. Chan, S. Zeng, G. Xu, J. Qu, et al., Nanocarbons for biology and medicine: sensing, imaging, and drug delivery, Chem. Rev. 119 (2019) 9559e9656. [130] W. Shao, P. Arghya, M. Yiyong, L. Rodes, S. Prakash, Carbon nanotubes for use in medicine: potentials and limitations, Syntheses Appl. Carbon Nanotubes Composites 13 (2013) 285e311.

CHAPTER 11

Dispersions of carbon nanotubes and its biomedical and diagnostic applications Lopamudra Giri1, Gowtham Kenguva1, Smruti Rekha Rout1, Mohammad A.S. Abourehab2, 3, Prashant Kesharwani4, 5 and Rambabu Dandela1 1

Department of Industrial and Engineering Chemistry, Institute of Chemical Technology Mumbai-Indian Oil Odisha Campus, Bhubaneswar, Odisha, India; 2Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia; 3Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt; 4Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India; 5University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India

11.1 Introduction Nanotechnology is a key enabler for the growth of novel substances and devices, beginning a new age of biomedical science [1e6]. The early diagnosis and treatment of disease are two potential applications of nanomaterials in biomedical engineering. Among various types of nanomaterials, carbon nanotubes (CNTs), in particular, have a unique set of qualities, including a strong chemical resistance, outstanding mechanical capabilities, and have low weight. Graphene, nanodiamonds, CNTs, and graphene and their associated compounds are the most often utilized carbon nanomaterials [7e12]. Among different nanomaterials, CNTs are expansively researched for medicinal administration in vitro and in vivo over the last decade, due to their exclusive electrical, mechanical, and optical properties [13e17]. With appropriate loading capacity, CNT-based systems have recently been used in tissue regeneration and stem cell-based clinical uses, such as cardiac treatment, bone production, muscle repair, and brain recovery. CNTs are also effective agents for biological sensing and imaging due to their distinctive optical features, including, maximum absorption in the NIR (Near-Infrared) region, photoluminescence, and significant Raman shift [18]. However, in order for CNTs to be employed more widely and potentially in therapeutic, their biological features, behavior, and efficiency must be well explored. Furthermore, CNTs should also have well-defined Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery ISBN 978-0-323-85199-2 https://doi.org/10.1016/B978-0-323-85199-2.00007-8

© 2023 Elsevier Ltd. All rights reserved.

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physiological, ecological, and safety characteristics when generated in large quantities [19,20]. Depending on the preparations, purification, and functionalization process utilized to produce CNTs, their size, shape, composition, and purity might vary greatly. As a result, the contact between biological entities and CNTs is much complicated and unexpected. Furthermore, CNTs are extremely hydrophobic, which significantly reduces their bioavailability. They have the potential to harm DNA and also the cytoplasmic membrane. The major drawbacks of CNTs are hard to obtain stable aqueous suspensions due to bundle-like frameworks with extraordinarily complicated topologies and a large number of van der Waals contacts which limits their functional use. Nanotube materials’ level of individualization and agglomeration in the cellular environment play a significant role in their pharmacological performance, according to the many strategies adopted to alter the surface of nanomaterials used in drug. Chemical modification by functionalization of CNTs has the advantage of keeping therapeutic, targeted, or imaging molecules stably involved to the nanotube backbone. Functionalized nanotubes have been used in a larger range in case of therapeutic models than coated nanotubes [21e23]. Toxicity data for CNTs is still inconsistent. It was evaluated a number of recent studies and come to the conclusion that a more systematic approach to determining CNTs toxicity is required. Multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) are fibrous materials produced from hexagonal crystal structures layers of graphite wrapped into a tube shape in one or several layers. Other features of CNTs, on the other hand, have been reported to be hazardous to human health. Many studies around the world, also found that CNTs have little developmental toxicity, and that interpretation has been complicated in certain cases by the study methods used in the characterization of CNTs in suspension [23e25]. Aside from their possible toxicity, another issue that could hamper CNTs’ use in nanomedicine is their bio persistence; such as characterization of CNTs in suspension. CNTs have been observed to stay for lengthy periods of time in the spleen and liver. Since Allen et al. discovered the possibility of CNT biodegradation via enzymatic activity in 2008, the parameters for advantageous biodegradation of these materials have been investigated. According to a study, both SWNTs and MWNTs are vulnerable to enzymatic oxidative degradation, which is more prominent in functionalized CNTs with surface defects and in SWNTs than in MWNTs [10,26].

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Figure 11.1 Schematic representation of different applications of carbon nanotubes in the various field.

Our objective is to give a comprehensive idea of these fascinating materials’ many uses while stressing the importance of their individual unique features. This chapter describes present methodologies for dispersions of carbon nanotubes, the adaptability of CNTs in many biomedical sectors (Fig. 11.1) such as drug/gene delivery, bio-imaging, biosensors, tissue engineering, and regenerative medicine, and Table 11.1 lists the applicability of these emerging areas of interest. The limitations are addressed, with a focus on toxicity, as well as all profound opportunities.

11.2 Significance of dispersion of carbon nanotubes CNTs are discovered in past year which unlocks new opportunities for the expansion of revolutionary great performance materials. Disaggregation and uniformity are properties of CNTs because prefer to self-associate into micro-scale clumps; hence, dispersion are essential problems to overcome in order to generate such high-performance materials. As a result, mechanical and electrical performance suffers as a result. Recognizing this issue, substantial research on the dispersion technologies is being developed utilizing

298

Table 11.1 Methodologies, dispersive materials used for dispersions of carbon nanotubes and its applications. Preparation method

Application

References

N-isopropylacrylamide and polyethyleneglycol methacrylate Gold nanostars

Ultra-sonication followed by stirring Seed-mediated growth process/ Hydrothermal method Bath sonication Sonication followed by aqueous two-phase extraction (ATPE). Ultra-sonication Probe Sonication followed by freeze drying Dielectrophoretic

Cancer therapy

[27]

Cancer therapy

[28]

Cancer therapy Cancer therapy Bioimaging

[29] [30] [31]

Tissue engineering Tissue engineering

[32] [33]

Tissue regeneration and cell therapy Tissue engineering Cell therapy

[34] [35] [36]

Biosensor

[37]

Biosensor Biosensor Biosensor Biosensor

[38] [39] [40] [41]

Folic acid (FA-GdN@CQDs P-glycoprotein (pgp) Polycarbodiimide polymers

DNA-functionalized silica nanoparticles Peptide (EFK8)

Gelatin methacryloyl (GelMA) hydrogels Gold (au) nanoparticles (NPs) Glycidyl methacrylate (GMA) functionalized quaternized chitosan (Qcsg) Ti-bonded b-cyclodextrin (b-CD) NiO poly(vinyl alcohol)-(PVA)-poly(acrylic acid) (PAA) CdS@ZnS QDs Bismuth molybdate (BMO)

Probe ultrasonication Ultra-sonication

Ultra-sonication followed by stirring Physical mixing Electrospinning technique Electrochemical Hydrothermal

