Pragya Yadav-DDL-MS

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Carbon Nanotube: A Versatile Carrier for Various Biomedical Applications Pragya Yadav*, Vaibhav Rastogi, Arun Kumar Mishra and Anurag Verma School of Pharmaceutical Sciences, IFTM University, Moradabad, Uttar Pradesh, 244001, India Abstract: With the development of nanotechnology different biological nanomaterials have been developed, out of these, Carbon Nanotubes (CNTs) have attracted attention of various scientists as a carrier for the delivery of therapeutic agents. CNTs are third allotropic form of carbon-fullerenes which consist of graphite sheet rolled up into a cylindrical tube. They have nanometer scale diameter with high aspect ratio. CNTs exist in two forms, single-wall (SWCNTs) and multiwall (MWCNTs) depending on the number of graphene layer they are surrounded with as well as to the method of synthesis. Techniques have been developed to produce nanotubes in sizeable quantities, including Electrical arc discharge, Laser ablation, Chemical vapor deposition, Gas phase catalytic processes, as they tend to strip carbon atoms off from the carbon bearing compounds. Various techniques are available for the evaluation of CNTs which mainly involve the estimation of purity, functionalization and structure variations. Presence of impurities in the CNTs makes them vulnerable to be used as carrier in drug delivery hence CNTs need to undergo purification. To integrate into biological system CNTs can be loaded with active molecules by forming stable covalent bonds or supramolecular assemblies based on non-covalent interactions. Functionalized CNTs have been remarkably shown to exert their potential benefits in drug delivery, drug targeting and various other fields such as diagnostic, tissue regeneration. This review attempts to highlight all aspects of CNTs such as their structure, characterization, synthesis and purification, functionalization, bio-compatible applications in the clinical science and toxicity related issues due to CNTs.

Keywords: Carbon nanotubes, nanotechnology, carrier, functionalization, electrical arc discharge, laser ablation, chemical vapor deposition, toxicity. INTRODUCTION Since the discovery of fullerenes, carbon molecules are cage-like hollow-sphere conformations, by Smalley, Kroto, Curl and co-workers in 1985, numerous ground breaking advances have been made in the field of carbon nanotechnology [1]. The word “nano” defines the scale that is used to describe systems applied in the field of nanoscience [2]. Nanotechnology is the convergence of engineering and molecular biology, leading to the development of structures, devices and systems that have novel functional properties with size ranging between 1-100 nm. Carbon materials on a nanometer scale have unique physicochemical properties that are due to their small size, surface area, chemical composition, surface structure, solubility, and shape [3]; these properties have thus far been utilized in various fields including drug and gene delivery, imaging and diagnostics [4,5]. CNTs have attracted tremendous attention as one of the most promising nanomaterials after the discovery of the third allotropic form of carbon-fullerene in 1991, Sumio Iijima identified a new structural form of this allotrope the cylindrical fullerene and named as carbon nanotubes [6]. This potential carbon skeleton opens an era of introducing CNTs in the form of drug delivery carrier and an abeyant imaging agent which has now far attracted many researchers for exploring a variety of biomedical applications owing to its unique *Address correspondence to this author at the School of Pharmaceutical Sciences, IFTM University, Moradabad, Uttar Pradesh, 244001, India; Tel: +91-9451284944; E-mail: [email protected] 2210-3031/14 $58.00+.00

structural properties and ability of the functional groups to undergo conjugation with the therapeutic drug entity [7]. The proceeding paragraphs will illustrate the detailed information about the structural alignment of CNTs, method of synthesis, characterization, eligibility to get functionalized with the drug and their numerous applications in biomedical science. CARBON NANOTUBES CNTs are graphene sheets rolled into a seamless cylinder that can be open ended or capped, having a high aspect ratio with diameters as small as 1nm and a length of several micrometers [8]. Depending on the number of sheets rolled into concentric cylinders, there are two broad categories of CNTs, namely, single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). SWCNTs are made up of single layer of graphene molecules whereas MWCNTs consist of several coaxial cylinders, each made of a single graphene sheet surrounding a hollow core (Fig. 1). Carbon atoms constituting CNT walls possess sp2 hybridization, which generate a delocalized electron cloud along the wall which is similar to graphene. Such an electron cloud is responsible for the π-π interactions existing between adjacent cylindrical layers in MWCNTs, although a fraction of interwall sp3 bonds can also be found [9]. SWCNTs can be either metallic or semiconducting depending upon the tube diameter and the chirality (the sheet direction in which graphite sheet (graphene) is rolled to form a nanotube). The hexagonal lattice structure of CNTs, gives rise to three types of SWCNTs. Based on the unit cell of a CNT it is possible to © 2014 Bentham Science Publishers

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Fig. (1). Molecular structures of CNT fragments: (left) SWCNT and (right) MWCNT. The distance between the MWCNT graphene layers is 0.34 nm.

identify (i) armchair nanotubes (chiral angle is 30°); (ii) zigzag nanotubes, (chiral angle is 0°); and (iii) chiral tubes, with chiral angles intermediate between 0° and 30°[10]. Comparison between these two types of CNTs is mentioned in Table 1.

three main approaches for the synthesis of CNTs including electric arc discharge, laser ablation and chemical vapor deposition (CVD) approach. Electric Arc Discharge Method

METHODS OF SYNTHESIS The production of CNTs results in the transformation of a carbon source into nanotubes, usually at high temperature and low pressure, wherein the synthesis conditions influence the characteristics of the final product. Presently, there are Table 1.

The method was originally intended for the synthesis of fullerenes, the allotropic form of carbon discovered in 1985 [22]. The arc discharge method (Fig. 2) involves the generation of an electric arc through the passage of electric current (in the range of 50-100 A, driven by an applied voltage of

Comparison Between SWCNTs and MWCNTs.

Single-walled carbon nanotubes (SWCNTs)

Multi-walled carbon nanotubes (MWCNTs)

Made up of single graphene layer wrapped into a cylindrical structure and their diameter varies from 0.4-3.0 nm and length ranges from 20-1000 nm [11].

Consist of several coaxial cylinders, each made of a single graphene sheet surrounding a hollow core. The outer diameter of MWCNTs ranges from 2 to 100 nm, while the inner diameter is in the range of 1-3 nm, and their length is 1 to several µm [12].