Emerging Applications of Carbon Nanotubes in Drug and Gene Delivery

Dispersive materials

Co2SnO4 0D/1d nanocomposite polymeric nanoparticles Polyaniline Poly(glutamic acid) (P-GLU) Tb@Mesoporous metal-oorganic frameworks AgeFe Reduced graphene oxide (RGO) N NiO b-cyclodextrin MoS2 Chitosan (CS)-dopamine (DA) Styrene-co-divinylbenzene Au 1-Butyl-3-methylimidazolium hexafluorophosphate [Cu(H(2)dimpy) Cl]PF6 poly(diallyldimethylammonium) chloride-Au nanocluster

Surface molecular imprinting technique

Biosensor

[42]

Ultra-sonication Hydrothermal In situ electropolymerization Ultra-sonication Physical mixing Ultra-sonication Ultra-sonication Solvothermal DC arc discharge evaporation Ultra-sonication Chemical vapor deposition (CVD) Ultra-sonication Ultra-sonication Solution-based method Ultra-sonication Ultra-sonication Ultra-sonication Ion-exchange method Ultra-sonication Ultra-sonication

Biosensor Biosensor Biosensor

[43] [44] [45]

Biosensor Biosensor Biosensor Biosensor Biosensor Biosensor

[46] [47] [48] [49] [50] [51]

Biosensor Biosensor

[52] [53]

Biosensor Biosensor Biosensor Biosensor Biosensor Biosensor Biosensor Biosensor Biosensor

[38] [54] [55] [56] [57] [58] [58] [59] [60]

Dispersions of carbon nanotubes and its biomedical and diagnostic applications

poly(1-vinyl-3-octylimidazole hexafluoride phosphorus)multi-walled carbon nanotubes@polydopamine-carboxyl single-walled carbon nanotubes DNA Covalent organic frameworks (COFs) Nano-Pt

299

Continued

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Table 11.1 Methodologies, dispersive materials used for dispersions of carbon nanotubes and its applications.dcont'd Preparation method

Application

References

ZIF-8 [Ni -(protoporphyrin IX)] NiNp GOx@PAVE, glucose oxidase (GOx), an amphiphilic copolymer PAVE containing photo-cross-linkable coumarin segments and carboxylic groups AuNPs, GQDs DOX (doxorubicin) Poly (e-caprolactone)/poly (N-vinyl-2-pyrrolidone) AgNPs

Ultra-sonication Dropcasting Ultra-sonication

Biosensor Biosensor Biosensor

[61] [62] [63]

Physical mixing Physical mixing Ultrasonic probe Modified chemical reaction process (NOT ACCESSABLE) NOT ACCESSABLE Ultra-sonication

Biosensor Drug delivery Drug delivery Antibacterial activity

[64] [65] [66] [67]

Antibacterial activity Antibacterial activity

[68] [69]

Ultra-sonication Ultra-sonication Ultra-sonication followed by stirring

Antibacterial activity Antibacterial activity Biodegradable membranes in reconstructive medicine Regulation of angiogenesis

[70] [71] [72]

Silver nanoparticles (Ag NPs) Silver nanoparticles (AgNPs)-co-doped polylactic acid (PLA) Silver nanodots (AgND) Ag PCL-polycaprolactone, PCLOH-polycaprolactone, PCLCOOH-polycaprolactone, PCLTI-polycaprolactone

Polyethyleneimine (PEI)

Incubation

[73]

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Dispersive materials

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301

both chemical and mechanical methods has been published in the literature. CNTs have an exclusive feature that make them ideal for use in polymerbased solutions, suspensions, composites, and melts. Good mechanical properties, such as elastic modulus and tensile strength, as well as outstanding flexibility, excellent electrical and thermal conductions, low percolation thresholds are among their distinguishing features [74]. CNTs integration has long been recognized as having enormous potential in a number of utilizations. CNTs disaggregation and uniform spreading in a number of substrates are required for successful use of their properties. Ultrasonication, high shear mixing, and procedures targeted at changing the surface chemistry of the tubes either noncovalently or covalently have all been suggested as ways to reduce nanotube clumping (adsorption). CNTs’ limited solubility in aqueous solutions is a significant characteristic that limits their biological use. CNTs, in particular, have a considerable agglomeration affinity, owing to strong surface contacts. Noncovalent functionalization of CNTs with biocompatible conjugates like phospholipid-polyethylene glycol (PEG) can improve their dispersion in aqueous solutions. Because noncovalent functionalization of CNTs does not alter the carbon lattice, the physicochemical properties of CNTs are largely preserved. Noncovalent functionalization should, result in increased stability, water solubility, biocompatibility in a variety of functional groups and biological solutions, that can be used in future bioconjugation [75].

11.3 Adopted techniques for dispersing CNTs The scattering of CNTs materials is critical for maintaining the nanotubes’ fundamental characteristics. Since CNTs suspensions clump rapidly and generate additional treatment difficulties, dissolvable functional material composites are required for post-processing. As a result, three criteria must be met simultaneously in order to create evenly dispersed carbon nanotubes. Firstly, damaging long strands’ intertwined bond configuration, secondly, bypassing the agglomerates’ high absorption strength and thirdly, stabilizing CNTs in their scattered form. In this section, numerous dispersion techniques such as physical (Di electrophoresis, ultra-sonication, ball milling, plasma and irradiation techniques, gel electrophoresis, ultracentrifuge, density gradient) and chemical (functionalization, ozonolysis, with porphyrins, polymers, peptides, amines, bromine, pyrene, etc.) are described in depth (Fig. 11.2).

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Figure 11.2 Representation of different techniques of preparation techniques for dispersion of CNTs categorizing into physical and chemical methods.

11.3.1 Physical methods There are several physical methods involved in the dispersion of CNTsbased material in order to optimize and develop techniques for their successful separation. The main disadvantage of this progression is the highenergy ultrasound in this process can shatter CNTs, decrease their dimension, diminish their aspect ratio, and inflict significant degradation to the carbon nanotubes, lowering the broad applicability. 11.3.2 Ultrasonication The process of using ultrasound radiation to churn particles in a medium for different reasons is known as ultrasonication. In the laboratory, an ultrasonic probe/horn or ultrasonic bath is often used for sonication. This is the most widely used technique for dispersing nanoparticles. The rationale behind this method is when ultrasound travels through a sequence of compressions, it induces dampened waves in the molecules of the substance whereby it travels. Individual nanoparticles positioned in the outside section of the nanoparticle bunches, or flocculate, are “peeled off” by these sonic booms, consequential in the detachment of individualized nanoparticles from the clusters [76]. CNTs manufacturing procedures frequently result in a combination of solid morphologies that are physically selfassemble into entities. In order to generate materials with unique mechanical features or transport properties, entangled or aggregated nanoparticles must frequently be disseminated in fluid solutions.

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Ultrasonication is an efficient way to distribute CNTs in liquids having low-viscosity including water, acetone, and ethanol. Conventional probe sonicators offer a variable amplitude range of 20%e70% and a power range of 100e1500 W. The probe is commonly constructed of titanium, which is a nonreactive metal. The majority of probes have a base unit and then taper down to a point with a diameter ranging from 1.6 to 12.7 mm. This implies that the power from the broad base is concentrated on the tip, resulting in a high-intensity probe. As a result of this setup, sonication may create a lot of heat very quickly. As a result, for CNTs dispersion in volatile solvents like acetone and EtOH the collections should be maintained in ice-bath and sonication should be performed in brief intervals. CNTs can be readily and badly destroyed if the ultrasonic irradiation is too vigorous and/or too protracted, particularly when a probe sonicator is used. CNTs’ graphene sheets are utterly wrecked in severe situations, and the nanotubes are transformed to unstructured carbon nanofibers [77,78]. A very effective purification and sonication procedure was also employed to create multi-walled carbon nanotubes (MWCNTs) buckypapers employing a simple, surfactant-free assembling method [79]. A minimum of 5 minutes of ultrasonic irradiation time was needed to accurately disperse MWCNTs in ethanol. The buckypaper made from purified CNTs has an outstanding humic acid (HA) removal rate (>93%) and a prolonged lifespan. 11.3.3 Ball milling Ball milling is a sort of crushing that is utilize to get incredibly fine powders out of substances. Due to the friction between the small, stiff balls in a covered container, a significant pressure is created locally during milling. Because of its ease of use, low cost and suppleness for bulk processing, ball milling is quickly becoming one of the most preferred technologies. The high-energy ball mill technique has been discovered as a hopeful tool for producing uniformly dispersed CNTs. The milling balls’ repeated contact forces on the CNTs, on the other hand, run the danger of causing significant damage to these nanomaterials. Milling conditions can induce damage to CNTs through openings in the carbonecarbon structure and sp3 disorders on the side walls. Despite these outstanding capabilities, the actual dispersion of these nanoparticles in metal matrices is a critical element that researchers have yet to solve. This is because metal matrices and CNTs have low wettability and strong van der Waals forces.

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To overcome these obstacles, scientists have performed experiments and developed a variety of dispersion techniques, including high energy ball milling, ultrasonic-assisted solvent dispersion, in situ synthesis molecule level mixing, cryomilling, and nanoscale dispersion, with varying degrees of achievement [80,81]. In a study, it was observed that the long-term ball milling of a sample assisted to debundle and embed the MWCNTs in the broken particles in form of dust, resulting in undeviating dispersion in the two-stage grinding. The subsequent low-speed, short-term high energy ball mill grinding is assisted in further dispersing the few remaining agglomerates within the matrix, resulting in a homogeneous dispersion of the nanotubes with minimal damage. CNTs are damaged, their crystallinity is lost, and their structural integrity is lost as an outcome of severe ball milling conditions. During dispersion processing of CNTs, optimizing the time of ball milling speed is critical to maintain their structural integrity [82]. Ball milling is a technique that generates high compression locally by colliding tiny, hard balls in a hidden container. Ball milling is effectively used on carbon constituents for a number of applications, including modifying the morphologies of cup-stacked CNTs. Although the effects of ball milling on changed carbon morphology are extensively established, nothing is known about in-situ functionalization of CNTs using this approach. CNTs are ball milled in the vicinity of chemicals, which improves their dispersibility while also introducing certain moiety to the CNT surface. Ball milling was employed to accomplish in situ amino derivatization of CNTs utilizing a straightforward chemo-mechanical technique [83], untangled and reduced than those crushed without the chemical. The findings indicate that CNTs grinded with ammonium bicarbonate (NH4HCO3) were more efficiently could be adjusted by selecting the right grinding period. As a result, this is a chemo-mechanical approach for amino functionalization of CNTs and the utilization of ball milling in presence of NH4HCO3 permitted in-situ amino functionalization of CNTs. Ball milling was used to present functional groups such as amine and amide to the CNT exterior, as well as improve the cutting impact of the ball milling to provide size-controllable CNTs. In this investigation, roughly 4 h was regarded sufficient to effectively shatter CNTs into small pieces in the presence of NH4HCO3. The current process for amino functionalization of CNTs is simple and straightforward, allowing for many applications in a number of sectors [83].

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11.3.4 Plasma and irradiation techniques To increase CNT dispersibility, several plasma procedures have been used, often without surfactants, with varying quantitative results. Without the use of any surfactants, CNTs microcapsules were set using oil in H2O Pickering emulsions. For many different response periods, CNTs were exposed with oxygen plasma (13.56 MHz radiofrequency) at 100 W power and 200 mTorr pressure. The oxygen plasma activation caused in the development of numerous hydrophilic groups on the CNTs, which increased their water dispersion. These plasma-treated CNTs self-assembled at the interface between the oil and water phases. The CNT nanocomposites generated using plasma functionalized CNTs were found to be homogeneous and had good CNT dispersion [84,85]. In a similar study, Plasma nanocoated CNTs for heat transfer nanofluids, MWCNTs were plasma-treated with argon, oxygen, and methaneeoxygen combination flash discharges before being disseminated in a water base fluid. It was discovered that utilizing nanoscale plasma coverings to plasma treat CNTs significantly enhanced dispersion and stabilized CNT suspension in the base fluid. With 0.01 vol% totaling of plasma-treated CNTs, a 25% improvement in thermal conductivity was reached, with a 20% increase in stability. The scientist noted that a high improvement in heat conductivity was obtained whenever plasmatreated CNTs were dispersed in water without the use of dispersing surfactants [86]. The dispersibility of CNTs with larger diameters was shown to be superior. Plasma treatment might increase CNT dispersibility, and if the CNTs were already acid-treated, plasma treatment might enhance the performance. DNA wrapping has been used to develop a new approach for very effective functionalization of SWCNTs. The quantity of single stranded deoxyribonucleic acid (ssDNA) essential for SWCNT alteration was reduced by a direction of magnitude after SWCNTs were exposed to gamma-irradiation (50 kGy). Electrostatic forces stabilized the gammairradiated SWCNTs that were functionalized with ssDNA. This early work reveals that gamma-irradiating SWCNTs with DNA can considerably increase their functionalization [87]. To examine the dispersion characteristics of MWCNTs, scientist proposes a new strategy for using a planetary ball mill to provide the best grinding conditions and a simple way for purifying MWCNTs. MWCNT dispersion properties in aqueous solutions without and with surfactant were examined under wet and dry grinding