Usually occur as hexagonal close-packed bundles in which the nanotubes are held together by van der Waals forces.

They are mainly monodispersed.

They can be either semi-conducting or metallic [13].

They are only semi-conducting [13].

They have better defined walls.

They more likely to have structural defects, resulting in a less stable nanostructure.

SWCNTs are produced by electric arc [14], laser ablation [15], chemical vapor deposition (CVD) [16] and gas-phase catalytic processes (HiPco or High-Pressure Co-Conversion) [17].

Most common techniques for their production are electric arc [18] and chemical vapor deposition (CVD) [19, 20].

Their growth requires a metal catalyst, usually Fe, Ni, Co, Y or Mo.

The growth of MWCNTs may require a catalyst.

Bulk synthesis is difficult as it requires proper control over growth and atmospheric condition [21].

Bulk synthesis is easy [21].

Less accumulation in body [21].

More accumulation in body [21].

Characterization and evaluation is easy [21].

They have very complex structure [21].

They can be easily twisted and are more pliable [21].

They cannot be easily twisted [21].

Carbon Nanotubes Based Drug Carrier and Their Applications

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Fig. (2). Schematic drawing of the direct-current arc discharge method for producing carbon nanotubes [24].

approximately 20 V between electrodes) across two closebut-non-contacting carbonaceous electrodes (separated approximately by 1 mm) in an inert gas atmosphere (filled at low pressure 50-700 mbar) [14, 23]. The method relies on the electrical breakdown of gas molecules located in the gap between two carbon electrodes. As a result, a continuous electric discharge from one electrode to the other occurs through the normally nonconductive gas. Such an extreme condition turns the gas into plasma- a fluid of negatively and positively charged particlesgenerating a temperature high enough to vaporize most materials. Accordingly, the electric arc vaporizes carbon, which reassembles in a variety of allotropic forms, including nanotubes [25]. The anode is hotter than the cathode due to electron collisions, so carbon vaporization occurs in the former. This results in dimensional changes in the anode, requiring its continuous adjustment to ensure that the separation with the cathode is maintained constant. Additionally, both the cathode and the usually smaller anode are water-cooled during the synthesis. The synthesized material can be typically found deposited on the cathode after several minutes of anode evaporation. The appearance of the grown material is the carbonaceous powder, which upon inspection using scanning electron microscopy (SEM) reveals the presence of needlelike structures (CNTs) in the carbon soot [23]. The two most important parameters to be taken care of in this method are: 1) the control of arcing current and 2) the optimal selection of inert gas pressure in the chamber [26]. The arc-discharge technique produces high quality MWCNTs and SWCNTs. The incorporation of a metal catalyst at either on the cathode or the anode is mandatory for the synthesis of SWCNTs using arc discharge method. The first SWCNTs synthesized by this method used iron or cobalt as catalyst, but only a low yield was achieved [27,28]. A difference with the formation of MWCNTs is that SWCNTs do not appear in the normal deposit on the cathode, but rather in other parts of the chamber [28]. Laser Ablation Method The process was developed by Smalley in 1995, with the intention of improving the purity of the final product [29]. In

this method intense pulse of laser light was directed on a carbon surface in a stream of helium gas. The evaporated material condenses to yield fullerenes. However, it was later noticed that the incorporation of a metal catalyst in the carbon target leads to the formation of SWCNT with a narrow diameter distribution and high yields. The general set-up for laser ablation is shown in Fig. 3. To produce single-walled nanotubes, the graphite target was doped with cobalt and nickel catalyst [15]. Briefly, the method relies on focusing an energetic laser beam on an ablation target that is majorly made of carbon and small amounts of transition metals, which results in a laser plume due to the vaporization target. Thus, the laser plume contains vaporized carbon and metallic nanoparticles that lead to the reassembling of carbon in the form of nanotubes [25]. There is a major difference between arc discharge method and laser ablation method that the former shown to produce both MWCNTs and SWCNTs, whereas the latter has only been shown to produce SWCNTs. Some of the major parameters that determine the amount of CNTs produced are the amount and type of catalysts, laser power and wavelength, temperature, pressure, type of inert gas present, the fluid dynamics near the carbon target, etc [30]. Chemical Vapour Deposition (CVD) This method involves the reaction and/or decomposition of one or more volatile carbon precursors on substrates containing catalytic metal nanoparticles to produce CNTs [16]. The general set-up for laser ablation is shown in Fig. 4. Historically, CVD had been widely used to catalytically decompose hydrocarbon gases such as methane and ethylene to obtain different carbon products such as carbon fibers [31]. CVD growth mechanism generally involves the dissociation of hydrocarbon molecules and saturation of carbon atoms in the catalyst metal nanoparticles. The precipitation of carbon from the saturated metal particles leads to the formation of carbon nanotubes. Use of catalyst reduces the need for high temperatures. Hydrogen from the decomposition process contributes to the activation and reactivation of the catalytic surface.


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Fig. (3). Schematic representation of Laser Ablation method used for carbon nanotube synthesis [24].

Fig. (4). Schematic representation of Chemical Vapour Deposition method used for carbon nanotube synthesis [24].

The literature available with chemical vapour deposition methods is really extensive, which also shows how this multivariable process can been adjusted in several manners to render various CVD-based methods such as thermochemical CVD (traditional), plasma enhanced CVD, aerosol assisted (AA-CVD), aerogel supported, high pressure CO disproportionation (HiPCO), alcohol catalytic CVD (AACCVD) in ambient or vacuum base pressure, the CoMoCAT ( Co-Mo catalyst) CVD process, and even a hybrid laser assisted thermal CVD (LCVD) among other [32].

Table 2. Different Purification Approaches Based on CNTs Synthesis Methods. CNTs synthesis method

CNTs PURIFICATION Purification processes are needed in order to satisfy the requirements of highly purified CNTs for the majority of applications so that the possibility of adverse effects associated with the impure CNTs will be nullify. The most common impurities are carbonaceous materials, whereas metals are the other types of impurities generally observed [26]. The type and amount of impurities depend upon the synthesis process used, and therefore they require the use of specific purification techniques [33]. The different approaches used in accordance with the impurities associated with the respective synthesis procedures are given in Table 2.

Purification approaches used

Arc Discharge Method

Purification by oxidation: 1. Gas phase purification [34] 2. Liquid phase purification [35]

Laser Ablation method

Approach proposed by Bandow et al. in which they used cationic surfactant and trapped SWCNTs on a membrane filter [36].