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situations at varied rotating speeds. For the optimal dispersion, the maximum absorbance of nanofluid was found to be 2.485 abs at a wavelength of 253 nm. Grinding wet at a high rotational speed of 500 rpm with CNT dispersion in aqueous systems using ultrasonication and surfactant produced the best dispersion characteristics [88]. 11.3.5 Chemical methods Nanodiamonds (NDs) and graphene oxide are two carbon allotropes that have been used to increase CNT dispersion. CNTs were dispersed using nanodiamond particles, enabling for the development of stable colloidal suspensions. High pressure or detonation NDs, more temperature NDs could be used to suspend MWCNTs and SWCNTs in deionized water. Electrostatic interactions may have caused negatively charged NDs to suspend CNTs in deionized water more favorably than positively charged elements. Graphene oxide (GO) sheets, which are classified as “so” 2D macromolecules with numerous aromatic regions and hydrophilic oxygen groups, can adsorb virgin MWNTs via p-stacking interactions, causing them to disperse and fractionate in aqueous conditions [89,90].The noncovalent functionalization and covalent functionalization are the two types of chemical functionalization processes. Covalent functionalization is the process of treating carbon nanotubes with a mixture of acids or other strong oxidizing agents to modify their surface morphology and add different active groups such as amino, hydroxyl, and carboxyl to increase aqueous solution dispersion. But this approach can cause significant disruption to the structural and mechanical features of CNTs. Moreover, to achieve completely functionalized CNTs, noncovalent functionalization involves combining CNTs with other molecules via a non-covalent interaction. This approach not only enhances CNTs dispersibility, but also preserves the structural as well as physical properties [91]. 11.3.6 Inorganic salts facilitate CNT dispersion There are only a few examples of CNT dispersion in salt. The properties of inorganic monovalent ions such as NaI and NaCl on the stability of CNT dispersion were investigated. Molecular dynamics simulations with all atoms were investigated and computed utilizing nonpolarizable contact models the mean force probable between two SWNTs as compared to the potential of NaCl/NaI in the occurrence of NaCl/NaI to the potential of mean. In purified H2O, there is a force between SWNTs [92]. The impacts

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of ions (NaI) on pristine CNT dispersions in N-methyl-2-pyrrolidone (NMP) were studied in an investigated, yielding a different set of results. In CNTeNMP dispersions, both particles (Naþ and I) are deprived from the CNT exterior for two reasons: (a) ion partial desolvation at the CNT surface has a substantial energy penalty, (b) NMP molecules produce a dense solvation layer at the CNT surface that prevents ions from approaching the CNT outward. The “osmotic” stress in the CNTeNMP system increases as the salt concentration rises, lowering the stability of CNT dispersions in NMP [93]. 11.3.7 CNT dispersion aided by polymers CNT dispersion techniques in polymers are now receiving a lot of interest. If one polymer can be dissolved in solvents, then its CNT composites can be distributed as well; clearly, this isn’t the sole criterion for polymer-aided CNT dispersion. While in the instance of the vast majority of basic polymers, for example, poly (vinyl alcohol) (PVA), It was discovered that amorphous carbon PVA composite thin films and nanotubes (a-CNTs) may be synthesized at low temperatures [94]. To increase distribution in water, the a-CNTs were first designed and synthesized using an acid wash. The stability of the synthesized a-CNTs in water was significantly increased. It was discovered that when the a-CNT percentage in the composites increases, the resistance value of the PVAea-CNT composites fall steadily over time. When compared to the standard PVA, the composite had a greater crystallinity and a greater a-CNT percentage. For the step-by-step manufacture of composites, many polymers can be employed. As a result, PEGylated MWNTs were synthesized in preparation for solution casting of poly (vinyl alcohol) PVA/MWNT nanocomposite sheets [95]. In an aqueous PVA solution, the surface modified MWNTs demonstrated high hydrodynamic stability and increased dispersion consistency. CNTs can also be dispersed using polyethylene oxide and its analogs. Molecular dynamics simulations (MDS) were used to examine CNT associations and distribution in a PEO or water solution for such a technique [96]. The bidirectional hosteguest association among functionalized PEO (AzoPEO) and pyrene-labeled hosts connected on the surfaces of tubes of the nanostructures through pep piling resulted in a sort of light-switchable “smart” SWCNTs. Not only the above SWCNT hybrids observed to be efficiently distributed in pure water, but they also showed tunable scattering states when irradiated with UV and visible light. Several conventional

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Figure 11.3 Different applications of single-walled carbon nanotube when functionalized with various bioactive agents.

polymers might be used to regulate the scattering state of SWNTs using such a reversible hosteguest contact mechanism [97]. When CNTs are functionalized, they become extremely soluble materials that can be derivatized with active molecules to make them compatible with biological systems. In comparison to nonfunctionalized CNTs, functional CNTs have a broader biological use [98] (Fig. 11.3).

11.4 The biomedicinal and diagnostic applications of dispersed carbon nanotubes 11.4.1 Biomedical implications of dispersed carbon nanotubes (CNTs) CNTs diverse combination of optical, mechanical, and electrical qualities has sparked interest in their usage in a variety of fields, particularly biomedicine. A few obstacles must be overcome before CNT may be used in biological applications. The first is connected to safety, and it entails using CNT of extremely high integrity to restrict the emission of harmful ions while operating in any biological system. This is a significant hurdle since highly pure CNT samples are seldom produced in large quantities,

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necessitating a trade-off in terms of quantity and quality. Some other major obstacles, which are not limited to the biomedical area, are more formulation-related. One of the most important is the ability to obtain excellent CNT distribution in solvents, particularly water. Another method is to restrict or prevent drying processes during CNT production because they are always hard to spread when beginning from a dry powder [99]. The interaction among biomolecules and SWNTs has indeed been engineered to use both covalent techniques and noncovalent, with the target of protecting the biomolecules’ functional capabilities. The bifunctional chemical 1-pyrenebutanoic acid succinimidyl ester, irreversibly adsorbs on the surface of hydrophobic graphene the SWCNT, that has been produced as a noncovalent method. 11.4.2 CNT as a vehicle for drugs and gene transport Nanomaterials are extensively explored transporters of therapeutic agents, in the last decades. A major technological push has resulted in the invention, even clinical support and fruitful testing, of a variety of nanotubes. However, because to a lack of worldwide regulatory criteria for testing the protection of nanoparticles and widespread worry regarding the toxicity, there is still a major lag in their commercialization [100]. Drug delivery is typically done to enhance the pharmacodynamic and pharmacokinetic qualities of existing medications, as well as to address issues for example lack of selectivity, drug aggregation, restricted solubility and poor biodistribution, as well as to prevent the natural destruction to tissue. CNTs have lately increased appeal as prospective drug carriers, therapeutic agents, and diagnostic instruments among the already existing delivery systems, which include micro-particles, emulsions, liposomes, and polymeric micelles [101]. Drug delivery systems can be engineered to reduce toxicity and negative side effects by minimizing drug degradation, increasing bioavailability, allowing targeting to specific cells, and reducing the overall amount of drug required. The capacity of f-CNT to penetrate cells suggests that it could be used as a carrier for the transport of tiny medicinal molecules. In the treatment of cancer and many infectious diseases, the creation of delivery systems capable of carrying one or more therapeutic drugs with recognition capacity, optical signals for imaging, and/or specialized targeting is of vital benefit. In order to do this, researchers devised a unique technique for multiple functionalizations of CNT with various chemicals. CNTs were covalently bonded to a fluorescent probe for measuring the

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material’s cellular absorption and an antibiotic moiety as the active chemical. f-CNTs are a special variant of drug delivery technology that can transport and translocate drug molecules into a variety of mammalian cells. These CNT conjugates known to show no cytotoxicity in vitro, it will be necessary to examine their metabolism, biodistribution, and clearance from the body [102]. Gene delivery is also a great factor that contributed by CNTs. CNTs have a lot of potential as carriers of physiologically active compounds. Engineering a novel gene delivery system using functionalized carbon nanotubes could be fascinating. Ammonium-functionalized carbon nanotubes (f-CNTs) can bind with plasmid DNA via electrostatic interactions, according to a study. These f-CNTs permeate cell membranes and are taken up into mammalian cells when they come into contact with them. The nanotubes are noncytotoxic, and plasmid DNA coupled with fCNTs is successfully transported to cells; gene expression levels up to 10 times higher than with DNA alone [103]. 11.4.3 CNT uses for biomedical imaging There are number of most recent developments in the usage of CNTs as multifunctional nanoprobes for biomedical imaging, including SWNTs and MWNTs. Because of their unique physicochemical properties, CNTs have become a popular tool in cancer detection and treatment. They are believed to be among the greatest potential nanoparticles since they can perceive malignant cells as well as act as vehicle to carry therapeutic compounds to the target location. Cancer has been a great cause of high mortality rate; it is increasingly important to get a timely diagnosis and complete therapy. Although there are realistic risks, diagnosing and treating with nanoparticles is beneficial. Because early identification is crucial for effective therapy, advancements in detection tools are incredibly significant. Imaging investigations with SWCNTs have exploded in popularity in recent years. Nanoparticles have a significant edge over traditional techniques in terms of multifunctionality. Therapeutic drugs, targeting ligands, and a variety of other biomedicines can all be incorporated into the nanostructured to enable targeted molecular imaging by overcoming biological and biophysical walls. SWCNT-based nanoplatform act as a multifunctional nanocarrier has significant capability for medical utilizations [104]. One of the additional advances in the use of CNTs as multimodal contrast agents in biomedical imaging was, fluorescence imaging of SWNTs in the NIR-II area in vivo has advanced dramatically in recent years,

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making it a promising imaging device with significant ability in biomedical investigation [105]. Surface enhanced Raman scattering can greatly improve the Raman scattering signals of metallic nanoparticles grown on SWNTs, enabling for considerably faster Raman imaging of biological samples. Several dyes with NIR absorbance at various wavelengths were loaded into vivo for multiplexed photoacoustic imaging [106,107]. In order to provide even ‘brighter’ fluorescence, improved samples of SWNT having much quantum yield and pure chiralities are needed. Furthermore, various SWNTs exhibit emission wavelengths and separated excitation have purified single chiralities, which could be relevant for multicolor NIR-II fluorescence imaging in the future. Other fluorescence enhancement approaches, such as gold substrate-based surface resonance improvement of SWNT fluorescence, could be used to boost imaging and detection sensitivity even more [108]. Raman scattering produces distinct peaks in different locations for different molecules, making it excellent for multiplexed sensing and imaging. In the G band, the vibration frequency of carbonecarbon bonds. The carbon atom mass influences the Raman peak of SWNTs. The G band peaks of SWNTs could be adjusted by switching from 12 to 13C carbon isotopes [109,110]. External labels, including such radioisotopes, can be added to expand the adaptability of SWNT-based imaging probes, in addition to adopting the intrinsic features of SWNTs. The 125 I could be utilized to follow the biodistribution of SWNTs in animals for the first time, according to Wang et al. Instead of 125I, 14C was used as an alternative technique [111]. In a report, a widely applicable tracking approach for studying the biodistribution of functionalized CNTs in vivo was developed. Taurine covalently functionalized multi-walled carbon nanotubes (tau-MWNTs) and Tween-80 wrapped multi-walled carbon nanotubes (Tween-MWNTs) were marked with 125I and their distribution in mice was studied. Tween-80 has a remarkable ability to inhibit the RES absorption of MWNTs. The distribution of (125)I-tau-MWNTs was highly similar to that of (14)C-taurine-MWNTs, indicating that the simple (125)I labeling approach is both trustworthy and effective [112]. 11.4.4 CNTs use for phototherapy Treatment using photothermal therapy (PTT) for cancer, ablates tumors by using heat generated by absorbed light energy. By releasing tumor antigens into the local milieu, PTT can activate a host anticancer immune response. PTT usually involves injecting a light absorber directly into a tumor to

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maximize selectivity and limit harmfulness. Photothermal therapy can be used alone to reduce tumor volume, or it can be combined with an anticancer agent to create chemo-photothermal removal. Photothermal therapy shown to be effective by itself, the tumors that it can treat are limited due to interaction from skull thickness or adipose tissue thickness on muscle or brain therapies [113]. For the future of cancer treatment, Hyaluronic Acid (HA)-conjugated carbon nanomaterial offers a lot of untapped promise. The hybrid systems discussed in the research showed reduction results of tumor size that were more effective compared to the drug alone, which was connected to HA receptor-mediated endocytosis and tumor selectivity observed in fluorescent imaging data [114]. 11.4.5 CNT-based biosensors Biomarker discovery has significant promise for the early identification of disease and metabolic dysfunction; in this perspective, highly advanced sensors can provide persistent diagnosis or therapy performance. The development of a system for accomplishing this is critical for both basic biology and commercial point-of-care and home diagnostics, in which limited processability and multifunctional analytical capacity are some of the most important properties that detection systems should have [115,116]. With the rise of nanostructured materials and the beginnings of nanoparticles with unique chemical and physical properties, a new class of biosensors known as nanobiosensors has emerged, which combines the benefits of nanomaterials, such as their small size and high surface/volume ratio, with the capabilities of “macro"-biosensors.

11.5 Conclusions CNTs’ exceptional electrochemical capabilities have cleared the way for their usage as platforms for the development of a variety of electrochemical biosensors with better analytical behavior. They have a unique combination of physical, chemical, electrical, and optical properties that make them one of the best materials for signal transduction in metabolites, analytes, and disease biomarkers. The strong mechanical properties of nanomaterials, together with their unique transport qualities and other multi-functional capabilities, provide CNT a significant promise for structural and functional applications. Despite the fact that several studies have been dedicated to the creation of CNTs for various applications, their implementation in commercial goods is still in its early stages. Before this novel class of material

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can be widely employed in real goods and systems, two significant connected challenges must be addressed first one is the lack of solubility and dispersion when mixed with polymer resins, and the second one includes the poor interfacial adhesion between CNTs and different polymers. Comparing the respective mechanical and functional performances of dispersion CNT-based nanocomposites the importance of it will reach the requirement most of research and industry.

Acknowledgments Rambabu Dandela thanks DST-SERB for Ramanujan fellowship (SB/S2/RJN-075/2016), Core research grant (CRG/2018/000782) and ICT-IOC start-up grant. The authors acknowledge ICT-IOC Bhubaneswar for providing necessary support.