Chemical Vapour deposition

Approach proposed by Xu et al. in which the purification processes included were sonication, oxidation and acid washing steps [37].

Apart from above mention approaches some other approaches are as follows: •

Ultrasonically assisted microfiltration used by Shelimov et al. for purifying SWCNTs from amorphous and crystalline carbon impurities and metal particles [38].


•

Boiling CNTs in nitric acid aqueous solutions to remove amorphous carbon and metal particles, an approach proposed by Dujardin et al. [39].

•

Harutyunyan et al. developed a scalable purification method for SWCNTs by using microwave heating in air followed by treatment with hydrochloric acid [40].

CNTs FUNCTIONALIZATION CNTs are materials practically insoluble, or hardly dispersed, in any kind of solvent. As drug carriers, the solubility of CNTs in aqueous solvent is a prerequisite for gastrointestinal absorption, blood transportation, secretion, and biocompatibility and so on; hence, CNT composites involved in therapeutic delivery system must meet this basic requirement. Addition of functional groups to the CNT side walls makes the tubular structures soluble in aqueous solutions, less harmful to the organism cells, and therefore biocompatible, allowing the advantage of their excellent physical properties to be used within biological environments [25]. The requirements of delivery systems can commonly classify into two categories: first to improve the efficacy, and second to reduce toxicity through enhancement of specificity [41]. Functionalization reduces the toxicity caused by the highly hydrophobic surface of the nanotubes. Recently, researches have found that carbon atoms in both SWCNTs and MWCNTs can exhibit chemical reactivity toward many reagents to some extent and so both CNTs can be considered as new macromolecular form of carbon which are particularly crucial to enable the functionalized CNTs to be used as a drug delivery system, for example integrin αvβ3 monoclonal antibody conjugated phospholipid-polyethylene functionalized SWCNTs were developed for effective and efficient targeting integrin αvβ3 – positive Human Neuronal Glioblastoma (U-87 MG) cells with low cellular toxicity [42]. Ren et al., developed a dual targeted PEGylated functionalized MWCNTs and loaded with angiopep2 (ANG) and doxorubicin, respectively to target low density lipoprotein receptorrelated protein receptor which is over expressed on the blood brain barrier (BBB) and C6 glioma cells and reveal better anti-glioma effect with good biocompatibility and low toxicity[43]. Triple functionalized SWCNTs were fabricated with an anticancer drug (Doxorubicin), a monoclonal antibody and a fluorescent marker (fluorescein) at the non-competitive binding sites on the SWCNTs for targeting the cancer cells


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[44]. A novel system based on polysaccharide sodium alginate and chitosan-modified SWCNTs was developed for controlled release of doxorubicin, having folic acid as a targeting ligand [45]. In another study, gonadotrophin-releasing hormone (GnRH) modified MWCNTs were fabricated to readily enter the prostate cancer cells (DU145 cells) and kill them effectively [46]. Functionalization can be divided into two main subcategories: non-covalent functionalization and covalent functionalization. Covalent Functionalization The chemical functionalization of CNT is based on two main approaches: (i) esterification or amidation of oxidized tubes (ii) side-wall covalent attachment of functional groups. (i) Esterification or Amidation of Oxidized Tubes CNT can be oxidised, giving CNTs hydrophilic group such as OH, COOH and so on. This treatment results in the opening of CNT end caps, generating carboxylic groups suitable for further derivatization [47]. In addition, carboxylic functions are created where defects of the nanotubes side walls are present. (ii) Side-wall Covalent Attachment of Functional Groups Addition of functional groups to the side walls of carbon nanotubes is frequently carried out by chemical reactions between functional organic molecules and the SWCNTs surface by using reactive species such as nitrenes, carbenes and radicals (Fig. 5) [48]. Non-covalent Functionalization The non covalent dispersion of CNT in solution allows preservation of their aromatic structure and thus their electronic characteristics. This can be done by the addition of hydrophilic polymers, biopolymers and surfactants. A series of anionic, cationic and non ionic surfactants have been already proposed to disperse nanotubes. Sodium dodecyl sulfate (SDS) and benzylalkonium chloride are other good examples of surfactants non-covalently aggregated to the nanotube side walls (Fig. 6) that facilitate dissolution of carbon nanotubes in water, besides providing an effective charge to the nanotube surface which enables their later puri-

Fig. (5). Covalent functionalization of SWCNT by addition of pyrene functional groups on side walls by 1,3 dipolar cycloaddition.


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Fig. (6). Non covalent functionalization of SWCNT by pyrene groups (directly added to the nanotube surface due to π-π stacking interactions).

fication. In the case of surfactants, non-covalent hybrids are formed after molecules with high surface activity are aggregated to the hydrophobic side walls of the SWCNTs. The adhesion between surfactants and nanotube walls becomes very strong due to the π-π stacking interactions resulted from the attachment of aromatic groups of the amphiphile surfactant to the aromatic network of the nanotube side walls, as evidenced in the case of adhesion of N-succinimidyl-1pyrenebutanoate. For the solubilization of CNT, polymers represent a good alternative to surfactants although they do not have better dispersion efficiency [49]. In this, polymer wrap around the tubes. Thus, solubility and conductivity properties are appreciably modified after wrapping the nanotube surface with polymers. For example, polyvinylpyrrolidone (PVP), having polar sides along its long chain, assists the dissolution of PVP/SWCNT aggregates in polar solvents. Similarly Star et al. have substituted poly(metaphenylenevinylene) to suspend SWCNT in organic solvents [50]. Biopolymers can also be used for the functionalization of CNT. Nucleic acids are certainly ideal candidates to form supramolecular complexes based on π- π stacking between the aromatic bases and the CNT surface. Indeed, Zhao et al. reported the DNA adsorption on a single-walled carbon nanotube (SWCNT) in an aqueous environment. The hydrophobic end groups of DNA are attracted to the hydrophobic surface of uncharged SWCNTs, while the hydrophilic backbone of DNA does not bind to the uncharged SWCNT [51,52].