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Index ‘Note: Page numbers followed by “f ” indicate figures and “t” indicate tables.’

A Agglomeration and aggregation, 17 Alkylating agents, 183e184 Allotropic forms, carbon, 120f Alzheimer disease (AD), 233e234, 277 Amidation, 98e100 Aminoglycosides, 131 Ammonium-functionalized carbon nanotubes (f-CNTs), 281e283, 309e310 Amphotericin B (AMB), 130 Antihypertensive therapy antihypertensives, 136 candesartan cilexetil and diltiazem hydrochloride, 137 highly water-soluble drug release, 137 transdermal DDS, 137 water-dispersible SWCNTs, 137e138 Anti-inflammatory APIs, 20e21 Anti-inflammatory therapy anti-inflammatory agents, 133 drug loading, 134 intra-articular corticosteroid injections, 136 ketoprofen delivery, 135 NSAID delivery ketoprofen, 135 membrane technology, 135e136 transdermal drug delivery, 134e135 oxidized MWCNTs, 134 Antimetabolites agents, 185e188 5-fluorouracil (5-FU), 188 gemcitabine (GEM), 187e188 MTX-FA-MWCNT platform, 190 MTX loading, 186e187 purines and pyrimidines, 187 Antimicrobial treatments, 130e133 aminoglycosides, 131 amphotericin B (AMB), 130

antimicrobial agent, 130 carbon nanotube (CNT) antimicrobial activity, 132f surface groups, 133 surfactant solutions, 133 SWCNTs length, 133 pazufloxacin mesylate, 131 tuberculosis (TB) treatment, 131e132 Antimicrotubule agents, 184e185 Antineoplastic APIs, 19e20 Antioxidant delivery gallic acid, 138e139 quercetin and rutin, 138 tocopheryl PEG succinate (TPGS), 138 Antioxidant properties, 263e264 Arc discharge, 42 Atom-transfer radical polymerization (ATRP), 84e85, 104e105

B Bacterial magnetic nanoparticles (BMPs), 237e238 Ballistic conduction, 261e262 Ball milling technique ammonium bicarbonate (NH4HCO3), 304 high energy, 303e304 long-term, 303e304 milling conditions, 303e304 b Cyclodextrins (b-CDs), 110e112 Bioelectronic system, 263e264 Bioimaging application, 3t Biomimetic carbon nanotubes Biosensing application, 3t Biosensors, 312 Bloodebrain barrier (BBB), drug delivery, 238e240 Brain tumors, 233e234

321

322

Index

C Cancer therapy CNTs-based nanocarriers alkylating agents, 183e184 antimetabolites agents, 185e188 antimicrotubule agents, 184e185 gene delivery, 194 hyperthermia therapy, 193e194 nuclear medicine imaging (NMI), 190e192 radiotherapy, 188e190 topoisomerase I/II inhibitors, 179e183 in vitro viability assays, 194e197 functional CNTs, 265e276, 267te274t nanotechnology systems advantages, 176e177 nanomaterials, 176e177 nanomedicine, 176e177 nanostructure-based products, 177 photothermal therapy (PTT), 311e312 sensor studies and clinical study, 267te274t Carbon allotropes, 10 Carbon-mediated nucleic acid delivery, 159f Carbon nanohorns, 219e220 Carbon-nanotubes-doped sericin scaffold (CNTs-SS), 242e244 Carboplatin, 213 Carboxylic acid-functionalized multiwalled CNTs (MWCNTs), 241e242 Cardiovascular APIs, 21e22 Catalytic chemical vapor deposition (CCVD), 64 Caveolin-mediated endocytosis process, 16 Cell transfection properties, 232e233 Central nervous system (CNS) illnesses, 233e234 Chemical vapor deposition (CVD) method, 43e44, 230e232 carbon-containing gas and process gas, 64e65 catalytic chemical vapor deposition (CCVD), 64

types, 64 Chirality, 12, 262 Cis-dichlorodiammine platinum eSWCNTs (CDDPeSWCNTs), 183e184 Cisplatin, 206e208, 207f emetogenicity, 213 nephrotoxicity, 212e213 ototoxicity, 213 Conjugated polymer (CPs), 107e108 Conventional chemotherapeutics, 206 Copper transporter 1, 211 Covalent functionalization, 95, 158e159, 306 advantages, 128 classification, 71e74 cyclobutadiene and benzene, 67e69, 69f end-functionalization, 128, 128f Huckel’s rule, 69e70 misalignment of pz orbitals, 67, 68f multistep acid treatments, 127 phenyl diazonium addition, 72e74 phenyl groups configuration, 74f sidewall functionalization, 128e129, 129f soft functionalization, 71e72 sp2 carbon geometry, 66e67, 67f stability, 66 structural defects, 70e71 COVID-19, 276e277 Cycloaddition, pristine carbon nanotubes (pCNTs), 102e105 Cytotoxicity, 211, 244e245

D Direct covalent sidewall functionalization, 158e159 Direct cycloaddition, 103 Dispersed carbon nanotubes ball milling, 303e304 biomedical imaging, 310e311 biomedical implications, 308e309 biosensors, 312 chemical methods, 306 criteria, 301 dispersive materials, 298te300t

Index

drugs and gene transport, 309e310 inorganic salts, 306e307 noncovalent functionalization, 297e301 photothermal therapy (PTT), 311e312 physical methods, 302 plasma and irradiation techniques, 305e306 polymers, 307e308 preparation techniques, 302f significance, 297e301 ultrasonication, 302e303 DNA/RNA aptamers, 168e169 DNA wrapping approach, 305e306 Docetaxel (DTX), 184e185 Double-walled carbon nanotubes (DWCNTs), 11, 146e147, 231f Drug delivery acetylcholine, 139 antihypertensive therapy, 136e138 anti-infective APIs, 22e23 anti-inflammatory APIs, 20e21 anti-inflammatory therapy anti-inflammatory agents, 133 drug loading, 134 ketoprofen delivery, 135 oxidized MWCNTs, 134 transdermal drug delivery, 134e135 antimicrobials treatments, 130e133 antineoplastic APIs, 19e20 antioxidant delivery, 138e139 cardiovascular APIs, 21e22 CNT preparation, 121, 121t conjugated CNT-liposomes, 23 drug delivery systems, 119 gene therapy, 23 multi-wall CNT (MWCNTs), 120 single-wall CNTs (SWCNTs), 120 surface engineering covalent modification, 124, 127e129 noncovalent functionalization, 125e127 surface functionalization, 124 theophylline, 139 Drug loading, carbon nanotubes agglomeration and aggregation, 17 amphiphilic/hydrophilic polymers, 13e14

323

cell type, 18e19 cellular uptake, 15e16 drug permeation, 15 endocytosis, 16 macropinocytosis mechanism, 16 mechanisms, 15 cisplatin delivery system, 14f endohedral modification, 14e15 mechanism, 13t size, 17 surface charge, 17e18