shows the main characterization parameters along with their measuring techniques. Electron Microscopy It is a useful technique for direct observations of impurities, local structures as well as defects in CNTs. The local structure of the CNT can be investigated at the nanometer and subnanometer level by TEM, SEM, AFM and STM. TEM is used to assess length, structural integrity, diameter, structural changes (caused by surface modification) and dispersion state of CNT [53]. TEM can also be used to detect and identify sample impurities (carbonaceous and metal nanoparticles). Metal nanoparticles are easily recognized like dark spots in TEM images [54], this is due to the differences in their electron transmission properties compared with the majority carbon atoms. SEM attached with energy dispersive X-ray (EDX) microanalysis (also called energy dispersive spectroscopy (EDS) can provide a quantitative estimation of metal content in microscopic surface regions of CNTs. Raman Spectroscopy

1) Estimation of metal content (metal catalysts)

The Raman bands CNT spectra provide information about the diameter, chirality, conductor or semiconductor character, crystallinity and functionalization degree of CNT [55,56]. Raman spectra of CNT consist of graphitic or Gband (due to highly ordered CNT sidewalls) and D-band (due to disorder in the sidewall structures). A quantitative measure of defect density in the CNT sidewall can be determined by determining ratios between these two bands (I D:I G). Consequently the structural changes as a result of functionalization can also be analysed by these ratios [53]. In addition, the diameters and electronic structures of CNTs can be determined by using the resonance Raman scattering [57].

2) Estimation of carbonaceous impurities.

Thermogravimetric Analysis (TGA)

3) Estimation of variation in the CNT structures, which include defects in the nanotube walls and tips, degree of functionalization, diameter distribution and length of tube etc.

TGA can be used to estimate CNT purity and the presence of organic molecules attached to CNT sidewalls. This is also used to assay metal impurities by burning CNT samples in air and examining the residual sample mass when all the organics have been volatalized [53,58]. The homogeneity of CNT samples can be evaluated by standard deviations of the oxidation temperature and metal content obtained in several

CHARACTERIZATION OF CARBON NANOTUBES The characterization of CNTs samples mainly comprises the following:

There are different analytical methods for the characterization (qualitative/quantitative) of CNT materials. Table 3


Table 3.


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Analytical Methods for the Characterization of CNT Samples.

Parameters

Technique

Analysis

Purity

TGA

Quantitative- residual mass after TGA in air at 5o C/min to 800oC.

SEM/TEM

Qualitative- amorphous carbon impurities.

EDS

Qualitative- metal content.

Raman

Qualitative-relative amount of carbon impurities and damage/disorder.

Thermal stability

TGA

Quantitative-burning temperature in TGA in air at 5oC/min to 800oC, dM=dT peak maximum.

Identification and quantification of functional groups

IR

Qualitative- Functional group identification.

Chemical derivatization (CD)

Quantitative-Direct quantification of targeted functional groups.

TGA

Quantitative-standard deviation of burning temperature and residual mass taken on 3-5 samples.

SEM/TEM

Qualitative-image comparison.

Ultra-sonication

Qualitative-time required to fully disperse (to the eye) low concentration CNT in DMF using standard settings.

UV/VIS/NIR

Quantitative-relative change in absorption spectra of sonicated low concentration CNT/ DMF solution.

AFM/STM

Quantitative-band gap, conductivity of a single CNT could be measured through DOS plot.

Homogeneity

Dispersability

Electronic property

In CD, a targeted oxygen-containing functional group reacts selectively and stoichiometrically with a specific derivatizing reagent that contains a unique chemical tag. After each derivatization reaction, the concentration of these chemical tags introduced on to the surface can be quantified.

separate TGA run since purer and less defective CNT samples show higher oxidation temperature (>500ºC) [59,60]. APPLICATIONS OF CNTS CNTs in Drug Delivery While attachment of drugs to suitable carriers significantly improve their bioavailability, owing to their increased residence time in blood circulation and enhanced solubility, the therapeutic efficacy of the drug can be improved by the site selective accumulation in the pathological zone of interest that sometimes were called therapeutic-effects-related sites. The unique capability of CNTs to penetrate cell membranes paves the road for using them as carriers to deliver therapeutic agents into the cytoplasm and, in many cases, in to the nucleus [61]. Functionalized CNTs represent an innovative drug delivery system, since they are capable to penetrate into the cell without altering their morphology [62,63]. Covalent and non-covalent functionalizations of therapeutic molecules to CNTs have attracted significant interest from biomedical researchers. It has been demonstrated that fluorescently labelled CNT were taken up by various cell types [63]. The mechanism of penetration is not yet completely elucidated. Two routes of internalization have been proposed. It has been found that f-CNT penetrates following a passive diffusion across the lipid bilayer similar to a ‘‘nanoneedle’’ able to perforate the cell membrane without causing cell death [64, 65]. Wu et al. reported that CNT functionalized with amphotericin B (AmB), one of the most effective antibiotic molecules for the treatment of chronic fungal infections, are rapidly internalized by mammalian cells with a reduced toxicity

in comparison to the drug administered alone [66]. CNTs are readily internalized by cells; and after surface modification, they exhibit low cytotoxicity over the period of a few days. In addition, they have a higher surface area to volume ratio than spheres, giving nanotubes the potential to be conjugated with more functional agents than spheres and to accommodate higher loadings of therapeutic agents [67]. For this reason CNTs have received a lot of attention in the delivery of drug in cancer. In a typical example reported by Dai and coworkers, a water soluble SWCNT-Paclitaxel (PTX) conjugate has been developed by conjugating PTX to functionalized polyethylene glycol SWCNTs via a cleavable ester bond. SWCNT-PTX has been found to be highly efficient in suppressing tumor growth when compared with clinical taxol in a murine 4T1 breast cancer cells, which has attributed to the extended blood circulation (due to PEGylation) and tenfold higher tumor PTX uptake by SWCNT delivery, probably through enhanced permeability and retention (EPR) effect [68]. In another approach, Hosmane and coworkers prepared a SWCNT substituted with a carborane cage for neutron boron capture therapy. After the administration of the conjugates the concentration of the boron atoms was mostly detected in tumor cells rather than in blood or other organs [69]. Similarly, in another study, the poorly water-soluble anticancer drug camptothecin has been loaded into polyvinyl alcohol-functionalized MWCNTs and reported to be potentially effective in treatment of breast and skin cancers [70]. SWCNTs, with all atoms exposed on their surface, have ultra-high surface area available for binding of aromatic molecules via supramolecular π-π stacking. Functionalized SWCNTs with or without targeting ligands can be loaded with doxorubicin (DOX), an aromatic chemotherapy drug


used for various types of cancers, by simply mixing of the two solutions at a slightly basic pH. Based on the UV-VISNIR absorption spectra, up to 4 grams of DOX can be loaded on 1 gram of SWCNTs. The toxicity of SWCNT-DOX is lower than that of free DOX. PEGylated MWCNTs functionalized and loaded with angiopep2 (ANG) and doxorubicin showed better anti-glioma effect on C6 glioma cells, compared with that of free doxorubicin [43]. Triple functionalized SWCNTs were fabricated with an anticancer drug (Doxorubicin), a monoclonal antibody and a fluorescent marker (fluorescein) at the non-competitive binding sites on the SWCNTs for targeting the cancer cells. Confocal laser microscopy reveals the Bovine serum albumin-antibody specific receptor mediated uptake of SWCNTs by the Human colon adenocarcinoma cell (WiDr cells) with subsequent targeting of Doxorubicin intracellularly to the nucleus [44]. A difunctionalized MWCNT has been developed by conjugating Folic acid and iron nanoparticles with oxidized MWCNTs (FA-MWCNT-Fe). The developed dual-targeted drug nanocarrier delivered DOX into HeLa cells with the assistance of an external magnetic field. It was concluded that the developed nanocarrier displayed biologic (active) and magnetic (passive) targeting characteristics toward HeLa cells and was found to be six-fold more efficient in delivering DOX into the cells when compared with ‘free’ DOX uptake [71]. An enhanced targeted delivery of daunorubicin (Dau) to acute lymphoblastic leukemia was achieved by Taghdisi et al. They developed a tertiary complex of Sgc8c aptamer, daunorubicin and SWCNT named as Dau-aptamerSWCNTs. Flow cytometric analysis showed that the tertiary complex was internalized effectively into Human T cell leukemia cell (MOLT-4 cells) but not to U266 myeloma cells [72].Yang et al., compared the in vitro and in vivo potential therapeutic effect of Gemcitabine (GEM) loaded magnetic MWCNTs (mMWCNTs) with that of Gemcitabine loaded magnetic-carbon particles (mACs). His findings reflected the high anti-tumor activity in human pancreatic cancer BxPC-3 cells of both the systems when compared along with free drug but mMWCNTs-GEM was proved to be superior to mACs-GEM in successful inhibition of lymph node metastasis after following subcutaneous administration under the impact of magnetic field [73]. CNTs in Gene Delivery Gene therapy is one of the most promising approache to treat a variety of different diseases, such as cancer and genetic disorders. Gene delivery is based on the development and use of viral and non-viral vector systems. The introduction of foreign DNA to cells is another major area for therapeutic delivery using CNTs. Both single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) have been modified with positively charged biomolecules such as ammonium group and cationic amino acid lysine, which lead to easy complex formation with genes of interest [74]. Non viral vectors are less efficient than viral vectors and short lived; however, they are far safer. Generally, functionalized SWCNTs have been suggested as suitable non-viral carriers of macromolecules and internalization of such macromolecules into living cells by

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CNTs has been reported to take place via energy-dependent endocytosis [8]. Guo and coworkers conjugated amino-functionalized MWCNTs (NH2-MWCNTs) to plasmid DNA and successfully achieved the delivery of a green fluorescence protein (GFP) gene into cultured human umbilical vein endothelial cells [75]. In another study, Cai et al. used nanotube spearing, utilizing the magnetic properties of nickel-embedded single-wall carbon nanotubes (SWCNTs), to deliver DNA plasmid vectors containing the sequence for EGFP (a fluorescent protein) to Bal17 and Mouse splenic B cells, which are non dividing cells, and therefore notoriously difficult to transfect [65]. In another example reported by Pantratto and coworkers, DNA was linked to functionalized CNTs via electrostatic interactions and delivered to cells. It was found that the gene expression levels were up to ten times higher than those achieved with DNA alone [64]. Reports have indicated that functionalized positively charged CNTs can condense DNA efficiently; however both surface area and charge density of nanotube are critical parameters that determine the interaction and electrostatic complex formation between CNTs and DNA of interest [74]. MWCNTs based targeting of the gene to the tumor cells was investigated by Pan et al. In this work, they fabricated MWCNTs modified with polyamidoamine dendrimer which were further conjugated with FITC-labelled antisense c-myc oligonucleotides (asODN). Human breast cancer cell line MCF-7 cells, MDAMB-435 cells were incubated with modified MWCNTs (asODN-dMNTs). Fluorescence developed by the FITC revealed the cellular uptake of asODN-dMNTs within 15 min. These composites inhibit the cell growth in time and dose dependent means and down regulated the expression of cmyc gene (over expression of this gene amplify the expression of HER2) and C-Myc protein [76]. A chemically functionalized SWCNT carrier has been developed for the effective delivery of SiRNA and SiRNA-MDM2 complexes to the breast carcinoma B-Cap-37 cells. Results proved the high efficiency of F-SWCNT in carrying SiRNA to the carcinoma cells and the new F-SWCNT-SiRNA-MDM2 complexes caused 44.53% inhibition of proliferating B-Cap-37 carcinoma cells for 72 hours by down regulating the expression of c-myc gene [77]. Li et al., developed a novel targeting SiRNA delivery system by using SWCNTs which were chemically functionalized with polyethylenimine and bound by DSPE-PEG 2000 maleimide for further conjugation with tumor targeting Asn-Gly-Arg (NGR) peptide. This novel system efficiently crosses human prostate cancer cell (PC-3 cell) membrane in vitro, induce more severe apoptosis and suppression in the proliferating cells. The combination of near-infrared photothermal therapy and RNAi significantly enhanced the antitumor activity without causing toxicity to other organs [78]. CNTs in Peptide Delivery CNTs not only deliver small drug molecules but can also deliver proteins. In a study, B-cell epitope of the foot-andmouth disease virus (FMDV) was covalently attached to the amine groups present on CNT, using a bifunctional linker. The peptides around the CNT adopt the appropriate secondary structure for recognition by specific monoclonal and polyclonal antibodies. The immunogenic features of peptide-