E Elastic modulus, 261 Electric arc discharge, 62e63 Electrolysis, 65 Electronic properties, 39e40, 262e263 Electron microscopy high-resolution transmission electron microscopy (HR-TEM) amorphous carbon fiber, 48, 49f ceria nanoparticles, 50f crystallinity, 48 metal nanoparticles, 48e49 scanning electron microscopy (SEM), 47, 48f Endohedral functionalization, 76e77 Endohedral modification, 160 Epichlorohydrin, 83e84 Esterification, 98e100 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxy succinimide (NHS) coupling reaction, 99e100

F Fluorination, 79, 100, 102f Fullerene, platinum based anticancer drug delivery, 220e221 Full width at half maximum (FWHM), 54e55 Functionalization carbon nanotube Ag nanoparticles, 82 amines compounds, 79e80 atom transfer radical polymerization (ATRP), 84e85

324

Index

Functionalization (Continued) carboxylic carbon nanotubes, 81 carboxylic groups, 78 chemical functionalization, 77 chitosan, 81e82 coupling and nucleophilic addition reactions, 83 covalent functionalization, 77e78 fluorination, 79 nitric acid, 80 organometallic point, 83e84 oxidation process, 79 paclitaxel (PTX) loaded PEG-gCNTs, 85e86 poly(acryloyl chloride) (PAC), 86 polyethylene glycol f-MWCNTs, 88 covalent functionalization. See Covalent functionalization endohedral functionalization, 76e77 non-covalent functionalization, 74e76 Functionalized carbon nanotubes (fCNTs) anticancer drugs, 122 antiviral and antibacterial agents, 122e123 chemical approaches, 258e259 covalent chemistry, 257e258 drug and gene delivery, 281e283 end defect functionalization, 258e259 functionalization, 257e258, 257f functionalizing agents, 259e260, 259f medical applications, 123f noncovalent functionalization, 257e258 nonviral vectors. See Nonviral vectors theranostics applications cancer, 265e276, 267te274t infectious diseases, 276e277 neurodegenerative diseases, 277 personalized medicine, 283e285 tissue generation, 124 Functionalized nanomaterials, 177 Functional neurosurgery, 240e241

G Gallic acid delivery, 138e139 Gas-phase oxidation, 97e98 Gene delivery, 309e310 therapeutically active gene interventions, 157

viral and nonviral vectors, 157 Gene therapy, 23 Graphene, platinum based anticancer drug delivery, 220e221 Graphene sheet folding directions, 41f Graphene structure, 256f

H Halogenation, pristine carbon nanotubes (pCNTs) chlorination/bromination, 100e102 fluorinations, 100 Heat capacity, 39 Hemotoxicity complement activation, 146e147 human serum proteins interactions, 146 surface-functionalized SWCNTs with acid groups (AF-SWCNT), 147 High-speed vibration milling technique (HSVM), 110e112 Hyaluronic Acid (HA)-conjugated carbon nanomaterial, 311e312 Hybrid nanomaterial-based drug delivery system, 219 Hydrothermal method, 65e66 Hyperthermia therapy MWCNT/AuNPs hybrids, 193e194 PTT and PDT strategies, 193, 193f

I Immunogenicity, 244e245 Infectious diseases, 276e277, 278te280t Inorganic salts, CNT dispersion, 306e307 Ischemic stroke treatment carbon-nanotubes-doped sericin scaffold (CNTs-SS), 242e244 multi-walled CNTs (MWCNTs), 241e242 MWCNTs-NGF combination, 242e244 numbness, 241e242 vertically aligned MWCNTs (VA-MWCNTs), 242e244 Isoniazid (INH), tuberculosis (TB) treatment, 131e132

Index

K

multiple functionalization, 218e219 wet chemical approach, 217 pulmonary toxicity, 144 Raman spectra, 51f, 52, 54f single stranded DNA (ssDNA) conjugation, 100 structural properties, 260 synthesis, 11 thermal conductivity, 261e262 thermogravimetrical analysis, 55e57 tuberculosis (TB) treatment, 131e132 X-ray diffraction, 54e55

Ketoprofen delivery, 135

L Lab-on-chip-devices, 3t Lactate dehydrogenase (LDH) assay, 196 Laser ablation, 42e43, 63e64, 230e232 Liquid-phase oxidation, 97e98

M Macropinocytosis mechanism, 16 Mechanical properties, 38e39, 261 Microtubules, 184 Mononuclear phagocyte system (MPS), 160e161 Morphology, 10 Multi-walled carbon nanotubes (MWCNTs), 40, 296 Ag-MWCNTs nanocomposites, 81e83 alkyne functionalized, 104e105, 106f amino-modified, 98e99 anti-inflammatory therapy, 134 antimicrobial activities, 132e133 antimicrobials treatments, 131 architectures, 229e230, 231f cancer, 265e276, 267te274t carboxylic acid-functionalized, 241e242 categories, 120 cytotoxicity, 144e145 diameter, 120 dispersion characteristics, 305e306 electric arc discharge, 62 Fast Fourier transform (FFT), 53f grafting polymers, 85 graphene structure, 256 HR-TEM images, 52, 53f intra-tumoral administration, 232e233 mannosylated, 131 MWCNT-based AMB conjugate, 130 MWNT-HCPT conjugates, 101f neurodegeneration, 236e237 platinum-based anticancer drug delivery, 217e219 carbon nanotube bottle, 218 carboplatin encapsulation, 217 cisplatin prodrug, 217 hybrid nanomaterials, 219

325

N Nanobiosensors, 312 Nanocarriers, 205, 238e239 Nanodiamonds (NDs), 306 Nanoimaging techniques, 240e241 Nanomedicine, 1e2, 205 Nanoneedle effect, 162 Nanoparticles, 1e2 Nanotechnology, 295 Neurodegeneration, 235e237 Neurodegenerative diseases, 277 Neurological disease prevention drug delivery, bloodebrain barrier, 238e240 functional neurosurgery, 240e241 ischemic stroke treatment, 241e244 neurodegeneration, 235e237 neuroprotection, 237e238 Neurological related conditions, 234f Neuronal tissue, 234e235 Neuroprotection, 237e238 Neuroregeneration, 236t N-methyl-2-pyrrolidone (NMP) dispersion, 306e307 Noncovalent CNT functionalization, 126f advantage, 125 characteristics, 126f helical model, 127 higher molecular weight systems, 125e126 thermodynamic model, 127 Noncovalent functionalization, 306 Noncovalent functionalization, 257e258

326

Index

Non-covalent modification, 159e160 Nonreceptor mediated endocytosis, 161f Nonviral vectors carbon-mediated nucleic acid delivery, 159f CNT-based gene vectors DNA/RNA aptamers, 168e169 endosomal entrapment, 160e161 enzymatic degradation, 162 mononuclear phagocyte system (MPS), 160e161 nanoneedle effect, 162 oligonucleotides (ODNs), 167e168 penetration pathways, 161f plasmid DNA delivery, 162e163 RNA interference (RNAi), 164e167 endohedral modification, 160 exohedral modification covalent modification, 158e159 non-covalent modification, 159e160 pristine CNTs (pCNTs), 158 Nuclear medicine imaging (NMI) positron emission tomography (PET), 191 single photon emission computed tomography (SPECT), 191 SPION-MWCNT hybrids, 192 Nucleic acid functionalization, 112