CNT conjugates were subsequently assessed in vivo [79]. Immunisation of mice with FMDV peptide-nanotube conjugates elicited high antibody responses as compared with the free peptide as complement activation is involved in immune response to antigens, this might support the enhancement of antibody response following immunisation with peptideCNT conjugates [80]. Wang et al. explored the potential of CD133 monoclonal antibody (anti-CD133) conjugated SWCNTs for therapeutic targeting of CD133 cancer stem like cells (CSCs). Glioblastoma (GBM)-CD133+ cells were selectively targeted and eradicated whereas GBM-CD133cells remained viable. Furthermore, anti-CD133-SWCNTs pretreated GBM-CD133+ cells were irradiated with nearinfrared laser for 2 days and showed no sign of sustainability of CSCs for tumor growth after xenotransplantation in nude mice [81]. From this report it is stated that, monoclonal antibody conjugated SWCNTs are capable of selectively targeting the CSCs as well as blocking their recurrence. MWCNTs have been used as cellular carriers of recombined ricin A chain protein toxin (RAT) for tumor targeting. The complexes of RAT and MWCNT were capable of translocating in the cytoplasm of various cell lines, including L929, HL7702, MCF-7, HeLa, and COS-7, and showed excellent performance of their biological functions, as evidenced by the effects of inducing cell apoptosis or death. Tumor targeting CNT constructs were synthesized by Mc Devitt et al. from a water soluble precursor CNTs functionalized with covalently conjugating multiple copies of tumor specific mAbs, radiometal ion chelates, and fluorescent probes. The experiments were designed to observe the target effects in a model of disseminated human lymphoma and in cells by flow cytometry and cell based immunoreactivity assays versus appropriate control cells. They demonstrated that the nanoconstructs were selectively reactive with human cancer cells [61]. Integrin αvβ3 monoclonal antibody conjugated phospholipid-PEG functionalized SWCNTs were developed which showed high targeting efficiency of modified SWCNTs on integrin αvβ3-positive Human Neuronal Glioblastoma (U-87 MG) cells with low cellular toxicity whereas showed the low targeting efficiency to integrin αvβ3-negative Human breast carcinoma cells (MCF-7cells) [42]. Proteins and peptides are the most versatile natural molecular bricks, due to their extensive chemical, conformational and functional diversity. The interaction between CNTs and proteins in biological media can affect the way cells interact with, recognize and process the nanoparticles and this has an important implication for safety considerations. However, the studies on the CNT-organic nanoparticle hybrid architectures are poorly developed comparatively. For example, there are not enough studies on the influence that the nanomaterial properties (such as composition, morphology, and surface chemistry) have on the structure and function of conjugated proteins. The most important parameter in all such studies is the type of CNTs used, which is determined by (i) the preparation and manufacturing process followed; (ii) the structural characteristics of the CNTs; (iii) the surface characteristics of the CNTs (iv) type of functionalization; and other parameters related to proteins or peptides such as their molecular structure (simple, complex, helical), molecular weight (low, high), length and hydrophobicity. It


9

is necessary to obtain the proper formulation of the complex with the therapeutic molecule to generate a vehicle capable of being transported in the blood if a systemic administration is needed and retaining a significant stability before reaching the target cell. In a recent reported literature by Saifuddin et al. [82], it was concluded that the structure and stability of proteins or enzymes after conjugation depends on the type of enzyme or protein and nanotubes, functional groups on the surface of the CNTs and the techniques of conjugation. Since CNTs possess large surface area, minimized mass transfer resistance and effective enzyme loading properties, therefore, various biomolecules such as proteins, nucleic acid and carbohydrates can be conjugated with it. They reviewed that proteins can interact with nanotubes with multiple types of interactions. In noncovalent approach, the protein molecules adsorbed onto the CNT by hydrophobic and electrostatic interactions. These interactions influence protein or enzyme structure and functions. Although noncovalent attachment preserves the unique properties of enzymes and CNTs but studies have revealed that structure of protein was distorted or partially unfolded due to strong interactions with the CNTs that results in the significant loss of alpha-helix content of proteins. For example, partially unfolding of protein was observed when Albumin Lysozyme was conjugated with SWCNTs. Apart from this, this approach also showed lower enzyme loading as compared to other methods [82]. On the other hand, covalent approach involves either direct covalent linking of proteins onto the CNTs or indirect covalent attachment using linking molecules. The covalent conjugation provides durable attachment of proteins but it diminishes the mechanical and electrical properties of CNTs due to the involvement of harsh oxidation step which disrupts the π -networks of CNT surfaces [82]. CNTs in Imaging CNT-based contrast agent may enhance molecular imaging by improving detection sensitivity and selectivity. The strategies developed for design of CNT-based contrast agents for biomedical imaging include encapsulation of medically relevant metal ions within their carbon sheath, the functionalization of the carbon sheath with a variety of imaging agents, and exploiting the intrinsic physical properties of the CNTs. The semiconducting SWCNTs exhibit photoluminescence in the near infrared (NIR) range. The emission ranges of 800-2000 nm cover the biological tissue transparency window, which is therefore suitable for the imaging of biological systems through the luminescence created by semiconducting SWCNTs [83]. The dynamic spectral capabilities of SWCNTs make them suitable for a wide variety of sensing and monitoring applications. For example, Weisman and co-workers tested the NIR fluorescence microscopy of SWCNTs in phagocytic cells. NIR fluorescence imaging revealed that there was no difference in population growth, adhesion, morphology and confluence between the control and the cultures containing SWCNTs. Detectable emission was seen only in cells incubated with SWCNTs [84,85]. As