O Oligonucleotides (ODNs), 167e168, 168f Oxaliplatin, 211e212 Oxidation techniques, 97e98 Oxidized single walled carbon nanohorns, 219e220 Oxygen plasma activation, 305 Ozonation, 79

P Paclitaxel (PTX), 184e185 Parkinson disease, 277 Pazufloxacin mesylate, 131 Peptide functionalization, 113 Permeability and retention (EPR) effect, 232e233 Personalized medicine, 283e285

Photothermal therapy (PTT), 3t, 311e312 Picoplatin, 208 Plasma and irradiation techniques, 305e306 Platinum-based anticancer drugs BBR3464 and 56MESS, 208 cisplatin, 206e208, 207f combinatorial therapy, 206 cytotoxicity, 211 DNA adduct, 212 drug delivery system carbon nanohorns, 219e220 carbon nanotubes (CNTs), 215f cisplatin, 215f graphene and fullerene, 220e221 multi-walled carbon nanotubes, 217e219 single-walled carbon nanotubes, 214e217 intercalators, 208 limitations, 212e213 mechanism of action, 211e212 nanocarrier-based targeted delivery, 206 neurotoxicity, 213 picoplatin, 208 platinum complexes, 209te210t rate of aquation, 211e212 Poly (vinyl alcohol) (PVA)-a-CNT composites, 307 Polybenzimidazoles (PBIs), 108e109 Poly(citric acid) (PCA)-functionalized MWCNTs, 185 Polyimides (PIs), 108e109 Polymer-aided CNT dispersion, 307e308 Positron emission tomography (PET), 191 Post-cycloaddition, 104e105 Pristine carbon nanotubes (pCNTs), 95 covalent modification, 96e106, 97f cycloaddition, 102e105 direct sidewall functionalization, 96 esterification and amidation, 98e100 halogenation, 100e102 oxidation, 97e98 radical addition, 105e106

Index

debundling, 96 dispersion, 96 noncovalent modification biomolecular coating, 112e113 polymer coating, 107e109 polysaccharide coating, 109e112 Purification acid treatment method, 46f ferrocene, 45 infrared (IR) spectroscopy, 45, 46f

Q Quantitative approaches bibliometric searches, 4e5 drug delivery system, 3e4 keyword search, 6e7 prisma flow diagram, 5f publication, 9f review documents, 5 trend topic growth, 7e8, 7f Quantum dots, 240e241

R Radical addition, 105e106 Radiotherapy nanomaterials, 188e189 radiation beam exposure, 188 radioisotope, 188 tumor clearance, blood compartment, 189 Raman spectroscopy inelastic light scattering, 50e51 intensity changes, 51e52 MWCNTs, 51f, 54f Receptor mediated endocytosis, 161f RNA interference (RNAi) PEI-g-GNR, 167 shRNA delivery, 166, 167f siRNA delivery vectors, 164e166, 166f surface-modified CNTs, 164 tumor-bearing nude mice model, 165f Rosiglitazone, 21 Russian doll model, 229e230

327

S Safety profile, CNTs, 24 SARS-CoV-2 virus, 276e277, 278te280t Scanning electron microscopy (SEM), 48f Schizophyllan (SPG), 109e110 Single-walled carbon nanotubes (SWCNT), 40, 296 acetylcholine loading, 139 antimicrobials treatments, 132e133 antioxidants delivery, 138e139 architecture, 229e230 bioactive agents, 308f biomedical imaging, 310 cancer, 265e276, 267te274t covalent functionalization, 142e143 covalent modification DielseAlder reaction, 103, 105f electric arc discharge, 62 fluorescence imaging, 310e311 forms, 120 functionalization, 128e129, 129f gamma-irradiation, 305e306 graphene structure, 256 hemotoxicity, 146 infectious diseases, 278te280t neurodegeneration, 236e237 noncovalent modification nucleic acid functionalization, 112 peptide functionalization, 113 polymer coating, 107e109 polysaccharide coating, 109e112 octadecylamine reaction, 125 platinum-based anticancer drug delivery, 214e217 carboplatin encapsulation, 216e217 cisplatin encapsulation, 216 platinum (IV) prodrug, 214e216, 217f structural properties, 260 SWCNTeAMB conjugate, 130 synthesis, 11 thermal conductivity, 261e262 Sonication-assisted oxidation, 97e98 Stem cells differentiation, 235e236

328

Index

Stimulated drug release, 21 Stroke, 241e242 Structural properties, carbon nanotubes, 260 Structure, 10 Surface-functionalized SWCNTs with acid groups (AF-SWCNT), 147 Synthesis of carbon nanotubes, 61f arc discharge, 42 chemical vapor deposition (CVD), 43e44 chemical vapor deposition (CVD) method, 64e65 electric arc discharge, 62e63 electrolysis, 65 elements needed, 42f hydrothermal method, 65e66 laser ablation, 42e43, 63e64

T Taurine covalently functionalized multi-walled carbon nanotubes (tau-MWNTs), 310e311 Telomerase reverse transcriptase siRNA (TERT), 194 Theranostics applications, 265f cancer, 265e276, 267te274t infectious diseases, 276e277 neurodegenerative diseases, 277 personalized medicine, 283e285 Therapeutically active gene interventions, 157 Therapeutic studies, 282t Thermal conductivity, 39, 261e262 Thermal properties, 39, 261e262 Thermogravimetrical analysis, 55e57 Tissue engineering study, 3t, 277e281, 282t Tocopheryl PEG succinate (TPGS), 138 Topoisomerase inhibitors, cancer therapy anthracyclines, 181 CPT-loaded multiwalled CNTs (MWCNTs), 180 daunorubicin (DAU) loading, 182e183

DNA double strain breaks, 179 DNA replication and apoptosis arrest, 180 etoposide (ETO), 181 folic acid (FA) conjugation, 182 10-hydroxycamptothecin (HCPT) loading, 180 irinotecan encapsulation, 180e181 polymer-coated MWCNTs, 180 Toxicity, 244e245, 296 and biosafety considerations, 284e285 CNT dimensions, 143e145 CNT surface modification, 140e143 covalently functionalized CNTs, 142e143 harmful effects, 140 hemotoxicity, 146e147 noncovalently functionalized CNTs, 141e142, 141f purity, 145 route of administration, 146 Toxicology, 24 Tween-80 wrapped multi-walled carbon nanotubes (Tween-MWNTs), 310e311

U Ultrasonication, carbon nanotubes dispersions, 302e303 UV stabilizing activity, 263e264

V Vertically aligned carbon nanotubes (VACNTs), 47e48, 48f Vertically aligned MWCNTs (VAMWCNTs), 242e244 Viability assay, tetrazolium salts, 195f

X X-ray diffraction, 54e55

Y Young’s modulus, 38e39, 261