reported by Weisman et al. NIR imaging studies suggest the effectiveness of SWCNTs as NIR probes for studying individual nanotubes in tissue specimens or inside living organisms during the course of tissue regeneration [86]. The advent of numerous non-invasive imaging modalities, such as X-ray, computed tomography, single photon emission-computed tomography, magnetic resonance imaging (MRI), ultrasound imaging, radio frequency (rf), and optical imaging now allow scientists and clinicians to acquire in vivo images of the anatomy and physiology of animals and humans. Thermoacoustic tomography/Photoacoustic tomography (TAT/PAT) synergizes the advantages of pureultrasound and pure-rf/optical imaging, allowing both satisfactory spatial resolution and high soft-tissue contrast. For instance, PAT is a unique non-invasive technology for imaging and quantifying the levels of vascularization and oxygen saturation in tumors. Pramanik et al. in recent in vivo studies in mice have shown that SWCNT enhanced PA imaging of the lymph nodes and vasculature with a high contrast to CNTs in regenerative medicine [87]. SWCNTs have also been proposed as contrast agents for hyperpolarized 13C MRI in a patent by Hurd et al. SWCNTs exposed to hyperpolarization techniques excite nuclear spin transitions in the 13C nuclei of the SWCNTs. The technology enhances the ability to detect 13C magnetic resonance signals and generate a clear image of 13C in living tissues in comparison to 1H MRI. In a patent by Takahashi et al. the ability of SWCNTs to emit electrons by field emission at room temperature was reported which claims the unique thermal properties of SWCNTs to control the cathode temperatures in such a manner that the energy emitted by the electron stream from the cathode is majorly converted to X-rays instead of thermal energy and hence increases the lifetime of cathode filament [85]. To image and track SWCNTs in vivo by positron emission tomography (PET), SWCNTs are labelled with a radioactive isotope. PET imaging provides three dimensional distribution information of radio labelled nanotubes in live mice at the real time. To obtain RGD conjugated radiolabeled SWCNTs, SWCNTs are first reacted with a mixture of sulfoSMCC and N-Hydroxysuccinimide (NHS) activated 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA), and then conjugated to RGD-SH. After removal of excess reagents, 64Cu radioactive isotope can then be complexed to the DOTA rings on the SWCNTs to achieve radiolabeling. The radiolabeled, targeted SWCNT bioconjugate can be used for in vivo PET imaging of mice bearing integrin αvβ3 positive e.g., U87MG human glioblastomas tumors. 510 million of U87MG cells should be injected subcutaneously on the shoulder of a nude mouse. The mice can be used 2-3 weeks after tumor inoculation. PET imaging should be carried out at 0.5 h, 2 h, 4 h, 6 h and 24 h post injection (p.i.). Mice may be sacrificed at 24 h p.i. when the blood circulation of nanotubes is ended [88]. CNTs have been incorporated in electrochemical sensors to detect proteins, neurotransmitters and small bio-molecules for medical applications. Several electrochemical methods are used in these sensors including direct electrochemical detection of DNA biosensors, indirect detection of an oxidation product using enzyme sensors, and detection of conductivity changes using CNT field effect transistors (FETs) [25]. For example Insulin-like Growth Factor 1 (IGF-1) is a 70-

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amino acid polypeptide with a high degree of homology to insulin. The level of IGF-1 positively associated with breast cancer risk in women. Real-time detection of breast cancer cells using antibody-conjugated CNT-FETs was investigated. In this study, the conjugation of IGF-1 receptorspecific antibodies to CNT-FET devices caused a conductance decrease. Subsequent addition of BT474 or MCF7 breast cancer cells dramatically increased the conductance, demonstrating the possibility of detecting specific cell surface antigens on cancer cells using functionalized CNT-FETs [89, 90]. For electrical detection of DNA, the assay sensitivity was found to be higher with alkaline phosphatase (ALP) enzyme linked to CNTs than with ALP alone. The sensitivity of the assay using SWCNT-DNA sensor obtained by integration of SWCNTs with single-strand DNAs (ssDNA) was considerably higher than traditional fluorescent and hybridization assays. This CNT-biosensor-linked assay can be modified for antigen detection by using specific antibodyantigen recognition. Thus, it could provide a fast and simple solution for molecular diagnosis in pathologies where molecular markers exist, such as DNA or protein. [91]. Dopamine is a neurotransmitter and a neurohormone belonging to the catecholamine family of phenolic compounds. Dopamine is used as a drug; it acts on the nervous system to increase heart rate and blood pressure. A deficiency of dopamine in the brain is believed to cause schizophrenia and Parkinson’s disease. Thus sensing of dopamine in brain tissue is vital in clinical diagnoses. The first ever reported biosensor using CNT-based electrodes was a dopamine biosensor. It consisted of a MWCNT paste electrode, which was used to detect dopamine in goat’s brain tissue homogenate by monitoring its reversible electrochemical oxidation using Differential Pulse Voltammetry (DPV). Cytochrome c is a highly conserved protein present in a wide spectrum of plant and animal species. It is a small protein that plays an important role in the mitochondrial electron transfer chain, which renders its detection important. The electrochemical response of cytochrome c is very poor at bare metal electrodes mainly due to denaturation at the electrode surface. Voltammetric detection of cytochrome c has been possible using a glassy carbon electrode (GCE) modified with activated SWCNTs [92]. CNTs in Tissue Regeneration CNTs can be ideal candidates in the field of regenerative medicine due to their excellent mechanical properties and special thin linear shape with suitable length close to the size of the triple helix of collagen fibrils. For example in neural tissue regeneration, Mattson and co-workers showed that functionalized MWCNTs can be used to support neuronal cell attachment and growth [93]. Furthermore, Parpura and co-workers revealed that CNTs functionalized with various bioactive molecules can improve neural regeneration activity including outgrowth, neurite branching, and attachment of growth cones. In addition, it was revealed that the high electrical conductivity of CNTs can enhance neuronal circuit activities [94]. Maurizio Prato et al. highlighted the exceptional ability of MWCNTs in interfering with nerve tissue growth. Electrophysiological studies associated to gene expression analysis were performed and the result indicates that spinal neurons plated on electro-conductive CNTs


showed a facilitated development. Hence, tissue scaffolds blended with conductive nanotubes may be exploited to promote cell differentiation and reparative pathways in neural regeneration strategies [95]. Sadeghizadeh et al. designed nerve guide conduits based on freeze-dried silk/single walled CNTs conjugated with fibronectin (SF-SWCNT-FN). The conduits were then implanted to 10 mm left sciatic nerve defects in rats. The histological assessment has showed that nerve regeneration has taken places in proximal region of implanted nerve after 5 weeks following surgery [96]. In a study by Hirata et al., Fibroblast Growth Factor (FGF)-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF [97]. Abarrategi et al. created SWCNT-incorporated chitosan scaffolds. They demonstrated the ability of these nanocomposites to support cell growth in vitro using C2Cl2 cells which are derived from the C2 myogenic cell line. Meng et al. created polyurethane MWCNT nanocomposites, and cultured fibroblast cells onto these nanofibrous scaffolds. Their results show favorable interactions between the cells and the polyurethane surface. These fibroblasts were capable of proliferating and secreting proteins. In vivo studies of SWCNT– PPF (Polypropylene fumarate) scaffolds implanted into rabbit tibia showed that after 12 weeks there was a change in the hard tissue response with increased levels of collagen in the extracellular matrix of these scaffolds. Inflammatory cells appeared within the scaffold after 4 weeks, but this level decreased after 12 weeks. The results also suggested that these SWCNT-PPF scaffolds were potentially bioactive and could promote osteogenesis [98]. TOXICITY RISKS DUE TO CNTS The key factors which are responsible for the potential toxicity of fibrous nanomaterials like CNTs are physical dimensions, surface properties, biopersistency, and residual metal content that generate reactive oxygen species [99]. The biopersistency (the property of material to persist and be retained the tissues or organ over time without normal physiological clearance and any changes in the physical or chemical characteristics of the fibers) is associated with the property of CNTs to agglomerate into the microscopic bundles or ropes due to van der Waals forces between the molecules [100,101]. Exposure of CNTs to living cells cause induction of inflammation, alteration in the antioxidant levels and protein expression, apoptosis, these all events lead to the nanotoxicity. The carcinogenicity and toxic effects of CNTs exposure on the respiratory system are mainly linked with the structure analogy (high aspect ratio and dimensions) of CNTs with other toxic fibres such as asbestos which are known to be extremely pathogenic. Takagi et al. demonstrated the carcinogenic potential of MWCNT to induce mesothelioma when administered intraperitoneally to p53 heterozygous mice. The result suggested the similarity of postulated carcinogenesis mechanism between asbestos with that of CNT


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[102]. In a similar study, Sakamoto et al. assessed the carcinogenicity of MWCNTs by mesothelioma development in intact male Fischer 344 rats, when administered intrascrotally [103]. Lam et al. investigated pulmonary toxicity of SWCNT in mice after intratracheal instillation. They observed dose dependent epitheloid granulomas and in some cases interstitial inflammation in mice after 7 days of instillation. These lesions were more pronounced after 90 days with peribronchial inflammation and necrosis that had extended into the alveolar septa [104]. Apart from the asbestos-like effects in lung toxicity mediated by MWCNT, presence of residual metal content also plays an important role in determining different cellular events to MWCNT. Therefore, in a study, Aldieri et al. exposed the murine alveolar macrophages to two different MWCNTs samples, which differed only in the presence or absence of iron. They showed that iron content is also responsible in promoting MWCNT toxicity, since iron-rich MWCNTs exerted cytotoxicity, genotoxicity and induced cellular oxidative stress as compared to pristine MWCNT [105]. CNTs induced pulmonary toxicity was also investigated by Ravichandran et al. In his study BALB/c mice were exposed to aerosolized SWCNT and MWCNT (5µg/g of mice) for 7 days in a nose-only exposure system and the pulmonary toxicities were assessed. The results showed that inhaled CNTs induce inflammation, fibrosis, alteration of oxidant and antioxidant levels and induction of apoptosis related proteins in the lung tissues to trigger cell death [106]. Although pulmonary toxicity and carcinogenicity are main toxicity of concern, but other toxicity mediated by CNTs has been also studied by various researchers. In this context, Urankar et al. reported that oropharyngeal aspiration of MWCNT aggravates cardiac ischemia and repurfusion injury and this may pose a significant risk to the cardiovascular system [107]. Since graphite and other carbon allotropes have been associated with the increase incidences of dermal toxicity like dermatitis and keratosis. Therefore potential of CNTs to cause dermal toxicity needs to be investigated. In a study, Shvedova et al. studied the adverse effects of SWCNTs using a cell culture of immortalized human epidermal kaeratinocytes (HaCaT). After 18 h of exposure, dermal toxicity was observed, indicated by formation of free radicals, antioxidant depletion, loss of cell viability also resulted in ultrastructural and morphological changes in cultured cell lines [108]. In another study, Patlolla et al. investigated the toxicity of purified MWCNTs in normal human dermal fibroblast cells using cell viability, DNA damage and apoptosis as the toxicological end points. The results clearly indicated a significant increase in cytotoxicity, genotoxicity and apoptosis in normal human dermal fibroblast cell line due to exposure of CNTs [109]. Patlolla et al. in his study reported the genetic damage cause by the exposure of MWCNTs. In this study, he compared the clastogenic/ genotoxic potential of functionalized and non functionalized MWCNTs in bone marrow cells of Swiss-Webster mice [110]. Therefore for the risk assessment there is a need of careful monitoring of the fate and toxicological profile after CNTs administration.


CONCLUSION In summary, carbon nanotubes are among those carbon nano materials which possess large surface area with chemically tuneable functional groups. Owing to great physical and mechanical properties, CNT represents themselves as a potent drug carrier as well as a great imaging candidate in medicine. CNTs potential to undergo functionalization with the therapeutic or sensing moieties through a series of chemical reactions has made them promising biocompatible nano candidate for the diagnosis and targeted treatment of refractory diseases such as cancer, CNS disorder and infectious diseases and also in the field of tissue regeneration. However, one major hurdle that needs to be addressed is the issue of toxicity, research on the potential toxicity of CNTs is still underway. Therefore, more rigorous in vitro and in vivo testing is not only worth pursuing, but very necessary.

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[8] [9] [10]

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CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

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ACKNOWLEDGEMENTS

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Authors are grateful to Prof. R.M. Dubey, Vice Chancellor of IFTM University for providing moral support as well as necessary facilities for the completion of this review manuscript and Department of Pharmaceutical Technology, IFTM University.

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LIST OF ABBREVIATIONS AFM

=

Atomic Force Microscopy

CD

=

Chemical Derivatization

CVD

=

Chemical Vapour Deposition

EDS

=

Energy Dispersive Spectroscopy

f-CNT

=

Functionalized-Carbon Nanotube

IR

=

Infrared

NIR

=

Near Infrared

SEM

=

Scanning Electron Microscopy

STM

=

Scanning Tunneling Microscope

[16] [17]

[18] [19] [20] [21]

SWCNT =

Single-Wall Nanotubes

TEM

=

Transmission Electron Microscopy

[22]

TGA

=

Thermogravimetric Analysis

[23]

Ultraviolet-Visible

[24]

UV/VIS =

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Received: ????????, 2014

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Accepted: ????????, 2014

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